Principles of Pharmacology - The Pathophysiologic Basis of Drug Therapy

P R IN C IP LE S o f P H A R M A C O LO G Y T H E P AT H O P H YS I O LO G I C B A S I S O F D R U G T H E R A P Y Fo u rt h Ed it io n

P R IN C IP LE S o f P H A R M A C O LO G Y T H E P AT H O P H YS I O LO G I C B A S I S O F D R U G T H E R A P Y

Fo u rt h Ed it io n

David E. Go lan, MD, PhD Editor-in-Chief Ehrin J. Arm s tro ng , MD, MS c April W. Arm s tro ng , MD, MPH Associate Editors

Acquisitions Editor: Matthew Hauber Product Development Editor: John Larkin Marketing Manager: Mike McMahon Production Project Manager: Bridgett Dougherty Design Coordinator: Holly McLaughlin Manufacturing Coordinator: Margie Orzech Prepress Vendor: Absolute Service, Inc. Fourth edition Copyright © 2017 Wolters Kluwer. Copyright © 2006, 2011 Wolters Kluwer Health/Lippincott Williams & Wilkins. All rights reserved. This book is protected by copyright. No part o this book may be reproduced or transmitted in any orm or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any in ormation storage and retrieval system without written permission rom the copyright owner, except or brie quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part o their o f cial duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via our website at lww.com (products and services). 9 8 7 6 5 4 3 2 1 Printed in China Library of Congress Cataloging-in-Publication Data Names: Golan, David E., editor. | Armstrong, Ehrin J., editor. | Armstrong, April W., editor. Title: Principles o pharmacology : the pathophysiologic basis o drug therapy / David E. Golan, editor in chie ; Ehrin J. Armstrong, April W. Armstrong, associate editors. Other titles: Principles o pharmacology (Golan) Description: Fourth edition. | Philadelphia : Wolters Kluwer Health, [2017] | Includes bibliographical re erences and index. Identif ers: LCCN 2015048962 | ISBN 9781451191004 Subjects: | MESH: Pharmacological Phenomena | Drug Therapy Classif cation: LCC RM301 | NLM QV 38 | DDC 615/.1—dc23 LC record available at http://lccn.loc.gov/2015048962 This work is provided “as is,” and the publisher disclaims any and all warranties, express or implied, including any warranties as to accuracy, comprehensiveness, or currency o the content o this work. This work is no substitute or individual patient assessment based on healthcare pro essionals’ examination o each patient and consideration o , among other things, age, weight, gender, current or prior medical conditions, medication history, laboratory data, and other actors unique to the patient. The publisher does not provide medical advice or guidance, and this work is merely a re erence tool. Healthcare pro essionals, and not the publisher, are solely responsible or the use o this work including all medical judgments and or any resulting diagnosis and treatments. Given continuous, rapid advances in medical science and health in ormation, independent pro essional verif cation o medical diagnoses, indications, appropriate pharmaceutical selections and dosages, and treatment options should be made and healthcare pro essionals should consult a variety o sources. When prescribing medication, healthcare pro essionals are advised to consult the product in ormation sheet (the manu acturer’s package insert) accompanying each drug to veri y, among other things, conditions o use, warnings, and side e ects and identi y any changes in dosage schedule or contraindications, particularly i the medication to be administered is new, in requently used, or has a narrow therapeutic range. To the maximum extent permitted under applicable law, no responsibility is assumed by the publisher or any injury and/or damage to persons or property, as a matter o products liability, negligence law or otherwise, or rom any re erence to or use by any person o this work. LWW.com

To our students and the patients they will serve

Contents Preface ........................................................................................... ix

S e c t io n IIB

Preface to the First Edition............................................................. xi

Principles of Autonomic and Peripheral Nervous System Pharmacology

Acknowledgments........................................................................ xiii

126

Contributors....................................................................................xv

10 Cholinergic Pharmacology............................................. 127

S E C T IO N I

11 Adrenergic Pharmacology ............................................. 150

Fundamental Principles of Pharmacology

1

1 Drug–Receptor Interactions .............................................. 2 Francis J. Alenghat and David E. Golan

2 Pharmacodynamics ........................................................... 17 Quentin J. Baca and David E. Golan

3 Pharmacokinetics .............................................................. 27 Quentin J. Baca and David E. Golan

4 Drug Metabolism ............................................................... 43 F. Peter Guengerich

5 Drug Transporters .............................................................. 56 Baran A. Ersoy and Keith A. Ho master

6 Drug Toxicity ....................................................................... 70 Michael W. Conner, Catherine Dorian-Conner, Vishal S. Vaidya, Laura C. Green, and David E. Golan Amber Dahlin and Kelan Tantisira

96

Quentin J. Baca, Joshua M. Schulman, and Gary R. Strichartz

S e c t io n IIC Principles of Central Nervous System Pharmacology

183

13 Pharmacology of GABAergic and Glutamatergic Neurotransmission................................ 184 Stuart A. Forman, Hua-Jun Feng, Janet Chou, Jianren Mao, and Eng H. Lo

14 Pharmacology of Dopaminergic Neurotransmission .......................................................... 206 15 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission ....................... 227 16 Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System..... 249 Susannah B. Cornes, Edmund A. Gri f n, Jr., and Daniel H. Lowenstein

17 General Anesthetic Pharmacology............................... 265

S e c t io n IIA 97

8 Principles of Cellular Excitability and Electrochemical Transmission......................................... 98 Elizabeth Mayne, Lauren K. Buhl, and Gary R. Strichartz

9 Principles of Nervous System Physiology and Pharmacology ...................................... 110 Joshua M. Galanter, Susannah B. Cornes, and Daniel H. Lowenstein

12 Local Anesthetic Pharmacology ................................... 167

Stephen J. Haggarty and Roy H. Perlis

S E C T IO N II

Fundamental Principles of Neuropharmacology

Nidhi Gera, Ehrin J. Armstrong, and David E. Golan

David G. Standaert and Victor W. Sung

7 Pharmacogenomics .......................................................... 87

Principles of Neuropharmacology

Alireza Atri, Michael S. Chang, and Gary R. Strichartz

Jacob Wouden and Keith W. Miller

18 Pharmacology of Analgesia ........................................... 288 Robert S. Gri f n and Cli ord J. Wool

19 Pharmacology of Drugs of Abuse ................................. 308 Peter R. Martin and Sachin Patel

S E C T IO N III Principles of Cardiovascular Pharmacology

335

20 Pharmacology of Cholesterol and Lipoprotein Metabolism.................................................. 336 Tibor I. Krisko, Ehrin J. Armstrong, and David E. Cohen vii

viii Contents

21 Pharmacology o Volume Regulation ........................... 358

David A. Barbie and David A. Frank

Hakan R. Toka and Seth L. Alper

22 Pharmacology o Vascular Tone ................................... 385 23 Pharmacology o Hemostasis and Thrombosis .......... 403 Ehrin J. Armstrong and David E. Golan

24 Pharmacology o Cardiac Rhythm ................................ 433 Ehrin J. Armstrong and David E. Clapham

25 Pharmacology o Cardiac Contractility ........................ 454 Ehrin J. Armstrong

S E C T IO N V I Principles o Inf ammation and Immune Pharmacology 782 42 Principles o Inf ammation and the Immune System......................................................... 783 Eryn L. Royer and April W. Armstrong

26 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure ............................................................. 469 James M. McCabe and Ehrin J. Armstrong

43 Pharmacology o Eicosanoids ....................................... 794 David M. Dudzinski and Charles N. Serhan

44 Histamine Pharmacology ............................................... 819 Elizabeth A. Brezinski and April W. Armstrong

S E C T IO N IV 497

27 Pharmacology o the Hypothalamus and Pituitary Gland .......................................................... 498 Anand Vaidya and Ursula B. Kaiser

28 Pharmacology o the Thyroid Gland ............................. 514 Anthony Hollenberg and William W. Chin

29 Pharmacology o the Adrenal Cortex ........................... 524 Rajesh Garg and Gail K. Adler

30 Pharmacology o Reproduction..................................... 541 Ehrin J. Armstrong and Robert L. Barbieri

31 Pharmacology o the Endocrine Pancreas and Glucose Homeostasis .............................................. 561 Giulio R. Romeo and Steven E. Shoelson

32 Pharmacology o Bone Mineral Homeostasis ............ 580 David M. Slovik and Ehrin J. Armstrong

45 Pharmacology o Hematopoiesis and Immunomodulation .................................................. 830 Andrew J. Wagner, Ramy A. Arnaout, and George D. Demetri

46 Pharmacology o Immunosuppression ........................ 844 Elizabeth A. Brezinski, Lloyd B. Klickstein, and April W. Armstrong

47 Integrative Inf ammation Pharmacology: Peptic Ulcer Disease ....................................................... 864 Dalia S. Nagel and Helen M. Shields

48 Integrative Inf ammation Pharmacology: Asthma ...... 877 Joshua M. Galanter and Stephen Lazarus

49 Integrative Inf ammation Pharmacology: Gout ........... 895 Ehrin J. Armstrong and Lloyd B. Klickstein

S E C T IO N V II Environmental Toxicology

904

50 Environmental Toxicology............................................... 905

S E C T IO N V Principles o Chemotherapy

41 Principles o Combination Chemotherapy ................... 770 Quentin J. Baca, Donald M. Coen, and David E. Golan

William M. Oldham and Joseph Loscalzo

Principles o Endocrine Pharmacology

40 Pharmacology o Cancer: Signal Transduction .......... 750

602

33 Principles o Antimicrobial and Antineoplastic Pharmacology ....................................... 603 Donald M. Coen, Vidyasagar Koduri, and David E. Golan

34 Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation ................. 622 Alexander J. McAdam and Donald M. Coen

35 Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis ...................................... 641 David W. Kubiak, Ramy A. Arnaout, and Sarah P. Hammond

Laura C. Green, Sarah R. Armstrong, and Joshua M. Galanter

S E C T IO N V III Fundamentals o Drug Development and Regulation

918

51 Drug Discovery and Preclinical Development ............ 919 John L. Vahle, David L. Hutto, and Maarten Postema

52 Clinical Drug Evaluation and Regulatory Approval........................................................ 933 Mark A. Goldberg and Alexander E. Kuta

53 Systematic Detection o Adverse Drug Events ........... 946 Jerry Avorn

36 Pharmacology o Fungal In ections .............................. 661 Chelsea Ma and April W. Armstrong

37 Pharmacology o Parasitic In ections .......................... 674 Louise C. Ivers and Edward T. Ryan

38 Pharmacology o Viral In ections .................................. 694 Jonathan Z. Li and Donald M. Coen

39 Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance ............................................ 723 David A. Barbie and David A. Frank

S E C T IO N IX Frontiers in Pharmacology

954

54 Protein Therapeutics ....................................................... 955 Quentin J. Baca, Benjamin Leader, and David E. Golan

55 Drug Delivery Modalities ................................................ 979 Joshua D. Moss and Robert Langer

Credit List .................................................................................... 987 Index............................................................................................ 991

Preface The editors are grate ul or many help ul suggestions rom readers o the f rst, second, and third editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy. The ourth edition eatures many changes to re ect the rapidly evolving nature o pharmacology and drug development. We believe that these updates will continue to contribute to the learning and teaching o pharmacology both nationally and internationally: ■

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Comprehensive updates o ull-color f gures throughout the textbook—about 450 in all. Every f gure has been updated and colorized, and over 50 f gures are new or substantially modif ed to highlight advances in our understanding o physiologic, pathophysiologic, and pharmacologic mechanisms. As in the f rst three editions, our collaboration with a single illustrator creates a uni orm “look and eel” among the f gures that acilitates understanding and helps the reader make connections across broad areas o pharmacology. Comprehensive updates and additions in the undamentals o pharmacology. Along with extensive updates in the chapters on drug–receptor interactions, pharmacodynamics, pharmacokinetics, drug metabolism, drug toxicity, and pharmacogenomics, a new chapter on drug transporters has been added. The f rst section o the textbook now provides a comprehensive ramework or the undamental principles o pharmacology that serve as the oundation or material in all subsequent chapters. Comprehensive updates o all 37 drug summary tables. These tables, which have been particularly popular with readers, group drugs and drug classes according to mechanism o action and list clinical applications, serious and common adverse e ects, contraindications, and therapeutic considerations or each drug discussed in the chapter. Comprehensive updates o all chapters, including new drugs approved through 2014–2015. We have ocused especially on newly discovered and revised mechanisms that sharpen our understanding o the physiology,

pathophysiology, and pharmacology o the relevant system. Sections throughout the book contain substantial amounts o new and updated material, especially the chapters on drug–receptor interactions; drug toxicity; pharmacogenomics; adrenergic pharmacology; local anesthetic pharmacology; the pharmacology o serotonergic and central adrenergic neurotransmission; the pharmacology o analgesia; the pharmacology o cholesterol and lipoprotein metabolism; the pharmacology o volume regulation; the pharmacology o vascular tone; the pharmacology o hemostasis and thrombosis; the pharmacology o the thyroid gland; the pharmacology o the endocrine pancreas and glucose homeostasis; the pharmacology o bone mineral homeostasis; the pharmacology o bacterial DNA replication, transcription, and translation; the pharmacology o bacterial and mycobacterial cell wall synthesis; the pharmacology o viral in ections; the pharmacology o cancer; the pharmacology o eicosanoids; the pharmacology o immunosuppression; the undamentals o drug development and regulation; and protein therapeutics. As with the third edition, we have recruited a panel o new, expert chapter authors who have added tremendous strength and depth to the existing panel o authors, and the editorial team has reviewed each chapter in detail to achieve uni ormity o style, presentation, and currency across the entire text. Finally, we would like to acknowledge the immeasurable contributions o the late Armen H. Tashjian, Jr., MD, to the conception, design, and implementation o this text. Armen was our riend, mentor, and close colleague, and his indomitable spirit lives on in this ourth edition o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy. David E. Golan, MD, PhD Ehrin J . Armstrong, MD, MSc April W. Armstrong, MD, MPH

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Preface t o t h e Firs t Ed it io n This book represents a new approach to the teaching o a f rst or second year medical school pharmacology course. The book, titled Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy, departs rom standard pharmacology textbooks in several ways. Principles of Pharmacology provides an understanding o drug action in the ramework o human physiology, biochemistry, and pathophysiology. Each section o the book presents the pharmacology o a particular physiologic or biochemical system, such as the cardiovascular system or the in ammation cascade. Chapters within each section present the pharmacology o a particular aspect o that system, such as vascular tone or eicosanoids. Each chapter presents a clinical vignette, illustrating the relevance o the system under consideration; then discusses the biochemistry, physiology, and pathophysiology o the system; and, f nally, presents the drugs and drug classes that activate or inhibit the system by interacting with specif c molecular and cellular targets. In this scheme, the therapeutic and adverse actions o drugs are understood in the ramework o the drug’s mechanism o action. The physiology, biochemistry, and pathophysiology are illustrated using clear and concise f gures, and the pharmacology is depicted by displaying the targets in the system on which various drugs and drug classes act. Material rom the clinical vignette is re erenced at appropriate points in the discussion o the system. Contemporary directions in molecular and human pharmacology are introduced in chapters on modern methods o drug discovery and drug delivery and in a chapter on pharmacogenomics.

This approach has several advantages. We anticipate that students will use the text not only to learn pharmacology but also to review essential aspects o physiology, biochemistry, and pathophysiology. Students will learn pharmacology in a conceptual ramework that osters mechanism-based learning rather than rote memorization, and that allows or ready incorporation o new drugs and drug classes into the student’s und o knowledge. Finally, students will learn pharmacology in a ormat that integrates the actions o drugs rom the level o an individual molecular target to the level o the human patient. The writing and editing o this textbook have employed a close collaboration among Harvard Medical School students and aculty in all aspects o book production, rom student– aculty co-authorship o individual chapters to student– aculty editing o the f nal manuscript. In all, 43 HMS students and 39 HMS aculty have collaborated on the writing o the book’s 52 chapters. This development plan has blended the enthusiasm and perspective o student authors with the experience and expertise o aculty authors to provide a comprehensive and consistent presentation o modern, mechanism-based pharmacology. David E. Golan, MD, PhD Armen H. Tashjian, J r., MD Ehrin J . Armstrong, MD, MSc Joshua M. Galanter, MD April W. Armstrong, MD, MPH Ramy A. Arnaout, MD, DPhil Harris S. Rose, MD FOUNDING EDITORS

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Acknowledgments The editors are grate ul or the support o students and aculty rom around the world who have provided encouragement and help ul suggestions. Stuart Ferguson continued his exemplary work as an executive assistant by managing all aspects o project coordination, including submission o chapter manuscripts, multiple layers o editorial revisions, coordination o f gure generation and revision, and delivery o the f nal manuscript. We are extraordinarily grate ul or his unwavering dedication to this project. Rob Duckwall did a superb job to update the ull-color f gures. Rob’s standardization and coloration o the f gures in this textbook re ect his creativity and expertise as a leading medical illustrator. His artwork is a major asset and highlight o this textbook. Quentin Baca electronically rendered the striking image on the cover o this textbook. We are most grate ul or his creativity and expertise. The editors would like to thank the publication, editorial, and production sta at Wolters Kluwer or their expert management and production o this handsome volume. David Golan would like to thank the many aculty, student, and administrative colleagues whose support and understanding were critical or the success ul completion o this project. Members o the Golan laboratory and aculty and sta in the Department o Biological Chemistry and

Molecular Pharmacology at Harvard Medical School and in the Hematology Division at Brigham and Women’s Hospital and the Dana-Farber Cancer Institute were gracious and supportive throughout. Deans Je rey Flier and John Czajkowski were especially supportive and encouraging. Laura, Liza, and Sarah provided valuable insights at many critical stages o this project and were constant sources o support and love. Ehrin Armstrong would like to thank colleagues at the University o Colorado and the Denver Veterans Administration Medical Center or providing academic support and guidance. Greg Schwartz and Jim Beck were especially encouraging. Ki any, Larry, and Ginger were a constant source o support and love throughout. April Armstrong would like to thank Drs. David Golan and Laura Green or their constant support over the years. She thanks her dedicated coauthors Eryn Royer, Elizabeth Brezinski, and Chelsea Ma or their hard work. She also thanks Drs. David Norris, David West, and Fu-Tong Liu or ostering her career. She is grate ul or the love o her amily—Amy, Yanni, and Susan. Credit lines identi ying the original source o a f gure or table borrowed or adopted rom copyrighted material, and acknowledging the use o noncopyrighted material, are gathered together in a list at the end o the book. We thank all o these sources or permission to use this material.

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Contributors Gail K. Adler, MD, PhD Associate Pro essor o Medicine Harvard Medical School Associate Physician Division o Endocrinology, Diabetes and Hypertension Department o Medicine Brigham and Women’s Hospital Boston, Massachusetts Francis J . Alenghat, MD, PhD Assistant Pro essor Department o Medicine, Section o Cardiology University o Chicago Chicago, Illinois Seth L. Alper, MD, PhD Pro essor o Medicine Harvard Medical School Renal Division and Molecular and Vascular Medicine Division Department o Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts April W. Armstrong, MD, MPH Associate Dean or Clinical Research Director o Clinical Research, Southern Cali ornia Clinical and Translational Science Institute (SC CTSI) Vice Chair, Department o Dermatology Associate Pro essor o Dermatology University o Southern Cali ornia Los Angeles, Cali ornia Ehrin J . Armstrong, MD, MSc Associate Pro essor o Medicine Division o Cardiology University o Colorado School o Medicine Denver, Colorado Sarah R. Armstrong, MS, DABT Consultant in Toxicology Amherst, Massachusetts

Ramy A. Arnaout, MD, DPhil Assistant Pro essor o Pathology Harvard Medical School Associate Director, Clinical Microbiology Department o Pathology Beth Israel Deaconess Medical Center Boston, Massachusetts Alireza Atri, MD, PhD Ray Dolby Endowed Chair in Brain Health Research Ray Dolby Brain Health Center Cali ornia Pacif c Medical Center San Francisco, Cali ornia Visiting Scientist in Neurology Harvard Medical School Boston, Massachusetts J erry Avorn, MD Pro essor o Medicine Harvard Medical School Chie , Division o Pharmacoepidemiology Brigham and Women’s Hospital Boston, Massachusetts Quentin J . Baca, MD, PhD Chie Resident in Anesthesia Department o Anesthesiology, Perioperative and Pain Medicine Stan ord University School o Medicine Palo Alto, Cali ornia David A. Barbie, MD Assistant Pro essor o Medicine Harvard Medical School Associate Physician Department o Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts

Robert L. Barbieri, MD Kate Macy Ladd Pro essor o Obstetrics, Gynecology and Reproductive Biology Department o Obstetrics, Gynecology and Reproductive Biology Harvard Medical School Chairman, Department o Obstetrics and Gynecology Brigham and Women’s Hospital Boston, Massachusetts Elizabeth A. Brezinski, MD Resident in Dermatology Harvard Combined Dermatology Residency Training Program Boston, Massachusetts Lauren K. Buhl, MD, PhD Clinical Fellow in Anaesthesia Harvard Medical School Resident in Anaesthesia Beth Israel Deaconess Medical Center Boston, Massachusetts Michael S. Chang, MD Assistant Pro essor o Orthopedic Surgery University o Arizona College o Medicine Complex Spine Surgeon Sonoran Spine Center Phoenix, Arizona William W. Chin, MD Bertarelli Pro essor o Translational Medical Science, Emeritus Harvard Medical School Boston, Massachusetts Chie Medical O f cer and Executive Vice President Pharmaceutical Research and Manu acturers o America Washington, DC

xv

xvi Contributors

J anet Chou, MD Instructor, Department of Pediatrics Harvard Medical School Assistant in Medicine Department of Immunology Children’s Hospital Boston Boston, Massachusetts David E. Clapham, MD, PhD Aldo R. Castañeda Professor of Cardiovascular Research Professor of Neurobiology Harvard Medical School Chief, Basic Cardiovascular Research Department of Cardiology Children’s Hospital Boston Boston, Massachusetts Donald M. Coen, PhD Professor of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts David E. Cohen, MD, PhD Robert H. Ebert Professor of Medicine and Health Sciences and Technology Director, Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology Harvard Medical School Director of Hepatology Division of Gastroenterology, Hepatology and Endoscopy Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts Michael W. Conner, DVM Vice President Theravance Biopharma, U.S., Inc. South San Francisco, California Susannah B. Cornes, MD Assistant Professor, Department of Neurology University of California, San Francisco Department of Neurology UCSF Medical Center San Francisco, California Amber Dahlin, PhD, MMSc Instructor in Medicine Harvard Medical School Associate Epidemiologist Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital Boston, Massachusetts

George D. Demetri, MD Professor of Medicine Department of Medical Oncology Co-Director, Ludwig Center Harvard Medical School Department of Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts Catherine Dorian-Conner, PharmD, PhD Consultant in Toxicology Half Moon Bay, California David M. Dudzinski, MD, J D Clinical Fellow in Medicine Harvard Medical School Fellow, Department of Cardiology Massachusetts General Hospital Boston, Massachusetts Baran A. Ersoy, PhD Instructor in Medicine Harvard Medical School Investigator Brigham and Women’s Hospital Boston, Massachusetts Hua-J un Feng, MD, PhD Instructor in Anaesthesia Harvard Medical School Assistant in Pharmacology Massachusetts General Hospital Boston, Massachusetts Stuart A. Forman, MD, PhD Associate Professor of Anesthesia Harvard Medical School Boston, Massachusetts David A. Frank, MD, PhD Associate Professor of Medicine Harvard Medical School Departments of Medicine and Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts J oshua M. Galanter, MD Assistant Professor, Department of Medicine University of California, San Francisco San Francisco, California Rajesh Garg, MD Assistant Professor of Medicine Harvard Medical School Associate Physician Division of Endocrinology, Diabetes and Hypertension Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts

Nidhi Gera, PhD Research Fellow Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts David E. Golan, MD, PhD Professor of Biological Chemistry and Molecular Pharmacology George R. Minot Professor of Medicine Dean for Basic Science and Graduate Education Special Advisor for Global Programs Harvard Medical School Senior Physician, Hematology Division, Brigham and Women’s Hospital and Dana-Farber Cancer Institute Department of Biological Chemistry and Molecular Pharmacology, Department of Medicine Harvard Medical School Boston, Massachusetts Mark A. Goldberg, MD Associate Professor of Medicine, Part-time Harvard Medical School Boston, Massachusetts Advisor Medical and Regulatory Strategy Synageva BioPharma Corp. Lexington, Massachusetts Laura C. Green, PhD, DABT President and Senior Toxicologist Green Toxicology, LLC Brookline, Massachusetts Edmund A. Gri f n, J r., MD, PhD Assistant Professor of Clinical Psychiatry Department of Psychiatry Columbia University Attending Psychiatrist New York-Presbyterian Hospital New York, New York Robert S. Gri f n, MD, PhD Clinical Assistant Professor of Anesthesiology Weill Cornell Medical College Assistant Attending Anesthesiologist Hospital for Special Surgery New York, New York F. Peter Guengerich, PhD Professor, Department of Biochemistry Vanderbilt University School of Medicine Nashville, Tennessee

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Stephen J . Haggarty, PhD Associate Pro essor o Neurology Harvard Medical School Director, Chemical Neurobiology Laboratory Center or Human Genetic Research Massachusetts General Hospital Boston, Massachusetts Sarah P. Hammond, MD Assistant Pro essor o Medicine Harvard Medical School Associate Physician Brigham and Women’s Hospital Boston, Massachusetts Keith A. Hoffmaster, PhD Director, Global Program Management Translational Clinical Oncology Novartis Institutes or Biomedical Research Cambridge, Massachusetts Anthony Hollenberg, MD Pro essor o Medicine Harvard Medical School Chie , Division o Endocrinology, Diabetes and Metabolism Beth Israel Deaconess Medical Center Boston, Massachusetts David L. Hutto, DVM, PhD, DACVP Corporate Senior Vice President and Chie Scientif c O f cer—Sa ety Assessment Charles River Laboratories, Inc. Wilmington, Massachusetts Louise C. Ivers, MD, MPH, DTM&H Associate Pro essor o Medicine Harvard Medical School Associate Physician Department o Medicine Brigham and Women’s Hospital Boston, Massachusetts Ursula B. Kaiser, MD Pro essor o Medicine Harvard Medical School Chie , Division o Endocrinology, Diabetes and Hypertension Brigham and Women’s Hospital Boston, Massachusetts Lloyd B. Klickstein, MD, PhD Head o Translational Medicine New Indications Discovery Unit Novartis Institutes or Biomedical Research Cambridge, Massachusetts

Vidyasagar Koduri, MD, PhD Clinical Fellow in Hematology/ Oncology Dana Farber Cancer Institute/Harvard Cancer Center Boston, Massachusetts Tibor I. Krisko, MD Instructor Department o Medicine Harvard Medical School Boston, Massachusetts Sta Gastroenterologist Department o Gastroenterology/ Medicine Boston VA Medical Center Jamaica Plain, Massachusetts David W. Kubiak, PharmD Adjunct Clinical Assistant Pro essor o Pharmacy Practice Massachusetts College o Pharmacy and Health Sciences Adjunct Assistant Pro essor o Pharmacology Massachusetts General Hospital Institute o Health Pro essions Adjunct Clinical Assistant Pro essor o Pharmacy Practice Northeastern University Bouvé College o Heath Sciences Co-Director o Antimicrobial Stewardship and Advanced Practice In ectious Diseases Pharmacy Specialist Brigham and Women’s Hospital Boston, Massachusetts Alexander E. Kuta, PhD Vice President and Head o US Regulatory A airs EMD Serono, Inc. Rockland, Massachusetts Robert Langer, ScD David H. Koch Institute Pro essor Departments o Chemical Engineering and Bioengineering Massachusetts Institute o Technology Cambridge, Massachusetts Senior Lecturer on Surgery Children’s Hospital Boston Boston, Massachusetts Stephen Lazarus, MD Pro essor o Medicine Division o Pulmonary and Critical Care Medicine Director, Training Program in Pulmonary and Critical Care Medicine University o Cali ornia, San Francisco San Francisco, Cali ornia

Contributors

Benjamin Leader, MD, PhD Chie Executive O f cer ReproSource Woburn, Massachusetts J onathan Z. Li, MD, MMSc Assistant Pro essor o Medicine Harvard Medical School Brigham and Women’s Hospital Boston, Massachusetts Eng H. Lo, PhD Pro essor o Radiology Harvard Medical School Director, Neuroprotection Research Laboratory Departments o Radiology and Neurology Massachusetts General Hospital Boston, Massachusetts J oseph Loscalzo, MD, PhD Hersey Pro essor o the Theory and Practice o Medicine Harvard Medical School Chairman, Department o Medicine and Physician-in-Chie Brigham and Women’s Hospital Boston, Massachusetts Daniel H. Lowenstein, MD Pro essor, Department o Neurology University o Cali ornia, San Francisco Director, UCSF Epilepsy Center UCSF Medical Center San Francisco, Cali ornia Chelsea Ma, MD Resident Physician Internal Medicine Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts J ianren Mao, MD, PhD Richard J. Kitz Pro essor o Anaesthesia Research Harvard Medical School Chie , Division o Pain Medicine Massachusetts General Hospital Boston, Massachusetts Peter R. Martin, MD Pro essor, Departments o Psychiatry and Pharmacology Vanderbilt University Director, Division o Addiction Psychiatry and Vanderbilt Addiction Center Vanderbilt University Medical Center Nashville, Tennessee

xviii Contributors

Elizabeth Mayne, MD, PhD Resident in Pediatrics and Child Neurology Department of Pediatrics Stanford University School of Medicine Palo Alto, California Alexander J . McAdam, MD, PhD Associate Professor of Pathology Harvard Medical School Medical Director Infectious Diseases Diagnostic Laboratory Boston Children’s Hospital Boston, Massachusetts J ames M. McCabe, MD Assistant Professor of Medicine University of Washington Director, Cardiac Catheterization Laboratory University of Washington Medical Center Seattle, Washington Keith W. Miller, MA, DPhil Edward Mallinckrodt Professor of Pharmacology Department of Anaesthesia Harvard Medical School Pharmacologist, Department of Anesthesia, Critical Care and Pain Medicine Massachusetts General Hospital Boston, Massachusetts J oshua D. Moss, MD Assistant Professor of Medicine Heart Rhythm Center University of Chicago Medical Center Chicago, Illinois Dalia S. Nagel, MD Clinical Instructor, Department of Ophthalmology Mount Sinai School of Medicine Attending Physician Department of Ophthalmology Mount Sinai Hospital New York, New York William M. Oldham, MD, PhD Instructor in Medicine Harvard Medical School Associate Physician Pulmonary and Critical Care Medicine Brigham and Women’s Hospital Boston, Massachusetts

Sachin Patel, MD, PhD Assistant Professor, Departments of Psychiatry and Molecular Physiology and Biophysics Vanderbilt University Medical Center Nashville, Tennessee Roy H. Perlis, MD, MSc Director, Center for Experimental Drugs and Diagnostics Center for Human Genetic Research and Department of Psychiatry Massachusetts General Hospital Associate Professor of Psychiatry Harvard Medical School Boston, Massachusetts Maarten Postema, PhD Director of Chemistry EISAI Inc. Andover, Massachusetts Giulio R. Romeo, MD Instructor in Medicine Harvard Medical School Staff Physician, Adult Diabetes Section Joslin Diabetes Center Staff Physician, Division of Endocrinology BIDMC Boston, Massachusetts Eryn L. Royer, BA Medical Student University of Colorado School of Medicine Aurora, Colorado Edward T. Ryan, MD Professor of Medicine Harvard Medical School Professor of Immunology and Infectious Diseases Harvard T.H. Chan School of Public Health Director, Tropical Medicine Massachusetts General Hospital Boston, Massachusetts J oshua M. Schulman, MD Assistant Professor of Dermatology University of California, Davis Director of Dermatopathology Sacramento VA Medical Center Sacramento, California

Charles N. Serhan, PhD Simon Gelman Professor of Anaesthesia (Biological Chemistry and Molecular Pharmacology) Department of Anesthesiology, Perioperative and Pain Medicine Harvard Medical School Director, Center for Experimental Therapeutics and Reperfusion Injury Brigham and Women’s Hospital Boston, Massachusetts Helen M. Shields, MD Professor of Medicine Harvard Medical School Physician, Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts Steven E. Shoelson, MD, PhD Professor of Medicine Harvard Medical School Associate Director of Research, Section Head, Cellular and Molecular Physiology Joslin Diabetes Center Boston, Massachusetts David M. Slovik, MD Associate Professor of Medicine Harvard Medical School Endocrine Unit Massachusetts General Hospital Boston, Massachusetts Chief, Division of Endocrinology Newton-Wellesley Hospital Newton, Massachusetts David G. Standaert, MD, PhD John N. Whitaker Professor and Chair, Department of Neurology University of Alabama at Birmingham Director, Division of Movement Disorders University Hospital Birmingham, Alabama Gary R. Strichartz, PhD Professor of Anaesthesia (Pharmacology), Harvard Medical School Director, Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine Brigham and Women’s Hospital Boston, Massachusetts

xix Contributors

Victor W. Sung, MD Associate Professor, Department of Neurology, Division of Movement Disorders The University of Alabama at Birmingham Birmingham, Alabama Kelan Tantisira, MD, MPH Associate Professor of Medicine Harvard Medical School Associate Physician Channing Division of Network Medicine and Division of Pulmonary and Critical Care Medicine Brigham and Women’s Hospital Boston, Massachusetts Hakan R. Toka, MD, PhD Assistant Professor of Medicine Division of Nephrology and Hypertension Eastern Virginia Medical School Norfolk, Virginia

J ohn L. Vahle, DVM, PhD, DACVP Senior Research Pathologist, Department of Toxicology and Pathology Lilly Research Laboratories Indianapolis, Indiana Anand Vaidya, MD Assistant Professor of Medicine (Endocrinology) Harvard Medical School Division of Endocrinology, Diabetes, and Hypertension Brigham and Women’s Hospital Boston, Massachusetts Vishal S. Vaidya, PhD Associate Professor of Medicine Head, Systems Toxicology Program, Laboratory of Systems Pharmacology Harvard Medical School Brigham and Women’s Hospital Associate Professor of Environmental Health Harvard T.H. Chan School of Public Health Boston, Massachusetts

Andrew J . Wagner, MD, PhD Assistant Professor, Department of Medicine Harvard Medical School Medical Director, Ambulatory Oncology Center for Sarcoma and Bone Oncology Dana-Farber Cancer Institute Boston, Massachusetts Clifford J . Woolf, MB, BCh, PhD Professor of Neurology and Neurobiology Harvard Medical School Director, F.M. Kirby Neurobiology Center Children’s Hospital Boston Boston, Massachusetts J acob Wouden, MD Radiologist, Washington Hospital Medical Staff Washington Hospital Healthcare Group Fremont, California

I Fundamental Principles of Pharmacology

B

C

1 Drug–Receptor Interactions Fra n c is J . Ale n g h a t a n d David E. Go la n

α INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–33 INTR CONFORMATION AND D CH CHE EMISTRY Y OF DRUGS AND D RE RECE CEPT PTOR ORS S ..................................2 IImp mpaa ctt of Drug Binding on the Receptor . . . . . . . . . . . . . . . . . . . 5 Membrane Effects on Drug–Receptor Inter erac acti tion ons . . . . . . . . . . 6 MOLECULAR AND CEL LLU LULA LAR R DETERMINANTS OF DR RUG SEL ELEC ECTI TIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 MAJ OR TYPES OF DRUG RECEPTORS . . . . . . . . . . . . . . . . . . . . . . . 6 Tra T rans nsme memb mbra rane ne Ion Cha hannels . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Transmembrane G Protein-Coupl pled ed Recep ec epto tors . . . . . . . . . . . . . 9 Tra rans nsme memb mbra rane ne Receptors with Linked Enzymatic Domains . . . 11 Receptor Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Receptor Tyrosine Phosphatases . . . . . . . . . . . . . . . . . . . . . . 12 Tyrosine y Kinase-Associated Receptors p . . . . . . . . . . . . . . . . . 12 Receptor Serine/Threonine Kinasess . . . . . . . . . . . . . . . . . . . 12 Receptor Guanylyl Cyclasess . . . . . . . . . . . . . . . . . . . . . . . . . . 12

INTRODUCTION Why is it that one drug a ects cardiac unction and another alters the transport o specif c ions in the kidney? Why do antibiotics e ectively kill bacteria but rarely harm patients? These questions can be answered by f rst examining the interaction between a drug and its specif c molecular target and then considering the role o that action in a broader physiologic context. This chapter ocuses on the molecular details o drug–receptor interactions, emphasizing the variety o receptors and their molecular mechanisms. This discussion provides a conceptual basis or the action o the many drugs and drug classes discussed in this book. It also serves as a background or Chapter 2, Pharmacodynamics, which discusses the quantitative relationships between drug–receptor interactions and pharmacologic e ect. Although drugs can theoretically bind to almost any three-dimensional target, most drugs achieve their desired (therapeutic ) e ects by interacting selectively with target molecules that play important physiologic or pathophysiologic roles. In many cases, selectivity o drug binding to receptors also determines the undesired (adverse ) e ects o a drug. In general, drugs are molecules that interact with specif c molecular components o an organism to cause biochemical and physiologic changes within that organism.

2

β

γ

GD G DP

Int n racee llular Recep nt e tors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 IInt ntrra ce cellllular Enzymes and Signal Traa ns nsdu duct c ion Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Transcription Factorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Structural Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Extracellular Targetss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Cell Surface Surf rfac acc e Ad Adhheesion sion si on R Receptors eceptors . . . . . . . . . . . . . . . . . . . . . . . . 14 PROCESSING OF SIGNALS RESULTING FROM DRUG–RECEPTOR INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . 14 CELLULAR REGULATION OF DRUG–RECEPTOR INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 DRUGS THAT DO NOT FIT THE DRUG–RECEPTOR MODEL. . . . . 16 CONCLUSION C ONCL ON CLUS USIO ION NA AND ND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . 16 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Drug receptors are macromolecules that, upon binding to a

drug, mediate those biochemical and physiologic changes.

CONFORMATION AND CHEMISTRY OF DRUGS AND RECEPTORS An understanding o why a drug binds to a particular receptor can be ound in the structure and chemical properties o the two molecules. This section discusses the basic determinants o receptor structure and the chemistry o drug–receptor binding. The discussion here ocuses primarily on the interactions o drugs that are small molecules with target receptors that are mainly macromolecules (especially proteins), but many o these principles also apply to the interactions o antibody- or other protein-based therapeutics with their molecular targets (see Chapter 54, Protein Therapeutics). Because many human and microbial drug receptors are proteins, it is use ul to review the our major levels o protein structure (Fig. 1-1). At the most basic level, proteins consist o long chains o amino acids, the sequences o which are determined by the sequences o the DNA that code or the proteins. A protein’s amino acid sequence is re erred to as its primary structure . Once a long chain o amino acids has been synthesized on a ribosome, many o the amino acids

C h a p t e r 1 Drug–Receptor Interactions

In te n t o n e n jo yin g h is n e w ly o u n d re tire m e n t, Mr. B h a s m a d e a p o in t o p la yin g te n n is a s o te n a s p o s s ib le d u rin g th e p a s t ye a r. Fo r th e p a s t 3 m o n th s , h o w e ve r, h e h a s n o te d in cre a s in g a tig u e . Mo re o ve r, h e is n o w u n a b le to f n is h a m e a l, d e s p ite h is typ ica lly vo ra cio u s a p p e tite . Wo rrie d a n d w o n d e rin g w h a t th e s e s ym p to m s m e a n , Mr. B s ch e d u le s a n a p p o in tm e n t w ith h is d o cto r. On p hys ica l e xa m in a tio n , th e p hys icia n n o te s th a t Mr. B h a s a n e n la rg e d s p le e n , e xte n d in g a p p ro xim a te ly 10 cm b e lo w th e le t co s ta l m a rg in ; th e p hys ica l e xa m is o th e rw is e w ith in n o rm a l lim its . Blo o d te s ts s h o w a n in cre a s e d to ta l w h ite b lo o d ce ll co u n t (70,0 0 0 ce lls /m m 3 ) w ith a n a b s o lu te in cre a s e in n e u tro p h ils , b a n d o rm s , m e ta m ye lo cyte s , a n d m ye lo cyte s , b u t n o b la s t ce lls (u n d i e re n tia te d p re cu rs o r ce lls ). Cyto g e n e tic a n a lys is o m e ta p h a s e ce lls d e m o n s tra te s th a t 90% o Mr. B’s m ye lo id ce lls p o s s e s s th e Ph ila d e lp h ia ch ro m o s o m e (in d ica tin g a tra n s lo ca tio n b e tw e e n ch ro m o s o m e s 9 a n d 22), co n f rm in g th e d ia g n o s is o ch ro n ic m ye lo id le u ke m ia . Th e p hys icia n in itia te s th e ra p y w ith imatinib, a h ig h ly s e le ctive in h ib ito r o th e BCR-Ab l tyro s in e kin a s e u s io n p ro te in th a t is e n co d e d b y th e Ph ila d e lp h ia ch ro m o s o m e . Ove r th e n e xt m o n th , th e ce lls

begin to interact with nearby amino acids in the polypeptide chain. These interactions, which are typically mediated by hydrogen bonding, give rise to the secondary structure o a protein by orming well-def ned con ormations such as the helix, pleated sheet, and barrel. As a result o their highly organized shape, these structures o ten pack tightly with one another, urther def ning the overall shape o the protein. Tertiary structure results rom the interaction o amino acids more distant rom one another along a single amino acid chain. These interactions include hydrogen bond and ionic bond ormation as well as the covalent linkage o sul ur atoms to orm intramolecular disulf de bridges. Finally, polypeptides may oligomerize to orm more complex structures. The con ormation that results rom the interaction o separate polypeptides is re erred to as the quaternary structure . Di erent portions o a protein’s structure generally have di erent a f nities or water, and this eature has an additional e ect on the protein’s shape. Because both the extracellular and intracellular environments are composed primarily o water, hydrophobic protein segments are o ten drawn to the inside o the protein or shielded rom water by insertion into lipid bilayer membranes. Conversely, hydrophilic protein segments are o ten located on a protein’s exterior sur ace. A ter all o this twisting and turning is completed, each protein has a unique shape that determines its unction, location in the body, relationship to cellular membranes, and binding interactions with drugs and other macromolecules. The site on the receptor at which the drug binds is called its binding site . Each binding site has unique chemical characteristics that are determined by the specif c properties o the amino acids that make up the site. The

3

co n ta in in g th e Ph ila d e lp h ia ch ro m o s o m e d is a p p e a r co m p le te ly ro m Mr. B’s b lo o d , a n d h e b e g in s to e e l w e ll e n o u g h to co m p e te in a s e n io rs te n n is to u rn a m e n t. Mr. B co n tin u e s to ta ke im a tin ib e ve ry d a y, a n d h e h a s a co m p le te ly n o rm a l b lo o d co u n t a n d n o a tig u e . He is n o t s u re w h a t th e u tu re w ill b rin g , b u t h e is g la d to h a ve b e e n g ive n th e ch a n ce to e n jo y a h e a lthy re tire m e n t.

Questions 1 . How does imatinib interrupt the activity o the BCR-Abl tyrosine kinase usion protein? 2 . Unlike imatinib, most o the older therapies or chronic myeloid leukemia (such as inter eron- ) had signif cant “ u-like” adverse e ects. Why did these therapies cause signif cant adverse e ects in most patients, whereas (as in this case) imatinib causes adverse e ects in very ew patients? 3 . Why is imatinib a selective therapy or chronic myeloid leukemia? Is this selectivity related to the lack o adverse e ects associated with imatinib therapy? 4 . How does the BCR-Abl protein a ect intracellular signaling pathways?

three-dimensional structure, shape, and reactivity o the site, and the inherent structure, shape, and reactivity o the drug, determine the orientation o the drug with respect to the receptor and govern how tightly these molecules bind to one another. Drug–receptor binding is the result o multiple chemical interactions between the two molecules, some o which are airly weak (such as van der Waals orces) and some o which are extremely strong (such as covalent bonding). The sum total o these interactions provides the specif city o the overall drug–receptor interaction. The avorability o a drug–receptor interaction is re erred to as the a f nity o the drug or its binding site on the receptor. This concept is discussed in more detail in Chapter 2. The chemistry o the local environment in which these interactions occur—such as the hydrophobicity, hydrophilicity, and pKa o amino acids near the binding site—may also a ect the a f nity o the drug–receptor interaction. The primary orces that contribute to drug–receptor a f nity are described below and in Table 1-1. van der Waals orces , resulting rom the polarity induced in a molecule by the shi ting o its electron density in response to the close proximity o another molecule, provide a weak attractive orce or drugs and their receptors. This induced polarity is a ubiquitous component o all molecular interactions. Hydrogen bonds have substantial strength and are o ten important or drug–receptor association. This type o bond is mediated by the interaction between positively polarized hydrogen atoms (which are covalently attached to more electronegative atoms such as nitrogen or oxygen) and negatively polarized atoms (such as oxygen, nitrogen, or sul ur that are covalently attached to less electronegative atoms such as carbon or hydrogen). Ionic interactions ,

4 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY Primary

Amino a cids Secondary

Be ta ple a te d s he e t

Alpha he lix

Tertiary

Be ta ple a te d s he e t

Alpha he lix

Quaternary

FIGURE 1-1. Levels of protein structure. Protein structure can be divided into our levels o complexity, re erred to as primary, secondary, tertiary, and quaternary structure. Primary structure is determined by the sequence o amino acids that make up the polypeptide chain. Secondary structure is determined by the interaction o positively polarized hydrogen atoms with negatively polarized atoms (such as oxygen) on the same polypeptide chain. These interactions result in a number o characteristic secondary patterns o protein con ormation, including the helix and pleated sheet. Tertiary structure is determined by the interactions o amino acids that are relatively ar apart on the protein backbone. These interactions, which include ionic bonds and covalent disulf de linkages (among others), give proteins their characteristic three-dimensional structure. Quaternary structure is determined by the binding interactions among two or more independent protein subunits.

which occur between atoms with opposite charges, are stronger than hydrogen bonds but less strong than covalent bonds. Covalent bonding results rom the sharing o a pair o electrons between two atoms on di erent molecules. Covalent interactions are so strong that, in most cases, they are essentially irreversible. Table 1-1 indicates the mechanism

o interaction and relative strength o each o these types o bonds. As noted above, the environment in which drugs and receptors interact also a ects the avorability o binding. The hydrophobic effect re ers to the mechanism by which the unique properties o the ubiquitous solvent water cause the interaction o a hydrophobic molecule with a hydrophobic binding site to be enhanced. Rarely is drug–receptor binding caused by a single type of interaction; rather, it is a combination of these binding interactions that provides drugs and receptors with the forces necessary to form a stable drug–receptor complex. In general, multiple weak orces comprise the majority o drug–receptor interactions. For example, imatinib orms many van der Waals interactions and hydrogen bonds with the ATP-binding site o the BCR-Abl tyrosine kinase. The sum total o these relatively weak orces creates a strong (high a f nity) interaction between this drug and its receptor (Fig. 1-2). Ionic and hydrophobic interactions exert orce at a greater distance than van der Waals interactions and hydrogen bonds; or this reason, the ormer interactions are o ten critical to initiate the association o a drug and receptor. Although relatively rare, covalent interactions between a drug and its receptor are a special case. The ormation o a covalent bond is o ten essentially irreversible, and in such cases, the drug and receptor orm an inactive complex. To regain activity, the cell must synthesize a new receptor molecule to replace the inactivated protein; and the drug molecule, which is also part o the inactive complex, is generally not available to inhibit other receptor molecules. Drugs that modi y their target receptors (o ten enzymes) through this mechanism are sometimes called suicide substrates . Aspirin is an example o such a drug; it irreversibly acetylates cyclooxygenases to reduce the production o prostaglandins (anti-in ammatory e ect) and thromboxanes (antiplatelet e ect) (see Chapter 43, Pharmacology o Eicosanoids). The molecular structure o a drug dictates the physical and chemical properties that contribute to its specif c binding to the receptor. Important actors include hydrophobicity, ionization state (pKa), con ormation, and stereochemistry o the drug molecule. All o these actors combine to determine the complementarity o the drug to the binding site. Receptor binding pockets are highly specif c, and small changes in the drug can have a large e ect on the a f nity o the drug–receptor interaction. For example, the stereochemistry o the drug has a great impact on the strength o the binding interaction. Warfarin is synthesized and administered as a racemic mixture (a mixture containing 50% o the righthanded molecule and 50% o the le t-handed molecule); however, the S enantiomer is our times more potent than the R because o a stronger interaction o the S orm with its binding site on vitamin K epoxide reductase. Stereochemistry can also a ect toxicity in cases where one enantiomer o a drug causes the desired therapeutic e ect and the other enantiomer causes an undesired toxic e ect, perhaps due to an interaction with a second receptor or to metabolism to a toxic species. Although it is sometimes di f cult or pharmaceutical companies to synthesize and puri y individual enantiomers on a large scale, a number o currently marketed drugs are produced as individual enantiomers in cases where one enantiomer has higher e f cacy and/or lower toxicity than its mirror image.

C h a p t e r 1 Drug–Receptor Interactions

5

TABLE 1-1 Relative Strength o Bonds between Receptors and Drugs BOND TYPE

MECHANISM

BOND STRENGTH

van der Waals

Shi ting electron density in areas o a molecule, or in a molecule as a whole, results in the generation o transient positive or negative charges. These areas interact with transient areas o opposite charge on another molecule.

Hydrogen

Hydrogen atoms bound to nitrogen or oxygen become more positively polarized, allowing them to bond to more negatively polarized atoms such as oxygen, nitrogen, or sul ur.

Ionic

Atoms with an excess o electrons (imparting an overall negative charge on the atom) are attracted to atoms with a def ciency o electrons (imparting an overall positive charge on the atom).

Covalent

Two bonding atoms share electrons.

Impact o Drug Binding on the Receptor How does drug binding produce a biochemical and/or physiologic change in the organism? In the case o receptors with enzymatic activity, the binding site o the drug is o ten the active site at which an enzymatic trans ormation is catalyzed, and the catalytic activity o the enzyme is inhibited by drugs that prevent substrate binding to the site or that covalently modi y the site. In cases where the binding site is not the active site o the enzyme, drugs can cause a change by preventing the binding o endogenous ligands to their receptor binding pockets. In many drug–receptor interactions, however, the binding o a drug to its receptor results in a change in the con ormation o the receptor. Altering the shape o the receptor can a ect its unction, including enhancing the a f nity o the drug or the receptor. Such an interaction is o ten re erred to as induced f t, because the receptor’s con ormation changes so as to improve the quality o the binding interaction.

A

The principle o induced f t suggests that drug–receptor binding can have pro ound e ects on the con ormation o the receptor. By inducing con ormational changes in the receptor, many drugs not only improve the quality o the binding interaction but also alter the action o the receptor. The change in shape induced by the drug is sometimes identical to that caused by the binding o an endogenous ligand. For example, exogenously administered insulin analogues all stimulate the insulin receptor to the same extent, despite their slightly di erent amino acid sequences. In other cases, drug binding alters the shape o the receptor so as to make it more or less unctional than normal. For example, imatinib binding to the BCR-Abl tyrosine kinase causes the protein to assume an enzymatically inactive con ormation, thus inhibiting the kinase activity o the receptor. Another way to describe the induced f t principle is to consider that many receptors exist in multiple con ormational states—such as inactive (or closed), active (or open), and desensitized (or inactivated)—and that the binding o a

B

C

Glu 286

Imatinib

Me t 290

Ile 313

Imatinib

P he 382

Ala 269

Gly 383 As p 381

Activa tion loop of kina s e

As n 368 Thr 315 P he 382

Le u 248

P he 317

As p 381 Tyr 253

Lys 271

As p 363

Arg 367

Tyr 393

Va l 256

Me t 318 Gly 321 Le u 370

FIGURE 1-2. Structural basis o specif c enzyme inhibition: imatinib interaction with the BCR-Abl kinase. A. The kinase portion o the BCR-Abl tyrosine kinase is shown in a ribbon ormat (gray). An analogue o imatinib, a specif c inhibitor o the BCR-Abl tyrosine kinase, is shown as a space-f lling model (blue ). B. Detailed diagram o the intermolecular interactions between the drug (shaded in purple ) and amino acid residues in the BCR-Abl protein. Hydrogen bonds are indicated by dashed lines, while van der Waals interactions (indicated by halos around the amino acid name and its position in the protein sequence) are shown or nine amino acids with hydrophobic side chains. C. The interaction o the drug (blue ) with the BCR-Abl protein (gray) inhibits phosphorylation o a critical activation loop (green-highlighted ribbon format), thus preventing catalytic activity.

6 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

drug to the receptor stabilizes one or more o these con ormations. Quantitative models that incorporate these concepts o drug–receptor interactions are discussed in Chapter 2.

Membrane E ects on Drug–Receptor Interactions The structure o the receptor also determines where the protein is located in relationship to cellular boundaries such as the plasma membrane. Proteins that have large hydrophobic domains are able to reside in the plasma membrane because o the membrane’s high lipid content. Many receptors that span the plasma membrane have lipophilic domains that are located in the membrane and hydrophilic domains that reside in the intracellular and extracellular spaces. Other drug receptors, including a number o transcription regulators (also called transcription actors ), have only hydrophilic domains and reside in the cytoplasm, nucleus, or both. Just as the structure o the receptor determines its location in relationship to the plasma membrane, the structure o a drug a ects its ability to gain access to the receptor. For example, many drugs that are highly water-soluble are unable to pass through the plasma membrane and bind to target molecules in the cytoplasm. Certain hydrophilic drugs are able to pass through transmembrane channels (or use other transport mechanisms) and gain ready access to cytoplasmic receptors. Drugs that are highly lipophilic, such as many steroid hormones, are o ten able to pass through the hydrophobic lipid environment o the plasma membrane without special channels or transporters and thereby gain access to intracellular targets. Drug-induced alterations in receptor shape can allow drugs bound to cell sur ace receptors to a ect unctions inside cells. Many cell sur ace receptors have extracellular domains that are linked to intracellular e ector molecules by receptor domains that span the plasma membrane and extend into the cytoplasm. In some cases, changing the shape o the extracellular domain can alter the con ormation o the membrane-spanning and/or intracellular domains o the receptor, resulting in a change in receptor unction. In other cases, drugs can cross-link the extracellular domains o two receptor molecules, orming a dimeric receptor complex that activates e ector molecules inside the cell. All o these actors—drug and receptor structure, the chemical orces in uencing drug–receptor interaction, drug solubility in water and in the plasma membrane, and the unction o the receptor in its cellular environment—con er substantial specif city on the interactions between drugs and their target receptors. This book discusses numerous examples o drugs that can gain access and bind to receptors, induce con ormational changes in the receptors, and thereby produce biochemical and physiologic e ects. Specif city o drug–receptor binding suggests that, armed with the knowledge o the structure o a receptor, one could theoretically design a drug that interrupts or enhances receptor activity. This process, known as rational drug design, could potentially increase the e f cacy and reduce the toxicity o drugs by optimizing their structure so that they bind more selectively to their targets. Rational drug design was f rst used to develop highly selective agents such as the antiviral protease inhibitor ritonavir and the antineoplastic tyrosine kinase inhibitor imatinib. Indeed, urther rounds o rational drug design have led to the development o second-generation

protease inhibitors and antineoplastics with high a f nity or the mutated drug targets that can evolve in patients who develop resistance to f rst-generation drugs. The rational drug design approach is discussed in greater detail in Chapter 51, Drug Discovery and Preclinical Development.

MOLECULAR AND CELLULAR DETERMINANTS OF DRUG SELECTIVITY The ideal drug would interact only with a molecular target that causes the desired therapeutic e ect but not with molecular targets that cause unwanted adverse e ects. Although no such drug has yet been discovered (i.e., all drugs currently in clinical use have the potential to cause adverse e ects as well as therapeutic e ects; see Chapter 6, Drug Toxicity), pharmacologists can take advantage o several determinants o drug selectivity in an attempt to reach this goal. Selectivity o drug action can be con erred by at least two classes o mechanisms, including (1) the cell-type specif city o receptor subtypes and (2) the cell-type specif city o receptor–e ector coupling. Although many potential receptors or drugs are widely distributed among diverse cell types, some receptors are more limited in their distribution. Systemic administration o drugs that interact with such localized receptors can result in a highly selective therapeutic e ect. For example, drugs that target ubiquitous processes such as DNA synthesis are likely to cause signif cant toxic side e ects; this is the case with many currently available chemotherapeutics or the treatment o cancer. Other drugs that target cell-type restricted processes such as acid generation in the stomach may have ewer adverse e ects. Imatinib, or example, is an extremely selective drug because the BCR-Abl protein is not expressed in normal (noncancerous) cells. In general, the more restricted the cell-type distribution o the receptor targeted by a particular drug, the more selective the drug is likely to be. Similarly, even though many di erent cell types may express the same molecular target or a drug, the e ect o that drug may di er in the various cell types because o di erential receptor–e ector coupling mechanisms or di erential requirements or the drug target in the various cell types. For example, although voltage-gated calcium channels are ubiquitously expressed in the heart, cardiac pacemaker cells are relatively more sensitive to the e ects o calcium channel blocking agents than are cardiac ventricular muscle cells. This di erential e ect is attributable to the act that action potential propagation depends mainly on the action o calcium channels in cardiac pacemaker cells, whereas sodium channels are more important than calcium channels in the action potentials o ventricular muscle cells. In general, the more the receptor–e ector coupling mechanisms di er among the various cell types that express a particular molecular target or a drug, the more selective the drug is likely to be.

MAJ OR TYPES OF DRUG RECEPTORS Given the great diversity o drug molecules, it might seem likely that the interactions between drugs and their molecular targets would be equally diverse. This is only partly true. In act, most o the currently understood drug–receptor interactions can be classif ed into six major groups. These groups comprise the interactions between drugs and (1) transmembrane

C h a p t e r 1 Drug–Receptor Interactions

A

B

C

α

β

7

D

γ

GDP

FIGURE 1-3. Major types of interactions between drugs and receptors. Most drug–receptor interactions can be divided into six groups, our o which are shown here. A. Drugs can bind to ion channels spanning the plasma membrane, causing an alteration in the channel’s conductance. B. Heptahelical receptors spanning the plasma membrane are unctionally coupled to intracellular G proteins. Drugs can in uence the actions o these receptors by binding to the extracellular sur ace or transmembrane region o the receptor. C. Drugs can bind to the extracellular domain o a transmembrane receptor and cause a change in signaling within the cell by activating or inhibiting an enzymatic intracellular domain (rectangular box) o the same receptor molecule. D. Drugs can di use through the plasma membrane and bind to cytoplasmic or nuclear receptors. This is o ten the pathway used by lipophilic drugs (e.g., drugs that bind to steroid hormone receptors). Additionally, drugs can bind to enzymes and other targets in the extracellular space and to cell sur ace adhesion receptors without the need to cross the plasma membrane (not shown).

ion channels; (2) transmembrane receptors coupled to intracellular G proteins; (3) transmembrane receptors with linked enzymatic domains; (4) intracellular receptors, including enzymes, signal transduction molecules, transcription actors, structural proteins, and nucleic acids; (5) extracellular targets; and (6) cell sur ace adhesion receptors (Fig. 1-3). Table 1-2 provides a summary o each major interaction type. Knowing whether and to what extent a drug activates or inhibits its target provides valuable in ormation about the interaction. Although pharmacodynamics (the e ects o drugs on the human body) is covered in detail in the next chapter, it is use ul to state brief y the major pharmacodynamic relationships between drugs and their targets be ore examining the molecular mechanisms o drug–receptor interactions. Agonists are molecules that, upon binding to their targets, cause a change in the activity of those targets. Full agonists bind to and activate their targets to the maximal extent possible. For example, acetylcholine binds to the nicotinic acetylcholine receptor and induces a con ormational change in the receptor-associated ion

channel rom a nonconducting to a ully conducting state. Partial agonists produce a submaximal response upon binding to their targets. Inverse agonists cause constitutively active targets to become inactive. Antagonists inhibit the ability of their targets to be activated (or inactivated) by physiologic or pharmacologic agonists. Drugs that directly block the binding site o a physiologic agonist are called competitive antagonists ; drugs that bind to other sites on the target molecule, and thereby prevent the con ormational change required or receptor activation (or inactivation), may be either noncompetitive or uncompetitive antagonists (see Chapter 2). As the mechanism o each drug– receptor interaction is outlined in the next several sections, it will be use ul to consider at a structural level how these di erent pharmacodynamic e ects could be produced.

Transmembrane Ion Channels Many cellular unctions require the passage o ions and other hydrophilic molecules across the plasma membrane.

TABLE 1-2 Six Major Types of Drug–Receptor Interactions RECEPTOR TYPE

SITE OF DRUG–RECEPTOR INTERACTION

SITE OF RESULTANT ACTION

EXAMPLES

Transmembrane ion channel

Extracellular, intrachannel, or intracellular

Cytoplasm

Amlodipine, diazepam, lidocaine, omeprazole

Transmembrane linked to intracellular G protein

Extracellular or intramembrane

Cytoplasm

Albuterol, loratadine, losartan, metoprolol

Transmembrane with linked enzymatic domain

Extracellular or intracellular

Cytoplasm

Erlotinib, insulin, nesiritide, sunitinib

Intracellular

Cytoplasm or nucleus

Cytoplasm or nucleus

Atorvastatin, doxycycline, levothyroxine, paclitaxel

Extracellular target

Extracellular

Extracellular

Dabigatran, donepezil, etanercept, lisinopril

Adhesion

Extracellular

Extracellular

Eptif batide, natalizumab

8 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Specialized transmembrane channels regulate these processes. The unctions o ion channels are diverse, including undamental roles in neurotransmission, cardiac conduction, muscle contraction, and secretion. Because o this, drugs targeting ion channels can have a substantial impact on major body unctions. Three major mechanisms are used to regulate the activity o transmembrane ion channels. In some channels, the conductance is controlled by ligand binding to the channel. In other channels, the conductance is regulated by changes in voltage across the plasma membrane. In still other channels, the conductance is controlled by ligand binding to plasma membrane receptors that are linked to the channel in some way. The f rst group o channels is re erred to as ligandgated, the second as voltage-gated, and the third as second messenger-regulated. Table 1-3 summarizes the mechanism o activation and unction o each channel type. Channels are generally highly selective or the ions they conduct. For example, action potential propagation in neurons o the central and peripheral nervous systems occurs as a result o the synchronous stimulation o voltage-gated ion channels that permit the selective passage o Na ions into the cell. When the membrane potential in such a neuron becomes su f ciently positive, the voltage-gated Na channels open, allowing a large in ux o extracellular sodium ions that urther depolarizes the cell. The role o ion-selective channels in action potential generation and propagation is discussed in Chapter 8, Principles o Cellular Excitability and Electrochemical Transmission. Most ion channels share some structural similarity, regardless o their ion selectivity, the magnitude o their conductance, or their mechanism o activation (gating) or inactivation. Ion channels are pore- orming macromolecules consisting o one or more protein subunits that pass through the plasma membrane. The ligand-binding domain can be extracellular, within the channel, or intracellular, whereas the domain that interacts with other receptors or modulators is most o ten intracellular. The structures o several ion channels have been determined to atomic resolution; the nicotinic acetylcholine (ACh) receptor provides an example o the structure o an important ligand-gated ion channel. This receptor consists o f ve subunits, each o which crosses the

plasma membrane (Fig. 1-4). Two o the subunits have been designated ; each contains a single extracellular binding site or ACh. In the ree (nonliganded) state o the receptor, the channel is occluded by amino acid side chains and does not allow the passage o ions. Binding o two molecules o acetylcholine to the receptor induces a con ormational change that opens the channel and allows ion conductance. Although the nicotinic ACh receptor appears to assume only two states, open or closed, many ion channels assume other states as well. For example, some ion channels are able to become refractory or inactivated. In this state, the channel’s permeability cannot be altered or a certain period o time, known as the channel’s re ractory period. The voltage-gated sodium channel undergoes a cycle o activation, channel opening, channel closing, and channel inactivation. During the inactivation (re ractory) period, the channel

A α

γ

α

Liga nd binding s ite s

B

α

O

Re ce ptor ga te clos e d

+

N

O

α

Ace tylcholine

Na + C

TABLE 1-3 Three Major Types of Transmembrane Ion Channels CHANNEL TYPE Ligand-gated Voltage-gated

Second messengerregulated

MECHANISM OF ACTIVATION

FUNCTION

Binding o ligand to channel

Altered ion conductance

Change in transmembrane voltage gradient

Altered ion conductance

Binding o ligand to transmembrane receptor with G protein-coupled cytosolic domain, leading to second messenger generation

Second messenger regulates ion conductance o channel

α

α

Na + Re ce ptor ga te ope n

FIGURE 1-4. Ligand-gated nicotinic acetylcholine receptor. A. The plasma membrane acetylcholine (ACh) receptor is composed o f ve subunits—two subunits, a subunit, a subunit, and a subunit. B. The subunit has been removed to show an internal schematic view o the receptor, demonstrating that it orms a transmembrane channel. In the absence o ACh, the receptor gate is closed, and cations (most importantly, sodium ions [Na ]) are unable to traverse the channel. C. When ACh is bound to both subunits, the channel opens, and sodium can pass down its concentration gradient into the cell.

C h a p t e r 1 Drug–Receptor Interactions

cannot be reactivated or a number o milliseconds, even i the membrane potential returns to a voltage that normally stimulates the channel to open. Some drugs bind with di erent a f nities to di erent states o the same ion channel. This state-dependent binding is important in the mechanism o action o some local anesthetic and antiarrhythmic drugs, as discussed in Chapters 12 (Local Anesthetic Pharmacology) and 24 (Pharmacology o Cardiac Rhythm), respectively. Two important classes o drugs that act by altering the conductance o ion channels are the local anesthetics and the benzodiazepines. Local anesthetics block the conductance o sodium ions through voltage-gated sodium channels in neurons that transmit pain in ormation rom the periphery to the central nervous system, thereby preventing action potential propagation and, hence, pain perception (nociception). Benzodiazepines also act on the nervous system, but by a di erent mechanism. These drugs inhibit neurotransmission in the central nervous system by potentiating the ability o the neurotransmitter gamma-aminobutyric acid (GABA) to increase the conductance o chloride ions across neuronal membranes, thereby driving the membrane potential urther away rom its threshold or activation.

Transmembrane G Protein-Coupled Receptors G protein-coupled receptors are the most abundant class o

receptors in the human body. These receptors are exposed at the extracellular sur ace o the plasma membrane, traverse the membrane, and possess intracellular regions that activate a unique class o signaling molecules called G proteins . (G proteins are so named because they bind the guanine nucleotides GTP and GDP.) G protein-coupled signaling mechanisms are involved in many important processes, including vision, ol action, and neurotransmission.

9

G protein-coupled receptors have seven transmembrane regions within a single polypeptide chain. Each transmembrane region consists o a single helix, and the helices are arranged in a characteristic structural moti that is similar in all members o this receptor class. The extracellular domain o this class o proteins usually contains the ligand-binding region, although some G protein-coupled receptors bind ligands within the transmembrane domain o the receptor. G proteins have and subunits that are noncovalently linked in the resting state. Stimulation o a G protein-coupled receptor causes its cytoplasmic domain to bind and activate a nearby G protein, whereupon the subunit o the G protein exchanges GDP or GTP. The -GTP subunit then dissociates rom the subunit, and the or subunit di uses along the inner lea et o the plasma membrane to interact with a number o di erent e ectors. These e ectors include adenylyl cyclase, phospholipase C, various ion channels, and other classes o proteins. Signals mediated by G proteins are usually terminated by the hydrolysis o GTP to GDP, which is catalyzed by the inherent GTPase activity o the subunit (Fig. 1-5). One major role o the G proteins is to activate the production o second messengers ; that is, signaling molecules that convey the input provided by the f rst messenger—usually an endogenous ligand or an exogenous drug—to cytoplasmic e ectors (Fig. 1-6). The activation o cyclases such as adenylyl cyclase , which catalyzes the production o the second messenger cyclic adenosine-3′,5′-monophosphate (cAMP), and guanylyl cyclase , which catalyzes the production o cyclic guanosine-3′,5′monophosphate (cGMP), constitutes the most common pathway linked to G proteins. In addition, G proteins can activate the enzyme phospholipase C (PLC), which, among other unctions, plays a key role in regulating the concentration o intracellular

Re ce ptor

Effe ctor

A 1 Agonis t binding

1 Agonis t unbinding 2 GTP hydrolys is

α

3 He te rotrime ric G prote in re cons titute d

2 GTP -GDP e xcha nge β

3 G prote in a ctiva tion

γ

GDP

Effe ctor a ctiva te d

GTP

C

Agonis t B

1 α -GTP diffus ion to e ffe ctor β

γ

α

α

2 Effe ctor a ctiva tion

GTP

β

γ

GTP

GDP

FIGURE 1-5. Receptor-mediated activation of a G protein and the resultant effector interaction. A. In the resting state, the and

subunits o a G protein are associated with one another, and GDP is bound to the subunit. B. Binding o an extracellular ligand (agonist) to a G protein-coupled receptor causes the exchange o GTP or GDP on the subunit. C. The subunit dissociates rom the subunit, which di uses to interact with e ector proteins. Interaction o the GTP-associated subunit with an e ector activates the e ector. In some cases (not shown), the subunit can also activate e ector proteins. Depending on the receptor subtype and the specif c G iso orm, G can also inhibit the activity o an e ector molecule. The subunit possesses intrinsic GTPase activity, which leads to hydrolysis o GTP to GDP. This leads to reassociation o the subunit with the subunit, and the cycle can begin again.

10 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

A

Agonis t Re ce ptor

β

TABLE 1-4 The Major G Protein Families and Examples of Their Actions

Ade nylyl cycla s e

αs

γ

GTP ATP

cAMP

PKA

G PROTEIN

ACTIONS

G-stimulatory (Gs )

Activates Ca 2 channels, activates adenylyl cyclase

G-inhibitory (Gi)

Activates K channels, inhibits adenylyl cyclase

Go

Inhibits Ca 2 channels

Gq

Activates phospholipase C

G12/13

Diverse ion transporter interactions

P rote in phos phoryla tion

B P LC

PIP2

DAG P KC

β

γ

(a ctive )

αq GTP

P KC IP3 Ca 2+

Ca 2 +

P rote in phos phoryla tion

FIGURE 1-6. Activation of adenylyl cyclase (AC) and phospholipase C (PLC) by G proteins. G proteins can interact with several di erent types o e ector molecules. The subtype o G protein that is activated o ten determines which e ector the G protein will activate. Two o the most common G subunits are G s and G q, which stimulate adenylyl cyclase and phospholipase C, respectively. A. When stimulated by G s , adenylyl cyclase converts ATP to cyclic AMP (cAMP). cAMP then activates protein kinase A (PKA), which phosphorylates a number o specif c intracellular proteins. B. When stimulated by G q, phospholipase C (PLC) cleaves the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG di uses in the membrane to activate protein kinase C (PKC), which then phosphorylates specif c cellular proteins. IP3 stimulates release o Ca 2 rom the endoplasmic reticulum into the cytosol. Calcium release also stimulates protein phosphorylation events that lead to changes in protein activation. Although not shown, the subunits o G proteins can also a ect certain cellular signal transduction cascades.

be grouped into f ve major amilies—G-stimulatory (Gs), G-inhibitory (Gi), Go, Gq, and G12/13. Examples o the e ects o these iso orms are shown in Table 1-4. The di erential unctioning o these G proteins, some o which may couple in di erent ways to the same receptor in di erent cell types, is likely to be important or the potential selectivity o uture drugs. The subunits o G proteins can also act as second messenger molecules, although their actions are not as completely characterized. One important class in the G protein-coupled receptor amily is the -adrenergic receptor group. The most thoroughly studied o these receptors have been designated 1, 2, and 3. As discussed in more detail in Chapter 11, Adrenergic Pharmacology, 1 receptors play a role in controlling heart rate; 2 receptors are involved in the relaxation o smooth muscle; and 3 receptors play a role in the mobilization o energy by at cells. Each o these receptors is stimulated by the binding o endogenous catecholamines, such as epinephrine and norepinephrine , to the extracellular domain o the receptor. Epinephrine binding induces a con ormational change in the receptor and thereby activates G proteins associated with the cytoplasmic domain o the receptor. The activated (GTP-bound) orm o the G protein activates adenylyl cyclase, resulting in increased intracellular cAMP levels and downstream cellular e ects. Table 1-5 indicates

TABLE 1-5 Tissue Localization and Action of -Adrenergic Receptors RECEPTOR

calcium. Upon activation by a G protein, PLC cleaves the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 triggers the release o Ca2 rom intracellular stores, thereby dramatically increasing the cytosolic Ca2 concentration and activating downstream molecular and cellular events. DAG activates protein kinase C, which then mediates other molecular and cellular events including smooth muscle contraction and transmembrane ion transport. All o these events are dynamically regulated, so that the di erent steps in the pathways are activated and inactivated with characteristic kinetics. A large number o G protein iso orms have been identif ed, each with unique e ects on its targets. Based on the primary sequence o the G subunit, these iso orms can

1

2

3

TISSUE LOCALIZATION

ACTION

Sinoatrial (SA) node o heart Cardiac muscle Adipose tissue

Increases heart rate

Bronchial smooth muscle Gastrointestinal smooth muscle Uterus Bladder Liver

Dilates bronchioles

Increases contractility Increases lipolysis

Pancreas

Constricts sphincters and relaxes gut wall Relaxes uterine wall Relaxes bladder Increases gluconeogenesis and glycolysis Increases insulin release

Adipose tissue

Increases lipolysis

C h a p t e r 1 Drug–Receptor Interactions

11

FIGURE 1-7. Major types of transmembrane receptors with linked A

Tyr

Tyr

P Tyr

Tyr P

Tyros ine kina s e a ctivity

Cytopla s mic prote in Tyr

P Tyr

B

Tyros ine phos pha ta s e a ctivity P Tyr

Tyr

enzymatic domains. There are f ve major categories o transmembrane receptors with linked enzymatic domains. A. The largest group is composed o receptor tyrosine kinases . A ter ligand-induced activation, these receptors dimerize and transphosphorylate tyrosine residues in the receptor and, o ten, on target cytosolic proteins. Examples o receptor tyrosine kinases include the insulin receptor and many growth actor receptors. B. Some receptors can act as tyrosine phosphatases. These receptors dephosphorylate tyrosine residues either on other transmembrane receptors or on cytosolic proteins. Many cells o the immune system have receptor tyrosine phosphatases. C. Some tyrosine kinase-associated receptors lack a def nitive enzymatic domain, but binding o ligand to the receptor triggers activation o receptor-associated protein 1 (termed nonreceptor tyrosine kinases ) that then phosphorylate tyrosine residues on certain cytosolic proteins. D. Receptor serine/threonine kinases phosphorylate serine and threonine residues on certain target cytosolic proteins. Members o the TGF- super amily o receptors are in this category. E. Receptor guanylyl cyclases contain a cytosolic domain that catalyzes the ormation o cGMP rom GTP. The receptor or B-type natriuretic peptide is one o the receptor guanylyl cyclases that has been well characterized.

some o the diverse tissue localizations and actions o the -adrenergic receptors.

Transmembrane Receptors with Linked Enzymatic Domains

C

Activa te d kina s e Ina ctive kina s e

Tyros ine kina s e a ctivity Tyr

P Tyr

D

S e r/Thr

P S e r/Thr

S e rine /thre onine kina s e a ctivity S e r/Thr

P S e r/Thr

E

The third major class o cellular drug targets consists o transmembrane receptors that transduce an extracellular ligand-binding interaction into an intracellular action through the activation o a linked enzymatic domain. The enzymatic domain may be part o the receptor itsel or part o a cytosolic protein that is recruited to the receptor in response to receptor activation. Such receptors play roles in a diverse set o physiologic processes, including cell metabolism, growth, and di erentiation. Receptors that have a linked enzymatic domain can be grouped into f ve major classes based on their cytoplasmic mechanism o action (Fig. 1-7). All o these receptors are single–membrane-spanning proteins, in contrast to the seven–membrane-spanning moti present in G protein-coupled receptors. Many receptors with enzymatic cytosolic domains orm dimers or multisubunit complexes to transduce their signals. Many receptors with linked enzymatic domains modi y proteins by adding or removing phosphate groups to or rom specif c amino acid residues. Phosphorylation is a ubiquitous mechanism of protein signaling. The large negative charge o phosphate groups can dramatically alter the threedimensional structure o a protein and thereby change that protein’s activity. In addition, phosphorylation is easily reversible, thus allowing this signaling mechanism to act specif cally in time and space.

Receptor Tyrosine Kinases Gua nylyl cycla s e a ctivity

GTP

cGMP

The largest group o transmembrane receptors with enzymatic cytosolic domains is the receptor tyrosine kinase amily. These receptors transduce signals rom many hormones and growth actors by phosphorylating tyrosine residues on the cytoplasmic tail o the receptor. This leads to recruitment and subsequent tyrosine phosphorylation o cytosolic signaling molecules. When aberrantly expressed or overexpressed, growth actor-responsive receptor tyrosine kinases (such as epidermal growth actor receptor [EGFR], HER2/neu, and vascular endothelial growth actor receptor [VEGFR]) are

12 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

associated with a wide array o cancers; these receptor tyrosine kinases are the targets o several monoclonal antibody and small-molecule inhibitor drugs (see Chapter 40, Pharmacology o Cancer: Signal Transduction). The insulin receptor is a well-characterized receptor tyrosine kinase. This receptor consists o two extracellular subunits that are covalently linked to two membranespanning subunits. Binding o insulin to the subunits results in a change in con ormation o the adjacent subunits, causing the subunits to move closer to one another on the intracellular side o the membrane. The proximity o the two subunits promotes a transphosphorylation reaction, in which one subunit phosphorylates the other (autophosphorylation). The phosphorylated tyrosine residues then act to recruit other cytosolic proteins, known as insulin receptor substrate (IRS) proteins. Type 2 diabetes mellitus may, in some cases, be associated with de ects in post-insulin receptor signaling; thus, understanding the insulin receptor signaling pathways is relevant or the potential design o rational therapeutics. The mechanism o insulin receptor signaling is discussed in more detail in Chapter 31, Pharmacology o the Endocrine Pancreas and Glucose Homeostasis.

Receptor Tyrosine Phosphatases

Just as receptor tyrosine kinases phosphorylate the tyrosine residues o cytoplasmic proteins, receptor tyrosine phosphatases remove phosphate groups rom specif c tyrosine residues. In some cases, this may be an example o receptor convergence (discussed later), where the di erential e ects o two receptor types can negate one another. However, receptor tyrosine phosphatases possess novel signaling mechanisms as well. Many receptor tyrosine phosphatases are ound in immune cells, where they regulate cell activation. These receptors are discussed urther in Chapter 46, Pharmacology o Immunosuppression.

Tyrosine Kinase-Associated Receptors

Tyrosine kinase-associated receptors constitute a diverse amily o proteins that, although lacking inherent catalytic activity, recruit active cytosolic signaling proteins in a ligand-dependent manner. These cytosolic proteins are also called (somewhat con usingly) nonreceptor tyrosine kinases . Ligand activation o cell sur ace tyrosine kinase-associated receptors causes the receptors to cluster together. This clustering event recruits cytoplasmic proteins that are then activated to phosphorylate other proteins on tyrosine residues. Thus, the downstream e ect is much like that o receptor tyrosine kinases, except that tyrosine kinase-associated receptors rely on a nonreceptor kinase to phosphorylate target proteins. Important examples o tyrosine kinase-associated receptors include cytokine receptors and a number o other receptors in the immune system. These receptors are discussed in detail in Chapter 46.

Receptor Serine/Threonine Kinases

Some transmembrane receptors are capable o catalyzing the phosphorylation o serine or threonine residues on cytoplasmic protein substrates. Ligands or such receptors are typically members o the trans orming growth actor (TGF- ) super amily. Many receptor serine/threonine kinases are important mediators o cell growth and di erentiation that have been implicated in cancer progression and metastasis. While there are many approved drugs that target cytosolic

serine/threonine kinases (see Intracellular Receptors below), drugs selective or receptor serine/threonine kinases are mainly in development.

Receptor Guanylyl Cyclases

As illustrated in Figure 1-6, the stimulation o G proteincoupled receptors may cause activation and release o G subunits, which, in turn, alter the activity o adenylyl and guanylyl cyclases. In contrast, receptor guanylyl cyclases have no intermediate G protein. Instead, ligand binding stimulates intrinsic receptor guanylyl cyclase activity, in which GTP is converted to cGMP. This is the smallest amily o transmembrane receptors. B-type natriuretic peptide, a hormone secreted by the ventricles in response to volume overload, acts via a receptor guanylyl cyclase. Nesiritide , a recombinant version o the native peptide ligand, is approved or the treatment o decompensated heart ailure (although it does not reliably improve outcomes), as discussed in Chapter 21, Pharmacology o Volume Regulation.

Intracellular Receptors The plasma membrane provides a unique barrier or drugs that have intracellular receptors. Many such drugs are small or lipophilic and are thus able to cross the membrane by di usion. Others require specialized protein transporters or acilitated di usion or active transport into the cell.

Intracellular Enzymes and Signal Transduction Molecules

Enzymes are common intracellular drug targets. Many drugs

that target intracellular enzymes exert their e ect by altering the enzyme’s production o critical signaling or metabolic molecules. Vitamin K epoxide reductase, a cytosolic enzyme involved in the post-translational modif cation o glutamate residues in certain coagulation actors, is the target o the anticoagulant drug warfarin. HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, is the target o atorvastatin and the other lipid-lowering statins. Many inhibitors o cytosolic signal transduction molecules are approved or in development. For example, inhibitors o the serine/threonine kinase mTOR (such as everolimus ) are used to prevent rejection o transplanted organs, to treat certain cancers, and to prevent restenosis in drug-eluting coronary stents. Many other intracellular kinases play important roles in cellular growth and di erentiation, and it is not surprising that “gain-o - unction” mutations in these proteins can lead to uncontrolled cell growth and cancer. Recall rom the introductory case that chronic myeloid leukemia is associated with the Philadelphia chromosome, which results rom a reciprocal translocation between the long arms o chromosomes 9 and 22. The mutant chromosome codes or a constitutively active tyrosine kinase re erred to as the BCR-Abl protein. (BCR and Abl are short or “break-point cluster region” and “Abelson,” respectively, the two chromosomal regions that undergo translocation with high requency in this orm o leukemia.) The constitutive activity o this kinase results in phosphorylation o a number o cytosolic proteins, leading to dysregulated myeloid cell growth and chronic myeloid leukemia. Imatinib is a selective therapy or chronic myeloid leukemia because it selectively targets the BCR-Abl protein; the drug inhibits BCR-Abl activity by neutralizing its ability to phosphorylate substrates. Imatinib was the f rst example o a drug targeted selectively to tyrosine kinases,

C h a p t e r 1 Drug–Receptor Interactions

and its success has led to the development o a number o drugs that act by similar mechanisms. Such drugs include second-generation drugs such as dasatinib and nilotinib that are used to treat CML patients with imatinib-resistant BCR-Abl iso orms, as well as the inhibitors o growth actor-responsive receptor tyrosine kinases discussed above. Indeed, the kinase targets o antineoplastic drugs are diverse. For instance, sorafenib targets both receptor tyrosine kinases and intracellular serine/threonine kinases, and vemurafenib is a recently approved late-stage melanoma treatment that targets a specif c mutant o the serine/threonine kinase B-RAF. As a f nal example, idelalisib is a recently approved phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor used to treat certain leukemias and lymphomas (see Chapter 40).

13

S te roid hormone

Hormone re ce ptor Cha pe rone

A

Nucle us

B

Transcription Factors

The transcription regulatory actors are important intracellular receptors that are targeted by lipophilic drugs. All proteins in the body are encoded by DNA. The transcription o DNA into RNA and the translation o RNA into protein are controlled by a diverse set o molecules. Transcription o many genes is regulated, in part, by the interaction between lipid-soluble signaling molecules and transcription regulatory actors. Because o the undamental role played by control o transcription in many biological processes, transcription regulators (also called transcription factors ) are the targets o some important drugs. Steroid hormones are a class o lipophilic drugs that di use readily through the plasma membrane and act by binding to transcription actors in the cytoplasm or nucleus (Fig. 1-8). Just as the shape o a transcription actor governs the drugs to which it binds, the shape also determines where on the genome the transcription actor attaches and which coactivator or corepressor molecules bind to it. By activating or inhibiting transcription, thereby altering the intracellular or extracellular concentrations o specif c gene products, drugs that target transcription actors can have pro ound e ects on cellular unction. The cellular responses to such drugs, and the e ects that result rom these cellular responses in tissues and organ systems, provide links between the molecular drug–receptor interaction and the e ects o the drug on the organism as a whole. Because gene transcription is a relatively slow and long-lasting process (minutes to hours), drugs that target transcription actors o ten require a longer period o time or the onset o action to take place, and have longer lasting e ects, than do drugs that alter more transient processes such as ion conductance (seconds to minutes).

Structural Proteins

Structural proteins are another important class o intracellu-

lar drug targets. For example, the antimitotic vinca alkaloids bind to tubulin monomers and prevent the polymerization o this molecule into microtubules. Inhibition o microtubule ormation arrests the a ected cells in metaphase, making the vinca alkaloids use ul antineoplastic drugs.

Nucleic Acids

Nucleic acids are a ourth subset o intracellular drug tar-

gets. Some small-molecule drugs bind directly to RNA or ribosomes; these include important antibiotics (such as doxycycline and azithromycin) that block translation in target microorganisms. DNA- and RNA-binding chemotherapeutic

DNA

C

FIGURE 1-8. Lipophilic molecule binding to an intracellular transcription factor. A. Small lipophilic molecules can diffuse through the plasma membrane and bind to intracellular transcription factors. In this example, steroid hormone binding to a cytosolic hormone receptor is shown, although some receptors of this class may be located in the nucleus before ligand binding. B. Ligand binding triggers a conformational change in the receptor (and often, as shown here, dissociation of a chaperone repressor protein) that leads to transport of the ligand–receptor complex into the nucleus. In the nucleus, the ligand–receptor complex typically dimerizes. In the example shown, the active form of the receptor is a homodimer (two identical receptors binding to one another), but heterodimers (such as the thyroid hormone receptor and the retinoid Xreceptor) may also form. C. The dimerized ligand– receptor complex binds to DNA and may then recruit coactivators or corepressors (not shown). These complexes alter the rate of gene transcription, leading to a change (either up or down) in cellular protein expression.

agents (such as doxorubicin) are mainstays o treatment or many cancers. Drugs composed o nucleic acids can also target nucleic acids. Antisense therapeutics (such as the recently approved drug mipomersen) bind target mRNA to block transcription o specif c proteins. With continued development o such antisense approaches and o related RNA inter erence (RNAi) therapeutics, such targeting could someday enable physicians to routinely modi y the expression levels o specif c gene transcripts. To date, technical challenges in delivering such therapeutics to their targets have limited their utility to specialized applications.

Extracellular Targets Many important drug receptors are enzymes with active sites located outside the plasma membrane. The extracellular environment consists o a milieu o proteins and signaling molecules. Many o these proteins serve a structural role, and others are used to communicate in ormation between cells. Enzymes that modi y the molecules mediating these important signals can in uence physiologic processes such as vasoconstriction and neurotransmission. One example o this class o receptors is the angiotensin converting enzyme (ACE), which converts angiotensin I to the potent vasoconstrictor angiotensin II. ACE inhibitors are drugs that inhibit

14 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

this enzymatic conversion and thereby lower blood pressure (among other e ects; see Chapter 21). Another example is acetylcholinesterase , which degrades acetylcholine a ter this neurotransmitter is released rom cholinergic neurons. Acetylcholinesterase inhibitors enhance neurotransmission at cholinergic synapses by preventing neurotransmitter degradation at these sites (see Chapter 10, Cholinergic Pharmacology). Some extracellular targets are not enzymes. For example, several proteins, including monoclonal antibodies, are used to target soluble cytokines and block them rom interacting with their endogenous receptors. One set o such drugs is the anti-TNF- agents, including etanercept, in iximab, adalimumab, and others, which are commonly used to treat autoimmune diseases such as rheumatoid arthritis (see Chapter 46).

Cell Sur ace Adhesion Receptors Cells o ten interact directly with other cells to per orm specif c unctions or to communicate in ormation. The ormation o tissues and the migration o immune cells to a site o in ammation are examples o physiologic processes that require cell–cell adhesive interactions. A region o contact between two cells is termed an adhesion, and cell–cell adhesive interactions are mediated by pairs o adhesion receptors on the sur aces o the individual cells. In many cases, several such receptor–counter-receptor pairs combine to secure a f rm adhesion, and intracellular regulators control the activity o the adhesion receptors by changing their a f nity or by controlling their expression and localization on the cell sur ace. Adhesion receptors also mediate adhesion o cells to the extracellular matrix. Several adhesion receptors involved in the in ammatory response are attractive targets or selective inhibitors. Inhibitors o a specif c class o adhesion receptors, known as integrins , have entered the clinic in recent years, and these drugs are used in the treatment o a range o conditions including thrombosis (abciximab, eptif batide ), in ammatory bowel disease (vedolizumab), and multiple sclerosis (natalizumab) (see Chapter 23, Pharmacology o Hemostasis and Thrombosis, and Chapter 46).

Liga nd 1

GDP

γ

Many cells in the body are continuously inundated with multiple inputs, some stimulatory and some inhibitory. How do cells integrate these signals to produce a coherent response? G proteins and other second messengers appear to provide important points o integration. As noted above, relatively ew second messengers have been identif ed, and it is unlikely that many more remain to be discovered. Thus, second messengers are an attractive candidate mechanism or providing cells with a set o common points upon which numerous outside stimuli could converge to generate a coordinated cellular e ect (Fig. 1-9). Ion concentrations provide another point o integration or cellular e ects because the cellular concentration o a particular ion is the result o the integrated activity o multiple ionic currents that both increase and decrease the concentration o the ion within the cell. For example, the contractile state o a smooth muscle cell is a unction o the intracellular calcium ion concentration, which is determined by several di erent Ca2 conductances. These conductances include calcium ion leaks into the cell and calcium currents into and out o the cytoplasm through specialized channels in the plasma membrane and smooth endoplasmic reticulum. Because the magnitude o cellular response is o ten considerably greater than the magnitude o the stimulus that caused the response, cells appear to have the ability to ampli y the e ects o receptor binding. G proteins provide an excellent example o signal amplif cation. Ligand binding to a G protein-coupled receptor activates a single G protein molecule. This G protein molecule can then bind to and activate many e ector molecules, such as adenylyl cyclase, which can then generate an even greater number o second messenger molecules (in this example, cAMP). Another example o signal amplif cation is “trigger Ca2 ” or calcium-induced calcium release, in which a small in ux

Ade nylyl cycla s e

Re ce ptor

αs β

PROCESSING OF SIGNALS RESULTING FROM DRUG–RECEPTOR INTERACTIONS

Re ce ptor

αs

αs

αi

αi

GTP

GTP

GTP

GTP

ATP

cAMP

cAMP

γ

β

Liga nd 2

αi GDP

ATP

Ne t re s ult = inte gra te d e ffe ct

FIGURE 1-9. Signaling convergence o two receptors. A limited number of mechanisms are used to transduce intracellular signal cascades. In some cases, this allows for convergence, where two different receptors have opposite effects that tend to negate one another in the cell. In a simple example, two different G protein-coupled receptors could be stimulated by different ligands. The receptor shown on the left is coupled to G s , a G protein that stimulates adenylyl cyclase to catalyze the formation of cAMP. The receptor shown on the right is coupled to G i, a G protein that inhibits adenylyl cyclase. When both of these receptors are activated simultaneously, they can attenuate or even neutralize each other, as shown. Sometimes, signaling through a pathway may alternate as the two receptors are sequentially activated.

C h a p t e r 1 Drug–Receptor Interactions

15

o Ca2 through voltage-gated Ca2 channels in the plasma membrane “triggers” the release o larger amounts o Ca2 rom intracellular stores into the cytoplasm.

CELLULAR REGULATION OF DRUG– RECEPTOR INTERACTIONS Drug-induced activation or inhibition o a receptor o ten has a lasting impact on the receptor’s subsequent responsiveness to drug binding. Mechanisms that mediate such e ects are important because they prevent overstimulation that could lead to cellular damage or adversely a ect the organism as a whole. Many drugs show diminishing e ects over time; this phenomenon is called tachyphylaxis . In pharmacologic terms, the receptor and the cell become desensitized to the action o the drug. Mechanisms o desensitization can be divided into two types: homologous , in which the e ects o agonists at only one type o receptor are diminished, and heterologous , in which the e ects o agonists at two or more types o receptors are coordinately diminished. Heterologous desensitization is thought to be caused by drug-induced alteration in a common point o convergence in the signaling pathways activated by the involved receptors, such as a shared e ector molecule. Many receptors exhibit desensitization. For example, the cellular response to repeated stimulation o -adrenergic receptors by epinephrine diminishes steadily over time (Fig. 1-10). -Adrenergic receptor desensitization is mediated by epinephrine-induced phosphorylation o the cytoplasmic tail o the receptor. This phosphorylation promotes the binding o -arrestin to the receptor; in turn, -arrestin inhibits the receptor’s ability to stimulate the G protein Gs. With lower levels o activated Gs present, adenylyl cyclase produces less cAMP. In this manner, repeated cycles o ligand–receptor binding result in smaller and smaller cellular e ects. Other molecular mechanisms have even more pro ound e ects, completely turning o the receptor to stimulation by ligand. The latter phenomenon, re erred to as inactivation, may also result rom phosphorylation o the receptor; in this case, the phosphorylation completely blocks the signaling activity o the receptor or causes removal o the receptor rom the cell sur ace. Another mechanism that can a ect the cellular response caused by drug–receptor binding is called refractoriness. Receptors that assume a refractory state ollowing activation require a period o time to pass be ore they can be stimulated again. As noted above, voltage-gated sodium channels, which mediate the f ring o neuronal action potentials, are subject to re ractory periods. A ter channel opening induced by membrane depolarization, the voltagegated sodium channel spontaneously closes and cannot be reopened or some period o time (called the refractory period). This inherent property o the channel determines the maximum rate at which neurons can be stimulated and transmit in ormation. The e ect o drug–receptor binding can also be in uenced by drug-induced changes in the number o receptors on or in a cell. One example o a molecular mechanism by which receptor number can be altered is called down-regulation. In this phenomenon, prolonged receptor stimulation by ligand induces the cell to endocytose and sequester receptors

A P hos phoryla tion by P KA a nd/or β ARK

P P P P

β-a rre s tin binding

P P

P P

β-a rre s tin

Agonis t

G prote in binding pre ve nte d

B S e que s tra tion

C

De gra da tion

Endos ome

Lys os ome

FIGURE 1-10.

-Adrenergic receptor regulation. Agonist-bound -adrenergic receptors activate G proteins, which then stimulate adenylyl cyclase activity (not shown). A. Repeated or persistent stimulation of the receptor by agonist results in phosphorylation of amino acids at the C-terminus of the receptor by protein kinase A (PKA) and/or -adrenergic receptor kinase ( ARK). -Arrestin then binds to the phosphorylated domain of the receptor and blocks Gs binding, thereby decreasing adenylyl cyclase (effector) activity. B. Binding of -arrestin also leads to receptor sequestration into endosomal compartments via clathrin-mediated endocytosis (not shown), effectively neutralizing -adrenergic receptor signaling activity. The receptor can then be recycled and reinserted into the plasma membrane. C. Prolonged receptor occupation by an agonist can lead to receptor down-regulation and eventual receptor degradation. Cells can also reduce the number of receptors by inhibiting the transcription or translation of the gene coding for the receptor (not shown).

in endocytic vesicles. This sequestration prevents the receptors rom coming into contact with ligands, resulting in cellular desensitization. When the stimulus that caused the receptor sequestration subsides, the receptors can be recycled to the cell sur ace and thereby rendered unctional again (Fig. 1-10). Cells also have the ability to alter the rates o synthesis or degradation o receptors and thereby to regulate the number o receptors available or drug binding. Receptor sequestration and alterations in receptor synthesis and degradation occur on a longer time scale than does phosphorylation and have longer lasting e ects as well. Table 1-6 provides a summary o the mechanisms by which the e ects o drug–receptor interactions can be regulated.

16 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 1-6 Mechanisms of Receptor Regulation MECHANISM

DEFINITION

Tachyphylaxis

Repeated administration of the same dose of a drug results in a diminishing effect of the drug over time

Desensitization

Decreased ability of a receptor to respond to stimulation by a drug or ligand Decreased response at a single type of receptor Decreased response at two or more types of receptor

Homologous Heterologous Inactivation

Loss of ability of a receptor to respond to stimulation by a drug or ligand

Refractory

After a receptor is stimulated, a period of time is required before the next drug–receptor interaction can produce an effect

Down-regulation

Repeated or persistent drug–receptor interaction results in removal of the receptor from sites where subsequent drug–receptor interactions could take place

DRUGS THAT DO NOT FIT THE DRUG– RECEPTOR MODEL Although most drugs interact with one o the basic receptor types outlined above, others act by nonreceptor-mediated mechanisms. Two examples are the osmotic diuretics and the antacids. Diuretics control uid balance in the body by altering the relative rates o water and ion absorption and secretion in the kidney. Many o these drugs act on ion channels. One class o diuretics, however, alters water and ion balance not by binding to ion channels or G protein-coupled receptors but by changing the osmolarity in the nephron directly. The sugar mannitol, which is used mainly to treat increased intracranial pressure, is secreted into the lumen o the nephron and increases the osmolarity o the urine to such a degree that water is drawn rom the peritubular blood into the lumen. This uid shi t serves to increase the volume o urine while decreasing the blood volume. Another class o drugs that does not f t the drug–receptor model is the antacids, which are used to treat gastroesophageal re ux disease and peptic ulcer disease. Unlike antiulcer agents that bind to receptors involved in the physiologic generation o gastric acid, antacids act nonspecif cally by absorbing or chemically neutralizing stomach acid. Examples o these agents include bases such as NaHCO3 and Mg(OH)2.

CONCLUSION AND FUTURE DIRECTIONS Although the molecular details o drug–receptor interactions vary widely among drugs o di erent classes and receptors o di erent types, the undamental mechanisms o action described in this chapter serve as paradigms or the principles o pharmacodynamics. The ability to classi y drugs based on their receptors and mechanisms o action makes it possible to simpli y the study o pharmacology, because the molecular mechanism o action o a drug can usually be linked to its cellular, tissue, organ, and system levels o action. In turn, it becomes easier to understand how a given drug mediates its therapeutic e ects and its unwanted or adverse e ects in a particular patient. The major aim o modern drug development is to identi y drugs that are highly selective by tailoring drug molecules to unique targets responsible or disease. As knowledge o drug development and the genetic and pathophysiologic basis o disease progresses, physicians and scientists will learn to combine the molecular specif city o a drug with the genetic and pathophysiologic specif city o the drug target to provide more and more selective therapies.

Acknowledgment We thank Jose B. Simon, Christopher W. Cairo, and Zachary S. Morris or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Alexander SP, Mathie A, Peters JA. Guide to Receptors and Channels (GRAC), 5th ed. Br J Pharmacol 2011;164(suppl 1):S1–S324. (Brie overviews o molecular targets or drugs, organized by types o receptors.) Katritch V, Cherezov V, Stevens RC. Structure- unction o the G proteincoupled receptor super amily. Annu Rev Pharmacol Toxicol 2013;53: 531–556. (Reviews recent structural insights into G protein-coupled receptors.) Kole R, Krainer AR, Altman S. RNA therapeutics: beyond RNA intererence and antisense oligonucleotides. Nat Rev Drug Discov 2012;11: 125–140. (Highlights early successes, therapeutic mechanisms, and remaining challenges in the development o RNA-based therapies.) Lagerström MC, Schiöth HB. Structural diversity o G protein-coupled receptors and signif cance or drug discovery. Nat Rev Drug Discov 2008;7:339–357. (Discusses the f ve amilies o G protein-coupled receptors, with an eye toward uture drug development.) Pratt WB, Taylor P, eds. Principles o drug action: the basis o pharmacology. 3rd ed. New York: Churchill Livingstone; 1990. (Contains a detailed discussion o drug–receptor interactions.) Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM. Molecular signatures o G protein-coupled receptors. Nature 2013; 494:185–194. (Comparative analysis o structures, ligand binding, and con ormational changes o G protein-coupled receptors.) Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 2009;9:28–39. (Discusses dysregulation o protein kinases in cancer and targeting o these molecules by drugs such as imatinib.)

Agon n is t C ompee titive a n ta g o n is t

C

D

2

Pharmacodynamics Q u e n t in J . Ba c a a n d David E. Go la n

Agonis nINTRODUCTION iiss t b iinding n ding g Compe C omp mpe p e ttiti itivveeNoncncom aompetiti nntta vege Rooni nnis itsorrtAntagonistss . . . . . . . . N o n c o m p e tit & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 18 . . . . . . . . . 22 ecep ec epto ep Nonreceptor Antagonists . . . . . . . . . . . . . . . . . . . . DRUG–RECEPTOR BI BIND N ING. . . . . . . . . . . . . . . . . . . bindin bindd in g Partial binding b bind i n d i n g Agonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 DOSE–RESPONSE RELA ATI TIONSHIPS . . . . . . . . . . . . . . . . . . . . . . . . 18 Graded Dose–Respponnse s e Relat elatiionships . . . . . . . . . . . . . . . . . . . 18 Quan Qu anta tall Dose–Response Re Rela lationships . . . . . . . . . . . . . . . . . . . 19 DRUG–RECEPTOR INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . 20 A i t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Agonists Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Competitive Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . 21

INTRODUCTION Pharma Pharmacodynamics Phar maco cody dyna nami mics cs is tthe he tterm erm er m us used ed ttoo de describe desc scri ribe be tthe he e ec ects ts o a drug on the body. These e ects are typically described in quantitative terms. The previous chapter considered the molecular interactions by which pharmacologic agents exert their e ects. The integration o these molecular actions into an e ect on the organism as a whole is the subject addressed in this chapter. It is important to describe the e ects o a drug quantitatively in order to determine appropriate dose ranges or patients, as well as to compare the potency, e f cacy, and sa ety o one drug to that o another.

DRUG–RECEPTOR BINDING The study o pharmacodynamics is based on the concept o drug–receptor binding. When either a drug or an endogenous ligand (such as a hormone or neurotransmitter) binds to its receptor, a response may result rom that binding interaction. When a su f cient number o receptors are bound (or “occupied”) on or in a cell, the cumulative e ect o receptor “occupancy” may become apparent in that cell. At some point, all o the receptors may be occupied, and a maximal response may be observed (an exception is the case o spare receptors; see below). When the response occurs in many cells, the e ect can be seen at the level o the organ or even the patient. But this all starts with the binding o drug or ligand to a receptor ( or the purpose o discussion, “drug” and “ligand” will be used interchangeably or the remainder o this chapter). A model that accurately describes the binding o drug to receptor would there ore be use ul in predicting the e ect o the drug at the molecular, cellular, tissue (organ), and organism (patient) levels. This section describes one such model.

Invee rs rsee Agonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2 Spare Recept ptor orss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 CONCEPTS IN THERAPEUTICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Therapeutic Index and Therapeutic Wi Window ndow d w . . . . . . . . . . . . . . 25 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . 26 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Consider the simp simplest plest case,, in whic which ch th thee re receptor rece cept ptor or iiss ei either eith ther er ree (unoccupied) or reversibly bound to drug (occupied). We ccan an ddescribe escr es crib ibee th this is ccase asee as ol as ollo lows ws:: ollows: kon

L

RÆ ¨ LR

Equation 2-1

koff

where L is ligand (drug), R is ree receptor, and LR is bound drug–receptor complex. At equilibrium, the raction o receptors in each state is dependent on the dissociation constant, Kd, where Kd ko /kon. Kd is an intrinsic property o any given drug–receptor pair. Although Kd varies with temperature, the temperature o the human body is relatively constant, and it can there ore be assumed that Kd is a constant or each drug–receptor combination. According to the law o mass action, the relationship between ree and bound receptor can be described as ollows: Kd

[L][R] , rearranged to [LR] [LR]

[L][R] Kd

Equation 2-2

where [L] is ree ligand concentration, [R] is ree receptor concentration, and [LR] is ligand–receptor complex concentration. Because Kd is a constant, some important properties o the drug–receptor interaction can be deduced rom this equation. First, as ligand concentration is increased, the concentration o bound receptors increases. Second, and not so obvious, is that as ree receptor concentration is increased (as may happen, or example, in disease states or upon repeated exposure to a drug), bound receptor concentration also increases. There ore, an increase in the effect of a drug can result from an increase in the concentration of either the ligand or the receptor. The remainder o the discussion in this chapter, however, assumes that the total concentration o receptors is a 17

18 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Ad m ira l X is a 66-ye a r-o ld re tire d s u b m a rin e ca p ta in w ith a 70 p a ck–ye a r s m o kin g h is to ry (tw o p a cks a d a y o r 35 ye a rs ) a n d a a m ily h is to ry o co ro n a ry a rte ry d is e a s e . He ta ke s d a ily a to rva s ta tin to re d u ce h is ch o le s te ro l le ve l a n d a s p irin to re d u ce h is ris k o co ro n a ry a rte ry o cclu s io n . On e d a y, w h ile w o rkin g in h is w o o d s h o p , Ad m ira l X b e g in s to e e l tig h tn e s s in h is ch e s t. Th e e e lin g ra p id ly b e co m e s p a in u l, a n d th e p a in ra d ia te s d o w n h is le t a rm . He ca lls 911, a n d a n a m b u la n ce tra n s p o rts h im to th e lo ca l e m e rg e n cy d e p a rtm e n t. A te r e va lu a tio n , it is d e te rm in e d th a t Ad m ira l X is h a vin g a n a n te rio r m yo ca rd ia l in a rctio n . Be ca u s e Ad m ira l X ca n n o t b e tra n s e rre d to a h o s p ita l w ith a ca rd ia c ca th e te riza tio n la b o ra to ry w ith in 120 m in u te s o f rs t m e d ica l co n ta ct, a n d h e h a s n o re la tive co n tra in d ica tio n s to th ro m b o lytic th e ra p y (s u ch a s u n co n tro lle d hyp e rte n s io n , h is to ry o s tro ke , o r re ce n t s u rg e ry), th e p h ys icia n in itia te s th e ra p y w ith b o th a th ro m b o lytic a g e n t, tis s u e -typ e p la s m in o g e n a ctiva to r (tPA), a n d a n a n tico a g u la n t, h e p a rin . Be ca u s e o th e ir lo w th e ra p e u tic in d ice s , im p ro p e r d o s in g o

constant, so that [LR] [R] 2-2 to be arranged as ollows: [Ro]

[R]

[LR]

[R] [R] Ê1 Ë

[Ro]. This allows Equation

[L][R] Kd [L] ˆ Kd ¯

Equation 2-3

Solving or [R] and substituting Equation 2-3 into Equation 2-2 yields: [LR]

[Ro] [L] , rearranged to [L] Kd

[LR] [Ro]

[L] [L] Kd

Equation 2-4

Note that the le t side o this equation, [LR]/[Ro], represents the raction o all available receptors that are bound to ligand. Figure 2-1 shows two plots o Equation 2-4 or the binding o two hypothetical drugs to the same receptor. These plots are known as drug–receptor binding curves . Figure 2-1A shows a linear plot, and Figure 2-1B shows the same plot on a semilogarithmic scale. Because drug responses occur over a wide range o doses (concentrations), the semilog plot is o ten used to display drug–receptor binding data. The two drug–receptor interactions are characterized by di erent values o Kd. In this case, KdA KdB. Notice rom Figure 2-1 that maximal drug–receptor binding occurs when [LR] is equal to [Ro], or [LR]/[Ro] 1. Also notice that, according to Equation 2-4, when [L] Kd, then [LR]/[Ro] Kd/2Kd ½. Thus, Kd can be def ned as the concentration o ligand at which 50% o the available receptors are occupied.

b o th o th e s e d ru g s ca n h a ve d ire co n s e q u e n ce s (h e m o rrh a g e a n d d e a th ). Th e re o re , Ad m ira l X is clo s e ly m o n ito re d , a n d th e p h a rm a co lo g ic e e ct o th e h e p a rin is m e a s u re d p e rio d ica lly b y te s tin g th e p a rtia l th ro m b o p la s tin tim e (PTT). Ad m ira l X’s s ym p to m s re s o lve ove r th e n e xt s e ve ra l h o u rs , a lth o u g h h e re m a in s in th e h o s p ita l o r m o n ito rin g . He is d is ch a rg e d a te r 4 d a ys in th e h o s p ita l; h is d is ch a rg e m e d ica tio n s in clu d e a to rva s ta tin , a s p irin , a te n o lo l, lis in o p ril, a n d clo p id o g re l o r s e co n d a ry p re ve n tio n o m yo ca rd ia l in a rctio n .

Questions 1 . How does the molecular interaction o a drug with its receptor determine the potency and e f cacy o the drug? 2 . Why does the act that a drug has a low therapeutic index mean that the physician must use greater care in its administration? 3 . What properties o certain drugs, such as aspirin, allow them to be taken without monitoring o plasma drug levels, whereas other drugs, such as heparin, require such monitoring?

DOSE–RESPONSE RELATIONSHIPS The pharmacodynamics o a drug can be quantif ed by the relationship between the dose (concentration) o the drug and the organism’s (patient’s) response to that drug. One might intuitively expect the dose–response relationship to be related closely to the drug–receptor binding relationship, and this turns out to be the case or many drug–receptor combinations. Thus, a use ul assumption at this stage o discussion is that the response to a drug is proportional to the concentration o receptors that are bound (occupied) by the drug. This assumption can be quantif ed by the ollowing relationship: response max response

[DR] [Ro]

[D] [D] Kd

Equation 2-5

where [D] is the concentration o ree drug, [DR] is the concentration o drug–receptor complexes, [Ro] is the concentration o total receptors, and Kd is the equilibrium dissociation constant or the drug–receptor interaction. (Note that the right side o Equation 2-5 is equivalent to Equation 2-4, with [D] substituted or [L].) The generalizability o this assumption is examined below. There are two major types o dose–response relationships— graded and quantal. The di erence between the two types is that graded dose–response relationships describe the e ect o various doses o a drug on an individual, whereas quantal relationships show the e ect o various doses o a drug on a population o individuals.

Graded Dose–Response Relationships Figure 2-2 shows graded dose–response curves or two hypothetical drugs that elicit the same biological response.

C h a p t e r 2 Pharmacodynamics

A Line a r

19

A Line a r

1.0

1.0 Drug A

Drug A Drug B

Drug B

[LR] 0.5 [R o ]

E 0.5 E MAX

0

0 KdA KdB

EC 50 (A) EC 50 (B)

[L]

B S e miloga rithmic

[L]

B S e miloga rithmic

1.0

1.0 Drug A

Drug A Drug B

[LR] 0.5 [R o ]

0

Drug B

E 0.5 E MAX

0 KdA KdB

[L]

EC 50 (A) EC 50 (B)

[L]

FIGURE 2-1. Ligand–receptor binding curves. A. Linear graphs o drug–

FIGURE 2-2. Graded dose–response curves. Graded dose–response

receptor binding or two drugs with di erent values o Kd. B. Semilogarithmic graphs o the same drug–receptor binding. Kd is the equilibrium dissociation constant or a given drug–receptor interaction—a lower Kd indicates a tighter drug–receptor interaction (higher a f nity). Because o this relationship, Drug A, which has the lower Kd, will bind a higher proportion o total receptors than Drug B at any given drug concentration. Notice that Kd corresponds to the ligand concentration [L] at which 50% o the receptors are bound (occupied) by ligand. [L] is the concentration o ree (unbound) ligand (drug), [LR ] is the concentration o ligand–receptor complexes, and [Ro] is the total concentration o occupied and unoccupied receptors. Thus, [LR]/[Ro] is the fractional occupancy o receptors, or the raction o total receptors that are occupied (bound) by ligand.

curves demonstrate the e ect o a drug as a unction o its concentration. A. Linear graphs o graded dose–response curves or two drugs. B. Semilogarithmic graphs o the same dose–response curves. Note the close resemblance to Figure 2-1: the raction o occupied receptors [LR]/[Ro] has been replaced by the ractional e ect E/Emax, where E is a quantif able response to a drug (e.g., an increase in blood pressure). EC50 is the potency o the drug, or the concentration at which the drug elicits 50% o its maximal e ect. In the f gure, Drug A is more potent than Drug B because it elicits a hal -maximal e ect at a lower concentration than Drug B. Drugs A and B exhibit the same e f cacy (the maximal response to the drug). Note that potency and e f cacy are not intrinsically related—a drug can be extremely potent but have little e f cacy, and vice versa. [L] is drug concentration, E is e ect, Emax is e f cacy, and EC50 is potency.

The curves are presented on both linear and semilogarithmic scales. The curves are similar in shape to those in Figure 2-1, consistent with the assumption that response is proportional to receptor occupancy. Two important parameters—potency and e f cacy—can be deduced rom the graded dose–response curve. The potency (EC50) o a drug is the concentration at which the drug elicits 50% of its maximal response. The e f cacy (E max) is the maximal response produced by the drug. In accordance with the assumption stated above, e f cacy can be thought o as the state at which receptor-mediated signaling is maximal and, there ore, additional drug will produce no additional response. This usually occurs when all the receptors are occupied by the drug. Some drugs, however, are capable o eliciting a maximal response when less than 100% o the drug’s receptors are occupied; the remaining receptors can be called spare receptors . This concept is discussed urther in the text

that ollows. Note again that the graded dose–response curve o Figure 2-2 bears a close resemblance to the drug–receptor binding curve o Figure 2-1, with EC50 replacing Kd and Emax replacing Ro.

Quantal Dose–Response Relationships The quantal dose–response relationship plots the fraction of the population that responds to a given dose of drug as a function of the drug dose. Quantal dose–response relationships describe the concentrations o a drug that produce a given e ect in a population. Figure 2-3 shows an example o quantal dose–response curves. Because o di erences in biological response among individuals, the e ects o a drug are seen over a range o doses. The responses are def ned as either present or not present (i.e., quantal, not graded). Endpoints such as “sleep/no sleep” or “alive at

20 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY Cumula tive % e xhibiting The ra pe utic e ffe ct

Toxic e ffe ct

Le tha l e ffe ct

50

Therapeutic effect

% Requiring dose to achieve Toxic effect Lethal effect

An agonist is a molecule that binds to a receptor and stabilizes the receptor in a particular conformation (usually, the active conformation). When bound by an agonist, a typical receptor is more likely to be in its active con ormation than its inactive con ormation. Depending on the receptor, agonists may be drugs or endogenous ligands. A use ul model or understanding the relationship between agonist binding and receptor activation is shown in Equation 2-6:

i

vi

d

u

a

l

s

r

e

s

p

o

n

d

i

n

g

100

Agonists

%

I

n

d

D 0 ED50

TD50

LD50

Dos e FIGURE 2-3. Quantal dose–response curves. Quantal dose–response curves demonstrate the average e ect o a drug, as a unction o its concentration, in a population o individuals. Individuals are typically observed or the presence or absence o a response (e.g., sleep or no sleep), and this result is then used to plot the percentage o individuals who respond to each dose o drug. Quantal dose–response relationships are use ul or predicting the e ects o a drug when it is administered to a population o individuals and or determining population-based toxic doses and lethal doses. These doses are called the ED50 (dose at which 50% o subjects exhibit a therapeutic response to a drug), TD50 (dose at which 50% o subjects experience a toxic response), and LD50 (dose at which 50% o subjects die). Note that ED50 is the dose at which 50% o subjects respond to a drug, whereas EC50 (as described in the previous f gure) is the dose at which a drug elicits a hal maximal e ect in an individual subject.

12 months/not alive at 12 months” are examples o quantal responses; in contrast, graded dose–response relationships are generated using scalar responses such as change in blood pressure or heart rate. The goal is to generalize a result to a population rather than to examine the graded e ect o di erent drug doses on a single individual. Types o responses that can be examined using the quantal dose–response relationship include e ectiveness (therapeutic e ect), toxicity (adverse e ect), and lethality (lethal e ect). The doses that produce these responses in 50% o a population are known as the median effective dose (ED50), median toxic dose (TD50), and median lethal dose (LD50), respectively.

DRUG–RECEPTOR INTERACTIONS Many receptors or drugs can be modeled as having two conormational states that are in reversible equilibrium with one another. These two states are called the active state and the inactive state . Many drugs unction as ligands or such receptors and a ect the probability that the receptor exists pre erentially in one con ormation or the other. The pharmacologic properties o drugs are o ten based on their e ects on the state o their cognate receptors. A drug that, upon binding to its receptor, avors the active receptor con ormation is called an agonist; a drug that prevents agonist-induced activation o the receptor is re erred to as an antagonist. Some drugs do not f t neatly into this simple def nition o agonist and antagonist; these include partial agonists and inverse agonists . The ollowing sections describe these pharmacologic classif cations in more detail.

R Æ D ¨

Ø≠ DR

Æ ¨

R* Equation 2-6

Ø≠ DR*

where D and R are unbound ( ree) drug and receptor concentrations, respectively, DR is the concentration o the agonist– receptor complex, and R* indicates the active con ormation o the receptor. For most receptors and agonists, R* and DR are unstable species that exist only brie y and are quantitatively insignif cant compared to R and DR*. There ore, in most cases, Equation 2-6 simplif es to D

RÆ ¨ DR**

Equation 2-7

Note that Equation 2-7 is identical to Equation 2-1, which was used or the analysis o drug–receptor binding. This suggests that, or most receptors, agonist binding is proportional to receptor activation. Some receptors, however, do have limited stability in the R* and/or DR con ormations; in these cases, Equation 2-6 must be revisited (see below). Equation 2-6 can also be used to illustrate quantitatively the concepts o potency and e f cacy. Recall that potency is the agonist concentration required to elicit a hal -maximal e ect, and e f cacy is the maximal e ect o the agonist. Assuming that a receptor is not active unless bound to a drug (i.e., R* is insignif cant compared to DR*), Equation 2-8 provides a quantitative description o potency and e f cacy: D

R

kon Æ ¨ koff Potency

DR

k Æ ¨ k

DR*

Equation 2-8

Efficacy

Here, k is the rate constant or receptor activation, and k is the rate constant or receptor deactivation. This equation demonstrates the relationship between potency (Kd ko /kon) → DR), as well as the relationand agonist binding (D R ← ship between e f cacy (k /k ) and the con ormational change → DR*). These required or activation o the receptor (DR ← relationships are intuitive when we consider that more potent drugs are those that have a higher a f nity or their receptors (lower Kd), and more e f cacious drugs are those that cause a higher raction o receptors to be activated.

Antagonists An antagonist is a molecule that inhibits the action of an agonist but has no effect in the absence of the agonist. Figure 2-4 shows one approach to classi ying the various types o antagonists. Antagonists can be divided into receptor and nonreceptor antagonists. A receptor antagonist binds to either the active site (agonist binding site) or an allosteric site on a receptor. Binding o an antagonist to the active site prevents the binding o the agonist to the receptor, whereas binding

C h a p t e r 2 Pharmacodynamics

21

Anta gonis ts

Re ce ptor a nta gonis ts

Active s ite binding Re ve rs ible

Irre ve rs ible

Compe titive a nta gonis t

Noncompe titive a ctive s ite a nta gonis t

Nonre ce ptor a nta gonis ts

Allos te ric binding Re ve rs ible

Irre ve rs ible

Noncompe titive a llos te ric a nta gonis t

Che mica l a nta gonis t

P hys iologic a nta gonis t

FIGURE 2-4. Antagonist classif cation. Antagonists can be categorized based on whether they bind to a site on the receptor or agonist (receptor antagonists) or interrupt agonist–receptor signaling by other means (nonreceptor antagonists). Receptor antagonists can bind either to the agonist (active) site or to an allosteric site on the receptor; in either case, they do not a ect basal receptor activity (i.e., the activity o the receptor in the absence o agonist). Agonist (active) site receptor antagonists prevent the agonist rom binding to the receptor. I the antagonist competes with the ligand or agonist site binding, it is termed a competitive antagonist; high concentrations o agonist are able to overcome competitive antagonism. Noncompetitive active site antagonists bind covalently or with very high a f nity to the agonist site, so that even high concentrations o agonist are unable to activate the receptor. Allosteric receptor antagonists bind to the receptor at a site other than the agonist site. They do not compete directly with agonist or receptor binding, but rather alter the Kd or agonist binding or inhibit the receptor rom responding to agonist binding. High concentrations o agonist are generally unable to reverse the e ect o an allosteric antagonist. Nonreceptor antagonists all into two categories. Chemical antagonists sequester agonist and thus prevent the agonist rom interacting with the receptor. Physiologic antagonists induce a physiologic response opposite to that o an agonist, but by a molecular mechanism that does not involve the receptor or agonist.

o an antagonist to an allosteric site either alters the Kd or agonist binding or prevents the con ormational change required or receptor activation. Receptor antagonists can also be divided into reversible and irreversible antagonists ; that is, antagonists that bind to their receptors reversibly and those that bind irreversibly. Figure 2-5 illustrates the general e ects o these antagonist types on agonist binding; more detail is provided in the ollowing sections. A nonreceptor antagonist does not bind to the same receptor as an agonist, but it nonetheless inhibits the ability o an agonist to initiate a response. At the molecular level, this inhibition can occur by inhibiting the agonist directly (e.g., using antibodies), by inhibiting a downstream molecule in the activation pathway, or by activating a pathway that opposes the action o the agonist. Nonreceptor antagonists can be divided into chemical antagonists and physiologic antagonists. Chemical antagonists inactivate an agonist be ore it has the opportunity to act (e.g., by chemical neutralization);

physiologic antagonists cause a physiologic e ect opposite

to that induced by the agonist.

Competitive Receptor Antagonists

A competitive antagonist binds reversibly to the active site of a receptor. Unlike an agonist, which also binds to the active site o the receptor, a competitive antagonist does not stabilize the con ormation required or receptor activation. There ore, the antagonist blocks an agonist rom binding to its receptor, while maintaining the receptor in the inactive con ormation. Equation 2-9 is a modif cation o Equation 2-7 that incorporates the e ect o a competitive antagonist (A). AR Æ ¨ A

RÆ ¨ DR*

D

Equation 2-9

In this equation, a raction o the ree receptor molecules (R) are unable to orm a drug (agonist)–receptor complex (DR*), because receptor binding to the antagonist results in the

Agonis t Agonis t binding s ite

A

Agonis t

Agonis t Allos te ric a nta gonis t binding s ite

Unbound re ce ptor

Compe titive a nta gonis t

B

C

Agonis t binding

Compe titive a nta gonis t binding

D

Noncompe titive a nta gonis t

Noncompe titive a nta gonis t binding

FIGURE 2-5. Types o receptor antagonists. A schematic illustrating the di erences between agonist (active) site and allosteric antagonists. A. The unbound inactive receptor. B. The receptor activated by agonist. Note the con ormational change induced in the receptor by agonist binding, or example, the opening o a transmembrane ion channel. C. Agonist site antagonists bind to the receptor’s agonist site but do not activate the receptor; these agents block agonist binding to the receptor. D. Allosteric antagonists bind to an allosteric site (di erent rom the agonist site) and thereby prevent receptor activation, even when the agonist is bound to the receptor.

22 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

e s n o

50

R

e

s

p

Agonis t + Anta gonis t

Equation 2-10

Noncompetitive Receptor Antagonists

Noncompetitive antagonists can bind to either the active site

or an allosteric site o a receptor (Fig. 2-4). A noncompetitive antagonist that binds to the active site o a receptor can bind either covalently or with very high a f nity; in either case, the binding is e ectively irreversible. Because an irreversibly bound active site antagonist cannot be “outcompeted,” even at high agonist concentrations, such an antagonist exhibits noncompetitive antagonism. A noncompetitive allosteric antagonist acts by preventing the receptor rom being activated, even when the agonist is bound to the active site. An allosteric antagonist exhibits noncompetitive antagonism regardless o the reversibility o its binding, because such an antagonist acts not by competing

B Noncompe titive a nta gonis t

Agonis t a lone

n

s

e

100

o

Agonis t + Anta gonis t

p s

Equation 2-10 is similar to Equation 2-4, except that the e ective Kd has been increased by a actor o (1 [A]/KA), where KA is the dissociation constant or binding o the antagonist to the receptor (i.e., KA [A][R]/[AR]). Because an increase in Kd is equivalent to a decrease in potency, the presence o a competitive antagonist (A) reduces the potency o an agonist (D) by a actor o (1 [A]/KA). Although the potency o an agonist decreases as the concentration o competitive antagonist increases, the e f cacy o the agonist is una ected. This occurs because the agonist concentration [D] can be increased to counteract (“outcompete”) the antagonist, thereby “washing out” or reversing the e ect o the antagonist. Figure 2-6A shows the e ect o a competitive antagonist on the agonist dose–response relationship. Note that the competitive antagonist has the e ect o shi ting the agonist dose–response curve to the right, causing a decrease in agonist potency while maintaining agonist e f cacy. Atorvastatin, the drug used in the case at the beginning o this chapter to lower Admiral X’s cholesterol, is an example o a competitive antagonist. Atorvastatin is a member o the HMG-CoA reductase inhibitor (statin) class o lipid-lowering drugs. HMG-CoA reductase is an enzyme that catalyzes the reduction o HMG-CoA, which is the rate-limiting step in cholesterol biosynthesis. The similarity between the chemical structures o statins and HMG-CoA allows the statin molecule to bind to the active site o HMG-CoA reductase and thereby to prevent HMG-CoA rom binding. This inhibition is reversible because no covalent bonds are ormed between the statin and the enzyme. Inhibition o HMG-CoA reductase decreases endogenous cholesterol synthesis and lowers the patient’s cholesterol levels. For a more detailed discussion o the mechanism o action o atorvastatin and other HMG-CoA reductase inhibitors, see Chapter 20, Pharmacology o Cholesterol and Lipoprotein Metabolism.

Anta gonis t a lone

0

e

[A]ˆ KA ¯

Agonis t a lone

50

R

[D]

[D] Kd Ê1 Ë

100

%

[DR] [Ro]

A Compe titive a nta gonis t

%

ormation o an antagonist–receptor complex (AR) instead. In e ect, the ormation o the AR complex sets up a second equilibrium reaction that competes with the equilibrium or agonist–receptor binding. Note that AR is incapable o undergoing a con ormational change to the active (R*) state o the receptor. Quantitative analysis yields the ollowing equation or agonist (D) binding to the receptor in the presence o a competitive antagonist (A):

0

Anta gonis t a lone

Agonis t or a nta gonis t conce ntra tion

FIGURE 2-6. Antagonist effects on the agonist dose–response relationship. Competitive and noncompetitive antagonists have di erent e ects on potency (the concentration o agonist that elicits a hal -maximal response) and e f cacy (the maximal response to an agonist). A. A competitive antagonist reduces the potency o an agonist, without a ecting agonist e f cacy. B. A noncompetitive antagonist reduces the e f cacy o an agonist. As shown here, most allosteric noncompetitive antagonists do not a ect agonist potency.

with the agonist or binding to the active site, but rather by preventing receptor activation. The reversibility o antagonist binding is nonetheless important, because the e ect o an irreversible antagonist does not diminish when the ree (unbound) drug is eliminated rom the body, whereas the e ect o a reversible antagonist can be “washed out” over time as it dissociates rom the receptor (see Equation 2-9). A receptor that is bound by a noncompetitive antagonist can no longer be activated by the binding o an agonist. There ore, the maximal response (e f cacy) o the agonist is reduced. A characteristic di erence between competitive and noncompetitive antagonists is that competitive antagonists reduce agonist potency, whereas noncompetitive antagonists reduce agonist e f cacy. This di erence can be explained by considering that a competitive antagonist continuously competes or receptor binding, e ectively reducing the receptor’s a f nity or an agonist without limiting the number o available receptors. In contrast, a noncompetitive antagonist removes unctional receptors rom the system, thereby limiting the number o available receptors. Figures 2-6A and 2-6B compare the e ects o competitive and noncompetitive antagonists on the agonist dose–response relationship. Aspirin is one example o a noncompetitive antagonist. This agent irreversibly acetylates cyclooxygenase, the enzyme

C h a p t e r 2 Pharmacodynamics

A 100

Butyl

He xyl

n

He ptyl

Partial Agonists A partial agonist is a molecule that binds to a receptor at its active site but produces only a partial response, even when all of the receptors are occupied (bound) by the agonist. Figure 2-7A shows a amily o dose–response curves or several ull and partial agonists. Each agonist acts by binding to the same site on the muscarinic acetylcholine (ACh) receptor. Note that butyl trimethylammonium (TMA) is not only more potent than longer chain derivatives at stimulating muscle contraction but also more e f cacious than some o the derivatives (e.g., the heptyl and octyl orms) at producing a greater maximal response. For this reason, butyl TMA is a full agonist at the muscarinic ACh receptor, whereas the octyl derivative is a partial agonist at this receptor. Because partial agonists and ull agonists bind to the same site on a receptor, a partial agonist can reduce the response produced by a ull agonist. In this way, the partial agonist can act as a competitive antagonist. For this reason, partial agonists are sometimes called partial antagonists or even mixed agonist-antagonists. It is interesting to consider how an agonist could produce a less-than-maximal response i a receptor can exist in only the active or the inactive state. This is an area o current investigation, or which several hypotheses have been proposed. Recall that Equation 2-6 was simplif ed to Equation 2-7 based on the assumption that R and DR* are much more stable than R* and DR. But what would happen i a drug (call it a partial agonist) could stabilize DR as well

Octyl

a r t %

C

o

n

50

0 10 -7

10 -6

10 -5

10 -4

10 -3

[D] (Mola r)

B 100

n

a

l

g

e

s

i

a

Morphine

50 Bupre norphine

A

Nonreceptor antagonists can be divided into chemical antagonists and physiologic antagonists. A chemical antagonist inactivates the agonist o interest by modi ying or sequestering it, so that the agonist is no longer capable o binding to and activating the receptor. Protamine is an example o a chemical antagonist; this basic protein binds stoichiometrically to the acidic heparin class o anticoagulants and thereby inactivates these agents (see Chapter 23, Pharmacology o Hemostasis and Thrombosis). Because o this chemical antagonism, protamine can be used to terminate the e ects o heparin rapidly. A physiologic antagonist either blocks a receptor that mediates the physiologic response o the receptor or agonist or activates a receptor that mediates a response physiologically opposite to that o the receptor or agonist. For example, in the treatment o hyperthyroidism, -adrenergic antagonists are used as physiologic antagonists to counteract the tachycardic e ect o excess thyroid hormone. Excess thyroid hormone produces tachycardia, at least in part, via up-regulation o cardiac -adrenoceptors, and blocking -adrenergic stimulation relieves the tachycardia (see Chapter 11, Adrenergic Pharmacology, and Chapter 28, Pharmacology o the Thyroid Gland).

%

Nonreceptor Antagonists

c

t

i

o

responsible or generating thromboxane A2 in platelets. In the absence o thromboxane A2 generation, platelet aggregation is inhibited. Because the inhibition is irreversible and platelets are not capable o synthesizing new cyclooxygenase molecules, the e ects o a single dose o aspirin last or 7 to 10 days (the time required or the bone marrow to generate new platelets), even though the ree drug is cleared rom the body much more rapidly.

23

0 0.01

0.1

ED50 (B)

ED50 (M)

10

[D] (mg/kg)

FIGURE 2-7. Full and partial agonist dose–response curves. There are many instances in which drugs that all act at the agonist site on the same receptor produce di erent maximal e ects. A. Various alkyl derivatives o trimethylammonium all stimulate muscarinic acetylcholine (ACh) receptors to cause muscle contraction in the gut, but they produce di erent maximal responses, even when all receptors are occupied. In this example, the butyl and hexyl trimethylammonium derivatives are ull agonists— although they have di erent potencies, they are both capable o eliciting a maximal response. Agonists that produce only a partial response, such as the heptyl and octyl derivatives, are called partial agonists . Note that the dose–response curves o these partial agonists plateau at values less than those o ull agonists. ACh acts as a ull agonist in this system (not shown). B. Partial agonists may be more or less potent than ull agonists. In this case, buprenorphine (ED50 0.3 mg/kg) is more potent than morphine (ED50 1.0 mg/kg), although it cannot achieve the same maximal response as the ull agonist. Buprenorphine is used clinically in the treatment o opioid addiction, where it is desirable to use a partial agonist that is less e f cacious than an addicting opioid such as heroin or morphine. Low concentrations o the partial agonist buprenorphine bind tightly to the opioid receptor and competitively inhibit the binding o the more e f cacious opioids. Very high doses o buprenorphine show a paradoxically diminished analgesic e ect that may be due to lower a f nity interactions o the drug with non–mu-opioid receptors (not shown).

24 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

as DR*? In that case, addition o the partial agonist would result in stabilization o some receptors in the DR orm and some receptors in the DR* orm. At ull receptor occupancy, some receptors would be in the active state and some in the inactive state, and the e f cacy o the drug would be reduced compared to that o a ull agonist (which stabilizes only DR*). In this ormulation, a ull agonist binds pre erentially to the active state o the receptor, a partial agonist binds with comparable a f nity to both the active and inactive states o the receptor, and an inverse agonist binds pre erentially to the inactive state o the receptor (see below). A second hypothesis or the action o partial agonists is that a receptor may have multiple DR* con ormations, each with a di erent intrinsic activity. Depending on the particular con ormations o the receptor that are bound by the agonist, a raction o the maximum possible e ect may be observed even when a partial agonist is bound to 100% o the receptors. This may be the case with the so-called selective estrogen receptor modulators (SERMs) such as raloxifene and tamoxifen (see Chapter 30, Pharmacology o Reproduction). Raloxi ene acts as a partial agonist at estrogen receptors in bone and an antagonist at estrogen receptors in breast. The crystal structure o raloxi ene bound to the estrogen receptor, when compared to that o estrogen bound to the estrogen receptor, reveals that the side chain o raloxi ene inhibits an helix o the estrogen receptor rom aligning in the active site (see Fig. 30-8). This may result in inhibition o some downstream e ects o the estrogen receptor, while maintaining other e ects. At a physiologic level, this would be observed as partial agonist activity in bone (see Fig. 30-7). A variation o the second hypothesis is that a receptor may have multiple DR* con ormations that di erentially activate signal transduction pathways in cells. Drugs that activate some but not all o these pathways are called biased agonists . Biased agonism has been demonstrated or experimental compounds interacting with G protein-coupled receptors and may be relevant or some partial agonists in clinical use. A study o partial agonists acting on ligand-gated ion channels has suggested yet another model, in which the receptor requires a “priming” con ormational change that must occur be ore activation o the receptor is possible. In this model, although a partial agonist may bind to the receptor with high a f nity, it is less e f cient than a ull agonist at inducing the “primed” con ormation o the receptor. Because this “primed” con ormation is a prerequisite or activation o the receptor, a partial agonist causes the receptor to spend less time in the open con ormation than a ull agonist does, and the partial agonist has lower e f cacy than the ull agonist. The relative potency o ull agonists and partial agonists may be clinically relevant (Fig. 2-7B). A partial agonist with high a f nity or its receptor (such as buprenorphine) may be more potent but less e f cacious than a ull agonist with lower a f nity or the same receptor (such as morphine). This characteristic is leveraged clinically when the partial agonist buprenorphine is used to treat opioid addiction. Buprenorphine, with its high a f nity or the mu-opioid receptor, can be administered to outcompete other opioids taken by a patient and can there ore help to prevent relapse o opioid addiction. Buprenorphine must be care ully administered to a patient who is currently addicted to ull-agonist opioids such as

heroin or morphine, because it can outcompete these opioids and cause withdrawal symptoms.

Inverse Agonists The action o inverse agonists can be understood by considering Equation 2-6 again. As noted above, in some cases, receptors can have inherent stability in the R* state. In these cases, there is intrinsic activity (“tone”) o the receptor system, even in the absence o an endogenous ligand or an exogenously administered agonist. An inverse agonist acts by abrogating this intrinsic (constitutive) activity of the free (unoccupied) receptor. Inverse agonists may unction by binding to and stabilizing the receptor in the DR (inactive) orm. This has the e ect o deactivating receptors that had existed in the R* orm in the absence o drug. The physiologic importance o receptors that have inherent stability in the R* state is currently under investigation; receptors with mutations that render them constitutively active may become attractive targets or inverse agonist approaches. Consider the similarities and di erences between the actions o inverse agonists and competitive antagonists. Both types o drug act to reduce the activity o a receptor. In the presence o a ull agonist, both competitive antagonists and inverse agonists act to reduce agonist potency. Recall, however, that a competitive antagonist has no e ect in the absence o an agonist, whereas an inverse agonist deactivates receptors that are constitutively active in the absence o an agonist. Using Equations 2-6 through 2-9 as models, these concepts can be summarized by stating that full agonists stabilize DR*, partial agonists stabilize both DR and DR* (or alternate forms of DR* or “primed” forms of DR), inverse agonists stabilize DR, and competitive antagonists “stabilize” R (or AR) by preventing full, partial, and inverse agonists from binding to the receptor.

Spare Receptors Recall that the initial discussion o drug–receptor binding assumed that 100% receptor occupancy is required or an agonist to exert its maximal e ect. Now, consider the possibility that a maximal response could be achieved with less than 100% receptor occupancy. Figure 2-8 shows an example o a drug–receptor binding curve and a dose–response curve that illustrate this situation. In this example, a maximal e ect is achieved at a lower dose o agonist than that required or receptor saturation, that is, the EC50 is less than the Kd or this system. This type o discrepancy between the drug–receptor binding curve and the dose–response curve signif es the presence o spare receptors . At least two molecular mechanisms are thought to be responsible or the spare receptor phenomenon. First, the receptor could remain activated a ter the agonist departs, allowing one agonist molecule to activate several receptors. Second, the cell signaling pathways described in Chapter 1, Drug–Receptor Interactions, could allow or signif cant amplif cation o a relatively small signal, and activation o only a ew receptors could be su f cient to produce a maximal response. The latter is true, or example, with many G protein-coupled receptors; activation o a single G s molecule can stimulate adenylyl cyclase to catalyze the ormation o dozens o molecules o cAMP. The presence o spare receptors alters the e ect o a noncompetitive antagonist on the system. At low antagonist

C h a p t e r 2 Pharmacodynamics 1.0

A Drug-re ce ptor binding curve

Agonis t only

1.0

[DR] [R o ]

E

0.5

25

E MAX

0.5

Agonis t + incre a s ing noncompe titive a nta gonis t

0 Kd

B Dos e -re s pons e curve

0

[D]

1.0

FIGURE 2-9. Effect of a noncompetitive antagonist on the agonist dose–

E E MAX

response curve in the presence of spare receptors. In a system without spare receptors, a noncompetitive antagonist causes e f cacy to decrease at all concentrations o the antagonist (see Fig. 2-6B). In a system with spare receptors, however, potency is decreased but e f cacy is una ected at low concentrations o the antagonist, because a su f cient number o unoccupied receptors is available to generate a maximal response. As increasing concentrations o antagonist bind noncompetitively to more and more receptors, the antagonist eventually occupies all o the “spare” receptors, and e f cacy is also reduced.

0.5

0 EC 50

Kd

[D] FIGURE 2-8. Comparison between a drug–receptor binding curve and a

CONCEPTS IN THERAPEUTICS Therapeutic Index and Therapeutic Window

dose–response curve in the presence of spare receptors. In the absence o spare receptors, there o ten exists a close correlation between a drug– receptor binding curve and a dose–response curve—the binding o additional drug to the receptor causes an incremental increase in response, and EC50 is approximately equal to Kd. In situations with spare receptors, however, a hal -maximal response is elicited when less than hal o all receptors are occupied (the term spare implies that occupation o every receptor with drug is not necessary to elicit a ull response). A. Drug–receptor binding curve. B. Dose–response curve or the same drug, in the presence o spare receptors. Note that the maximal response occurs at a lower agonist concentration than does maximal binding, and EC50 Kd. These two relationships conf rm the presence o spare receptors. D is drug, R is receptor, and [DR]/[Ro] is ractional receptor occupancy. E is response (e ect), Emax is maximal response (e f cacy), and E/Emax is ractional response. EC50 is potency, and Kd is the equilibrium dissociation constant or drug–receptor binding.

The therapeutic window is the range o doses (concentrations) o a drug that elicits a therapeutic response, without unacceptable adverse e ects (toxicity), in a population o patients. For drugs that have a small therapeutic window, plasma drug levels must be monitored closely to maintain e ective dosing without exceeding the level that could produce toxicity. The next chapter discusses some o the techniques used in clinical therapeutics to maintain plasma concentrations o drugs within the therapeutic window. The therapeutic window can be quantif ed by the therapeutic index (TI) (sometimes called the therapeutic ratio), commonly def ned as

concentrations, the noncompetitive antagonist binds receptors that are not required to produce a maximal response; there ore, the e f cacy o the agonist is not decreased. The potency o the agonist is a ected, however, because potency is proportional to the raction o available receptors that must be occupied to produce a 50% response. A noncompetitive antagonist reduces the number o available receptors, thereby increasing the raction o receptors that must be bound at any agonist concentration to produce the same response. At high antagonist concentrations, the noncompetitive antagonist binds not only the “spare” receptors but also receptors that are required to produce a maximal response, and the e f cacy and potency o the agonist are both decreased. Figure 2-9 illustrates this concept.

where TD50 is the dose o drug that causes a toxic response in 50% o the population, and ED50 is the dose o drug that is therapeutically e ective in 50% o the population. The TI provides a single number that quantif es the relative sa ety margin o a drug in a population o people. A large TI represents a large (or “wide”) therapeutic window (e.g., a thousand- old di erence between the therapeutic and toxic doses), and a small TI represents a small (or “narrow”) therapeutic window (e.g., a two old di erence between the therapeutic and toxic doses). In the case at the beginning o this chapter, the potential or toxicity associated with the use o heparin and tPA is indicated by the low TIs o these drugs. For example, the dose o heparin that can cause major bleeding in a patient is o ten

Therapeutic Index (TI)

TD50 ED50

Equation 2-11

26 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 2-1 Summary of Agonist and Antagonist Action CLASSES OF AGONISTS AGONIST CLASS

ACTION

Full agonist

Activates receptor with maximal e f cacy

Partial agonist

Activates receptor but not with maximal e f cacy

Inverse agonist

Inactivates constitutively active receptor

CLASSES OF ANTAGONISTS ANTAGONIST CLASS

EFFECTS ON AGONIST POTENCY

EFFECTS ON AGONIST EFFICACY

Competitive antagonist

Yes

No

Binds reversibly to active site o receptor; competes with agonist binding to this site

Noncompetitive active site antagonist

No

Yes

Binds irreversibly to active site o receptor; prevents agonist binding to this site

Noncompetitive allosteric antagonist

No

Yes

Binds reversibly or irreversibly to site other than active site o receptor; prevents con ormational change required or receptor activation by agonist

less than twice the dose needed or a therapeutic e ect; heparin can there ore be def ned as having a therapeutic index o less than two. For this reason, patients treated with heparin must have their PTT, a marker o the coagulation cascade, monitored every ew hours. Aspirin’s high TI is indicative o its relative sa ety. Note that the pharmacologic e ect o heparin was monitored periodically in the case, whereas aspirin could be administered without the need to monitor its plasma drug level.

CONCLUSION AND FUTURE DIRECTIONS Pharmacodynamics is the quantitative study o the e ects o drugs on the body. Several tools have been developed to compare the e f cacy and potency o drugs, including the graded and quantal dose–response relationships. The ormer is used to examine the e ects o various drug doses on an individual, whereas the latter is used to examine the e ects o various drug doses on a population. The therapeutic window and therapeutic index are used to compare the concentrations o drugs that produce therapeutic e ects and toxic (adverse) e ects. In the study o pharmacodynamics, drugs can be divided into two broad classes—agonists and antagonists. Most agonists cause a receptor to maintain its con ormation in the active state, whereas antagonists prevent activation o the receptor by agonists. Antagonists are urther divided according to the molecular location o their e ect (i.e., receptor or nonreceptor), the site at which they bind to the receptor (i.e., active site or allosteric site), and the mode o their binding to the receptor (i.e., reversible or irreversible). Table 2-1 provides a summary o the various types o agonists and antagonists presented in this chapter.

ACTION

Elucidation o the molecular basis or receptor activation by ull agonists and partial agonists is likely to lead to new opportunities or drug discovery. For example, it has been shown that persistent activation o some G protein-coupled receptors (GPCRs) requires the binding o both an agonist and a G protein to the GPCR. This knowledge may be use ul in designing new drugs that modulate the unction o specif c GPCRs with greater selectivity.

Acknowledgment We thank Harris S. Rose or his valuable contributions to this chapter in the First and Second Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Cowan A, Doxey JC, Harry EJ. The animal pharmacology o buprenorphine, an oripavine analgesic agent. Br J Pharmacol 1977;60:547–554. (Provides an experimental demonstration o the variation in potency and e f cacy o ull and partial agonists.) Kenakin T, Williams M. Def ning and characterizing drug/compound unction. Biochem Pharmacol 2014;87:40–63. (Summarizes how the complex drug–receptor interactions o partial agonists, inverse agonists, biased agonists, and allosteric antagonists help to in orm drug discovery.) Lape R, Colquhoun D, Sivilotti LG. On the nature o partial agonism in the nicotinic receptor super amily. Nature 2008;454:722–727. (Suggests a mechanistic model or the e ect o partial agonists on ligand-gated ion channels.) Le P. The two-state model o receptor activation. Trends Pharmacol Sci 1995;16:89–97. (Provides the theoretical grounding or Equation 2-6; discusses quantitative treatment o drug–receptor interactions.) Pratt WB, Taylor P, eds. Principles o drug action: the basis o pharmacology. 3rd ed. New York: Churchill Livingstone; 1990. (Contains an in-depth discussion o pharmacodynamics.) Sprang SR. Cell signaling: binding the receptor at both ends. Nature 2011;469:172–173. (Summarizes the f nding that persistent activation o some GPCRs requires binding o both agonist and G protein molecules to the receptor.)

Free e

Bo u n d F re e

Bo

3 Pharmacokinetics Q u e n t in J . Ba c a a n d David E. Go la n

INTRODUCTIO ON & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27–28 PHYSIOLOGIC C BARRIERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Biological Membran M nes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Traversin inng the Membrane . . . . 28 Memb Me mbrraane Diffus . . . . 28 Central Nervous r System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 ABSORPTION N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Administratio ioon Routes and Rationale . . . . . . . . . . . . . . . . . . . . . 30 Enterall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Parenterall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Mucous Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Transdermall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Local, Regional, and Systemic Factors Affecting Absorption. . . . 31 DIST STRI RIBU BUTI TION ON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Volume of Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Plasma Protein Binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Modeling the Kinetics of Drug Distribution . . . . . . . . . . . . . . . . 33

P rote t iin in-boun bbound nd drug dr d rug ug

INTRODUCTION Even the most promising o pharmacologic therapies will ail in clinical trials i the drug is unable to reach its target organ at a concentration su f cient to have a therapeutic e ect. Many o the characteristics that render the human body resistant to harm by oreign invaders and toxic substances also limit the ability o modern drugs to combat pathologic processes within a patient. An appreciation o the many actors that a ect a drug’s ability to act within a patient and the dynamic nature o these actors over time is vitally important to the clinical practice o medicine. All drugs must meet certain minimal requirements to achieve clinical e ectiveness. A success ul drug must be able to cross the physiologic barriers that limit the access o oreign substances to the body. Drug absorption may occur by a number o mechanisms that are designed either to exploit or to breach these barriers. A ter absorption, the drug uses distribution systems within the body, such as the blood and lymphatic vessels, to reach its target organ in an appropriate concentration. The drug’s ability to act on its target is also limited by several processes within the patient. These processes all broadly into two categories: metabolism, in which the body typically inactivates the drug through enzymatic degradation (primarily in the liver), and excretion, in which the drug is eliminated rom the body (primarily

METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Oxidation/Reduction Reactions . . . . . . . . . . . . . . . . . 34 Conjugation/Hydrolysis Reactions . . . . . . . . . . . . . . . . . . . . . . . 344 EXCRETION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Rena Re nall Ex Excr cret ettioon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Bililiaa ry Excretion Bi reti et . . . . . . . . . . . . . . . . . . . . . . . . . 37 CLINICAL APPLICA APP ATI TION ONS S OF PHARMACOKINETICS . . . . . . . . . . 37 Clearance nc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Metabolism and Excretion Kineticss . . . . . . . . . . . . . . . . . . . . 37 Met Half-Life lf. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Factors Altering Half-Lifee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Therapeutic Dosing and Frequency . . . . . . . . . . . . . . . . . . . . . . 39 T Loading Dosee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Maintenance Dosee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . 41 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

M ta Me t bo b o lite bolit lite s lit ((a ctive ctiv ti e a nd nd inaa ctive ina cctiv tivee )

by the kidneys and liver, and in the eces). This chapter presents a broad overview o the pharmacokinetic processes o absorption, distribution, metabolism, and excretion (o ten abbreviated as ADME; Fig. 3-1), with a conceptual emphasis on basic principles that, when applied to an un amiliar situation, should enable the student or physician to understand the pharmacokinetic basis o drug therapy.

PHYSIOLOGIC BARRIERS A drug must overcome physical, chemical, and biological barriers to reach its molecular and cellular sites o action. The epithelial lining o the gastrointestinal tract and other mucous membranes is one sort o barrier; additional barriers are encountered a ter the drug enters the blood and lymphatics. Most drugs must distribute rom the blood into local tissues, a process that may be impeded by structures such as the blood–brain barrier. Typically, drugs leave the intravascular compartment at the level o the postcapillary venules, where there are gaps between the endothelial cells through which the drug can pass. Drug distribution occurs mainly through passive di usion, the rate o which is a ected by local ionic and cellular conditions. This section describes the major physical, chemical, and biological barriers to drug transport in the body and the properties o drugs that a ect their ability to overcome these barriers. 27

28 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Mr. W is a 66-ye a r-o ld te ch n o lo g y co n s u lta n t w h o m a ke s re q u e n t trip s a s p a rt o h is jo b in th e te le co m m u n ica tio n s in d u s try. His o n ly m e d ica l p ro b le m is ch ro n ic a tria l f b rilla tio n , a n d h is o n ly ch ro n ic m e d ica tio n is warfarin. On th e la s t n ig h t o a co n s u ltin g trip a b ro a d , h e a tte n d s a la rg e d in n e r e a tu rin g ke b a b s a n d o th e r o o d s h e d o e s n o t o te n e a t. Th e n e xt d a y, h e d e ve lo p s p ro u s e , wa te ry, o u ls m e llin g d ia rrh e a . A p hys icia n m a ke s a d ia g n o s is o tra ve le r ’s d ia rrh e a a n d p re s crib e s a 7-d a y co u rs e o trimethoprim-sulfamethoxazole. Mr. W e e ls e n tire ly w e ll 2 d a ys in to th e co u rs e o a n tib io tics , a n d 4 d a ys la te r (w h ile s till ta kin g h is a n tib io tics ), h e m e e ts w ith s o m e clie n ts a t a n o th e r la vis h d in n e r. Mr. W a n d h is g u e s ts b e co m e in toxica te d a t th e d in n e r, a n d Mr. W s tu m b le s a n d a lls o n th e cu rb a s h e is le a vin g th e re s ta u ra n t. Th e n e xt d a y, Mr. W h a s a m a rke d ly s w o lle n rig h t kn e e th a t

Biological Membranes All human cells are limited by a lipid bilayer membrane. The membrane lipids consist mainly o phospholipids, sterols (especially cholesterol), and glycolipids. The amphiphilic nature o the membrane lipids and the aqueous intracellular and extracellular environments cause the membrane to assume a structure with a hydrophobic core and two hydrophilic sur aces. In addition to lipid components, biological membranes contain proteins that may span the membrane (transmembrane proteins) or be exposed only at the extracellular or intracellular membrane sur ace. The membrane’s

Re ce ptors Fre e

Bound

Tis s ue re s e rvoirs Fre e

Bound

S ys te mic circula tion Abs orption

Free drug

P rote in-bound drug

Excre tion

Me ta bolite s (a ctive a nd ina ctive )

Me ta bolis m

FIGURE 3-1. Drug absorption, distribution, metabolism, and excretion (ADME). The basic principles o pharmacokinetics a ect the amount o ree drug that ultimately reaches the target site. To elicit an e ect on its target, a drug must be absorbed and then distributed to its target be ore being metabolized and excreted. At all times, ree drug in the systemic circulation is in equilibrium with tissue reservoirs, plasma proteins, and the target site (which usually consists o receptors); only the raction o drug that binds to specif c receptors will have a pharmacologic e ect. Note that metabolism o drug can result in both inactive and active metabolites; active metabolites may also exert a pharmacologic e ect, either on the target receptors or sometimes on other receptors.

re q u ire s e va lu a tio n in a lo ca l e m e rg e n cy d e p a rtm e n t. Phys ica l e xa m in a tio n a n d im a g in g s tu d ie s a re co n s is te n t w ith a m o d e ra te -s ize d h e m a rth ro s is o th e rig h t kn e e . La b o ra to ry s tu d ie s s h o w a m a rke d ly e le va te d in te rn a tio n a l n o rm a lize d ra tio (INR), w h ich is a s ta n d a rd ize d m e a s u re o p ro th ro m b in tim e a n d , in th is clin ica l s e ttin g , a s u rro ga te m a rke r o r p la s m a wa r a rin le ve l. Th e e m e rg e n cy p hys icia n a d vis e s Mr. W th a t h is wa r a rin le ve l is in th e s u p ra th e ra p e u tic (toxic) ra n g e a n d th a t th is e e ct is like ly d u e to a d ve rs e d ru g –d ru g in te ra ctio n s invo lvin g h is wa r a rin , h is a n tib io tics , a n d h is re ce n t a lco h o l in toxica tio n .

Questions 1 . How does a patient with well-established therapeutic levels o a chronic medication suddenly develop clinical mani estations o drug toxicity? 2 . Could this situation have been avoided? I so, how?

semipermeable lipid bilayer structure provides a barrier to the transport o molecules and has important implications or drug therapy.

Traversing the Membrane

The hydrophobic core of a biological membrane presents the major barrier to drug transport. Small nonpolar molecules, such as steroid hormones, are able to di use easily through membranes. However, passive di usion is ine ective or the transport o many large polar molecules and drugs. Some transmembrane proteins in the human solute carrier (SLC) super amily—which includes 52 amilies o proteins such as organic anion transporters, organic cation transporters, peptide transporters, and nucleoside transporters—allow transport o polar drugs and molecules across the membrane. Transmembrane carrier proteins may be specif c or a drug and related endogenous molecules; upon binding o the drug to the extracellular sur ace o the protein, the protein undergoes a con ormational change that may be energyindependent (facilitated diffusion) or require energy (active transport). This con ormational change allows the bound drug access to the interior o the cell, where the drug molecule is released rom the protein. Alternatively, some drugs bind to specif c cell sur ace receptors and trigger endocytosis , a process in which the cell membrane involutes around the molecule to orm a vesicle rom which the drug is subsequently released into the cell interior.

Membrane Diffusion

In the absence o other actors, a drug will enter a cell until the intracellular and extracellular concentrations o the drug are equal. The rate o di usion depends on the concentration gradient o the drug across the membrane and on the thickness, area, and permeability o the membrane. Fick’s law o di usion states that the net drug ux across the membrane is: Flux

(C2

C1) (Area Permeability) Thicknessmembrane

Equation 3-1

C h a p t e r 3 Pharmacokinetics

[1,000]

[1]

HA

A-

+

29

where HA is the protonated orm o Drug A. For example, consider the hypothetical case o a weakly acidic drug with a pKa o 4. In the stomach, which has a pH o approximately 1, Equation 3-2 becomes:

H+

pKadrug

S toma ch pH~1

pHstomach

log

[HA] , [A ]

which simplifies to: Ga s tric Mucos a l Ba rrie r

3 P la s ma pH~7

HA [1,000]

A-

+

H+

1,000

[1,000,000]

sider a hypothetical drug with pKa 4. Although this drug is a weak acid, in the highly acidic environment of the stomach, it is largely protonated. If the stomach pH is approximately 1, then for every 1,001 molecules of drug, 1,000 molecules are protonated (and neutral) and only 1 is deprotonated (and negatively charged). The protonated, neutral form of the drug is able to diffuse across the gastric mucosal barrier into the blood. Because the blood plasma has a pH of approximately 7 (it is actually 7.4), and the drug has a pKa of 4, the vast majority of drug now exists in the deprotonated (negatively charged) form: for every 1,001 molecules of drug, only 1 molecule is protonated (and neutral), while 1,000 molecules are deprotonated (and negatively charged). The negatively charged form of the drug is no longer able to diffuse across the lipid bilayers of the gastric mucosa, and the drug is effectively trapped in the plasma.

where C1 and C2 are the intracellular and extracellular concentrations o the drug, respectively. This def nition applies to an ideal situation where there is an absence o complicating actors such as ionic, pH, and charge gradients across the membrane. In vivo, however, these additional actors a ect the ability o a drug to enter cells. For example, a higher concentration o drug outside the cell would ordinarily avor net drug entry into the cell, but i both the cell interior and the drug are negatively charged, then net drug entry into the cell may be impeded. In contrast, a negatively charged cell interior could avor entry o a positively charged drug. Net di usion o acidic and basic drugs across lipid bilayer membranes may also be a ected by a charge-based phenomenon known as pH trapping, which depends on the drug’s acid dissociation constant (pKa) and the pH gradient across the membrane. For weakly acidic drugs, such as phenobarbital and aspirin, the protonated, electrically neutral orm o the drug is predominant in the highly acidic environment o the stomach. The uncharged orm o the drug can pass through the lipid bilayers o the gastric and duodenal mucosa, speeding the drug’s absorption (Fig. 3-2). The weakly acidic drug is then e ectively trapped as it is deprotonated to its electrically charged orm in the more basic environment o the plasma. In quantitative terms, the pKa o a drug represents the pH value at which one-hal o the drug is present in its ionic orm. The Henderson–Hasselbalch equation describes the relationship between the pKa o an acidic or basic Drug A and the pH o the biological medium containing the drug: pKa

pH

[HA] , [A ]

and finally:

FIGURE 3-2. pH trapping across lipid bilayers. In the example shown, con-

[HA] logg [A ]

log

Equation 3-2

[HA] . [A ]

The protonated orm o the drug is present at 1,000 times the concentration o the deprotonated orm, and 99.9% o the drug is in the neutral orm. Conversely, in the plasma, where the pH is approximately 7.4, more than 99.9% o the drug is in the deprotonated orm (see Fig. 3-2).

Central Nervous System The central nervous system (CNS) presents special challenges to pharmacologic therapy. Unlike most other anatomic regions, the CNS is particularly well insulated rom oreign substances. The blood–brain barrier uses specialized tight junctions to prevent the passive di usion o most drugs rom the systemic to the cerebral circulation. There ore, drugs designed to act in the CNS must either be su f ciently small and hydrophobic to traverse biological membranes easily or use existing transport proteins in the blood–brain barrier to penetrate central structures. Hydrophilic drugs that ail to target acilitated or active transport proteins in the blood–brain barrier cannot penetrate the CNS. The blood–brain barrier can be bypassed using intrathecal drug delivery, in which drugs are delivered directly into the cerebrospinal uid (CSF). Although this approach can be used, or example, to treat in ectious meningitis or to provide spinal anesthesia or a cesarean delivery, the intrathecal route is impractical or drugs that must be taken regularly by a patient.

ABSORPTION The human body presents ormidable obstacles to invasion by microorganisms. The integument has a keratinized outer layer and de ensins in the epithelium. Mucous membranes are protected by mucociliary clearance in the trachea, lysozyme secretion rom lacrimal ducts, acid in the stomach, and base in the duodenum. These nonspecif c de ense mechanisms present barriers to drug absorption and may limit the drug’s bioavailability at target organs. Bioavailability, or the raction o administered drug that reaches the systemic circulation, may depend on the route by which the drug is administered, the chemical orm o the drug, and a number o patient-specif c actors such as gastrointestinal and hepatic transporters and enzymes. Bioavailability is def ned quantitatively as:

Bioavailability

Quantity of drug reaching systemic circulation Quantity of drug administered

Equation 3-3

Ora l, s ubcuta ne ous , or intra mus cula r: 100% bioa va ila bility

Intra ve nous

This def nition o bioavailability is based on the act that most drugs reach their molecular and cellular sites of action directly from the systemic circulation. Intravenously administered drugs are injected directly into the systemic circulation; or these drugs, the quantity administered is equivalent to the amount reaching the systemic circulation, and the bioavailability is, by def nition, 1.0. In contrast, incomplete gastrointestinal absorption and “f rst-pass” hepatic metabolism (see below) typically cause the bioavailability o an orally administered drug to be less than 1.0 (Fig. 3-3).

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30 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Ora l, s ubcuta ne ous , or intra mus cula r: 50% bioa va ila bility

Time

FIGURE 3-3. Bioavailability after administration of a single dose of drug. An intravenously administered drug is immediately available in the circulation. In this example, the drug is then distributed to other body compartments (see Fig. 3-7) and eliminated by f rst-order kinetics (see Fig. 3-6). In contrast, other routes o administration (e.g., oral, subcutaneous, and intramuscular) demonstrate slower entry o drug into the blood. In addition, nonintravenous routes o administration must take into account bioavailability— or example, many orally administered drugs are incompletely absorbed or undergo f rst-pass metabolism in the liver. I a drug has 100% bioavailability, the total amount o drug reaching the systemic circulation will be the same or all routes o drug administration, but nonintravenous routes will require a longer period o time to reach a peak concentration o drug in the plasma. I the bioavailability o an oral, subcutaneous, or intramuscular dosage orm is less than 100%, then the dose o the drug would have to be increased in order or the total amount o drug reaching the systemic circulation to be the same as that o an intravenous dose. Note that the total amount o drug reaching the systemic circulation can be quantif ed by integrating the area under the curve (AUC) o the plasma drug concentration versus time plot. Thus, although di erent routes o administration (e.g., oral, subcutaneous, and intramuscular) can have di erent rates o drug absorption and there ore di er in the kinetics o plasma drug concentration over time (Fig. 3-4), i these routes exhibit the same level o bioavailability, then they will have the same AUC.

Administration Routes and Rationale New drugs are designed and tested in a dosage orm that is administered by a specif c route. Routes o administration are o ten chosen to take advantage o transport molecules and other mechanisms that permit the drug to enter body tissues. This section discusses the advantages and disadvantages o drug administration by enteral (oral), parenteral, mucous membrane, and transdermal routes (Table 3-1).

Enteral

Enteral drug administration, or the administration o a drug by mouth, is the simplest o drug routes. The enteral route o administration exploits existing weaknesses in human barrier de enses, but it exposes the drug to harsh acidic (stomach) and basic (duodenum) environments that could limit its absorption. This route provides many advantages or the patient: oral drugs are easily and conveniently sel administered, and these dosage orms are less likely than other methods to introduce systemic in ection as a complication o treatment. An orally administered drug must be stable during its absorption across the gastrointestinal tract epithelium. Gastrointestinal epithelial cell junctions make paracellular transport across an intact epithelium di f cult. Instead, ingested substances (such as drugs) must usually traverse the cell membrane at both apical and basal sur aces be ore entering the blood. The e f ciency o this process is determined by drug size and hydrophobicity and sometimes by the presence

TABLE 3-1 Routes of Drug Administration ROUTE

ADVANTAGES

DISADVANTAGES

Enteral (e.g., aspirin)

Simple, inexpensive, convenient, painless, no in ection

Drug exposed to harsh gastrointestinal (GI) environments and f rst-pass metabolism, requires GI absorption, slow delivery to site o pharmacologic action

Parenteral (e.g., morphine)

Rapid delivery to site o pharmacologic action, high bioavailability, not subject to f rst-pass metabolism or harsh GI environments

Irreversible, in ection, pain, ear, skilled personnel required

Mucous membrane (e.g., nitroglycerin)

Rapid delivery to site o pharmacologic action, not subject to f rst-pass metabolism or harsh GI environments, o ten painless, simple, convenient, low in ection, direct delivery to a ected tissues possible

Few drugs have chemical characteristics or ormulations that allow them to be administered via this route

Transdermal (e.g., nicotine)

Simple, convenient, painless, excellent or continuous or prolonged administration, not subject to f rst-pass metabolism or harsh GI environments

Requires highly lipophilic drug, slow delivery to site o pharmacologic action, may be irritating

C h a p t e r 3 Pharmacokinetics

o carriers through which the drug may enter and/or exit the cell. In general, hydrophobic and neutral drugs cross cell membranes more e f ciently than hydrophilic or charged drugs, unless the membrane contains a carrier molecule that acilitates the transport o hydrophilic substances. Upon traversing the gastrointestinal epithelium, drugs are carried by the portal system to the liver be ore entering the systemic circulation. While the portal circulation protects the body rom the systemic e ects o ingested toxins by delivering these substances to the liver or detoxif cation, this system may complicate drug delivery. All orally administered drugs are subjected to f rst-pass metabolism in the liver. In this process, liver enzymes may inactivate a raction o the ingested drug. Any drug that exhibits signif cant f rst-pass metabolism must be administered in a quantity su f cient to ensure that an e ective concentration o active drug exits the liver into the systemic circulation, rom which it can reach the target organ. Drugs administered by nonenteral routes are not subjected to f rst-pass liver metabolism.

Parenteral

Parenteral administration, in which a drug is introduced directly into the systemic circulation, cerebrospinal uid, vascularized tissue, or some other tissue space, immediately overcomes barriers that can limit the e ectiveness o orally administered drugs (Table 3-2). Tissue administration results in a rate o onset o drug action that di ers among the various body tissues, depending on the rate o blood ow to the tissue. Subcutaneous (SC) administration o a drug into poorly vascularized adipose tissue results in a slower onset o action than injection into well-vascularized intramuscular (IM) spaces. Drugs that are soluble only in oil-based solutions are o ten administered intramuscularly. Direct introduction o a drug into the venous (intravenous [IV]) or arterial (intra-arterial [IA]) circulation or into the cerebrospinal uid (intrathecal [IT]) results in the drug reaching its target organ the astest. Unlike subcutaneous and intramuscular injections, intravenous injection is not typically limited in the amount o drug that can be delivered. Continuous intravenous in usions can allow tight control over peak and steady-state plasma concentrations during drug delivery. Parenteral administration may be associated with several potential disadvantages, including an increased risk o

TABLE 3-2 Routes o Parenteral Drug Administration PARENTERAL ROUTE

ADVANTAGES

DISADVANTAGES

Subcutaneous (e.g., lidocaine)

Slow onset, may be used to administer oil-based drugs

Slow onset, small volumes

Intramuscular (e.g., haloperidol)

Intermediate onset, may be used to administer oilbased drugs

Can affect lab tests (creatine kinase), intramuscular hemorrhage, painful

Intravenous (e.g., morphine)

Rapid onset, controlled drug delivery

Peak-related drug toxicity

Intrathecal (e.g., methotrexate)

Bypasses blood– brain barrier

Infection, highly skilled personnel required

31

in ection and the requirement or administration by a health care pro essional. The onset o action o parenterally administered drugs is o ten rapid, potentially resulting in increased toxicity when such drugs are administered too rapidly or in incorrect doses. These disadvantages must be weighed against the advantages o parenteral administration (such as speed o onset and control o the delivered dose) and the urgency o the indication or pharmacologic therapy.

Mucous Membrane

Administration o drugs across mucous membranes can potentially provide rapid absorption, low incidence o in ection, convenience o administration, and avoidance o harsh gastrointestinal environments and f rst-pass metabolism. Sublingual, ocular, pulmonary, nasal, rectal, urinary, and reproductive tract epithelia have all been used to deliver drugs in the orm o liquid drops, rapidly dissolving tablets, aerosols, and suppositories (among other dosage orms). The mucous membranes are highly vascular, permitting the drug to enter the systemic circulation rapidly and to reach its target organ with minimal delay. Drugs may also be administered directly into the target organ, resulting in virtually instantaneous onset o action. This is advantageous in critical conditions such as acute asthma, where drugs such as -adrenergic agonists are administered via aerosol directly into the airways.

Transdermal

A limited number o drugs have su f ciently high lipophilicity that passive di usion across the skin is a viable route o administration. Transcutaneously administered drugs are absorbed rom the skin and subcutaneous tissues directly into the blood. This route o administration is ideal or a drug that must be slowly and continuously administered over extended periods. There is no associated risk o in ection, and drug administration is simple and convenient. The success o transdermal nicotine, estrogen, and scopolamine patches demonstrates the utility o this route o administration (see Chapter 55, Drug Delivery Modalities, or more details on transdermal drug delivery).

Local, Regional, and Systemic Factors A ecting Absorption The rate and extent o absorption o a drug are a ected by local, regional, and systemic actors. In general, a large or rapidly administered dose creates a high local concentration o a drug. A large concentration gradient between the site o administration and the surrounding tissue drives the distribution o the drug into the nearby tissue and/or vasculature. Any actor that decreases the concentration gradient at the site o administration will diminish the driving orce o the gradient and may reduce the amount o drug that is distributed into the local tissues. Regional blood ow has the greatest e ect in this regard; in a highly per used region, drug molecules crossing into that compartment are rapidly removed. This e ect maintains the drug concentration at a low level in the compartment, allowing the driving orce or entry o new drug molecules into the compartment to remain high (see Equation 3-1). For example, volatile general anesthetics are administered via inhalation. The lungs are highly per used, and the anesthetic is removed rapidly rom the lungs into the systemic circulation. Anesthetic does not accumulate in the local circulation, and a concentration

32 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 3-3 Drug Distribution to Different Body Compartments COMPARTMENT

EXAMPLES

Total body water

Small water-soluble molecules (e.g., ethanol)

Extracellular water

Larger water-soluble molecules (e.g., mannitol)

Blood plasma

Highly plasma protein-bound molecules, very large molecules, highly charged molecules (e.g., heparin)

Adipose tissue

Highly lipid-soluble molecules (e.g., propo ol)

Bone and teeth

Certain ions (e.g., uoride, strontium)

B C

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Time FIGURE 3-4. Effect of rate of absorption on peak plasma concentration of drug and on duration of drug action. The duration o action and peak plasma concentration o a drug can be a ected markedly by the drug’s absorption rate. In this example, three drugs with identical bioavailability, volume o distribution, and clearance are administered in identical doses. The drugs demonstrate di erent rates o absorption—Drug A is absorbed quickly, Drug C is absorbed slowly, and Drug B’s absorption rate is between those o Drugs A and C. Drug A reaches the highest peak plasma concentration, since all o the drug is absorbed be ore signif cant elimination can take place. Drug C is absorbed slowly and never achieves a high plasma concentration, but it persists in the plasma or longer than Drugs A or B because absorption continues during the elimination phase. It should be noted that the hypothetical Drugs A, B, and C could all be the same drug administered by three di erent routes. For example, curve A could represent intravenous glucocorticoid administration, curve B could be an intramuscular injection, and curve C could be a transdermal ormulation o the same drug.

gradient promoting di usion o anesthetic into the blood is maintained (see Chapter 17, General Anesthetic Pharmacology, or more details). In an individual with greater body mass, both the increased sur ace area or absorption and the larger tissue volumes available or distribution tend to remove a drug rom the site o administration aster and increase the rate and extent o drug absorption. The rate o drug absorption a ects both the local concentration o a drug (including its plasma concentration) and its duration o action (Fig. 3-4).

DISTRIBUTION Absorption o a drug is a prerequisite or establishing adequate plasma drug levels, but the drug must also reach its target organ(s) in therapeutic concentrations to have the desired e ect on a pathophysiologic process. Drug distribution is achieved primarily through the circulatory system; a minor component is contributed by the lymphatic system. Once a drug has been absorbed into the systemic circulation, it is then capable o reaching any target organ (with the possible exception o sanctuary compartments such as the central nervous system and testes). The concentration o drug in the plasma is typically used to def ne and monitor therapeutic drug levels, because the concentration o drug in the target organ is o ten di f cult to measure. Even in cases where the plasma concentration o a drug is very di erent rom the

tissue concentration, the e ect o the drug in the target tissue o ten correlates well with the plasma drug concentration. Organs and tissues vary widely in their capacity to take up di erent types o drugs (Table 3-3) and in the proportion o systemic blood ow they receive (Table 3-4). In turn, these actors a ect the concentration o the drug in the plasma and determine the amount o drug that must be administered to achieve the desired plasma drug concentration. The ability o nonvascular tissues and plasma proteins to take up and/or bind the drug must be accounted or in designing dosing regimens to achieve and maintain therapeutic drug levels.

Volume of Distribution The volume of distribution (Vd) describes the extent to which a drug partitions between the plasma and tissue compartments. In quantitative terms, Vd represents the uid volume that would be required to contain the total amount o absorbed drug in the body at a concentration equivalent to that in the plasma at steady state: Vd

Dose [Drug]plasma

Equation 3-4

The volume o distribution is an extrapolated volume based on the concentration o drug in the plasma, not a physical volume. Thus, Vd is low or drugs that are retained

TABLE 3-4 Total and Weight-Normalized Tissue Blood Flow in an Adult BLOOD FLOW (mL/min)

ORGAN MASS (kg)

NORMALIZED BLOOD FLOW (mL/min/kg)

Liver

1,700

2.5

680

Kidneys

1,000

0.3

3,333

Brain

800

1.3

615

Heart

250

0.3

833

Adipose

250

10.0

25

Other (muscle, etc.)

1,400

55.6

25

Total

5,400

70.0

—

ORGAN PERFUSED

C h a p t e r 3 Pharmacokinetics

primarily within the vascular compartment and high or drugs that are highly distributed into adipose and other nonvascular compartments. For very highly distributed drugs, the volume o distribution is o ten much greater than the volume o total body water, re ecting the low concentration o drug in the vascular compartment at steady state. Some drugs have very large volumes o distribution; examples include amiodarone (4,620 liters [L] or a 70-kg person), azithromycin (2,170 L), chloroquine (9,240 L), and digoxin (645 L), among others. The capacity o the blood and the various organs and tissues to take up and retain a drug depends on both the volume (mass) o the tissue and the concentrations o specif c and nonspecif c binding sites or the drug within that tissue. A drug that is taken up in large quantities by tissues such as adipose will pre erentially distribute out o the systemic circulation and into these tissues at steady state. In many cases, these tissues must be saturated be ore plasma levels o such drugs can increase su f ciently to a ect the drug’s target organ. Thus, or drugs o equal potency, a drug that is more highly distributed among body tissues generally requires a higher initial dose to establish a therapeutic plasma concentration than does a drug that is less highly distributed.

A

33

S ite of pha rma cologic a ction Va s cula r s pa ce

Cle a ring orga n

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S ite of pha rma cologic a ction Va s cula r s pa ce

Plasma Protein Binding

The capacity o adipose tissue to absorb a drug increases the tendency o the drug to di use rom the blood into nonvascular compartments, but this tendency can be counteracted to some extent by plasma protein binding o the drug. Albumin is the most abundant plasma protein ( 4 g/dL) and is the protein responsible or most drug binding. Many drugs bind with low a f nity to albumin through both hydrophobic and electrostatic orces. Plasma protein binding tends to reduce the availability o a drug or di usion or transport into the drug’s target organ because, in general, only the ree or unbound orm o the drug is capable o di usion across membranes (Fig. 3-5). Plasma protein binding may also reduce the transport o drugs into nonvascular compartments such as adipose tissue. Because a highly protein-bound drug tends to remain within the vasculature, such a drug o ten has a relatively low volume o distribution (typically, 7 to 8 L or a 70-kg person). Theoretically, plasma protein binding could be important as a mechanism or some drug–drug interactions. Coadministration o two or more drugs that bind to plasma protein could result in a higher-than-expected plasma concentration o the ree orm o either or both drugs as the coadministered drugs compete or the same binding sites. The increased ree drug concentration could potentially cause increased therapeutic and/or toxic e ects o the drug. In such cases, the dosing regimen o one or both o the drugs would need to be adjusted to keep the ree drug concentration in the therapeutic range. In practice, however, it has been di f cult to demonstrate clinically signif cant drug–drug interactions caused by competitive binding o drugs to plasma proteins, possibly because o the increased clearance o the ree drugs as they are displaced rom their plasma protein binding sites (see below). An important exception is the contraindication to the use o the antibiotic ceftriaxone in neonates with hyperbilirubinemia, since ce triaxone displaces bilirubin rom its binding sites on albumin and thereby exacerbates the hyperbilirubinemia.

Cle a ring orga n

Drug A

Drug A bound to a lbumin

Drug B

Drug B bound to a lbumin

Albumin

FIGURE 3-5. Protein binding and drug trapping. A drug that is bound to albumin or other plasma proteins cannot di use rom the vascular space into surrounding tissues. A. Drugs that do not bind plasma proteins appreciably (shown here as Drug A) di use readily into tissues. This results in both a high level o binding to the site o pharmacologic action (usually receptors) and a high rate o elimination (represented by f ux through a clearing organ). Examples o such drugs include acetaminophen, acyclovir, nicotine, and ranitidine. B. In contrast, or drugs that exhibit high levels o binding to plasma proteins (shown here as Drug B), a higher total plasma drug concentration is required to ensure an adequate concentration o ree (unbound) drug in the circulation, since only a small raction o the drug can di use into the extravascular space. Examples o such drugs include amiodarone, f uoxetine, naproxen, and war arin. It should be emphasized that plasma protein binding is only one of many variables that determine drug distribution. Drug molecule size, lipophilicity, and rate o metabolism are other important parameters that must be taken into account when considering the pharmacokinetics o a particular drug.

Modeling the Kinetics of Drug Distribution Most drugs are distributed rapidly rom the systemic circulation (intravascular compartment) to other compartments in the body. This distribution phase results in a sharp decrease in the plasma drug concentration shortly a ter intravenous

Dis tribution pha s e

Elimina tion pha s e

to have both greater muscle mass and greater proportional muscle blood ow. As a third example, an obese person typically exhibits higher capacity or drug uptake into adipose tissue. More complicated approaches to modeling the kinetics o drug distribution throughout the body can include an exhaustive number o compartments. Some approaches model each organ or vascular bed individually to describe more precisely the drug concentration at specif c target sites over time.

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34 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

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METABOLISM

Time FIGURE 3-6. Drug distribution and elimination after intravenous administration. Immediately a ter intravenous administration o a drug, the plasma drug concentration declines rapidly as the drug distributes rom the vascular compartment to other body compartments. This rapid decline is ollowed by a slower decline as the drug is metabolized and excreted rom the body. Both drug distribution and elimination display f rst-order kinetics, as demonstrated by linear kinetics on a semilogarithmic plot.

administration o a drug bolus. Even a ter the drug equilibrates among its tissue reservoirs, the plasma drug concentration continues to decline because o drug elimination rom the body. However, the plasma drug concentration decreases more slowly during the elimination phase, in part due to a “reservoir” o drug in the tissues that can di use back into the blood to replace the drug that has been eliminated (Figs. 3-6 and 3-7). The tendency or a drug to be taken up by adipose and muscle tissue during the distribution phase determines a set o dynamic equilibria among drug concentrations in the various body compartments. As shown in Figure 3-8, the rapid decline o plasma drug concentration a ter administration o an intravenous bolus o drug can be approximated by using a our-compartment model consisting o the blood and vesselrich, muscle-rich, and adipose-rich tissues. The vessel-rich group is the f rst extravascular compartment in which the concentration o drug increases, because the high blood ow received by this group kinetically avors drug entry into this compartment. However, the muscle-rich group and adiposerich group o ten have a higher capacity or taking up drug than the vessel-rich group, with the adipose-rich group accumulating the greatest amount o drug at the slowest rate. The capacity o a compartment or a drug and the rate o blood ow to the compartment also a ect the rate at which the drug exits rom the compartment. Drugs tend to exit f rst rom the vessel-rich group, ollowed by the muscle group and then the adipose group. A complex and dynamic pattern o changing blood concentrations may develop, and the pattern is specif c or each drug. The pattern may also be patient-specif c, depending on actors such as the size, age, and f tness level o the patient. For example, an older patient typically has less skeletal muscle mass than a younger patient, decreasing the contribution o muscle uptake to changes in the plasma concentration o drug. An opposite e ect may be seen in an elite athlete, who would be expected

Several organs are capable o metabolizing drugs to some extent, using enzymatic reactions that are discussed in Chapter 4, Drug Metabolism. The kidneys, gastrointestinal tract, lungs, skin, and other organs all contribute to systemic drug metabolism. However, the liver contains the greatest diversity and quantity o metabolic enzymes, and the majority o drug metabolism occurs there. The ability o the liver to modi y drugs depends on the amount o drug that enters the hepatocytes. Highly hydrophobic drugs can generally enter cells readily (including liver cells), and the liver pre erentially metabolizes hydrophobic drugs. However, the liver contains a multitude o transporters in the human solute carrier (SLC) super amily that allow entry o some hydrophilic drugs into hepatocytes as well. Hepatic enzymes chemically modi y a variety o substituents on drug molecules, thereby either rendering the drugs inactive or acilitating their elimination. These modif cations are collectively re erred to as biotransformation. Biotrans ormation reactions are classif ed into two types, termed oxidation/reduction reactions and conjugation/hydrolysis reactions . (Although biotrans ormation reactions are o ten called phase I and phase II reactions, in this book we typically use the more precise terms oxidation/reduction and conjugation/hydrolysis; see Chapter 4.)

Oxidation/Reduction Reactions Oxidation/reduction reactions modi y the chemical structure o a drug; typically, a polar group is added or uncovered. The most common pathway, the microsomal cytochrome P450 enzyme system in the liver, mediates a large number o oxidative reactions. Some drugs may be administered in inactive (prodrug) orm and are altered metabolically by oxidation/ reduction reactions to the active (drug) orm in the liver. This prodrug strategy can acilitate oral bioavailability, decrease gastrointestinal toxicity, and/or prolong the elimination hal li e o a drug.

Conjugation/Hydrolysis Reactions Conjugation/hydrolysis reactions hydrolyze a drug or conjugate a drug to a large, polar molecule in order to inactivate the drug or, more commonly, to enhance the drug’s solubility and excretion in the urine or bile. Occasionally, hydrolysis or conjugation can result in the metabolic activation o prodrugs. The most commonly added groups include glucuronate, sul ate, glutathione, and acetate. As described in more detail in the next chapter, the e ects o oxidation/reduction and conjugation/hydrolysis reactions on a particular drug also depend on the presence o other drugs that are being taken concomitantly by the patient. Certain classes o drugs, such as barbiturates, are power ul

35

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C

Extra va s cula r volume

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Blood

FIGURE 3-7. Schematic model of drug distribution and elimination. A two-compartment pharmacokinetic model can be used to describe drug distribution and elimination a ter administration o a single intravenous dose. The drug concentration rises rapidly as the drug is added to the f rst compartment. A. In the absence o elimination, the initial rise in drug concentration is ollowed by a rapid decline to a new plateau as the drug equilibrates (distributes) between the two compartments. B. I the distribution o the drug is conf ned to the blood volume, then the plasma drug concentration declines more slowly as the drug is eliminated rom the body. In both cases, as the concentration o drug in the plasma decreases, the orces driving (A) drug distribution and (B) elimination decrease, and the absolute amount o drug distributed or eliminated per unit time decreases. There ore, the kinetics o both distribution and elimination appear as straight lines on a semilogarithmic plot; this is the def nition o f rst-order kinetics. Note that the hal -time or drug elimination is generally longer than the hal -time or drug distribution. C. When drug distribution and elimination are occurring simultaneously, the decline o plasma drug concentration with time is represented by the sum o the two processes. Note that the curve in (C) is the sum o the two f rst-order processes shown in (A) and (B). In the schematics on the le t o the f gure, the volume in the “Blood” compartment represents plasma drug concentration, the volume in the “Extravascular volume” compartment represents tissue drug concentration, the dropper above the “Blood” compartment represents absorption o drug into the systemic circulation, and the drops below the “Blood” compartment represent elimination o drug by metabolism and excretion.

inducers o enzymes that mediate oxidation/reduction reactions; other drugs are capable o inhibiting these enzymes (see Table 4-3). An understanding o these drug–drug interactions is an essential prerequisite to the appropriate dosing o drug combinations. Physicians and researchers have begun to elucidate the important role o genetic di erences among individuals in the various transporters and enzymes responsible or drug absorption, distribution, excretion, and especially metabolism. For example, an individual’s complement o cytochrome

P450 enzymes in the liver and their specif c genetic polymorphisms determine the rate and extent to which that individual can metabolize numerous therapeutic agents. This topic is discussed in detail in Chapter 7, Pharmacogenomics.

EXCRETION Oxidation/reduction and conjugation/hydrolysis reactions enhance the hydrophilicity o a hydrophobic drug and its metabolites, enabling such drugs to be excreted along a f nal

36 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

n

t

1.0 Mus cle

0.8 VRG

0.6

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0.2

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Adipos e

0.4

0.1 0.1

1.0

10

100

1000

Time (minute s )

FIGURE 3-8. Four-compartment model o drug distribution. A ter administration o an intravenous bolus, the drug is delivered to various tissues via the systemic circulation. The raction o the administered dose is initially highest in the vascular compartment (blood), but the blood raction subsequently alls rapidly as the drug is distributed to the other tissue compartments. The most vessel-rich tissues (i.e., the tissues that are supplied by the highest raction o the cardiac output) are generally the f rst to accumulate drug. However, the tissue compartments also vary in their capacity or taking up drug. Because the mass o the muscle group is larger than that o the vessel-rich group (VRG), the muscle group has a larger uptake capacity. But because the muscles are less well per used than the vessel-rich group, this e ect is mani ested only a ter the drug has begun to distribute to the VRG. The most poorly per used group is the adipose-rich group, but this group has the highest capacity to accumulate drug. The peak level o drug in the adipose group is not as high as that in the muscle-rich group, because a signif cant amount o drug has been eliminated by metabolism or excretion be ore the adipose group begins to accumulate drug. A ter the administration o drug has been completed, the reverse pattern is seen—the drug leaves f rst rom the vessel-rich group and then rom the muscle and adipose groups, respectively. This pattern emphasizes that adipose tissue can provide a signif cant reservoir o drug even a ter drug administration is discontinued. The drug in this example is thiopental, a barbiturate that can be used to induce general anesthesia.

common pathway with drugs that are intrinsically hydrophilic. Most drugs and drug metabolites are eliminated rom the body through renal and biliary excretion. Renal excretion is the most common mechanism o drug excretion, and it relies on the hydrophilic character o a drug or metabolite. Only a relatively small number o drugs are excreted primarily in the bile or through respiratory and dermal routes. Many orally administered drugs are incompletely absorbed rom the upper gastrointestinal tract, and residual drug is eliminated by ecal excretion.

Renal Excretion Renal blood ow comprises about 25% o total systemic blood ow, ensuring that the kidneys are continuously exposed to any drug ound in the blood. The rate o drug elimination through the kidneys depends on the balance o drug f ltration, secretion, and reabsorption rates (Fig. 3-9). The a erent arteriole introduces both ree (unbound) drug and plasma protein-bound drug into the glomerulus. Typically, however, only the ree drug orm is f ltered into the renal tubule. There ore, renal blood ow, glomerular f ltration rate, and drug binding to plasma protein all a ect the amount o drug that enters the tubule at the glomerulus. Enhancing

blood ow, increasing glomerular f ltration rate, and decreasing plasma protein binding cause a drug to be excreted more rapidly. Renal excretion plays the primary role in the clearance o many drugs; examples include vancomycin, atenolol, and ampicillin. Drugs such as these can accumulate to toxic levels in patients with compromised renal function and in elderly patients (who often manifest some degree of renal compromise). For example, in individuals with normal kidney unction, the typical dosing interval or vancomycin is 12 hours. In severe kidney disease, however, therapeutic levels o the drug may persist or 7 days a ter a single intravenous dose. Urinary drug concentration rises in the proximal tubule because o passive di usion o uncharged drug molecules, acilitated di usion o charged or uncharged molecules, and active secretion o anionic and cationic molecules rom the blood into the urinary space. The secretory mechanisms are generally not specif c or the drugs; rather, drug secretion takes advantage o molecular similarities between the drug and naturally occurring substances such as organic anions (transported by OAT amily and other proteins) and cations (transported by OCT amily and other proteins). Penicillin is an example o a drug that is eliminated largely by active transport in the proximal tubule. The extent o plasma protein binding appears to have a relatively small e ect on

P roxima l tubule

P e ritubula r ca pilla ry Tubular Secretion

Glomerular Filtration

1

2

Affe re nt a rte riole

Drug in blood

3 Tubular Reabsorption

Effe re nt a rte riole

4

Urine

FIGURE 3-9. Drug f ltration, secretion, and reabsorption in the kidney. Drugs may be (1) f ltered at the renal glomerulus, (2) secreted into the proximal tubule, (3) reabsorbed rom the tubular lumen and transported back into the blood, and (4) excreted in the urine. The relative balance o f ltration, secretion, and reabsorption rates determines the kinetics o drug elimination by the kidney. Enhancing blood ow, increasing glomerular f ltration rate, and decreasing plasma protein binding all cause a drug to be excreted more rapidly, because all these changes result in increased f ltration o drug at the glomerulus. Some drugs, such as penicillin, are actively secreted into the proximal tubule. Although reabsorption can decrease the elimination rate o a drug, many drugs exhibit pH trapping in the distal tubule and are there ore e f ciently excreted in the urine. For drugs that are dependent on the kidney or elimination, compromised renal unction can result in higher plasma drug concentrations, and the dose and requency o drug administration must be altered accordingly.

C h a p t e r 3 Pharmacokinetics

drug secretion into the proximal tubule, because the highly e f cient transporters that mediate active tubular secretion rapidly remove ree (unbound) drug rom the peritubular capillaries and thereby alter the equilibrium between ree and protein-bound drug at these sites. The urinary concentration o a drug may all as the drug is reabsorbed in the proximal and distal tubules. Reabsorption is limited primarily by pH trapping, as described above. The renal tubular uid is typically acidic in and beyond the proximal tubule, which tends to avor trapping o the ionic orm o weak bases. Because this region o the tubule contains transporter proteins that are di erent rom those in preceding segments o the nephron, ionic drug orms resist acilitated di usional reabsorption, and their excretion is thereby enhanced. Drug reabsorption in the tubule can be enhanced or inhibited by chemical adjustment o the urinary pH. Changing the rate o urine ow through the tubules can also modi y the rate o drug reabsorption. An increased rate o urine output tends to dilute the drug concentration in the tubule and to decrease the amount o time during which acilitated di usion can occur; both o these e ects tend to decrease drug reabsorption. For example, aspirin is a weak acid that is excreted by the kidney. Aspirin overdose is treated by administering sodium bicarbonate to alkalinize the urine (and thus trap aspirin in the tubule) and by increasing the urine ow rate (and thus dilute the tubular concentration o the drug). Both o these clinical maneuvers result in aster elimination o the drug.

37

Clearance The clearance o a drug is the pharmacokinetic parameter that most signif cantly limits the time course o action o the drug at its molecular, cellular, and organ targets. Clearance can be conceptualized in two complementary ways. First, it is def ned as the rate o elimination o the drug rom the body relative to the concentration o the drug in plasma. Alternatively, clearance is the rate at which plasma would have to be cleared o the drug to account or the observed kinetics o change o the total amount o drug in the body, assuming that all the drug in the body is present at the same concentration as that in the plasma. There ore, clearance is expressed in units o volume/time, as ollows: Metabolism Excretion [Drug]plasma

Clearance

Equation 3-5

where metabolism and excretion are expressed as rates (amount/time). Although metabolism and excretion are distinct physiologic processes, the pharmacologic endpoint is equivalent— a reduction in circulating levels o active drug. As such, metabolism and excretion are o ten re erred to collectively as clearance mechanisms, and the principles o clearance can be applied to both: Clearancetotal

Clearancerenal

Clearancehepatic

ClearanceOther

Equation 3-6

Biliary Excretion Drug reabsorption also plays an important role in biliary excretion. Some drugs are secreted rom the liver into the bile by members o the ATP binding cassette (ABC) super amily o transporters, which includes seven amilies o proteins such as the multidrug resistance (MDR) amily. Because the bile duct enters the gastrointestinal tract in the duodenum, such drugs must pass through the length o the small and large intestine be ore being eliminated. In many cases, these drugs undergo enterohepatic circulation, in which they are reabsorbed in the small intestine and subsequently retained in the portal and then the systemic circulation. Drugs such as steroid hormones, digoxin, and some cancer chemotherapeutic agents are largely excreted in the bile.

CLINICAL APPLICATIONS OF PHARMACOKINETICS The dynamic interactions among drug absorption, distribution, metabolism, and excretion determine the plasma concentration o a drug and dictate the ability o the drug to reach its target organ in an e ective concentration. O ten, the desired duration o drug therapy exceeds that achievable by a single dose, and multiple doses are needed to provide a relatively constant plasma concentration o drug within the limits o e f cacy and toxicity. The results o clinical trials o drugs under development, as well as clinical experience using US Food and Drug Administration (FDA)-approved drugs, suggest standard doses o a drug in the average patient. However, pharmacokinetic and other di erences among patients (such as disease status and pharmacogenomic prof le) must also be considered in designing a dosing regimen or a drug or drug combination in the individual patient.

Metabolism and Excretion Kinetics

The rate o drug metabolism and excretion by an organ is limited by the rate o blood ow to that organ. The majority o drugs demonstrate f rst-order kinetics when used in standard therapeutic doses; that is, the amount o drug that is metabolized or excreted in a given unit o time is directly proportional to the concentration o drug in the systemic circulation at that time. Because the clearance mechanisms or most drugs are not saturated under ordinary circumstances, increases in plasma drug concentration are matched by increases in the rate o drug metabolism and excretion (see Equation 3-5). The f rst-order elimination rate (where elimination includes both metabolism and excretion) ollows Michaelis-Menten kinetics: E

Vmax Km

C C

Equation 3-7

where Vmax is the maximum rate o drug elimination, Km is the drug concentration at which the rate o elimination is ½ Vmax, C is the concentration o drug in the plasma, and E is the elimination rate (Fig. 3-10). Because elimination is usually a f rst-order process, a semilogarithmic plot o plasma drug concentration versus time typically shows a straight line during the elimination phase (see Fig. 3-6). A small number o drugs (e.g., phenytoin and ethanol) demonstrate saturation kinetics , in which the clearance mechanisms become saturated at or near the therapeutic concentration o the drug. Once saturation occurs, the clearance rate ails to increase with increasing plasma drug concentrations (zero-order kinetics ). This can result in dangerously elevated plasma concentrations o the drug, which can cause toxic (or even lethal) e ects.

where Vd is the volume o distribution and 0.693 is an approximation o ln 2. Thus, all o the actors outlined above that a ect the volume o distribution and clearance o a drug also a ect the hal -li e o the drug. A decrease in drug clearance or increase in volume o distribution tends to prolong the elimination hal -li e and thereby enhance the e ect o the drug on the target organ. The hal -li e must be care ully considered in designing any dosing regimen, as the e ects rom a drug with a long hal -li e may last or a number o days. For example, the hal -li e o chloroquine is more than 1 week and that o amiodarone is more than 1 month.

Vma x

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38 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Factors Altering Half-Life Km

P la s ma drug conce ntra tion FIGURE 3-10. Michaelis-Menten kinetics. Drug elimination typically ollows Michaelis-Menten (f rst-order) kinetics. The rate o drug elimination increases as the plasma drug concentration increases, until the elimination mechanisms become saturated and reach a maximal elimination rate (Vmax) at high plasma concentrations. The Michaelis-Menten constant, Km, is the drug concentration at which the drug elimination rate is ½ Vmax.

The extent to which an organ contributes to drug clearance is quantif ed by its extraction ratio, which compares the drug levels in plasma immediately be ore entering and just a ter exiting the organ: Extraction

Cin

Cout Cin

Equation 3-8

where C is the concentration. An organ that contributes substantially to drug clearance is expected to have a higher extraction ratio (closer to 1) than an organ that does not participate substantially in drug clearance (closer to zero). For example, the liver extraction ratio is high or drugs that have substantial f rst-pass metabolism.

Half-Life By decreasing the concentration o active drug in the blood, drug metabolism and excretion shorten the time during which a drug is capable o acting on a target organ. The elimination half-life o a drug is def ned as the amount of time over which the drug concentration in the plasma decreases to one-half of its original value. Knowledge o a drug’s elimination hal -li e allows the clinician to estimate the requency o dosing required to maintain the plasma concentration o the drug in the therapeutic range (see below). There are many potentially con ounding actors in any clinical situation, and it is use ul to consider here the simplest o cases. Because most drugs are eliminated by f rst-order kinetics, the body can o ten be modeled as a single compartment with a volume that is equivalent to the volume o distribution. In this model, the elimination hal li e (t1/2) depends only on the volume o distribution and clearance o the drug: t1/2

0.693 Vd Clearance

Equation 3-9

Physiologic and pathologic changes in the volume o distribution must be considered when determining the appropriate drug dose and dosing interval (Table 3-5). As patients age, their skeletal muscle mass decreases, which could decrease the volume o distribution. In contrast, an obese person has an increase in the capacity or drug uptake by adipose tissue, and a drug that distributes into at may need to be given in a higher dose in order to reach therapeutic plasma drug levels. As a third example, i drug dosing is based on total body weight but the adipose group does not take up the drug, then potentially toxic drug levels could be reached in an obese individual. Finally, some drugs may partition pre erentially into pathologic uid spaces such as ascites or a pleural e usion, causing long-term toxicity i the drug dosage is not adjusted accordingly. Physiologic and pathologic processes may also a ect drug clearance. For example, the cytochrome P450 enzymes responsible or drug metabolism in the liver can be induced, increasing the rate o drug inactivation, or inhibited, decreasing the rate o drug inactivation. Specif c P450 enzymes are induced by some drugs (such as carbamazepine , phenytoin, prednisone , and rifampin) and inhibited by others (such as

TABLE 3-5 Factors Affecting Drug Half-Life FACTORS AFFECTING HALF-LIFE

MOST COMMON EFFECT ON HALF-LIFE

Effects on Volume of Distribution Aging (decreased muscle mass → decreased distribution)

Decreased

Obesity (increased adipose mass → increased distribution)

Increased

Pathologic uid (increased distribution)

Increased

Effects on Clearance Cytochrome P450 induction (increased metabolism)

Decreased

Cytochrome P450 inhibition (decreased metabolism)

Increased

Cardiac ailure (decreased clearance)

Increased

Hepatic ailure (decreased clearance)

Increased

Renal ailure (decreased clearance)

Increased

C h a p t e r 3 Pharmacokinetics

cimetidine , ciprof oxacin, diltiazem, and f uoxetine ); see

Table 4-3 or a list o notable inducers and inhibitors o specif c enzymes. Organ ailure is another critical actor in determining appropriate dosing regimens. Hepatic ailure may both alter liver enzyme unction and decrease biliary excretion. Decreased cardiac output reduces the amount o blood that reaches clearance organs. Renal ailure decreases drug excretion because o decreased drug f ltration and secretion into the renal tubules. In summary, hepatic, cardiac, and renal ailure can each lead to a decreased ability to inactivate or eliminate a drug and thereby increase the elimination hal -li e o the drug.

Therapeutic Dosing and Frequency The basic principles o pharmacokinetics—absorption, distribution, metabolism, and excretion—inf uence the design o an optimal dosing regimen or a drug. Absorption determines the potential route(s) o administration and helps to

determine optimal drug dose. For two drugs with the same potency, the more highly absorbed drug—as evidenced by a higher bioavailability—generally requires a lower dose than the more poorly absorbed drug. In contrast, a more highly distributed drug—as evidenced by a higher volume o distribution—generally necessitates higher drug dosing. The elimination rate o a drug in uences its hal -li e and thereby determines the requency o dosing required to maintain therapeutic plasma drug levels. In general, therapeutic dosing o a drug seeks to maintain the peak (highest) plasma drug concentration below the toxic concentration and the trough (lowest) drug concentration above the minimally e ective level (Fig. 3-11). This can be accomplished most e f ciently using continuous drug delivery by intravenous (continuous in usion), subcutaneous (continuous pump or implant), transcutaneous (dermal patch), oral (sustained-release tablet), and other routes o administration, as described in more detail in Chapter 55. In many cases, however, the dosing regimen must also consider

n

B Therapeutic Dosing with Loading Dose

t e

n

t

c n c g m

3

6

9

a l

0

Da ys

9

12

Da ys

n

n

o i a

t

2.8

Toxic ra nge

2.0

t e

n

t

r

t

i

o

Toxic ra nge

r

a

6

D Subtherapeutic Dosing

n

n

c

2.1

1.5

g

c

o

e c

3

1s t dos e

o

n

S ubthe ra pe utic ra nge

0

12

c

u

1.4

The ra pe utic ra nge

1.0

d

d

r

g

0.5

s

0

P

P

l

S ubthe ra pe utic ra nge

r

u

The ra pe utic ra nge

d a

d a a

s

m

0.5

C Toxic Dosing

a

The ra pe utic ra nge

0.5

0

1s t dos e

3

6

Da ys

9

S ubthe ra pe utic ra nge

P

l

S ubthe ra pe utic ra nge

l

0

a

a

s

m

0.7

s

a

1.0

r

u

The ra pe utic ra nge

1s t dos e

m

1.5

o

c

e

n

1.0

r

u

g

c

o

n

1.5

0

P

Toxic ra nge

2.0

r

a

t

i

o

Toxic ra nge

r

a

t

i

o

n

A Therapeutic Dosing

2.0

39

12

0

0

1s t dos e

3

6

9

12

Da ys

FIGURE 3-11. Therapeutic, subtherapeutic, and toxic drug dosing. From a clinical perspective, drug concentrations in plasma can be divided into subtherapeutic, therapeutic, and supratherapeutic or toxic ranges. The goal o most drug-dosing regimens is to maintain the drug at concentrations within the therapeutic range (re erred to as the therapeutic window). A. The f rst several doses o a drug are typically subtherapeutic as the drug equilibrates to its steady-state concentration (approximately our elimination hal -lives are required to achieve steady state). Appropriate drug dosing and dosing requency result in steady-state drug levels that are therapeutic, and the maximal and minimal concentrations o the drug remain within the therapeutic window. B. I the initial (loading) dose is larger than the maintenance dose, the drug reaches therapeutic concentrations more rapidly. The magnitude o the loading dose is determined by the volume o distribution o the drug. C. Excessive maintenance doses or dosing requency result in drug accumulation and toxicity. D. Insu f cient maintenance doses or dosing requency result in subtherapeutic steady-state drug concentrations. In all our panels, the drug is administered once daily, distributed very rapidly to the various body compartments, and eliminated with f rst-order kinetics.

40 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Continuous infus ion Infre que nt la rge dos e s Fre que nt s ma ll dos e s

8

6

4

2

P

l

a

s

m

a

d

r

u

g

c

o

n

c

e

n

t

r

a

t

i

o

n

(

m

g

/

L

)

The ra pe utic ra nge

0 0

Time

FIGURE 3-12. Fluctuations in steady-state drug concentration depend on dosing frequency. The same average steady-state plasma drug concentration can be achieved using a variety o di erent drug doses and dosing intervals. In the example shown, the same total amount o a drug is administered by three di erent dosing regimens: continuous in usion, requent small doses, and in requent large doses. The smooth curve represents the e ect o a continuous in usion o drug. Discontinuous dosing results in uctuations above and below the continuous-in usion curve. Note that all three dosing regimens have the same time-averaged plasma drug concentration at steady state (4 mg/L) and the same area under the curve, but the discontinuous regimens result in peaks and troughs above and below the target drug concentration. I these peaks and troughs all above or below the boundaries o the therapeutic window (as in the in requent large-dose regimen), then clinical outcome can be adversely a ected. For this reason, requent small-dose regimens are generally more e f cacious and better tolerated than in requent large-dose regimens. However, this concern must be balanced against the convenience o (and improved patient adherence with) less requent (e.g., once daily) dosing regimens.

patient convenience. Frequent small doses (usually oral) can be administered to achieve minimal variation in steady-state plasma drug concentration, but this strategy subjects the patient to the inconvenience o requent drug administration. Less requent dosing requires the use o higher doses and leads to greater uctuations in peak and trough drug levels; this type o regimen is more convenient or the patient but also more likely to cause problems due to excessive (toxic) or insu f cient (subtherapeutic) drug levels (Fig. 3-12). Optimal dosing regimens typically maintain the steadystate plasma drug concentration within the therapeutic window or that drug. Because steady state is reached when the rate o drug input is equal to its output, the steady-state drug concentration is a ected by drug bioavailability, clearance, dose, and dosing interval (the requency o administration): Csteady state

Bioavailability Dose Intervaldosing Clearance

Equation 3-10

where C is the plasma concentration o the drug. Immediately a ter the initiation o drug therapy, the rate o drug entry into the body (kin) is much greater than the elimination rate (kout); there ore, the drug concentration in the blood increases. Assuming that elimination ollows f rst-order kinetics, the rate o elimination also increases as the plasma

drug concentration increases, because the elimination rate is proportional to the plasma drug concentration. Steady state is reached when the two rates (kin and kout) are equal. Because kin is a constant, the approach to steady state is governed by kout, the composite rate for all drug clearance mechanisms. (kout can also be called ke, the composite rate or drug elimination.) In most dosing regimens, drug levels accumulate a ter each successive dose, and the steady state is reached only when the amount o drug entering the system is equal to the amount being removed rom the system (see Fig. 3-11). Clinically, this principle must be remembered when the dosing regimen is altered, because approximately our elimination hal -lives must pass be ore the new steady state is reached. The concept o context-sensitive half-life describes the dynamic hal -li e o elimination o drug rom its target site rom the beginning o dosing to the time at which steady state is reached. This concept is important clinically, particularly or continuous in usions o drugs such as opioid analgesics or intravenous anesthetics such as propo ol. O ten, these drugs are administered as continuous in usions or periods o time that are insu f ciently long to reach steady state. In these cases, the hal -li e o elimination o drug rom a target site depends on the duration o drug administration. This concept can be used clinically to predict (1) the response o patients to drugs that are not at steady state and (2) the amount o time required or the drug e ects to clear a ter discontinuation o the drug in usion. The steady-state plasma drug concentration can be altered by the addition o a new drug to a patient’s treatment regimen. In the case o Mr. W, the addition o trimethoprimsul amethoxazole inhibited the metabolism o war arin, decreasing the clearance rate o the latter drug and causing its steady-state concentration to reach supratherapeutic levels. This e ect was exacerbated by Mr. W’s acute intoxication with ethanol, which also inhibited war arin metabolism. Assuming that Mr. W’s weight is approximately 70 kg, he is taking 5 mg o war arin every 24 hours, and the bioavailability o war arin is 0.93, then his initial steady-state plasma war arin concentration can be calculated as ollows: Csteady state

0.93 5 mg 24 h 0.192 L/ h

1.01 mg/L

where the clearance value o 0.192 L/hour is determined rom the hal -li e and volume o distribution o the drug (see Equations 3-9 and 3-10). When his war arin clearance was decreased by the addition o trimethoprim-sul amethoxazole and ethanol, the steady-state plasma concentration o war arin increased to toxic levels. This situation could have been avoided by measuring Mr. W’s INR several days a ter the addition o trimethoprim-sul amethoxazole (and adjusting his war arin dose, i necessary) and by cautioning him to temper his alcohol consumption while taking war arin (one to two drinks on an occasional basis).

Loading Dose

A ter administration o a drug by any route, the plasma concentration o the drug initially increases. Distribution o drug rom the vascular (blood) compartment to body tissues then causes the plasma drug concentration to decrease. The rate and extent o this decrease are signif cant or drugs with high volumes o distribution. I the administered dose o drug ails to take account o the volume o distribution, instead

41

The ra pe utic ra nge

u

g

c

o

n

c

e

n

t

r

a

t

i

o

Toxic ra nge

77 L

269.5 mg

s

m

3.5 mg/ L

a

where Vd is the volume o distribution and C is the desired steady-state plasma concentration o the drug. In the absence o a loading dose, approximately our elimination hal -lives are required or the tissue distribution and plasma concentration o a drug to reach steady state. Use o a loading dose circumvents this process by providing a su f cient amount o drug to attain an appropriate (therapeutic) drug concentration in the blood and tissues a ter only one or two doses o drug. For example, lidocaine has a volume o distribution o 77 L in a 70-kg person. Assuming that a steady-state plasma concentration o 3.5 mg/L is needed to control ventricular arrhythmias, the appropriate loading dose o lidocaine in this person can be calculated as: Doseloading

a

Equation 3-11

Csteady state

l

Vd

P

Doseloading

d

r

accounting only or the blood volume, then therapeutic drug levels will not be reached promptly. Initial (loading) doses o drug are o ten administered to compensate or drug distribution into the tissues. Such doses may be much higher than would be required i the drug were retained in the vascular compartment. Loading doses may be used to achieve therapeutic levels o drug (i.e., levels at the desired steady-state concentration) with only one or two doses o drug:

n

C h a p t e r 3 Pharmacokinetics

S ubthe ra pe utic ra nge

Time FIGURE 3-13. Saturation kinetics and drug toxicity. Drug elimination typically ollows f rst-order Michaelis-Menten kinetics, increasing as the plasma drug concentration increases. At optimal dosing, the steady-state plasma drug concentration remains within the therapeutic range (bottom curve). However, excessive drug dosing may saturate the body’s capacity to eliminate the drug, or example, by overwhelming the hepatic cytochrome P450 enzyme system (top curve). In this case, the elimination rate o the drug does not increase with increasing plasma drug concentration (i.e., elimination ollows zero-order rather than f rst-order kinetics). Continued administration o the drug results in drug accumulation, and the plasma drug concentration may reach toxic levels.

Maintenance Dose

Once steady-state drug concentrations are achieved in the plasma and the tissues, subsequent doses need to replace only the amount o drug that is lost through metabolism and excretion. The maintenance dose rate o a drug is dependent on the drug clearance, according to the principle that rate in rate out at steady state: Dosemaintenance

Clearance

Csteady state

Equation 3-12

Administration o a dose rate greater than the calculated maintenance dose rate would provide a drug input greater than the drug clearance, and the drug could accumulate to toxic levels within the tissues. In Mr. W, the calculated maintenance dose or war arin is: Dosemaintenance

0.192 L /h 0.194 mg/h

1.01 mg/ L

CONCLUSION AND FUTURE DIRECTIONS This chapter has provided an overview o the pharmacokinetic processes o absorption, distribution, metabolism, and excretion (ADME). An understanding o the actors that determine a drug’s ability to act in an individual patient and the changing nature o these actors over time is vitally important to the sa e and e f cacious use o drug therapy. The key equations governing the relationships among dosing, clearance, and plasma drug concentration (Table 3-6) are important to consider when making therapeutic decisions about drug regimens.

4.65 mg/day

The appropriate maintenance dose or Mr. W is there ore 4.65 mg/day. Because war arin is only 93% bioavailable, Mr. W should take 5 mg/day to maintain an adequate steadystate plasma concentration. (Note also that, because war arin has a low therapeutic index and toxic levels o the drug can lead to potentially atal hemorrhage, the biological activity o war arin should be monitored care ully by periodic measurement o the INR.) For a small number o drugs, the body’s capacity to eliminate the drug (e.g., through hepatic metabolism) may become saturated at therapeutic or only slightly supratherapeutic plasma drug concentrations. In these cases, the kinetics o drug elimination may change rom f rst-order to zero-order (also called saturation kinetics ; see above). Continued administration o drug results in rapid drug accumulation in the plasma, and drug concentrations may reach toxic levels (Fig. 3-13).

TABLE 3-6 Summary of Key Pharmacokinetic Relationships Initial concentration

Steady-state concentration

Elimination half -life

=

=

=

Loading dose Volume of distribution Fraction absorbed × Maintenance dose Dosing interval × Clearance 0.693 × Volume of distribution Clearance

42 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

At present, the clinical applicability o pharmacokinetics is mainly based on drug e ects that have been observed in a population o individuals. There are nearly inf nite major and minor variations among individuals, however, and these variations in uence the e ects o drug therapy. For example, clear di erences in pharmacokinetics are present among persons o di erent ages, genders, body mass, f tness levels, ethnicities, genomic makeup, and disease states. For some drugs, advances in therapeutic drug monitoring have enabled the real-time determination o plasma drug concentrations. A more extraordinary revolution in pharmacokinetics is o ered by pharmacogenomics . Future drug therapy may involve the administration o drugs that have been engineered specif cally or the patient who is receiving them. Knowledge o a patient’s genomic makeup could enable drug therapies to exploit strengths and compensate or weaknesses in a host o patient-specif c variables. For example, genetic tests or variants o the P450 enzymes that metabolize war arin are now available, and clinical trials are underway to study whether pharmacogenetic testing can better predict dosing requirements to maintain therapeutic levels o this drug. This topic is discussed in Chapter 7. Finally, it should be noted that the use o proteins and other macromolecules as drugs presents unique pharmacokinetic opportunities and challenges compared to the use o small molecules as drugs. Some o the challenges include protein absorption and stability, protein distribution to sites o therapeutic action, and protein clearance by enzymatic degradation and other mechanisms. The study o the mechanisms involved in determining the pharmacokinetics o protein therapeutics is in its in ancy compared to the considerable knowledge that exists regarding the pharmacokinetics

o small molecules and provides opportunities or discovery and optimization o macromolecular therapies. This topic is discussed in more detail in Chapter 54, Protein Therapeutics.

Acknowledgment We thank John C. LaMattina or his valuable contributions to this chapter in the First and Second Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Ezan E. Pharmacokinetic studies o protein drugs: past, present and uture. Adv Drug Deliv Rev 2013;65:1065–1073. (Overview o the opportunities and challenges presented by the pharmacokinetics o protein therapeutics.) Godin DV. Pharmacokinetics: disposition and metabolism o drugs. In: Munson PL, ed. Principles o pharmacology. New York: Chapman & Hall; 1995. (A solid introductory text, this chapter illustrates the various aspects o pharmacokinetics with many examples o specif c drugs.) Hediger MA, Clémençon B, Burrier RE, Bru ord EA. The ABCs o membrane transporters in health and disease (SLC series): introduction. Mol Aspects Med 2013;34:95–107. (Reviews and introduces a special issue on the 52 amilies o proteins in the human solute carrier super amily.) Klaasen CD, Aleksunes LM. Xenobiotic, bile acid, and cholesterol transporters: unction and regulation. Pharmacol Rev 2010;62:1–96. (Reviews the unction, regulation, and substrates o ABC-super amily, SLC-superamily, and other transporters that mediate the cellular uptake and e ux o drugs and other molecules.) Pratt WB, Taylor P, eds. Principles o drug action: the basis o pharmacology. 3rd ed. New York: Churchill Livingstone; 1990, Chapters 3 and 4. (This text provides a comprehensive treatment o pharmacokinetics and pharmacokinetic principles.) Rees DC, Johnson E, Lewinson O. ABC transporters: the power to change. Nat Rev Mol Cell Biol 2009;10:218–227. (Reviews the molecular mechanisms o ABC-super amily transporters.)

H2 O

O2 -

1

4

P 4 50 5 -Fe 3+ R -H H

R-OH RO

(paa re (p re nt n t dr drug ug)) ug

( xi (o xidi d iizz e d d rug g)

4 Drug Metabolism

R-OH

B

(oxiidiz diz e d d rug g) H2 O

H

H 0

H2 O

Fe 3+

INTRODUCTION ON & CA CASE CAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 CAS 4 –44 44 6 SITES T0E OF DRUG RUG UG MET TA ABOLISM LISM. . . . . . . . . . . . . . . . . . . . He . . . me . . . . . . 43 PATHWAYS AT T O OFRRUG META T BOLISM . . . . . . . . . . . . . . . . . . . . . . . 44 RDRUG R-H H ME Oxidatio Oxidati ion/Reduction Reductio Rea eact ctio ionns . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3 3+ + FConju Fe juugati tion/Hydrolysis Reac acti t ons . . . . . . . . . . . . . . . . . . . . . . . 47 Dru D rugg Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Induction and Inhib hibit itio ionn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Active and Toxic Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

H2 O

5

2H+

Our ues are exposed on a daily basis to xenobiotics — Ou tissues R or oreign orei ign ssubstances bsttHaanc nces c tthat hatt are nott nat naturally t rall ll oound ndd iinn tthe he bbody. odd Most drugs are xenobiotics that are used to modulate bodily unctions or therapeutic ends. Drugs and other environmental chemicals that enter the body are modif ed by a vast array o enzymes. The biochemical trans ormations per ormed by these enzymes can alter the compound to render it benef cial, harm ul, or simply ine ective. The processes by which biochemical reactions alter drugs within the body are collectively called drug metabolism or drug biotrans ormation. The previous chapter introduced the importance o renal clearance in the pharmacokinetics o drugs. Although the biochemical reactions that alter drugs to renally excretable orms are an essential part o drug metabolism, drug metabolism encompasses more than this one unction. Drug biotrans ormation can alter drugs in our important ways: ■

■ ■

R-H (pa re nt drug))

H2 O INDIVIDUA NDIVID VID IDUAL L FAC FACT FA AC CTORS C AFFE ECT CTIN ING DRUG METABOLISM. . . . . . 50 macog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 1 PPharmacogenomics Race and Ethnicity thn hnn y . . . . . . . . . . . R-H . . .H. . . . . . . . . . . . . . . . . . . . . . . 533 R RAgee a nd n Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 5 Diet and Enviro iron onnnment mFe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 F 33+ Metabolic Drug ug Interact Interacti Interactio tions i ns . . . . . . . . . . . . . .Flavoprote . . . . . .p. . . .t . .in . . 53 Diseases Affecting Drug Metabolism M . . . . . . . . ((re . .e. .duce . . . . .d) . ). . . 54 54 CONCLUSION AND FUTURE DIRECTIONS D CTIOe -S . . . . . . . . . . . . . . . . . . 544 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544

2

INTRODUCTION NT T 0-

■

F. Pe t e r Gu e n g e rich

An active drug may be converted to an inactive drug. An active drug may be converted to an active or toxic metabolite. An inactive prodrug may be converted to an active drug. An unexcretable drug may be converted to an excretable metabolite (e.g., to enhance renal or biliary clearance).

This chapter presents the major processes o drug metabolism. Following the case is an overview o the sites o drug metabolism, ocusing principally on the liver. The two major types o biotrans ormation are then discussed; these are o ten termed phase I and phase II reactions , although the terminology is imprecise and it incorrectly implies a temporal order o the reactions. (In addition, phase III is sometimes used to describe the process o drug transport.) In this chapter,

NA ADP +

Flavoprote in NADP H on/h xidi dl ) to d) i(on n/hydr /hidiz yddzroly reoly we use oxidation/reductionn and conjugation/hydrolysis describe desc de scri ribe be tthese hese he se pprocesses roce ro cess sses es m more oree ac oor accurately. accu cura rattelly. Th The chapt chapter h ter concludes with a discussion o th tthe actors ctors ors that tthaa can lead to di erR-H b li among iindividuals. di iidd l ences iin ddrug metabolism

SITES OF DRUG METABOLISM The liver is the main organ o drug metabolism. This act f gures prominently in the phenomenon known as the f rstpass e ect. Orally administered drugs are o ten absorbed in the gastrointestinal (GI) tract and transported directly to the liver via the portal circulation (Fig. 4-1). In this manner, the liver has the opportunity to metabolize drugs be ore they reach the systemic circulation and, there ore, be ore they reach their target organs. The f rst-pass e ect must be taken into account when designing dosing regimens because, i hepatic metabolism is extensive, the amount o drug that reaches the target tissue is much less than the amount (dose) that is administered orally (see Chapter 3, Pharmacokinetics). Certain drugs are inactivated so e f ciently upon their f rst pass through the liver that they cannot be administered orally and must be given parenterally. One such drug is the antiarrhythmic lidocaine, which has a bioavailability o only 3% when taken orally (see Chapter 12, Local Anesthetic Pharmacology). Although the liver is quantitatively the most important organ in metabolizing drugs, every tissue in the body is capable o drug metabolism to some degree. Particularly active sites include the skin, the lungs, the gastrointestinal tract, and the kidneys. The gastrointestinal tract deserves special mention because this organ, like the liver, can contribute to the f rst-pass e ect by metabolizing orally administered drugs be ore they reach the systemic circulation. 43

44 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Ms . B is a 32-ye a r-old Ca u ca s ia n w om a n w h o co m p la in s o s o re th ro a t a n d d i f cu lty s wa llo w in g o r th e p a s t 5 d a ys . Ph ys ica l e xa m in a tio n re ve a ls cre a m y w h ite le s io n s o n th e to n g u e th a t a re id e n tif e d a s o ra l th ru s h , a u n ga l in e ctio n . He r h is to ry in clu d e s s e xu a l a ctivity w ith m u ltip le p a rtn e rs , in co n s is te n t u s e o co n d o m s , a n d co n tin u o u s u s e o o ra l co n tra ce p tive s o r th e p a s t 14 ye a rs . Th e p re s e n ta tio n s u g g e s ts a d ia g n o s is o HIV-1 in e ctio n , w h ich is co n f rm e d b y p o lym e ra s e ch a in re a ctio n (PCR) a n a lys is . Ms . B h a s a lo w CD4 T-ce ll co u n t a n d is im m e d ia te ly s ta rte d o n a s ta n d a rd a n ti-HIV re g im e n th a t in clu d e s th e p ro te a s e in h ib ito r s a q u in a vir. He r o ra l th ru s h re s o lve s w ith a to p ica l a n tiu n ga l a g e n t. De s p ite a g g re s s ive th e ra p y, h e r CD4 ce ll co u n t co n tin u e s to d e cre a s e , a n d s h e p re s e n ts

to h e r p hys icia n s e ve ra l m o n th s la te r w ith a tig u e a n d a p e rs is te n t co u g h . Fu rth e r inve s tiga tio n le a d s to a d ia g n o s is o tu b e rcu lo s is .

Questions 1 . One o the f rst-line drugs in the treatment o tuberculosis is ri ampin, which decreases the e ectiveness o HIV protease inhibitors. What is the mechanism o this drug–drug interaction? 2 . Isoniazid is another drug commonly used in the treatment o tuberculosis. Why does Ms. B’s ethnic background give her physician reason or concern when considering the use o this drug? 3 . What dietary interactions should be taken into consideration when prescribing medications to treat Ms. B’s HIV in ection?

PATHWAYS OF DRUG METABOLISM PO

S ubcuta ne ous

Tra ns de rma l

GI

IV

P orta l ve in Live r

Conta ins firs t-pa s s me ta bolite s S ys te mic circula tion

Othe r orga ns

Drugs and other xenobiotics undergo biotrans ormation beore excretion rom the body. Many pharmaceuticals are lipophilic, enabling the drug to di use across cell membranes, such as those o the intestinal mucosa or o the target tissue. Un ortunately, the same chemical property that enhances bioavailability o drugs may also make renal excretion di f cult, because clearance by the kidney requires that these drugs be made more hydrophilic so that they can dissolve in the aqueous urine. Thus, biotrans ormation reactions o ten enhance the hydrophilicity o compounds to render them more susceptible to renal excretion. Biotrans ormation reactions have been classically divided into two main types: oxidation/reduction (phase I) and conjugation/hydrolysis (phase II). Oxidation reactions typically trans orm the drug into more hydrophilic metabolites by adding or exposing polar unctional groups such as hydroxyl (-OH) or amine (-NH2) groups (Table 4-1). Such metabolites are o ten pharmacologically inactive and, without urther modif cation, may be excreted. Some products o oxidation and reduction reactions, however, require urther modif cations prior to excretion. Conjugation (phase II) reactions modi y compounds through attachment o hydrophilic groups, such as glucuronic acid, to create more polar conjugates (Table 4-2). It is important to note that these conjugation reactions occur independently o oxidation/reduction reactions, and the enzymes involved in oxidation/reduction and conjugation/hydrolysis reactions o ten compete or substrates.

FIGURE 4-1. Portal circulation and the f rst-pass e ect. Drugs administered by mouth (per os, or PO) are absorbed in the GI tract and then delivered, via the portal vein, to the liver. This pathway allows the liver to metabolize drugs be ore they reach the systemic circulation, a process responsible or the f rst-pass e ect. In contrast, drugs that are administered intravenously (IV), transdermally, or subcutaneously enter the systemic circulation directly and can reach their target organs be ore hepatic modif cation. The f rst-pass e ect has important implications or bioavailability; the oral ormulation o a drug that undergoes extensive f rst-pass metabolism must be administered in a much larger dose than the equivalent IV ormulation o the same drug.

C h a p t e r 4 Drug Metabolism 45

TABLE 4-1 Oxidation and Reduction Reactions REACTION CLASS

STRUCTURAL FORMULA

REPRESENTATIVE DRUGS

I. Cytochrome P450-Dependent Oxidations 1. Aliphatic Hydroxylation

Barbiturates Digitoxin Cyclosporine

OH R R

OH

R O

2. Aromatic Hydroxylation

Propranolol Phenytoin

HO R

R

3. N-Dealkylation

Methamphetamine Lidocaine

O

R1

N H

+

R1

R2

NH2

H

4. O-Dealkylation

R2

Codeine

O

R

O

R2

+

OH

R

5. S-Oxidation

H

R2

Phenothiazine Cimetidine

O

S

R1

6. N-Oxidation R

R2

NH2

7. Desulfuration

S

R1

S R1

Quinidine

H N

R

R2

OH

Thiopental

O R1

R2

R2

8. Epoxide Formation

Carbamazepine

O R1

R2

R2

R1

II. Cytochrome P450-Independent Oxidations 1. Alcohol Dehydrogenation/ Aldehyde Dehydrogenation

R

OH

R

OH

O R

3. Decarboxylation

R

H

2. Oxidative Deamination

+

NH2

R

R

NH3

Histamine Norepinephrine

H

OH

R

Ethanol Pyridoxine

O

O

OH

+ CO 2

Levodopa

O

III. Reductions 1. Nitro Reduction

O2N

H2 N R

R

2. Dehalogenation

R

3. Carbonyl Reduction

R

X

O R1

R2

R1

Nitrofurantoin Chloramphenicol

H

Halothane Chloramphenicol

OH

Methadone Naloxone R2

46 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 4-2 Hydrolysis and Conjugation Reactions REACTION CLASS

STRUCTURAL FORMULA

REPRESENTATIVE DRUGS

I. Hydrolysis 1. Ester Hydrolysis

O

O

R1

O

2. Amide Hydrolysis

+

R2

R1

O

R2

HO

OH

Procaine Aspirin Succinylcholine Procainamide Lidocaine Indomethacin

O

R1

N H

3. Epoxide Hydrolysis

+

R2

R1

R2

H2 N

OH

Carbamazepine (epoxide metabolite)

OH

O R1

R2

R2

R1

OH

II. Conjugation 1. Glucuronidation

COOH

COOH

R

OH

+

OH

OH

O UDP

O

OH

O

OH

O R

3. Glycine Conjugation

CoA

S O

R

O

O

R

+

OH

Isoniazid Sulfonamides

O

+

OH

R

OH

OH

2. Acetylation

O

Diazepam Digoxin Ezetimibe

Salicylic acid

O

H2 N

H N

R

OH

OH

O

4. Sulfate Conjugation R

R

5. Glutathione Conjugation (and processing to mercapturic acids)

NH2

+

HO 3 S

OH

+

HO 3 S

O O

ADP

R

ADP

R

H N

S O3H

O

S O3H

O R

+ HOOC

X

COOH O

HS

O HOOC

NH2

H N

N H

Ethacrynic acid Dichloroacetic acid Acetaminophen (metabolite) Chlorambucil

NH2

H N

N H

Estrone Methyldopa

COOH O

S R HOOC R

6. N-Methylation R1

7. O-Methylation

H N

HO

S

H N O

R2 R

O

HO

8. S-Methylation

R

N

R1

Methadone Norepinephrine

R2

R

Catecholamines

HO

SH

R

S

Thiopurines

C h a p t e r 4 Drug Metabolism 47

Oxidation/Reduction Reactions

Conjugation/Hydrolysis Reactions

Oxidation reactions involve membrane-associated enzymes expressed in the endoplasmic reticulum (ER) o hepatocytes and, to a lesser extent, o cells in other tissues. The enzymes that catalyze these phase I reactions are typically oxidases; the majority o these enzymes are heme protein monooxygenases o the cytochrome P450 class. Cytochrome P450 enzymes (sometimes abbreviated CYP) are also known as microsomal mixed-function oxidases and are involved in the metabolism o approximately 75% o all drugs used today. (The term P450 re ers to the 450-nm absorption peak characteristic o these heme proteins when they bind carbon monoxide.) The net result o a cytochrome P450-dependent oxidation reaction is:

Conjugation and hydrolysis reactions provide a second set o mechanisms or modi ying compounds or excretion (Fig. 4-3). Although hydrolysis o ester- and amide-containing drugs is sometimes included among the phase I reactions (in the older terminology), the biochemistry o hydrolysis is more closely related to conjugation than to oxidation/reduction. Substrates or these reactions include both metabolites o oxidation reactions (e.g., epoxides) and compounds that already contain chemical groups appropriate or conjugation, such as hydroxyl (-OH), amine (-NH2), or carboxyl (-COOH) moieties. These substrates are coupled by trans er enzymes to endogenous metabolites (e.g., glucuronic acid and its derivatives, sul uric acid, acetic acid, amino acids, and the tripeptide glutathione) in reactions that o ten involve high-energy intermediates (Table 4-2). The conjugation and hydrolysis enzymes are located in both the cytosol and the endoplasmic reticulum o hepatocytes (and other tissues). In most cases, the conjugation process makes the drug more polar. Virtually all o the conjugated products are pharmacologically inactive, with some important exceptions (e.g., morphine glucuronide). Some conjugation reactions are important clinically in the case o neonates, who have not yet ully developed the capacity to carry out this set o reactions. UDP-glucuronyl trans erase (UDPGT) is responsible or conjugating bilirubin in the liver and acilitating its excretion. A relative def ciency o this enzyme at the time o birth puts in ants at risk or neonatal jaundice, which results rom increased serum levels o unconjugated bilirubin. Neonatal jaundice is a problem because neonates have not only underdeveloped activity o this enzyme but also an undeveloped blood–brain barrier. Unconjugated bilirubin is water-insoluble and very lipophilic; it binds readily to the unprotected neonatal brain and is capable o causing signif cant damage to the central nervous system. This pathologic condition is known as bilirubin encephalopathy or kernicterus . Neonatal hyperbilirubinemia (unconjugated) can be treated with phototherapy with 450-nm light, which converts circulating bilirubin to an isomer that is more rapidly excreted. Another e ective treatment is the administration o small doses o the barbiturate phenobarbital, which power ully up-regulates the expression o the enzyme UDPGT and thereby reduces serum levels o unconjugated bilirubin. This example illustrates a recurring theme: understanding drug metabolism can help predict both adverse and potentially advantageous drug–drug interactions. It is important to note that conjugation and hydrolysis reactions do not necessarily constitute the last step o biotrans ormation. Since the conjugation o these highly polar moieties occurs intracellularly, they o ten require active transport across cellular membranes to be excreted (active transport o the parent drug can also occur). Moreover, some conjugation products may be subjected to urther metabolism.

Drug

O2

NADPH

H Æ

Drug-OH

H2O

NADP

Equation 4-1

The reaction proceeds when the drug binds to the oxidized (Fe3 ) cytochrome P450 to orm a complex, which is then reduced in two sequential oxidation/reduction steps as outlined in Figure 4-2A. Nicotinamide adenine dinucleotide phosphate (NADPH) donates the electrons in both o these steps via a avoprotein reductase. In the f rst step, the donated electron reduces the cytochrome P450–drug complex. In the second step, the electron reduces molecular oxygen to orm an activated oxygen–cytochrome P450–drug complex. Finally, as the complex becomes more active through rearrangement, the reactive oxygen atom is trans erred to the drug, resulting in the ormation o the oxidized drug product and recycling oxidized cytochrome P450 in the process. The mechanism o these reactions is illustrated in Figure 4-2B. Most liver cytochrome P450 oxidases exhibit broad substrate specif city (Table 4-1). This is due in part to the activated oxygen o the complex, which is a power ul oxidizing agent that can react with a variety o substrates. The names o the cytochrome P450 enzymes are sometimes designated by “P450” ollowed by the number o the P450 enzyme amily, capital letter o the sub amily, and an additional number to identi y the specif c enzyme (e.g., P450 3A4). Many o the P450 enzymes have partially overlapping specif cities that together allow the liver to recognize and metabolize a wide array o xenobiotics. Together, P450-mediated reactions account or more than 95% o oxidative biotrans ormations. Other pathways may also oxidize lipophilic molecules. A pertinent example o a non-P450 oxidative pathway is the alcohol dehydrogenase pathway that oxidizes alcohols to their aldehyde derivatives as part o the overall process o excretion. These enzymes are also the basis or the toxicity o methanol. Methanol is oxidized by alcohol dehydrogenase to ormaldehyde, which can do considerable damage to some tissues. The optic nerve is particularly sensitive to ormaldehyde, and methanol toxicity can cause blindness. Another important non-P450 enzyme is monoamine oxidase (MAO). This enzyme is responsible or the oxidation o amine-containing endogenous compounds such as catecholamines and tyramine (see Chapter 11, Adrenergic Pharmacology) and some xenobiotics, including drugs.

Drug Transport Although many drugs are su f ciently lipophilic to cross cell membranes passively, it is now appreciated that many drugs need to be transported actively into cells. This act has signif cant consequences or oral bioavailability (transport into enterocytes or active excretion into the intestinal lumen), hepatic metabolism (transport into hepatocytes or enzymatic metabolism and or excretion into bile), and renal

48 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY NADP +

NADP H

Fla voprote in (re duce d)

Fla voprote in (oxidize d)

A

2 +e -

RH RH

P 450-Fe2+

P 450-Fe 3+

+e -

3

O2 P 450-Fe 2+

RH H2 O

O2-

1

4

P 450-Fe3+ R-H

R-OH

(pa re nt drug)

(oxidize d drug)

H

R-OH

B

(oxidize d drug)

H 0

H2 O

R-H (pa re nt drug)

H2 O

Fe3+

6

0

H2 O He me

R-H

1

R-H

Fe3+

Fe3+ e-

H2 O

5

2

+

2H

00 Fe2+

e(from NADP H)

NADP +

Fla voprote in (oxidize d)

NADP H

R-H

R-H

4

Fla voprote in (re duce d)

Fe2+ 0-

0 0 Fe2+

R-H

0

R-H

3

O2

Fe3+

FIGURE 4-2. Cytochrome P450-mediated drug oxidation. Many drug metabolism reactions involve a system o hepatic P450 microsomal enzymes that catalyze the oxidation o drugs. A. An overview o the reaction involves a set o oxidation/reduction steps in which an iron moiety in the P450 enzyme acts as an electron carrier to trans er electrons rom NADPH to molecular oxygen. The reduced oxygen is then trans erred to the drug, resulting in an additional -OH group on the now-oxidized drug ( or this reason, P450 enzymes are sometimes re erred to colloquially as “oxygen guns” or even “nature’s blowtorch”). The addition o the -OH group results in increased drug hydrophilicity and an increased rate o drug excretion. B. The detailed mechanism o the P450 reaction can be divided into six steps: (1) drug complexes with oxidized cytochrome P450; (2) NADPH donates an electron to the avoprotein reductase, which reduces the P450-drug complex; (3 and 4) oxygen joins the complex, and NADPH donates another electron, creating the activated oxygen–P450 substrate complex; (5) iron is oxidized, with the loss o water; and (6) the oxidized drug product is ormed. There are multiple P450 enzymes; each has a somewhat di erent specif city or substrates (such as drugs). Five o the human P450s (1A2, 2C9, 2C19, 2D6, and 3A4) account or approximately 90% o the oxidative metabolism o drugs.

C h a p t e r 4 Drug Metabolism 49 D-glucurona te

D

D-a ce ta te

Drug or pha s e I drug me ta bolite OH

D

D-glycine D-s ulfa te

NH2

Excre tion

D-gluta thione

D

D-me thyl

FIGURE 4-3. Conjugation reactions. In these reactions, a drug (represented by D) or drug metabolite (represented by D-OH and D-NH2) is conjugated to an endogenous moiety. Glucuronic acid, a sugar, is the most common group that is conjugated to drugs, but conjugations of acetate, glycine, sulfate, glutathione, and methyl groups are also common. The addition of one of these moieties makes the resulting drug metabolite more hydrophilic and often enhances drug excretion. (Methylation, an important exception, does not increase drug hydrophilicity.) Transport mechanisms also play a major role in the elimination of drugs and their metabolites.

clearance (transport into proximal tubular cells and excretion into the tubular lumen). Several important molecules mediate these processes. The multidrug resistance protein 1 (MDR1), or P-glycoprotein, which is a member o the ABC amily o e ux transporters, actively transports compounds back into the intestinal lumen. This process limits the oral bioavailability o several important drugs, including digoxin and HIV-1 protease inhibitors. The metabolism o drugs rom the portal circulation (i.e., the f rst-pass e ect) o ten requires the transport o compounds into hepatocytes via the organic anion transporting polypeptide (OATP) and the organic cation transporter (OCT) amily o proteins. These transporters are particularly relevant or the metabolism o several 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), which are used in the treatment o hypercholesterolemia. For example, metabolism o the

A

HMG-CoA reductase inhibitor pravastatin is dependent on the transporter OATP1B1, which transports the drug into hepatocytes. Drug uptake into hepatocytes via OATP1B1 is thought to be the rate-limiting step in the clearance o pravastatin rom the bloodstream. The uptake o pravastatin on its f rst pass through the liver also provides a potential advantage by keeping the drug out o the systemic circulation, rom which it could be taken up by muscle cells and thereby cause toxic e ects such as rhabdomyolysis. The organic anion transporter (OAT) amily o transporters is responsible or renal secretion o many clinically important anionic drugs, such as -lactam antibiotics, nonsteroidal anti-in ammatory drugs (NSAIDs), and antiviral nucleoside analogs.

Induction and Inhibition The use o phenobarbital to prevent neonatal jaundice demonstrates that drug metabolism can be in uenced by the expression levels o drug-metabolizing enzymes. Although some P450 enzymes are constitutively active, others can be induced or inhibited by various compounds. Induction or inhibition can be incidental (a side e ect o a drug) or deliberate (the desired e ect o therapy). The primary mechanism o P450 enzyme induction is an increase in the expression o the enzyme chie y through increased transcription, although augmented translation and decreased degradation can also have minor roles. The induction o P450 enzymes by a wide array o drugs re ects the biology o xenobiotic receptors that act as the body’s surveillance system to metabolize potentially toxic compounds. Drugs, environmental pollutants, industrial chemicals, and even oodstu s can enter hepatocytes and bind to several di erent xenobiotic receptors, such as the pregnane X receptor (PXR), constitutively active/androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) (Fig. 4-4).

D

D

D

Extra ce llula r

Cytopla s m

D

D

A P 450 e nzyme

P 450

OH

D

D

D

D

C

I

P 450

P 450

FIGURE 4-4. Conceptualization of P450 induction and inhibition. Drugs can Nucle us

Coa ctiva tor A

RxR P XR

P 450 tra ns cription

both induce the expression and inhibit the activity of P450 enzymes. Some drugs can induce the synthesis of P450 enzymes (left panel). In this example, Drug A activates the pregnane Xreceptor (PXR), which heterodimerizes with the retinoid Xreceptor (RXR) to form a complex with coactivators and initiate transcription of the P450 enzyme. Induction can also occur via the constitutively active/androstane receptor (CAR) or the aryl hydrocarbon receptor (AhR) (not shown). Drug D enters the cell and is hydroxylated by a P450 enzyme (right panel). The P450 enzyme can be inhibited by a second drug acting as a competitive inhibitor (Drug C) or an irreversible inhibitor (Drug I). The mechanism by which a drug inhibits P450 enzymes is not necessarily predictable from the drug’s chemical structure; the mechanism can only be determined experimentally. In addition, metabolites of Drugs A, C, and I can play a role in enzyme induction and inhibition (not shown).

50 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

These molecules are nuclear hormone receptors; when a xenobiotic compound binds to and activates the receptor, the complex is translocated to the nucleus, where it binds to the enhancer regions o various biotrans ormation enzymes, promoting increased expression o P450 enzymes via transcription. By a similar mechanism, nuclear hormone receptor activation can also increase the expression o drug transporters that aid in clearing the compounds rom the body, such as MDR1 and OATP1. There are multiple consequences o P450 enzyme induction. First, a drug can increase its own metabolism. For example, carbamazepine , an antiepileptic drug, not only induces P450 3A4 but also is metabolized by P450 3A4. Hence, carbamazepine increases its own metabolism by inducing P450 3A4. Second, a drug can increase the metabolism o a co-administered drug. For example, P450 3A4 is responsible or metabolizing almost 50% o all clinically prescribed drugs. Should such a drug be co-administered with carbamazepine, its metabolism would also be increased. This situation can be problematic, because the increased P450 3A4 activity can reduce drug concentrations below their therapeutic levels i standard doses o the drugs are administered. In Ms. B’s case, the administration o rifampin in conjunction with her HIV therapy could be detrimental, because ri ampin induces P450 3A4 and thereby increases the metabolism o protease inhibitors such as saquinavir, thus reducing the therapeutic e ectiveness o the protease inhibitor. Third, induction o P450s or some o the other biotrans ormation enzymes can result in the production o toxic levels o reactive drug metabolites, resulting in tissue damage or other adverse e ects. Just as certain compounds can induce P450 enzymes, other compounds can inhibit these enzymes. An important consequence of enzyme inhibition is the decreased metabolism of drugs that are metabolized by the inhibited enzyme. Such inhibition can both allow drug levels to reach toxic concentrations and prolong the presence o active drug in the body. Enzyme inhibition can be achieved in several di erent ways (Fig. 4-4). For example, ketoconazole , a widely used anti ungal drug, has a nitrogen moiety that binds to the heme iron in the active site o P450 enzymes; this binding prevents the metabolism o co-administered drugs by competitive inhibition. An example o irreversible inhibition is secobarbital, a barbiturate, which alkylates and permanently inactivates the P450 complex. On occasion, the inhibition o P450 enzymes can be used to therapeutic advantage. For example, the protease inhibitor ritonavir is highly e ective against HIV but has signif cant gastrointestinal adverse e ects that limit its use as a chronic treatment. However, because ritonavir is a potent inhibitor o P450 3A4, it can be used clinically in doses that are below the threshold or gastrointestinal adverse e ects but high enough to inhibit P450 3A4. Inhibition o P450 3A4 “boosts” the e ective concentrations o other HIV protease inhibitors that are metabolized by this P450 enzyme. For example, lopinavir cannot achieve therapeutic levels as a single agent because o extensive f rstpass metabolism, but co-administration with ritonavir allows lopinavir to reach therapeutic concentrations. Drug transporters can also be induced or inhibited by other drugs. For example, macrolide antibiotics can inhibit MDR1, and this inhibition can lead to increased serum levels o drugs, such as digoxin, that are excreted by MDR1.

MDR1 is also transcriptionally regulated by PXR. Consequently, drugs that induce up-regulation o P450 enzymes via the PXR pathway (e.g., P450 3A4) concomitantly increase transcription o the MDR1 drug transporter. A detailed list o compounds that can inhibit or induce the common P450 enzymes can be ound in Table 4-3. This list is not meant to be exhaustive, but only to highlight common medications that are metabolized by the same P450 enzymes. New drugs are extensively tested or drug interactions, both in the laboratory (in vitro) and in clinical trials, as required by the US Food and Drug Administration.

Active and Toxic Metabolites Knowing the routes by which therapeutic agents are metabolized can a ect the choice o drug to prescribe in a particular clinical situation. This is true both when the metabolite is active, in which case the administered agent may be acting as a prodrug, and when the agent has toxic metabolites (see Chapter 6, Drug Toxicity). Prodrugs are inactive compounds that are metabolized by the body into their active, therapeutic forms. One example o a prodrug is the selective estrogen receptor modulator tamoxifen; this drug has little activity until it is hydroxylated to become 4-hydroxytamoxi en, a metabolite that is 30- to 100- old more active than the parent compound. Another example is the angiotensin II receptor antagonist losartan; the potency o this drug is increased ten old upon oxidation o its alcohol group to a carboxylic acid by P450 2C9. The strategy o selective prodrug activation can be used or therapeutic benef t in cancer chemotherapy. One example o this strategy is the use o mitomycin C, a naturally occurring compound that is activated to a power ul DNA alkylating agent a ter it is reduced by several enzymes including a cytochrome P450 reductase. Mitomycin C selectively kills hypoxic cancer cells in the core o solid tumors because (1) these cells have increased levels o the cytochrome P450 reductase that activates mitomycin C and (2) reoxidation o the drug is inhibited under hypoxic conditions. Other examples o toxic metabolites, including the important case o acetaminophen, are discussed in Chapter 6.

INDIVIDUAL FACTORS AFFECTING DRUG METABOLISM For a number o reasons, the rates o biotrans ormation reactions may vary greatly rom one person to another. The most important o these actors are discussed below.

Pharmacogenomics The e ects o genetic variability on drug metabolism are an important part o the new science o pharmacogenomics (see Chapter 7, Pharmacogenomics). Certain populations exhibit polymorphisms or mutations in one or more enzymes o drug metabolism, changing the rates o some o these reactions and eliminating others altogether. These pharmacogenetic di erences must be taken into account in therapeutic decision making and drug dosing. Current research uses new technology (e.g., SNP analysis, gene microchips) to understand how genetic di erences in the enzymes o drug metabolism a ect patient responses to various drugs. Such approaches are already employed extensively in pharmaceutical development and are beginning to be applied in clinical practice.

C h a p t e r 4 Drug Metabolism 51

TABLE 4-3 Some Pharmacologic Substrates, Inhibitors, and Inducers o Cytochrome P450 Enzymes P450 ENZYME

SUBSTRATES

INHIBITORS

INDUCERS

P450 3A4

Anti-HIV agents Indinavir Nelf navir Ritonavir Saquinavir Benzodiazepines Alprazolam Midazolam Triazolam Calcium channel blockers Diltiazem Felodipine Ni edipine Verapamil Immunosuppressants Cyclosporine Tacrolimus Macrolide antibiotics Clarithromycin Erythromycin Statins Atorvastatin Lovastatin Others Finasteride Loratadine Losartan Quinidine Sildenaf l Tadalaf l

Anti ungal agents (azoles) Itraconazole Ketoconazole Anti-HIV agents Delavirdine Indinavir Ritonavir Saquinavir Calcium channel blockers Diltiazem Verapamil Macrolide antibiotics Clarithromycin Erythromycin Troleandomycin (not azithromycin) Others Cimetidine Grape ruit juice Mi epristone Ne azodone Nor oxacin

Antiepileptics Carbamazepine Oxcarbazepine Phenobarbital Phenytoin Anti-HIV agents E avirenz Nevirapine Ri amycins Ri abutin Ri ampin Ri apentine Others St. J ohn’s wort

P450 2D6

5-HT reuptake inhibitors Fluoxetine Paroxetine Antiarrhythmic agents Flecainide Mexiletine Propa enone Antidepressants Amitriptyline Clomipramine Desipramine Imipramine Nortriptyline Antipsychotics Haloperidol Perphenazine Risperidone Venla axine Beta-adrenergic antagonists Alprenolol Bu uralol Carvedilol Metoprolol Penbutolol Propranolol Timolol Opioids Codeine Dextromethorphan

5-HT reuptake inhibitors Fluoxetine Paroxetine Antiarrhythmic agents Amiodarone Quinidine Antidepressants Clomipramine Antipsychotics Haloperidol

None identif ed

continues

52 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 4-3 Some Pharmacologic Substrates, Inhibitors, and Inducers o Cytochrome P450 Enzymes (continued) P450 ENZYME

SUBSTRATES

INHIBITORS

INDUCERS

P450 2C19

Antidepressants Clomipramine Imipramine Proton pump inhibitors Lansoprazole Omeprazole Pantoprazole Others Clopidogrel Propranolol R-War arin

Proton pump inhibitors Omeprazole Others Fluoxetine Ritonavir Sertraline

Norethindrone Prednisone Ri ampin

P450 2C9

Angiotensin II receptor antagonists Irbesartan Losartan Nonsteroidal anti-inf ammatory drugs (NSAIDs) Ibupro en Supro en Others S-War arin Tamoxi en

Anti ungal agents (azoles) Fluconazole Miconazole Others Amiodarone Phenylbutazone

Ri ampin Secobarbital

P450 2E1

General anesthetics En urane Halothane Iso urane Methoxy urane Sevo urane Others Acetaminophen Ethanol

Disulf ram

Ethanol Isoniazid

P450 1A2

Antidepressants Amitriptyline Clomipramine Clozapine Imipramine Others R-War arin Tacrine

Quinolones Cipro oxacin Enoxacin Nor oxacin O oxacin Others Fluvoxamine

Char-grilled meat Cruci erous vegetables Insulin Omeprazole Tobacco smoke

For example, most pharmaceutical companies avoid development o a drug that is metabolized primarily by a highly polymorphic enzyme, because such polymorphisms may lead to wide interindividual variability in drug response. One clinically important example o pharmacogenetic variability involves the plasma enzyme cholinesterase. One in every 2,000 Caucasians carries a genetic alteration in cholinesterase, which metabolizes the muscle relaxant succinylcholine (among other unctions). This altered orm o the enzyme has an approximately 1,000- old reduced a f nity or succinylcholine, resulting in slowed elimination and prolonged circulation o the active drug. Should a su f ciently high plasma concentration o succinylcholine be reached, respiratory paralysis and death can occur unless the patient is supported with artif cial respiration until the drug is cleared. A similar situation can occur with isoniazid, one o the drugs considered or treatment o Ms. B’s tuberculosis. Genetic variability, in the orm o a widespread autosomal recessive trait that results in decreased synthesis o an enzyme, causes the metabolism o this drug to be slowed in

certain subsets o the US population. The enzyme at issue is N-acetyltrans erase, which inactivates isoniazid by an acetylation (conjugation) reaction. The “slow acetylator” phenotype is expressed by 45% o whites and blacks in the United States and by some Europeans living in high northern latitudes. The “ ast acetylator” phenotype is ound in more than 90% o Asians and in Inuits in the United States. Blood levels o isoniazid are elevated our old to six old in slow acetylators relative to ast acetylators. Moreover, because the ree drug acts as an inhibitor o P450 enzymes, slow acetylators are more susceptible to adverse drug interactions. I Ms. B expresses the slow acetylator phenotype and her dose o isoniazid is not decreased accordingly, then the addition o isoniazid to her drug regimen could potentially have a toxic e ect. A third example involves clopidogrel, an antiplatelet drug that promotes blood vessel patency a ter strokes or coronary angioplasty. The loss o e f cacy o this medication may lead to re-stenosis or thrombosis o a vessel or stent, o ten with severe consequences. Clopidogrel is a prodrug that is

C h a p t e r 4 Drug Metabolism 53

metabolized to its active orm via P450 enzymes, including P450 2C19. Polymorphisms o P450 2C19 have recently been associated with both decreased antiplatelet e ect and increased cardiovascular morbidity. In addition, because many proton pump inhibitors are also metabolized by P450 2C19, co-administration o clopidogrel with one o these commonly prescribed medications may lead to a decrease in the plasma levels o active clopidogrel.

Race and Ethnicity Some genetic aspects o race and/or ethnicity a ect drug metabolism. In particular, di erences in drug action among races/ethnicities have been attributed to polymorphisms in specif c genes. For example, P450 2D6 is unctionally inactive in 8% o Caucasian individuals but in only 1% o Asians. Moreover, A rican Americans have a high requency o a P450 2D6 allele that encodes an enzyme o reduced activity. These observations are clinically relevant, in that P450 2D6 is responsible or the oxidative metabolism o about 20% o drugs—including many beta-antagonists and tricyclic antidepressants—and or the conversion o codeine to morphine. In some cases, a polymorphism in the target gene is the basis or racial di erences in drug action. The activity o the enzyme vitamin K epoxide reductase (VKORC1), which is the target o the anticoagulant warfarin, is a ected by single nucleotide polymorphisms (SNPs) that render an individual either more or less sensitive to war arin and that dictate administration o lower or higher doses o the drug, respectively. In one study, Asian American populations were ound to be enriched in haplotypes (inherited combinations o individual base/SNP di erences) associated with increased sensitivity to war arin, while A rican American populations exhibited haplotypes associated with increased resistance to war arin. Perhaps the most prominent example o a race-based therapeutic is the combination o f xed-dose isosorbide dinitrate and hydralazine (also known as BiDil). This combination o vasodilators was reported to cause a 43% decrease in mortality in A rican Americans with heart ailure. Although the biochemical basis o this e ect is not known, these clinical data demonstrate that race may be a key consideration in choosing drug treatments and doses.

Age and Gender Drug metabolism can also di er among individuals as a result o age and gender di erences. Many reactions o biotrans ormation are slowed in both young children and the elderly. At birth, neonates are capable o carrying out many but not all oxidative reactions; however, most o these drugmetabolizing enzyme systems mature gradually over the f rst 2 weeks o li e and throughout childhood. Recall that neonatal jaundice results rom a def ciency o the bilirubin-conjugating enzyme UDPGT. Another example o a conjugating enzyme def ciency that puts in ants at risk or toxicity is the so-called gray baby syndrome . Haemophilus inf uenzae in ections in inants were once treated with the antibiotic chloramphenicol; excretion o this drug requires an oxidative trans ormation ollowed by a conjugation reaction. The oxidation metabolite o chloramphenicol is toxic; i this metabolite ails to undergo conjugation, it can build up in the plasma and may reach toxic concentrations. Toxic levels o the metabolite can cause neonates to experience shock and circulatory collapse, leading to the pallor and cyanosis that give the syndrome its name.

In the elderly, a general decrease in metabolic capacity is observed. As a result, particular care must be taken in prescribing drugs or this segment o the population. The elderly’s decline in metabolic capacity has been attributed to age-related decreases in liver mass, hepatic blood ow, and possibly hepatic enzyme activity. Another therapeutic consideration is that the elderly are requently taking multiple medications, with a consequent increase in the risk o drug– drug interactions. There is some evidence or gender di erences in drug metabolism, although the mechanisms are not well understood and data rom experimental animals have not been particularly illuminating. Decreased oxidation o ethanol, estrogens, benzodiazepines, and salicylates has been reported anecdotally in women relative to men and may be related to androgenic hormone levels.

Diet and Environment Both diet and environment can alter drug metabolism by inducing or inhibiting enzymes o the P450 system. An interesting example is grape ruit juice. The psoralen derivatives and avonoids in grape ruit juice inhibit both P450 3A4 and MDR1 in the small intestine. Inhibition o P450 3A4 signif cantly decreases the f rst-pass metabolism o co-administered drugs that are also metabolized by this enzyme, and inhibition o MDR1 signif cantly increases the absorption o coadministered drugs that are substrates or export (e ux) by this enzyme. The grapefruit juice effect is important when grape ruit juice is ingested together with drugs that are acted upon by these enzymes. Such drugs include some protease inhibitors, macrolide antibiotics, HMG-CoA reductase inhibitors (statins), and calcium channel blockers. Saquinavir is one o the protease inhibitors that is both metabolized by P450 3A4 and exported by MDR1. In the case that opens this chapter, Ms. B should be alerted to the act that the simultaneous ingestion o grape ruit juice and saquinavir could inadvertently lead to toxic serum levels o the protease inhibitor. Herbal medications can also have signif cant e ects on the P450 system. One such example is St. J ohn’s wort, a popular herbal preparation used or mood stabilization. Many observational studies have noted that St. John’s wort can induce P450 expression and thereby decrease the e f cacy o other drugs. Compounds rom herbs and spices may also inhibit P450s. One example is piperine (the essential chemical in black pepper), which has been shown to inhibit P450 3A4 and the MDR protein in animal models; the clinical importance o this e ect remains uncertain. Because many endogenous substances used in the conjugation reactions are ultimately derived rom the diet (and also require energy or the production o the appropriate co actors), nutrition can a ect drug metabolism by altering the pool o such substances available to the conjugating enzymes. Pollutant exposures can produce similarly dramatic e ects on drug metabolism; one common example is the AhR-mediated P450 enzyme induction by polycyclic aromatic hydrocarbons in cigarette smoke.

Metabolic Drug Interactions Drugs can potentially a ect oral bioavailability, plasma protein binding, hepatic metabolism, and renal excretion o co-administered drugs. Among the categories o drug–drug

54 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

interactions, the e ects on biotrans ormation have special clinical importance. The concept o P450 enzyme induction and inhibition has already been introduced. A common clinical situation that must take this type o drug–drug interaction into consideration is the prescription o certain antibiotics to women who are already using hormonal contraception. For example, enzyme induction by the antibiotic rifampin can cause estrogen-based hormonal contraception to be ine ective at standard doses because P450 3A4 is induced by ri ampin and this is the main enzyme involved in the metabolism o the common estrogenic component 17 -ethynylestradiol. In this situation, other means o birth control should be recommended during the course o riampin therapy. Ms. B should be made aware o this interaction i ri ampin is added to her therapeutic regimen. Another phenomenon associated with enzyme induction is tolerance , which can occur when a drug induces its own metabolism and thus reduces its e f cacy over time (see the discussion o carbamazepine above and the discussion o tolerance in Chapter 19, Pharmacology o Drugs o Abuse). Because drugs are o ten prescribed in combination with other pharmaceuticals, care ul attention should be paid to drugs that are metabolized by the same hepatic enzymes. The concomitant administration o two or more drugs that are metabolized by the same enzyme will generally result in higher serum levels o the drugs. The mechanisms o drug– drug interaction can involve competitive substrate inhibition, allosteric inhibition, or irreversible enzyme inactivation; in any case, drug levels can increase acutely, possibly leading to deleterious results. For example, erythromycin is metabolized by P450 3A4, but the resulting nitrosoalkane metabolite can orm a complex with P450 3A4 and inhibit the enzyme. This inhibition can lead to potentially atal drug–drug interactions. A notable example is the interaction between erythromycin and cisapride , a drug that stimulates GI tract motility. Toxic concentrations o cisapride can inhibit hERG potassium channels in the heart and thereby induce potentially atal cardiac arrhythmias; or this reason, cisapride was withdrawn rom the market in 2000. Be ore its withdrawal, cisapride was o ten well tolerated as a single agent. However, because cisapride is metabolized by P450 3A4, when the activity o P450 3A4 was compromised due to the concomitant administration o erythromycin or another inhibitor o P450 3A4, serum cisapride concentrations could increase to levels associated with arrhythmia induction. In other cases, drug interactions may be benef cial. For example, as noted above, the ingestion o methanol (a component o wood alcohol) can result in blindness or death because its metabolites ( ormaldehyde, an embalming agent, and ormic acid, a component o ant venom) are highly toxic. One treatment or methanol poisoning is the administration o ethanol, which competes with methanol or oxidation by alcohol dehydrogenase (and, to a lesser extent, by P450 2E1). The resulting delay in oxidation allows the methanol to be cleared renally be ore its toxic byproducts can orm in the liver.

Diseases Affecting Drug Metabolism Many disease states can a ect the rate and extent o drug metabolism in the body. Because the liver is the main site o biotrans ormation, many liver diseases signif cantly compromise drug metabolism. Hepatitis, cirrhosis, cancer, hemochromatosis, and atty inf ltration o the liver each impair

cytochrome P450s and other hepatic enzymes that are crucial to drug metabolism. As a result o this slowed metabolism, the levels o the active orms o many drugs may be signif cantly higher than intended and thereby cause toxic e ects. Thus, the doses o many drugs may need to be lowered or individuals with hepatic disease. Concomitant cardiac disease can also a ect drug metabolism. The rate o metabolism o many drugs, such as the antiarrhythmic lidocaine and the opioid morphine, is dependent on drug delivery to the liver via the bloodstream. Because blood ow is commonly compromised in cardiac disease, there must be heightened awareness o the potential or supratherapeutic levels o drugs in patients with heart ailure. In addition, some antihypertensive agents selectively reduce blood ow to the liver and can thereby increase the hal li e o a drug such as lidocaine, leading to potentially toxic levels. Thyroid hormone regulates the basal metabolic rate o the body, which, in turn, a ects drug metabolism. Hyperthyroidism can increase the rate o metabolism o some drugs, whereas hypothyroidism can do the opposite. Other conditions, such as pulmonary disease, endocrine dys unction, and diabetes, are also thought to a ect drug metabolism, but the mechanisms or these e ects are not yet completely understood.

CONCLUSION AND FUTURE DIRECTIONS This chapter has reviewed a number o issues relating to drug metabolism, including the sites o biotrans ormation, the transport and enzymatic metabolism o drugs at those sites, and individual actors that can a ect those reactions. The case o Ms. B illustrates the clinical implications o drug metabolism, including the possible in uences o ethnicity and drug–drug interactions on pharmacologic therapy. Understanding drug metabolism, and particularly the interactions o drugs within the body, allows the principles o biotrans ormation to be applied in the design and use o therapeutics. As pharmacogenomics and rational drug design lead pharmacology research into the uture, increased understanding o biotrans ormation will also render the pharmacologic treatment o disease more individualized, e f cacious, and sa e. This topic is discussed in Chapter 7.

Acknowledgment We thank Cullen Taniguchi or his valuable contributions to this chapter in the Second and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Burchard EG, Ziv E, Coyle N, et al. The importance o race and ethnic background in biomedical research and practice. N Engl J Med 2003;348:1170– 1175. (Current understanding regarding ethnic variability in response to drug administration.) Gong IY, Kim RB. Impact o genetic variation in OATP transporters to drug disposition and response. Drug Metab Pharmacokinet 2013;28:4 –18. (Review of the crucial role played by drug transporters in drug metabolism.) Johansson I, Ingelman-Sundberg M. Genetic polymorphism and toxicology— with emphasis on cytochrome P450. Toxicol Sci 2011;120:1–13. (Review of toxicity issues with some drugs and the role of P450 variations.) Katsanis SH, Javitt G, Hudson K. Public health. A case study o personalized medicine. Science 2008;320:53–54. (A discussion of aspects of the use of personalized medicine, including roles of P450 genes.)

C h a p t e r 4 Drug Metabolism 55 Kirchheiner J, Seeringer A. Clinical implications of pharmacogenetics of cytochrome P450 drug metabolizing enzymes. Biochim Biophys Acta 2007;1770:489–494. (Discussion o clinical issues with several drugs and P450s.) Mega JL, Close SL, Wiviott SD, et al. Cytochrome P450 polymorphisms and response to clopidogrel. N Engl J Med 2009;360:354–362. (Example o P450 genetic polymorphisms and clinical e f cacy o clopidogrel.) Rendic S, Guengerich FP. Survey of human oxidoreductases and cytochrome P450 enzymes involved in the metabolism of xenobiotic and natural chemicals. Chem Res Toxicol 2015;28:38–42. (Analysis o ractions o drugs metabolized by di erent cytochrome P450 enzymes.) Seden K, Dickinson L, Khoo S, Back D. Grapefruit-drug interactions. Drugs 2010;70:2373–2407. (Review o P450 interactions with grape ruit.) Shi S, Klotz U. Drug interactions with herbal medicines. Clin Pharmacokinet 2012;51:77–104. (Review o P450 interactions with herbal medicines.)

Wienkers L, Pearson P, eds. Handbook o drug metabolism. 2nd ed. New York: Marcel Dekker; 2009. (Collection o articles on aspects o drug metabolism.) Wilke RA, Lin DW, Roden DM, et al. Identifying genetic risk factors for serious adverse reactions: current progress and challenges. Nat Rev Drug Discov 2007;6:904–916. (Review o current status o use o genetics or predicting adverse reactions.) Wilkinson GR. Drug metabolism and variability among patients in drug response. N Engl J Med 2005;352:2211–2221. (An excellent basic review o the P450 system and drug–drug interactions.) Zhang D, Zhu M, Humphreys WG, eds. Drug metabolism in drug design and development: basic concepts and practice. Hoboken, NJ: John Wiley & Sons; 2007. (Drug metabolism as it pertains to development o new pharmaceuticals.)

E NT2 T2

OATP OA ATP MRP 3 ENT1, ENT E T2

MDR1 MD M R11 (P -gp)

OA O A AT TP 1A2, TP 1A2 OATP A 2B1

BCRP MRP P4

5

O Tα OS OS Tβ OC O C T1

d

o B lo

o

Drug Transporters

lo B

d

MRP 5

E NT2 ENT2

OA ATP 22B1 o

B lo

o

d

lo B

Ba ra n A. Ers oy a n d Ke it h A. Ho ffm OAT3 A as te r

d

OA O AT2

OS Tα -OS Tβ

NTCP

OATP OA A 1B3 MRP 6

OATP A 1B1

O AT1 OA OCT O OCT2 OC

ENT1,, ENT2

B ile

INTRODUCTION & CASE CA A AS SE S E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 56–5 6 6–57 6– 2 E f ux Tra ransporters . .OA . .A.T7 . . . . . . . . . . . . . . . . . . . MDR1 . . . . . . . . . . . . . 62 OAT7 (P -gp) ) g OA O ATP P ATP-Binding 4C1 4C Cassette (ABC) (AB BC) BC BC) CT Transp Tra Tr ran ra nnsp nspo ns sppo p rter Family amily ly . . . . . . . . . 62 6 UPTAKE AND EFFLUX UX X TR TRA RAN R ANS AN ANS NS SP P PO ORTE O ORTER R S . . . . . . . . . . . . . . 58 5 8 M MDR MDR1 MD DR R1 1 (P (P -gp) gp) O AT2 OAT2 OA AT2 Sollute te Car arri r er (SLC) Family Faa ily Fami y . . . . . .BS . . .EP . . . . . . . . . . . . . . . . . . 64 ENT1 Uptake Trans nsport rters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 MATE1 MA ATE2 TE2-K MATE1, ATE1 MA OCT1 VES Organic Anion-Transporting nns or ort rtin rting iing nngg Po Poly P Polypeptide Polyp oly ly CLINICAL PERSPECTIVES V SO ON ND DRU RU UG T UG TRANS TR S SP P POR PORTER O OR ORT R RTERS TERS TE ER RS . . . . . . . . 64 OCTN2 2 nsp MRP 2, MRP 4 (OATP) Family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599 Drug–Drug Interactio tioons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 6M MAT MA AT TE1 BCRP P Organic Anion Transporte ter e r (O er O OA A AT T) Fa F Fam am mil mi mily illy y . . . . . . . . . . . . . . . . 59 5 Drug Inter erence nce cee wit with w th En th Endogen Endo E nd ndogenou enous ouss M Metabol eeta etabo etabolite tab ta boolit olilit liit Traa ccking ck cki kiing kkin king g . . . 66 Orga Or gani nicc Ca Cati tion on Transporter (OCT) OCT O CT C T) F Family Fam aam m y . . . . . . . . . . . . . . . 59 mily Pharmacogenom ogggeno e om omi miics ccss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 MRP MR P4 Bile Acid Transporte INTE TEGR GRAT ATION N OF D DR DRUG RUG M T TRANSPORTER ER RS SCI SC C CIEN NC NCE P EPe rs T1 T1, 1 , P EP T2 . . . . . . . . . . . . . . . . . . . . . . . 59 IN MRP M P2 Peptide Transpporterr (PEP (PEPT) T) Fam amil ilyy . . . . . . . . . . . . . . . . . . . . . 61 INTO DRUG DIS DISCOVERY ISCOV ISC SCOVE SC ERY AND DEVELOPMENT DEVELO LOP OPM OP PM T . . . . . . . . . . . . . . . 67 67 MRP M 3 URA URAT1 UR AT1 T1 Concentrative and Equilibrative Nucleo oside ssid iid ide d de ENT1 CONCLUSION AND ND D FUTURE DIRECTIONS DIREC DIRECT CTI CT TIO . . . . .EN . . .T1 . . . . . . . . . . . 67 Transs po port rter er (CN CNT T an andd EN ENT T) Families OAT4 OA AT44. . . . . . . . . . . . . . . . . 61 Suggested Reading ngg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Glucose Transporterss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 U

n ri

e

Kidne y pro ximal Kid i l ttubule b l c e ll

INTRODUCTION Membrane transporters are proteins that acilitate the transport o soluble small molecules across lipid bilayers. These transporters regulate the tissue and plasma distribution as well as the excretion o drugs and endogenous compounds and there ore a ect their pharmacokinetic prof le (i.e., absorption, distribution, metabolism, and excretion). Transporters are generally classif ed as uptake or e f ux proteins depending on the direction in which they move substrates across the membranes o cells. They include several protein amilies that exhibit broad substrate specif city and tissue distribution. The unction o transporters depends on the tissue in which they are expressed as well as their subcellular localization to the apical or basolateral membrane (Fig. 5-1). Whereas transporters expressed in organs o elimination (e.g., liver or kidney) can acilitate the clearance o drugs and/or metabolites rom the systemic circulation, e ux transporters in non-eliminating organs such as the central nervous system and placenta primarily unction to minimize the exposure o the brain and the developing etus, respectively, to potentially harm ul exogenous substrates. In some cases, intestinal e ux transporters expressed at the apical membrane o the enterocyte can limit the oral bioavailability o drugs; in other cases, uptake transporters expressed at this membrane can aid in the systemic absorption o compounds, thereby enhancing their oral bioavailability. Transporter unction in the liver o ten acilitates drug clearance, but e f cient active uptake into the liver a ter an oral dose can also a ect drug bioavailability by contributing to f rst-pass hepatic metabolism and elimination. Because drugs can both 56

He pato He t c yte t inhibit and induce transporters, concurrent administration o multiple drugs may alter the pharmacokinetics and pharmacodynamics o the other compounds depending on transporter a f nity, specif city, and alternate mechanisms o drug disposition. There ore, identi ying the specif c transporter(s) or which a novel drug is a substrate (or inhibitor) may not only help to devise optimization and ormulation strategies that improve drug bioavailability and e f cacy, but may also prevent adverse e ects by anticipating and limiting drug– drug interactions. There are over 400 transporters in the human genome. This chapter provides an overview o this e ective but complex system by ocusing on a select number o well-characterized transporters involved in drug disposition and in the ux o endogenous substrates and on those that may become the sites o drug–drug interactions. The Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC) has approved standard acronyms or both solute carrier (SLC) and ATP-binding cassette (ABC) amilies o transporters. However, many drug transporters, especially those discussed in greater detail in this chapter, were initially cloned and named based on their pharmacologic attributes such as substrate specif city and association with drug resistance. For instance, the ABCB1 gene was originally named and is more commonly re erred to as P-glycoprotein (P-gp) because it was initially identif ed as a glycoprotein that controls cell membrane permeability (P). Ensuing studies have also led to its characterization as a multidrug resistance (MDR) protein encoded by the MDR1 gene. There ore, P-gp, MDR1, and ABCB1 are all synonyms or the same transporter. In this chapter, the transporters are re erred to using popular names, with HGNC nomenclature

C h a p t e r 5 Drug Transporters

Mr. H is a 47-ye a r-o ld m ild ly ove rw e ig h t m a n w h o co m e s to th e wa lk-in clin ic co m p la in in g o m u s cle p a in a n d w e a kn e s s in h is a rm s a n d le g s . He h a s b e e n ta kin g a s ta tin m e d ica tio n o r th e p a s t 4 ye a rs a n d h a s b e e n a b le to m a n a g e h is ch o le s te ro l le ve ls e e ctive ly w ith th is m e d ica tio n a n d d ie t m o d if ca tio n s . Wh e n a s ke d a b o u t a ny re ce n t ch a n g e s to h is m e d ica tio n s o r d ie t, h e in d ica te s th a t h is p rim a ry ca re p hys icia n s ta rte d h im 3 w e e ks a g o o n m e d ica l th e ra p y to h e lp co n tro l h is trig lyce rid e le ve ls . In a d d itio n to th e re ce n t m u s cle p a in , Mr. H h a s n o tice d a m ild ra s h o n h is to rs o ove r th e p a s t 3 w e e ks s in ce

57

s ta rtin g th e n e w m e d ica tio n p re s crib e d to h e lp co n tro l h is trig lyce rid e s .

Questions 1 . Without knowing the speci c medications that Mr. H is taking, which drug–drug interactions do you suspect might be contributing to his new symptoms? 2 . Consultation with Mr. H’s primary care physician reveals that Mr. H is also taking a xed-dose combination o lopinavir/ritonavir to manage his HIV in ection. How might this complicate the drug–drug interaction suspected in question 1?

Intestinal epithelial cell in

e

Brain capillary endothelial cell

In

te

st

ENT1, ENT2 MDR1 (P -gp)

MCT1

BCRP B

AS BT

ra

MRP 2

in

P EP T1, P EP T2

ENT2

OATP MRP 3 ENT1, ENT2

MDR1 (P -gp)

OATP 1A2, OATP 2B1

BCRP MRP 4

OS Tα OS Tβ OCT1

MRP 5

o

lo

d

B

B

d

lo

o

OS Tα -OS Tβ

NTCP ENT2

OATP 2B1 d

lo B

o

o d

OAT1 OCT2 ENT1 OCTN2

MDR1 (P -gp) MATE1, MATE2-K MRP 2, MRP 4

MRP 6

OATP 1B1

OATP 4C1

ENT1, ENT2 MDR1 (P -gp)

OAT7 OAT2

BS EP

OCT1 BCRP

B ile

OAT2

OATP 1B3

B lo

OAT3

MRP 4

P EP T1, P EP T2 n ri U e

URAT1

MRP 3

MATE1

MRP 2 ENT1

OAT4

Kidney proximal tubule cell

Hepatocyte

FIGURE 5-1. Major transporters of drugs and endogenous compounds in the intestine, kidney, liver, and blood–brain barrier. Uptake transporters are shown in blue and e f ux transporters are shown in red. Bidirectional transport is denoted by double arrows. See text or abbreviations.

58 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

and other acronyms stated in parentheses. Additional details on nomenclature and synonyms/previous names can be ound at http://www.genenames.org/gene amilies/ABC and http://www.genenames.org/gene amilies/SLC.

UPTAKE AND EFFLUX TRANSPORTERS Drugs and endogenous compounds cross cellular membrane barriers by simple di usion, passive transport, or active transport. Simple di usion (also called passive diffusion) can occur when small, partially water-soluble solutes such as certain polar lipids pass reely through a membrane bilayer, driven by their concentration gradient across the membrane (Fig. 5-2). However, many molecules require the assistance o membrane transporters to move across membranes. Solute carriers acilitate the passive transport o substrates down their concentration gradient (Fig. 5-3). In contrast, the transport o substrates against their concentration gradient requires active transporters (Fig. 5-4). Primary active transport utilizes the energy o hydrolysis o adenosine triphosphate (ATP); the substrate f rst binds to the transporter, and substrate transport across the membrane is completed upon ATP-mediated activation o the transporter (Fig. 5-4A). Secondary active transport depends on the creation o an inward sodium gradient by the ATP-mediated activation o the Na /K -ATPase pump. The inward sodium gradient drives the coupled transport (co-transport) through a solute carrier o sodium (down its electrochemical gradient) and o a second solute (against its concentration gradient) (Fig. 5-4B). This mechanism is called secondary active transport because the transport through the solute carrier does not directly require ATP hydrolysis but is coupled to a primary ATPase. Tertiary active transport also requires an ATPase-dependent sodium gradient. However, in this case, sodium in ux acilitates the di usion and intracellular accumulation o an anion such as bicarbonate via a secondary active transporter. In the f nal step o tertiary active transport, the solute carrier exports the intracellular anion in exchange or the uptake o an extracellular organic anion (Fig. 5-4B). With the

Extra ce llula r s pa ce

P rote in cha nne l

Ce ll me mbra ne Ca rrie r prote ins

Intra ce llula r s pa ce

FIGURE 5-3. Passive transport. In this process, protein channels or carrier proteins acilitate the transport o substrates down their concentration gradient.

exception o OST -OST (which transports bile acids; see below), all known uptake transporters lack ATPase activity and belong to the solute carrier (SLC) amily. Secondary or tertiary active transport mechanisms are required or cellular absorption o many drugs, nutrients, and endogenous metabolites. The next section outlines the transporters that have essential roles in the uptake o drugs and endogenous A Extra ce llula r s pa ce Na +/K+ ATP a s e

Ce ll me mbra ne P

ATP

P

K+

ADP

P

K+

Intra ce llula r s pa ce

B

Na+

K+

Extra ce llula r s pa ce

Na +/K+ ATP a s e ATP

Na+

HCO 3 -

S ymporte r

OA-

HCO 3 -

Antiporte r

ADP Na +

K+ P rima ry

Ce ll me mbra ne

Na +

Na +

Na +

HCO3-

S e conda ry

HCO3-

OA-

Te rtia ry

FIGURE 5-4. Active transport. The transport o substrates against their con-

Intra ce llula r s pa ce Time

FIGURE 5-2. Simple diffusion. In this process, small, partially watersoluble solutes pass reely through a membrane bilayer, driven by their concentration gradient across the membrane. This process is also called passive diffusion.

centration gradient requires active transporters. A. Primary active transport. In this process, the energy o hydrolysis o adenosine triphosphate (ATP) is used to transport solutes against their concentration gradients; or example, the Na /K -ATPase pump drives sodium (outward) and potassium (inward) against their concentration gradients. B. Secondary and tertiary active transport. In secondary active transport, the creation o an inward sodium gradient by the ATPase-mediated activation o the Na /K -ATPase drives the coupled transport (co-transport) through a solute carrier (symporter) o sodium (down its concentration gradient) and a second solute (against its concentration gradient). Here, the second solute is bicarbonate (HCO3 ). In tertiary active transport, a second solute carrier (antiporter) acilitates the export o the second solute (here, HCO3 ) in exchange or the uptake o an extracellular organic anion (OA ). Font size ref ects the relative concentration o the solute in the extracellular space and the intracellular space.

C h a p t e r 5 Drug Transporters

compounds in key tissues that are involved in the absorption, distribution, and elimination o drugs. These tissues include the intestine, liver, kidney, and endothelial cells o the blood–brain barrier.

Uptake Transporters Organic Anion-Transporting Polypeptide (OATP) Family

OATPs (SLCO, ormer SLC21) are expressed in all epithelial cells. These transporters acilitate the uptake o large hydrophobic and amphiphilic organic compounds, such as bile acids, thyroid hormones, conjugated steroids, and eicosanoids (Table 5-1). Although OATPs are primarily responsible or the transport o anionic compounds, some members o the OATP amily transport bulky type II organic cations such as rocuronium. The accumulation o substrates such as bile acids within the liver is a concentrating process, and OATPs move substrates into cells by a tertiary active transport mechanism. O the 11 amily members, 5 have been implicated in the transport o xenobiotics. OATP1B1, OATP1B3, and OATP2B1 mediate the uptake o drugs (e.g., statins ) across the sinusoidal membrane o hepatocytes, whereupon the drugs can be metabolized by enzymes such as cytochrome P450s (CYPs) and/or secreted into the bile or back into the systemic circulation (Fig. 5-1). Inhibition o hepatic OATPs has been implicated as a potential mechanism o drug–drug interactions. The potential or these interactions has resulted in revised dosing guidelines or some statins when they are administered with drugs that inhibit OATPs (e.g., cyclosporine , gemf brozil, lopinavir/ritonavir). OATP1A2 is ubiquitously expressed and contributes primarily to drug absorption rom the intestinal lumen into intestinal epithelial cells. OATP4C1 acilitates the uptake o drugs such as digoxin rom the circulation into kidney proximal tubule cells, rom which the drugs are eliminated via the urine (Fig. 5-1). In the introductory case, Mr. H was most likely started on a drug that inhibited the hepatic uptake o his statin, and thereby increased the systemic bioavailability o the statin, via a transporter-related drug–drug interaction. Fibrates such as gemf brozil are commonly used to reduce triglyceride levels when statins and diet modif cation prove insu f cient. Concurrent administration o statins with gemf brozil can cause myopathy, however, in part due to inhibition o OATP1B1-mediated hepatic uptake o statins by gemf brozil. This drug–drug interaction results in increased blood levels o the statin and consequent systemic toxicity. Mr. H was also taking a f xed dose o lopinavir/ritonavir, which is an inhibitor o OATP1B1- and OATP1B3-mediated drug uptake into the liver; this combination o HIV protease inhibitors may have urther contributed to drug–drug interactions and exacerbated systemic adverse e ects o Mr. H’s statin a ter initiation o treatment or hypertriglyceridemia.

Organic Anion Transporter (OAT) Family

OATs, which belong to the SLC22A amily, mediate the cellular uptake o small organic anions such as conjugated steroids, biogenic amines, and cGMP as well as a broad range o xenobiotics such as antivirals, antibiotics, ACE inhibitors, and anticancer drugs (Table 5-1). Despite their classif cation as uptake transporters, OAT iso orms 1–4 and 7 have essential roles in drug clearance by acilitating the

59

uptake o drugs rom the systemic circulation into the liver and kidneys, where they can be metabolized and excreted (Fig. 5-1). Uptake o anions into cells against their electrochemical gradient requires OATs to unction as tertiary active transporters. OAT1, OAT3, and OAT4 exchange intracellular 2-oxoglutarate, and OAT7 exports intracellular short-chain atty acids such as butyrate in exchange or their extracellular substrates. OAT1, OAT2, and OAT3 clear many organic anions rom the systemic circulation into the kidney proximal tubule, rom which the anions are eliminated in the urine. OAT2 and OAT7 are expressed mainly on the sinusoidal membrane o hepatocytes. Unlike OAT1, OAT2, and OAT3, OAT4 and the urate anion exchanger 1 (URAT1) are expressed on the apical (brush border) membrane o the kidney proximal tubule, where they mediate the reabsorption o uric acid rom urine. There ore, drugs that inhibit OAT4 and URAT1 may decrease blood uric acid levels and thereby provide therapeutic benef t (e.g., in the treatment o gout by the URAT1 inhibitor probenecid; see Chapter 49, Integrative In ammation Pharmacology: Gout) and may potentially cause enhanced elimination o OAT4 substrates.

Organic Cation Transporter (OCT) Family

Like OATs, OCTs belong to the SLC22A amily and contribute to the renal clearance o xenobiotics such as antiviral drugs. OCTs also mediate the transport o a diverse group o small organic cations such as catecholamines, hormones, and neurotransmitters (Table 5-1). Transport o cations by OCTs occurs down the electrochemical gradient o the solute and does not depend on ATP hydrolysis or ion exchange; instead, the transport is thought to be driven by di erences in membrane potential. OCT iso orms can have overlapping substrates, and the transport o solutes can be bidirectional depending on the electrochemical gradient. There is strong evidence or the roles o OCT1, OCT2, and OCT3 in drug disposition (Table 5-1; Fig. 5-1). OCT1 is highly expressed in the sinusoidal (basolateral) membrane o hepatocytes. OCT2 is expressed mostly in the kidney proximal tubule and it contributes to the uptake o metabolites rom the blood into the tubule. In contrast, OCT3 exhibits broad tissue distribution; its highest expression is in the intestine, liver, and kidney, where it acilitates intestinal absorption and hepatic and renal secretion o drugs, respectively. All three transporters mediate the uptake o a wide array o therapeutic agents, including sedatives, antidepressants, -blockers, and antidiabetic drugs such as met ormin. OCTs are also an important site o drug–drug interactions. In certain cases, OCT-mediated renal uptake can contribute to the adverse e ects o nephrotoxic drugs, which can be prevented by the concomitant administration o an OCT inhibitor.

Bile Acid Transporters

A signif cant raction o bile acids are recycled via three main transporter mechanisms in the liver and gastrointestinal (GI) tract. Na /taurocholate co-transporting polypeptide (NTCP, SLC10A1) is exclusively expressed at the sinusoidal membrane o hepatocytes and is a key mechanism in the transport o conjugated and unconjugated bile acids rom the circulation into the liver (Fig. 5-1). Whereas OATPs are responsible or the sodium-independent uptake o bile acids, NTCP is responsible or sodium-dependent secondary active bile acid transport that is coupled to the activation o Na /K -ATPase. In addition to bile acids, NTCP mediates

60 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 5-1 Uptake Transporters ORGAN/LOCATION (SEE FIG. 5-1)

ENDOGENOUS SUBSTRATES

OATP1B1 (SLCO1B1)

Hepatocytes (sinusoidal)

OATP1B3 (SLCO1B3)

TRANSPORTER

DRUG SUBSTRATES

INHIBITORS

Steroid hormones, thyroid hormones, bilirubin glucuronide, bilirubin, bile acids, prostaglandin E2

Repaglinide, valsartan, olmesartan, cerivastatin, pitavastatin, rosuvastatin, temocaprilat, enalapril

Saquinavir, ritonavir, lopinavir, ri ampicin, cyclosporine, gem brozil, clarithromycin

Hepatocytes (sinusoidal)

Steroid hormones, bile acids

Pitavastatin, rosuvastatin, exo enadine, valsartan, telmisartan, olmesartan, enalapril, erythromycin, valsartan

Ri ampicin, cyclosporine, ritonavir, lopinavir, erythromycin

OATP1A2 (SLCO1A2)

Brain, kidney, liver, intestine, endothelium

Bile salts, cholic acid, DHEAS, prostaglandin E2, taurocholate, bilirubin, conjugated steroids, peptides

Aliskiren, erythromycin, exo enadine, imatinib, levof oxacin, lopinavir, methotrexate, rosuvastatin, pitavastatin, ouabain, saquinavir, sul obromophthalein, unoprostone, acebutolol, atenolol, atrasentan, celiprolol, sotalol, talinolol, tebipenem, digoxin

Naringin, hesperidin, quercetin, ritonavir, lopinavir, saquinavir, ri ampicin, ri amycin, verapamil, apigenin

OATP2B1 (SLCO2B1)

Hepatocytes (sinusoidal), placenta, heart, brain, kidney, lung, small intestine, endothelium

Bile acids, steroid hormones, taurocholate

Glyburide, rosuvastatin, exo enadine, bosentan, ri ampicin

Ri ampin, cyclosporine, naringin, hesperidin, quercetin

OAT1 (SLC22A6)

Kidney proximal tubule, placenta

Uric acid, olate, cyclic nucleotides, prostaglandins E2 and F2

Ade ovir, cido ovir, zidovudine, lamivudine, zalcitabine, acyclovir, teno ovir, ciprof oxacin, cephaloridine, methotrexate, pravastatin

Probenecid, novobiocin

OAT3 (SLC22A8)

Kidney proximal tubule, choroid plexus, blood– brain barrier

Uric acid, bile acids, prostaglandins

Nonsteroidal anti-inf ammatory drugs (NSAIDs), exo enadine, methotrexate, ce tizoxime, ce aclor

Probenecid, novobiocin

OCT1 (SLC22A1)

Hepatocytes (sinusoidal), intestine (apical), neurons

Choline, acetylcholine, monoamine neurotransmitters

Met ormin, oxaliplatin, acyclovir, ganciclovir

Quinine, quinidine, disopyramide, cimetidine, atropine, prazosin

OCT2 (SLC22A2)

Kidney proximal tubule, neurons

Choline, acetylcholine, monoamine neurotransmitters, creatinine, bile acids

Met ormin, pindolol, procainamide, ranitidine, amantadine, amiloride, oxaliplatin, varenicline, cisplatin, debrisoquine, propranolol, guanidine, D-tubocurarine, pancuronium

Cimetidine, pilsicainide, cetirizine, testosterone, quinidine, ri ampicin, naringin, ritonavir

OCT3 (SLC22A3)

Liver, kidney, placenta, small intestine

Creatinine, guanidine, neurotransmitters, hormones

Atropine, prazosin, diphenhydramine, ranitidine, amantadine, ketamine, memantine, phencyclidine, nicotine, clonidine, dizocilpine, met ormin, cimetidine, verapamil, procainamide, D-amphetamine

Cimetidine, quinidine, ri ampicin, prazosin, phenoxybenzamine, corticosterone, progesterone, -estradiol

PEPT1 (SLC15A1)

Kidney proximal tubule, intestinal enterocytes

Dipeptides and tripeptides

Cephalexin, ce adroxil, bestatin, enalapril, captopril, valacyclovir, -lactam antibiotics, ACE inhibitors

Glycylproline, 4 -aminomethylbenzoic acid

PEPT2 (SLC15A2)

Kidney proximal tubule, choroid plexus, lung

Dipeptides, tripeptides

Cephalexin, ce adroxil, ubenimex, valacyclovir, enalapril, captopril, -lactam antibiotics, ACE inhibitors

Zo enopril, osinopril, ce adroxil, captopril, losartan

C h a p t e r 5 Drug Transporters

ORGAN/LOCATION (SEE FIG. 5-1)

ENDOGENOUS SUBSTRATES

NTCP (SLC10A1)

Hepatocytes (sinusoidal)

ASBT (SLC10A2) OST -OST

TRANSPORTER

DRUG SUBSTRATES

INHIBITORS

Taurocholate, bile salts, steroids, thyroid hormones

Rosuvastatin

Cyclosporine, gemf brozil, propranolol, urosemide, ketoconazole, ri amycin, glibenclamide, ritonavir, bosentan, e avirenz, saquinavir

Intestine

Taurocholate, bile acids

Dimeric bile acid analogues

Dihydropyridine, calcium channel blockers, statins

Intestine (apical), hepatocytes (sinusoidal)

Bile acids

Digoxin, rosuvastatin

Ri amycin SV

61

Data compiled rom: Giacomini KM, Huang SM, Tweedie DJ , et al.; International Transporter Consortium. Membrane transporters in drug development. Nat Rev Drug Discov 2010;9:215–236. Knowledge Center, Solvo Biotechnology; http://www.solvobiotech.com/knowledge-center. König J , Müller F, Fromm MF. Transporter and drug–drug interactions: important determinants o drug disposition and e ects. Pharmacol Rev 2013;65:944–966. US Food and Drug Administration. Drug development and drug interactions: table o substrates, inhibitors, and inducers. http://www. da.gov/drugs/developmentapprovalprocess/developmentresources/druginteractionslabeling/ucm093664.htm.

partial uptake o some statins (e.g., rosuvastatin; Table 5-1). The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) is expressed at the apical membrane o the epithelial cells o the distal small intestine and mediates the uptake o bile acids rom the intestinal lumen (Fig. 5-1, Table 5-1). OST -OST is the only uptake transporter that does not belong to the SLC amily. It comprises a heterodimer o two di erent subunits and can unction as either an e ux transporter or an uptake transporter depending on the electrochemical gradient o its substrates. However, the major unction o the transporter is to contribute to the enterohepatic recirculation o bile acids: OST -OST mediates the e ux o bile acids and conjugated steroids rom intestinal epithelia into the circulation, rom which the bile acids are taken up by hepatocytes (Fig. 5-1, Table 5-1).

Peptide Transporter (PEPT) Family

PEPT amily transporters (SLC15A) are proton-driven symporters that are highly expressed in the intestine and kidney. PEPT1 has a key role in the absorption o dietary nitrogen, in the orm o dipeptides and tripeptides, rom the lumen o the small intestine into enterocytes. Peptide-like metabolites or drugs such as -lactam antibiotics and ACE inhibitors exhibit high bioavailability due largely to PEPT1-mediated absorption (Table 5-1). Both PEPT1 and PEPT2 are expressed in the kidney, where they mediate reuptake o small peptides at the apical membrane o the kidney proximal tubule and regulate systemic nitrogen balance (Fig. 5-1).

Concentrative and Equilibrative Nucleoside Transporter (CNT and ENT) Families

CNT (SLC28) amily members CNT1, CNT2, and CNT3 mediate sodium-dependent uptake o nucleosides by epithelial cells. CNT1 and CNT2 specif cally transport pyrimidine and purine nucleosides, respectively, whereas CNT3 is capable o transporting both classes o nucleosides. In contrast, ENT (SLC29) amily members provide bidirectional transport o purine and pyrimidine nucleosides. The direction o ENT-mediated transport depends on the concentration

gradient o the nucleosides and unctions to equilibrate extracellular and intracellular nucleoside levels. Whereas intracellular nucleoside accumulation contributes to nucleotide synthesis, CNT- and ENT-mediated nucleoside uptake may also limit the activation o extracellular nucleoside signaling events, such as the activation o adenosine receptors. In addition to endogenous nucleosides, CNTs and ENTs transport nucleoside analogues such as cytotoxic anticancer (e.g., gemcitabine ) and antiviral (e.g., zidovudine ) drugs. There ore, actors or compounds that reduce the expression or activity o these transporters on target tissues may reduce the e f cacy o nucleoside anticancer and antiviral drugs.

Glucose Transporters

The glucose transporter amily (GLUT, SLC2) regulates the distribution o glucose between the plasma and tissues. Among the several amily members, GLUT1–4 iso orms are the most studied and most relevant to glucose metabolism. GLUT1 has broad tissue expression and is responsible or sustaining basal cellular glucose levels with a slow uptake rate. GLUT2 is expressed in the organs that participate in the regulation o plasma glucose levels, such as kidney, liver, intestine, and pancreas. GLUT2 mediates the uptake o dietary glucose in the intestine. Because it has relatively low a f nity or glucose, GLUT2 acts as a sensor or glucose levels within the pancreatic beta cells that secrete insulin in response to increases in plasma glucose. GLUT2 exhibits bidirectional transport capability, allowing or the in ux or e ux o glucose across the cell membrane. The e ux transporter activity o GLUT2 at the sinusoidal membrane o hepatocytes is essential or the transport o glucose produced in the liver into the plasma in order to maintain plasma glucose homeostasis during starvation or asting. GLUT3 is enriched in neurons; it exhibits high relative a f nity or glucose in order to provide a constant in ux o glucose rom the circulation into neuronal cells, even at low plasma glucose concentrations. GLUT4 mediates insulin-sensitive glucose uptake and storage in the adipose tissue and striated muscle. Under conditions o low plasma insulin concentrations, such

62 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

as asting, GLUT4 is sequestered in intracellular vesicles and does not transport glucose. Insulin stimulation causes GLUT4 to translocate to the plasma membrane, where it initiates glucose uptake (see Fig. 31-4). Sodium glucose transport protein (SGLT, SLC5) amily members 1 and 2 are symporters that transport glucose against its concentration gradient via a secondary active mechanism. SGLT1 at the intestinal brush border and SGLT1 and SGLT2 at the kidney proximal tubule brush border mediate glucose absorption rom the intestinal lumen and the renal f ltrate, respectively. In turn, GLUT2 e ux activity at the apical membrane o these tissues acilitates glucose transport rom the cells into the circulation. SGLT2 is responsible or more than 90% o glucose reuptake rom the renal f ltrate, and inhibition o SGLT2 reduces plasma glucose levels. SGLT2 has been targeted or pharmacologic intervention in the management o diabetes. Dapaglif ozin selectively inhibits SGLT2 over SGLT1; this selective inhibition acilitates reduction o plasma glucose levels without impairing SGLT1-mediated intestinal glucose absorption, potentially limiting adverse e ects such as diarrhea that may be associated with elevated intestinal glucose levels.

E f ux Transporters Oral drug administration generally provides the most convenient and a ordable route or the systemic delivery o therapeutic agents; high, reproducible oral bioavailability minimizes variability in drug exposure across patient populations. A ter oral administration, drugs typically are absorbed rom the intestinal lumen, delivered into the mesenteric blood supply, and then circulated through the hepatic portal system via the portal vein be ore entering the systemic circulation (see Chapter 3, Pharmacokinetics). E ux transporters can a ect oral bioavailability (1) by limiting the amount o drug that is absorbed across the enterocyte and/or (2) by transporting drug rom the liver into the bile and thereby contributing to a f rst-pass e ect. Whereas e ux transporters expressed on the canalicular membrane o the hepatocyte acilitate the excretion o xenobiotics into the bile, e ux transporters expressed on the sinusoidal (basolateral) membrane can promote the ux o drug and metabolites back into the systemic circulation (Fig. 5-1). Compounds eliminated into the bile across the canalicular membrane o the hepatocyte are concentrated in the gall bladder and released into the small intestine, where both parent drug and possibly metabolites can be absorbed across the intestine (a process known as enterohepatic recirculation) or eliminated into the eces. However, the same f rst-pass mechanisms (i.e., absorption, e ux, metabolism, and biliary excretion) that limited the compound’s ability to reach the systemic circulation and the peripheral tissues still exist. The oral bioavailability o compounds that undergo extensive enterohepatic recirculation may appear extremely low; however, in cases where the target tissue is either the liver (e.g., statins) or intestine, these compounds may have desired pharmacologic e ects despite limited systemic exposure. For drugs that are higha f nity substrates or intestinal and hepatic transporters, alternative routes o administration (such as intravenous and subcutaneous) can circumvent the f rst-pass e ect and possibly increase the systemic exposure. However, even when drug delivery is used to avoid a f rst-pass e ect, the drugs that reach the systemic circulation must nevertheless transit

through the hepatobiliary system and may still be cleared rapidly due to e f cient drug e ux processes. Compounds in the systemic circulation can also be eliminated in the kidney via active e ux into the urine. Because the transport o compounds rom within the intracellular space is o ten against a concentration gradient, the process requires active transport; the majority o e ux pumps belong to the ATPbinding cassette (ABC) amily o active transporters. This section outlines the e ux transporters that have major roles in drug disposition as well as those that acilitate the transport o endogenous compounds.

ATP-Binding Cassette (ABC) Transporter Family

ABC transporters constitute the largest transporter superamily. They are active transporters that mediate e ux o various substrates—such as phospholipids, steroids, and drugs—out o cells against their concentration gradient. The super amily is divided into seven amilies, o which only members o the ABCB, ABCC, and ABCG amilies have essential roles in drug disposition. ABCA and ABCD proteins transport only endogenous substrates and regulate cellular cholesterol and atty acid metabolism, and ABCE and ABCF amily members lack transmembrane domains and are not involved in the transport o drugs or endogenous compounds across the membranes o cells. P-Glycoprotein and Bile Salt Export Pump (ABCB Family)

The ABCB amily, also known as multidrug resistance/ transporters associated with antigen processing (MDR/ TAP), consists o 11 members and includes perhaps the most studied drug transporter, P-glycoprotein (P-gp, also known as MDR1 or ABCB1). P-gp was initially discovered and characterized as a protein that mediates anticancer drug resistance. Its expression is elevated in many cancer cells, and P-gp contributes to multidrug resistance against antineoplastic therapeutics. P-gp is also expressed in the apical membrane o the small intestine, liver, kidney, endothelial cells o the blood–brain barrier, and placenta (Fig. 5-1), and it both limits exposure o substrates to certain organs and serves as a mechanism to eliminate xenobiotics rom the body. P-gp has broad substrate specif city and it exhibits high a f nity or cationic and amphiphilic compounds such as phospholipids (Table 5-2). Although P-gp may have evolved as a de ense mechanism to protect organisms against exogenous toxins, other ABCB amily members such as MDR3 are important or phospholipid homeostasis in cell membranes. The bile salt export pump (BSEP, ABCB11) is an ABCB e ux transporter that is expressed at high levels on the hepatocyte canalicular membrane (Fig. 5-1). BSEP acilitates transport o bile salts and bile salt conjugates such as taurocholate rom hepatocytes into the bile, and bile ow rate is largely regulated by the activity o BSEP. BSEP has also been shown to export statins such as pravastatin into the bile; by this mechanism, such drugs may be cleared rom the liver (Table 5-2). Although its interaction with pravastatin suggests that BSEP may play a role in drug disposition, a more immediate concern is that a xenobiotic can inhibit BSEP and potentially contribute to cholestasis and intracellular accumulation o bile acids in hepatocytes. Clinical studies suggest that the endothelin receptor antagonist bosentan inhibits BSEP and may thereby induce cholestasis due to intracellular accumulation o cytotoxic bile salts.

C h a p t e r 5 Drug Transporters

63

TABLE 5-2 E f ux Transporters ORGAN/LOCATION (SEE FIG. 5-1)

ENDOGENOUS SUBSTRATES

P-gp (MDR1, ABCB1)

Intestine (apical), kidney proximal tubule, hepatocytes (canalicular), blood–brain barrier

MDR3 (ABCB4)

TRANSPORTER

DRUG SUBSTRATES

INHIBITORS

Steroids, phospholipids, bilirubin, bile acids

Digoxin, loperamide, quinidine, vinblastine, talinolol, berberine, irinotecan, doxorubicin, paclitaxel, exo enadine, seliciclib, telithromycin, clarithromycin

Cyclosporine, quinidine, tariquidar, verapamil, ketoconazole, nel navir, ritonavir, tacrolimus, valspodar, saquinavir, elacridar, reserpine

Liver (canalicular)

Phosphatidylcholine

Digoxin, paclitaxel, vinblastine

Verapamil, cyclosporine

BSEP (ABCB11)

Liver (canalicular)

Bile acids, taurocholate

Pravastatin, vinblastine

Bosentan, cyclosporine, ri ampin, glibenclamide, glyburide

BCRP (ABCG2)

Intestine, liver (canalicular), breast, placenta, blood–brain barrier, stem cells

Uric acid, vitamins, dietary f avonoids, porphyrins, estrone 3-sul ate

Daunorubicin, doxorubicin, topotecan, irinotecan, methotrexate, imatinib, rosuvastatin, sul asalazine, nucleoside analogues

Elacridar, imatinib, novobiocin, estrone, 17 -estradiol, ritonavir, omeprazole

MRP2 (ABCC2)

Intestine, liver, kidney, brain

Bilirubin, cholecystokinin, estrone 3-sul ate, glutathione and glucuronide conjugates

Glutathione and glucuronide conjugates, indinavir, cisplatin, methotrexate, etoposide, mitoxantrone, valsartan, olmesartan

Cyclosporine, delavirdine, e avirenz, emtricitabine, benzbromarone

MRP3 (ABCC3)

Intestine (brush border), liver (sinusoidal), kidney, placenta, adrenal gland

Bile salts, estradiol17 -glucuronide, leukotriene C4

Etoposide, methotrexate, teniposide, exo enadine, glucuronide conjugates, acetaminophen, vincristine

Delavirdine, e avirenz, emtricitabine, lamivudine, teno ovir, indomethacin, urosemide, probenecid, nevirapine

MRP4 (ABCC4)

Prostate, kidney, placenta, liver, blood–brain barrier

Taurocholate, cAMP, cGMP, urate, DHEAS, prostaglandins E1 and E2

Acyclovir, ritonavir, teno ovir, topotecan, PMEA, methotrexate, urosemide, ce tizoxime, ce azolin, 6-mercaptopurine

Indomethacin, MK571, diclo enac, celecoxib, sul npyrazone, quercetin

MATE1 (SLC47A1)

Kidney proximal tubule, liver (canalicular), skeletal muscle

Creatinine, guanidine, nucleosides

Met ormin, cephalexin, acyclovir, ganciclovir, exo enadine, oxaliplatin

Quinidine, cimetidine, verapamil, procainamide

MATE2-K (SLC47A2)

Kidney proximal tubule

Estrone sul ate, creatinine

Met ormin, cimetidine, procainamide

Cimetidine, quinidine, pramipexole

Data compiled rom same sources as in Table 5-1.

Multidrug Resistance-Associated Proteins (ABCC Family)

The ABCC amily, also known as the multidrug resistanceassociated protein (MRP/CFTR) family, consists o 9 members that exhibit high specif city or organic anions such as glutathione- and glucuronide-conjugated drugs. ABCC amily members are localized to the apical and basolateral membranes o hepatocytes, enterocytes, kidney proximal tubule, endothelial cells o the blood–brain barrier, and placenta (Fig. 5-1). Similar to P-gp, MRPs are abundantly expressed in tumors and cause resistance to anticancer drugs. MRP2 mediates hepatobiliary clearance o drugs including methotrexate and statins. In addition to its expression on the canalicular membrane o hepatocytes, MRP2 is expressed on the apical membrane o the intestine and the kidney proximal tubule. In contrast, MRP3 and MRP4 are expressed on the basolateral membrane o the liver and are responsible or the transport o compounds rom the liver back into the blood.

Several pathophysiologic consequences o hereditary MRP def ciency have been reported, the most notable o which is Dubin-J ohnson syndrome . In this syndrome, MRP2 def ciency results in the clinical mani estation o conjugated hyperbilirubinemia. MRPs transport not only exogenous molecules but also a wide array o endogenous compounds such as leukotrienes, bilirubin glucuronides, prostaglandins, cAMP, cGMP, and steroids (Table 5-2). Moreover, MRPs likely have a role in the disposition and elimination o drug metabolites, especially charged anionic glucuronide and sul ate conjugates. The cystic f brosis transmembrane conductance regulator (CFTR) is also a member o the ABCC amily. CFTR transports chloride ions across the membrane o mucus-secreting epithelial cells in the lung, digestive system, pancreas, reproductive system, and other organs and tissues. Mutations in CFTR disrupt chloride transport, which leads to impaired mucus ormation and ow, and are the cause o cystic f brosis in humans.

64 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Breast Cancer Resistance Protein (ABCG Family)

Among the f ve ABCG amily members, breast cancer resistance protein (BCRP, ABCG2) is the only one implicated in the disposition o drugs and xenobiotics (Table 5-2). BCRP is expressed in several tissues, including the intestinal tract, blood–brain barrier, liver canalicular membrane, kidney proximal tubule, testis, and placenta (Fig. 5-1). The substrate specif city o BCRP overlaps with that o P-gp; as such, BCRP can enhance the xenobiotic barrier unction where these transporters are co-expressed. Despite its name, BCRP has relatively low levels o expression in breast cancers, but it has been shown to con er drug resistance in several other tumor types (e.g., leukemia, lung cancer, and melanoma). In addition to its role in drug disposition, BCRP exports urea into the urinary tract and mediates secretion o vitamins into breast milk. Other ABCG amily members are involved in endogenous unctions (see Chapter 20, Pharmacology o Cholesterol and Lipoprotein Metabolism). ABCG5 and ABCG8 orm a heterodimer that controls biliary sterol and lipid secretion in the canalicular membrane o the liver as well as clearance o sterols in the small intestine. ABCG1 promotes e ux o cholesterol rom macrophages to high-density lipoprotein (HDL) particles. ABCA and ABCD Families

ABCA and ABCD amily members mediate the transport o endogenous substrates. For example, ABCA1 o the ABCA amily mediates cellular lipid e ux by transporting cholesterol and phospholipids out o hepatocytes and macrophages, contributing to the biogenesis o HDL. ABCD transporters are expressed on the membrane o peroxisomes and regulate atty acid uptake by these organelles. This suggests that some transporters may have evolved to regulate tra f cking o endogenous compounds between plasma and tissues and to eliminate toxic byproducts o cellular metabolism, whereas a subset o transporters may have adapted to also promote the elimination o nutrients, xenobiotics, and their metabolites.

Solute Carrier (SLC) Family Multi-Antimicrobial Extrusion Protein (MATE) Family

Multi-antimicrobial extrusion proteins, also known as multidrug and toxic compound extrusion proteins (MATEs), are a amily o multidrug e ux transporters that exhibit a f nity or organic cations. They unction as proton/drug antiporters that import a proton into the intracellular medium as a counterion or the export o a drug molecule out o the cell. MATE1 (SLC47A1) is expressed on the canalicular membrane o hepatocytes and on kidney proximal tubule cells, where it exports exogenous compounds into bile and urine, respectively (Fig. 5-1). MATE2-K (SLC47A2) is expressed only in the kidney proximal tubule, where it mediates clearance o exogenous substrates alongside MATE1 (Fig. 5-1). Both transporters exhibit selectivity or compounds that are substrates or OCTs, such as metformin and tetraethylammonium. Their endogenous substrates include organic cations such as guanidine and creatinine (Table 5-2).

CLINICAL PERSPECTIVES ON DRUG TRANSPORTERS The pharmacokinetic, pharmacodynamic, and toxicologic properties o drugs are characterized and optimized initially

through a comprehensive series o in silico predictions, in vitro cell culture, and membrane preparation studies and then through in vivo animal models and clinical trials. Membrane uptake and e ux transporters can in uence the absorption, distribution, and excretion o xenobiotics and may there ore have substantial impact on the e f cacy and sa ety o drugs. Drugs can act as substrates or transporters and may also alter the activity o transporters through inhibition or induction o transport processes, which could result in the impaired or enhanced transport o endogenous substrates. Moreover, much as in the case o drug-metabolizing enzymes, the concomitant administration o several drugs can lead to drug–drug interactions i one agent alters the transport o the other. This could potentially reduce the clearance o the co-administered drug, a ect the drug’s oral bioavailability, and lead to adverse e ects or lack o e f cacy due to unexpected changes in drug exposure. Understanding the specif c transporters that govern the absorption, distribution, and clearance o new therapeutic agents will help anticipate and minimize the unexpected impact o these mechanisms on drug disposition and potential drug–drug interactions. There are apparent species di erences in drug transporters as well as lack o direct orthologs or some human transporters (e.g., OATP1B1 and OATP1B3), and several human transporters have there ore been cloned and expressed in cell lines. Transgenic mouse models expressing human transporter genes have also been created to enable preclinical studies designed to help predict clinical outcomes. These models, together with known substrates and inhibitors o drug transporters (Tables 5-1 and 5-2), can aid in understanding the clinical relevance o these mechanisms or drug disposition.

Drug–Drug Interactions Since the discovery o P-gp as a multidrug resistance gene against anticancer drugs, many other hydrophobic and cationic drugs have been identif ed as its substrates (Table 5-2). The cardiac glycoside digoxin is considered to be a prototypical P-gp substrate, and the potential drug–drug interactions due to inhibition o P-gp by novel drug entities can be determined by measuring the plasma levels o digoxin upon co-administration o the novel drugs. Drugs that inhibit P-gp, such as the antiarrhythmic agent quinidine and the antiviral drug ritonavir, elevate intestinal uptake and reduce biliary and renal clearance o digoxin in vivo and thereby increase plasma levels o digoxin (Table 5-3). Because P-gp is the primary transporter preventing the entry o exogenous compounds into the central nervous system, substrates or P-gp are o ten unable to cross the blood–brain barrier. Inhibition o P-gp at the blood–brain barrier has been reported in some preclinical studies, but the ability to inhibit P-gp at the human blood–brain barrier does not yet have proven clinical relevance, likely because the compounds are unable to achieve high enough unbound concentrations in the systemic circulation to inhibit blood–brain barrier P-gp in vivo. Like P-gp, BCRP was initially characterized as a multidrug resistance gene expressed in neoplastic cell lines. In animal studies, BCRP not only limits the e f cacy o anticancer drugs such as topotecan but also substantially reduces the intestinal uptake o non-cancer drugs such as atorvastatin. Elacridar is a potent BCRP inhibitor that can be used to test whether new drugs are cleared by BCRP or to increase the bioavailability o known BCRP substrates. For example,

C h a p t e r 5 Drug Transporters

65

TABLE 5-3 Clinically Observed Drug–Drug Interactions Due to Transporters TRANSPORTER

INHIBITOR OR *INDUCER

AFFECTED DRUG

PHARMACOKINETIC CHANGES DUE TO AFFECTED DRUG

OATP1B1 (SLCO1B1)

Lopinavir/ritonavir Cyclosporine Rifampin (single dose)

Bosentan Pravastatin Glyburide

AUC↑ 5-48-fold AUC↑ 9.9-fold, Cmax↑ 7.78-fold AUC↑ 2.3-fold

OATP1B3 (SLCO1B3)

Cyclosporine Cyclosporine Lopinavir/ritonavir

Pitavastatin Rosuvastatin Rosuvastatin

AUC↑ 4.6-fold, Cmax↑ 6.6-fold AUC↑ 7.1-fold AUC↑ 2.1-fold, Cmax↑ 4.65-fold

OATP1A2 (SLCO1A2)

Grapefruit juice Orange juice Apple juice Naringin

Fexofenadine Fexofenadine Fexofenadine Aliskiren

AUC↓ 2.7-fold, Cmax↓ 2.63-fold AUC↓ 3.3-fold, Cmax↓ 3-fold AUC↓ 3.7-fold, Cmax↓ 3.57-fold AUC↓ 1.6-fold, Cmax↓ 2.44-fold

OATP2B1 (SLCO2B1)

Orange juice Apple juice

Aliskiren Aliskiren

AUC↓ 2.6-fold AUC↓ 2.6-fold

OAT1 (SLC22A6)

Probenecid Probenecid Probenecid

Cephradine Cidofovir Acyclovir

AUC↑ 3.6-fold AUC↑ 1.5-fold, CLr↓ 1.47-fold AUC↑ 1.4-fold, CLr↓ 1.47-fold

OAT3 (SLC22A8)

Probenecid

Furosemide

AUC↑ 2.9-fold

OCT2 (SLC22A2)

Cimetidine Cimetidine Cimetidine Cimetidine Cimetidine

Dofetilide Pindolol Metformin Varenicline Pilsicainide

AUC↑ 1.5-fold, CLr↓ 1.5-fold AUC↑ 1.5-fold, CLr↓ 1.5-fold AUC↑ 1.4-fold, CLr↓ 1.37-fold AUC↑ 1.3-fold AUC↑ 1.3-fold, CLr↓ 1.39-fold

P-gp (MDR1, ABCB1)

Dronedarone Quinidine Ritonavir Ranolazine Clarithromycin *Rifampin *St. J ohn’s wort *Rifampin *St. J ohn’s wort *Tipranavir/ritonavir *Tipranavir/ritonavir

Digoxin Digoxin Digoxin Digoxin Digoxin Digoxin Digoxin Talinolol Talinolol Loperamide Saquinavir/ritonavir

AUC↑ 2.6-fold, Cmax↑ 1.75-fold AUC↑ 1.7-fold, CLr↓ 1.5-2-fold AUC↑ 1.86-fold, CLr↓ 1.54-fold AUC↑ 1.6-fold, Cmax↑ 1.46-fold AUC↑ 1.7-fold, Cmax↑ 1.75-fold AUC↓ 1.4-fold, Cmax↓ 1.6-fold AUC↓ 1.4-fold, Cmax↓ 1.56-fold AUC↓ 1.5-fold, Cmax↓ 1.6-fold AUC↓ 1.3-fold AUC↓ 2-fold AUC↓ 5-fold

BCRP (ABCG2)

Elacridar

Topotecan

AUC↑ 2.4-fold, Cmax↑ 3.8-fold

the oral administration o elacridar with topotecan substantially increases plasma concentrations o topotecan (Table 5-3). Similar to the limited signif cance o P-gp inhibition at the human blood–brain barrier, BCRP inhibition at the blood–brain barrier is thought to have limited e ect on drug distribution to the brain. Among the uptake transporters, OAT1 and OAT3 are implicated in the transport and clearance via the kidney proximal tubule o a broad range o drugs such as antivirals, antibiotics, statins, and anticancer drugs. Probenecid is a well-established inhibitor o both transporters and, when co-administered with urosemide or methotrexate, increases the bioavailability o these OAT substrates. Probenecid is also used to prevent renal injury by blocking OAT1-mediated renal uptake o the nephrotoxic antiviral drug cidofovir (Table 5-3). OCTs and MATEs o ten work in concert to acilitate drug transport across the liver and kidney (Fig. 5-1, and see the ollowing discussion). These two transporter amilies also share the common inhibitors cimetidine (histamine receptor

antagonist), pyrimethamine (antiprotozoal and antimalarial dihydro olate reductase inhibitor), and the chemotherapeutic tyrosine kinase inhibitors imatinib and erlotinib (Tables 5-1 and 5-2). Although cimetidine has been largely supplanted by proton pump inhibitors in the treatment o acid re ux, it may prove use ul or the study o drug–drug interactions in vivo. Both cimetidine and pyrimethamine exhibit higher selectivity and increased potency or MATE compared to OCT amily members; there ore, any drug–drug interaction involving these drugs could depend on the dose o the coadministered inhibitors. OCTs acilitate drug uptake rom the blood into the liver and kidney, and MATEs contribute to drug secretion rom these tissues into the bile and urine, respectively. There ore, or example, low-dose cimetidine coadministration, which would inhibit only MATE-mediated excretion, may result in increased metformin localization to the liver and kidneys. As a second example, high-dose cimetidine co-administration inhibits not only MATE-mediated drug excretion but also OCT2-mediated renal uptake o the

66 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

anticancer drug cisplatin rom the blood, thereby increasing cisplatin plasma levels and protecting the kidneys against the nephrotoxic adverse e ects o this drug. Inhibition o drug uptake into the liver and kidney can result in increased plasma levels and improved e f cacy o the drug. This generalization does not hold, however, i the site o action o the drug is the liver or kidney. For example, the liver is the target tissue or statins such as atorvastatin, pravastatin, and rosuvastatin, where these agents inhibit HMG-CoA reductase to reduce cholesterol synthesis. The uptake o statins into hepatocytes is mediated by OATP1B1 and OATP1B3 (Fig. 5-1). Co-administered drugs that inhibit these uptake transporters impair statin uptake by the liver and increase statin plasma concentration (Table 5-3). This may not only lead to reduced e f cacy o the statin in the liver but also increase the risk o adverse e ects such as rhabdomyolysis; the combination o these e ects could decrease the therapeutic index o the drug. Foods as well as drugs can inter ere with transporter activity. Grape ruit juice was initially identif ed as an inhibitor o the drug-metabolizing enzyme CYP3A4. However, recent studies show that grape ruit juice and other ruit juices such as orange juice and apple juice may also impair OATPmediated uptake transport. Naringin, the active component o grape ruit juice, inhibits OATP1A2 and OATP1B1 and leads to reduced drug bioavailability in clinical studies. In addition, the hesperidin and quercetin constituents o orange juice and apple juice, respectively, may be responsible or inhibiting OATP1A2 and OATP2B1. Although co-administration o certain ruit juices with drugs that are OATP substrates may reduce drug bioavailability, clinical e ects have been observed only when the drugs are taken together with large volumes o these juices (Table 5-3). In vitro studies have started to explore the potential or oods to alter the unction o other transporters, but these observations have yet to translate into clinical outcomes. For example, grape ruit juice has been shown to inhibit P-gp activity in vitro, but clinical studies do not demonstrate inhibition o P-gp when grape ruit juice is co-administered with digoxin. Drugs are also capable o inducing the expression o transporters. Similar to the induction o P450 enzyme expression, drug transporter expression can be regulated by the nuclear pregnane X receptor (PXR), constitutive androstane receptor (CAR), arnesoid X receptor (FXR), and vitamin D receptor. Relative to the e ect o transporter inhibition, the overall impact o transporter induction on clinically relevant drug interactions is minimal, and most drug interactions involving induction have been attributed to the induction o P-gp in the intestine. St. J ohn’s wort and ri ampin are strong PXR agonists, and the co-administration o digoxin with St. John’s wort or ri ampin results in reduced digoxin plasma levels (Table 5-3). Similarly, ri ampin preadministration reduces plasma concentrations o talinolol and carvedilol, likely due to P-gp induction. In all o these cases, enhanced expression o P-gp would be expected to reduce the systemic exposure o the drug and thereby reduce the e f cacy o the drug.

Drug Inter erence with Endogenous Metabolite Tra cking Several drugs have been implicated in the disruption o endogenous metabolite tra f cking and homeostasis, most likely

TABLE 5-4 Adverse Drug Reactions Due to Transporter–Drug Interactions TRANSPORTER

DRUG

ADVERSE REACTION

P-gp (MDR1, ABCB1)

Cyclosporine Tacrolimus Loperamide

Nephrotoxicity Nephrotoxicity Respiratory depression

BSEP (ABCB11)

Bosentan Cyclosporine Glibenclamide Rifampin Troglitazone

Cholestatic liver injury Cholestatic liver injury Cholestatic liver injury Cholestatic liver injury Cholestatic liver injury

MRP2 (ABCC2)

Irinotecan Methotrexate

Diarrhea Nephrotoxicity

BCRP (ABCG2)

Irinotecan

Myelosuppression

OAT1 (SLC22A6)

Adefovir Cidofovir Tenofovir

Nephrotoxicity Nephrotoxicity Nephrotoxicity

OCTs

Metformin

Hyperlactacidemia, lactic acidosis

OCT2 (SLC22A2)

Cisplatin

Nephrotoxicity

OATP1B1 (SLCO1B1)

Mycophenolate mofetil

Leukopenia, anemia, thrombocytopenia, diarrhea, nausea, vomiting, infection Myopathy

Simvastatin

Adapted from Table 4 of König J , Müller F, Fromm MF. Transporter and drug–drug interactions: important determinants of drug disposition and effects. Pharmacol Rev 2013;65:944–966.

stemming rom the inhibitory e ect o these drugs on transporters. For example, inhibition o the bile salt transporter BSEP could impair bile ormation and ow, resulting in elevated hepatic bile acids and leading to cholestatic liver injury (Table 5-4). URAT1 and OAT4 are essential or establishing systemic uric acid balance, and inhibition o URAT1 has been exploited as a treatment or gout. However, compounds such as losartan can inhibit both URAT1 and OAT4 and may thereore cause undesirable adverse e ects such as the ormation o kidney stones. The HIV protease inhibitor ritonavir blocks GLUT4, and this could result in hyperglycemia in prediabetic patients treated with ritonavir. Pharmacologic inhibition o SGLT2 results in osmotic diuresis due to the increased glucose load in the urine and has been linked to increased urination and thirst in clinical studies. One such SGLT2 inhibitor, dapaglif ozin, may cause hypotension and light-headedness due to excessive dehydration. Additional adverse e ects related to transporter–drug interactions are listed in Table 5-4.

Pharmacogenomics Genotype-dependent alterations in drug-metabolizing enzymes (such as CYPs) are well documented. Similar to CYPs, uptake and e ux transporters are subject to single nucleotide polymorphisms (SNPs), which can result in altered pharmacokinetics or certain drugs such as statins and antidiabetics. The OATP1B1 polymorphism N130D has

C h a p t e r 5 Drug Transporters

been shown to result in elevated serum bilirubin levels and reduced hepatic uptake o OATP1B1 drug substrates, such as statins. Patients who carry one or two copies o the V174A variant o OATP1B1 exhibit 2- and 12- old increased risk o developing myopathy, respectively, in the setting o daily simvastatin treatment. The Q141K variant o BCRP is also associated with altered statin pharmacokinetics; increased plasma levels o the drug are due to reduced e ux activity at the intestinal lumen. The A270S variant o OCT2 has been implicated in reduced renal uptake and clearance o the antidiabetic drug metformin. Because such SNPs represent common variants that occur in more than 1% o the population, a large number o patients could potentially be a ected by altered pharmacokinetics. There ore, genetic analysis o specif c drug transporters could provide a valuable advance toward personalized medicine.

INTEGRATION OF DRUG TRANSPORTER SCIENCE INTO DRUG DISCOVERY AND DEVELOPMENT Progress in our understanding o mechanisms o drug transport aids in designing drugs with optimal transport properties, predicting drug–drug and drug–disease interactions, and determining optimal routes o administration (Box 5-1). A new drug entity may be optimized or maximum potency against its biological target by structural modif cation o moieties on the molecule and pharmacologic testing o the derivative compounds in cell culture or in silico systems. However, the e f cacy o a potential drug candidate with exquisite in vitro potency may be severely impeded in clinical trials i it does not also have an optimal pharmacokinetic prof le. For drug candidates that are substrates or one or more o the major drug transporters, the transporter(s) can in uence the pharmacokinetic prof le o the drug by limiting or enhancing the ability o the drug to reach its biological target in vivo. There ore, in drug discovery, an understanding o whether a compound (or compound class) is a substrate or inhibitor o a drug transporter can aid in early optimization o the compound’s chemical structure. For oral administration, it is important to take into account the contribution o intestinal drug e ux transporters (e.g., P-gp, BCRP, and MRP2) relative to the contribution o passive intestinal permeability, since these transporters can reduce overall intestinal permeability and contribute to low oral bioavailability. Drug disposition can be improved not only by avoiding e ux channels but also by optimizing or transport by uptake channels. Because the target organ o statins is the liver, optimal uptake rom the plasma into hepatocytes would be expected to improve the pharmacodynamics o these HMGCoA reductase inhibitors. The substrate pre erence o CNTs/ ENTs and PEPTs or nucleosides and peptides, respectively, has been exploited to enhance the absorption o drugs that otherwise exhibit high clearance rates. The oral bioavailability o ganciclovir is ten old less than its optimized peptide conjugate (prodrug) valganciclovir, which is a substrate or PEPT1. As a f nal example, the chemotherapeutic e f cacy o a cytotoxic nucleoside analogue such as gemcitabine may depend on its cellular uptake into tumors by ENT1. Patients whose tumors exhibit low levels o ENT1 expression respond poorly to treatment with gemcitabine.

67

The identif cation o transporters that may have primary roles in the uptake and elimination o new drug entities is key to understanding the disposition as well as the potential or drug–drug interactions o a novel therapeutic agent. Advances in drug transporter research have also contributed to awareness by regulatory agencies that transport is an important consideration in the evaluation o new drugs. The US Food and Drug Administration (FDA), European Medicines Agency (EMA), and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA) have o ered updated clinical and nonclinical guidelines on drug transporters to consider in drug development. Increased awareness and recognition o transporter-dependent drug–drug interactions has led to the inclusion o these interactions in drug labels, along with the precautions that must be taken in the setting o multidrug therapies. The International Transporter Consortium (ITC) has proposed decision trees that could guide and help interpret preclinical studies assessing the potential role o specif c transporters in the disposition o new drug entities and extrapolate whether additional clinical studies or drug–drug interactions are warranted. There is now general consensus around the importance o testing the transport o new drugs—especially by the well-established transporters P-gp, BCRP, OCT2, OAT1, OAT3, OATP1B1, and OATP1B3—in order to better predict clinical outcomes and drug–drug interactions.

CONCLUSION AND FUTURE DIRECTIONS Signif cant advances have been made in our understanding o drug–transporter interactions in recent years, and the majority o key transporters essential in drug disposition and drug–drug interactions have been identif ed. The impact (or lack thereo ) o many o these transporters has been tested and demonstrated clinically, and both drug development strategies and regulatory guidelines continue to be established and ref ned based on our evolving scientif c understanding o drug transporters. As listed in Tables 5-1 and 5-2, transporter inhibitors that are currently available or use in vitro and in vivo exhibit overlapping specif cities. This limits the use o nonspecif c inhibitors to screen or interactions in cell culture and animal models. Discovery o more selective inhibitors would help to establish and standardize in vitro and in vivo techniques and should lead to consistent characterization o drug disposition and drug–drug interactions or novel therapeutics. These e orts should be assisted by computational approaches that predict transporter–substrate interactions based on the chemical structures o the molecules using molecular docking simulations as well as established crystal structures o transporters. With the continued and expanding use o polypharmacy to treat complex diseases, there is an urgent need to understand the potential or transporter-mediated drug interactions together with metabolic drug interactions. Emerging in vitro and in vivo tools to study drug disposition, such as 3-D tissue-engineered organ models, coupled with our growing insights into the basic science o drug transporters, should enable better predictions o the clinical impact o transporter-related mechanisms on the patient.

68 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

B O X 5 - 1 Dru g De ve lo p m e n t Ca s e A pharmaceutical lab is developing a new drug or the treatment o hypercholesterolemia. The approach is to inhibit a key enzyme involved in the cholesterol biosynthesis pathway. This enzyme, HMG-CoA reductase, is localized to the liver and it serves as the rate-controlling enzyme o the mevalonate pathway, the metabolic pathway that produces cholesterol and other isoprenoids. The main strategy or the program is to utilize the liver’s capacity or active uptake o xenobiotics in order to target the liver selectively. Data rom recent studies with the company’s lead compounds have recently been presented. Figure A shows the uptake o our compounds into suspended rat hepatocytes. Each o the compounds was incubated with the cells at 37°C or up to 30 seconds. The project team decides to conduct ollow-up studies in rats. These in vivo studies show signi cant di erences among the compounds in bioavailability a ter oral administration (%F), acute hyperbilirubinemia with Compound A, and a transient increase in serum bile acids with Compound D (Figure B). Given the high in vitro potency o the our compounds (Figure B), the pharmacologist on the project team is also keen to determine their in vivo e cacy in rats. Results show that, when administered at 5 mg/kg orally, the e cacy o the compounds is rank-ordered as ollows: A (most e cacious) D B C (least e cacious).

Questions and Discussion 1. Based on the data shown in Figure A, by which mechanism is each compound likely entering the hepatocytes? Compound A and Compound B could be taken up by multiple uptake transporters (e.g., OATPs, OATs, or OCTs) that are expressed on the sinusoidal membrane o hepatocytes. Transporter-mediated uptake could be tested by measuring the uptake o each compound into suspended hepatocytes in the presence o selective transporter inhibitors or by measuring the uptake o the compounds in cell lines that express a specif c transporter compared to the uptake in a control cell line. Compound C and Compound D likely enter 3500

2500 Compound A 2000 1500 Compound B 1000

A

c

c

u

m

u

l

a

t

i

o

n

(

p

m

o

l

/

m

g

)

3000

500 0

Compounds C a nd D 0

5

10

15

20

25

30

Time (s e conds )

FIGURE A. Accumulation o our lead HMG-CoA reductase inhibitors in suspended rat hepatocytes over 30 seconds.

In vitro Compound pote ncy (nM)

Ora l bioa va ila bility (%F)

In vivo obs e rva tions

A

35

3.1

Ele va te d conjuga te d bilirubin

B

41

28

No a dve rs e e ve nts

C

0.2

2.2

No a dve rs e e ve nts

D

121

97

Incre a s e d s e rum bile a cids

FIGURE B. In vitro and in vivo data or Compounds A, B, C, and D a ter oral administration o 30 mg/kg in rats.

the cell through passive di usion or could be transported by a low-a f nity, high-capacity transporter at a very slow uptake rate. 2. Why is the bioavailability o Compounds A and C low, whereas the bioavailability o Compound D is high? Compound A most likely undergoes enterohepatic recirculation, and only a limited amount o the drug makes it into the systemic circulation. Compound C exhibits both low hepatic uptake and low bioavailability, so the majority o the compound might simply not be absorbed in the intestine and instead be eliminated in the eces. Compound D has minimal uptake into hepatocytes and nearly complete oral bioavailability. Complete absorption o Compound D across the intestine, with a minimal f rst-pass hepatic transport or metabolism e ect, would explain the high oral bioavailability in rats. 3. Assuming that Compound A is acidic and is charged at physiologic pH, which speci c active-transport mechanisms (uptake and/or e f ux) are likely responsible or the transport o Compound A rom the plasma into the bile? I Compound A is acidic and charged at physiologic pH (anionic), then it is more likely to be transported into hepatocytes by uptake transporters that exhibit relatively high a f nity or anionic substrates, such as OATP1B1. MRP2 may mediate its excretion into bile, since this e ux transporter also has relatively high a f nity or anionic substrates. 4. What are potential mechanisms by which Compounds A and D cause hyperbilirubinemia and cholestasis, respectively? Impaired clearance o bilirubin via the hepatobiliary route can result in hyperbilirubinemia. Compound A increases conjugated bilirubin, suggesting that the compound has minimal impact on the transport o bilirubin into the hepatocyte and limited impact on the metabolism o bilirubin to bilirubin glucuronide. Compound A likely inhibits a mechanism involved in the excretion o conjugated bilirubin into the bile, likely via MRP2. MRP2 inhibition studies in

C h a p t e r 5 Drug Transporters

69

B O X 5 - 1 Dru g De ve lo p m e n t Ca s e (co n tin u e d ) trans ected cell lines, combined with in vivo studies in rats with cannulated bile ducts, would help to support this hypothesis. Compound D does not appear to be a substrate or hepatic transport based on the suspended hepatocyte data; nonetheless, the compound could inhibit transporters involved in bile acid disposition. Inhibition o either NTCP at the sinusoidal membrane or BSEP at the canalicular membrane o hepatocytes would explain the in vivo results. Further studies o uptake inhibition o bile acids (e.g., taurocholate) into suspended hepatocytes, or inhibition o BSEP (measured by taurocholate transport) in BSEP-expressing vesicles or in sandwich-cultured hepatocytes, would help to determine the mechanism that is more likely to be involved. 5. The team is allowed to advance only one of the four lead compounds into clinical trials. Which compound would be the preferred agent for clinical-trial testing? Compound A should be advanced into clinical studies. This compound exhibits the highest e f cacy in vivo, likely due to its e ective uptake into the liver—which is the target site o action or the inhibition o HMG-CoA reductase. Compound A is likely to be associated with a relatively low level o systemic adverse e ects due to its enterohepatic recirculation, and the observed hyperbilirubinemia in rats could potentially be avoided by dose adjustment. Given the likely involvement o hepatic transporters in Compound A disposition, Compound A may need to be studied urther in patients with hepatic impairment in order to understand

any potential impact on sa ety and e f cacy. Compound D exhibits low hepatic uptake and high bioavailability, suggesting that higher levels o systemic exposure would be required to achieve a pharmacologic e ect comparable to that o Compound A. The need or higher systemic exposure would warrant a care ul assessment o o -target e ects that could contribute to dose-limiting toxicities in the clinic. Moreover, the observation o altered bile acid disposition in rats suggests that urther investigation o the mechanism o cholestasis would be warranted, as well as evaluation o the potential or translation to humans i the compound were to move urther into development. Compound B exhibits good hepatic uptake and in vitro potency. Although the lack o adverse e ects makes it a potential candidate or advancement to clinical trials, its e f cacy in vivo is signif cantly lower than that o Compounds A and D. Higher doses o Compound B may be permissible due to the apparent lack o toxicity, but the physicochemical properties o the compound will at some point limit the dose that can be absorbed/administered. Compound C is the least promising candidate due to its low hepatic uptake and low in vivo e f cacy. For all o the compounds, the potential or species di erences in transporter activity should be considered (both as substrates and inhibitors), and analogous studies with human transporters should be conducted to better predict the likelihood that the results o the rodent studies will translate aith ully to the clinic. ■

Suggested Reading Brouwer KL, Keppler D, Hoffmaster KA, et al.; International Transporter Consortium. In vitro methods to support transporter evaluation in drug discovery and development. Clin Pharmacol Ther 2013;94:95–112. (In vitro methods or the identif cation o drug transporters involved in the disposition o new drug entities.) Giacomini KM, Huang SM, Tweedie DJ, et al.; International Transporter Consortium. Membrane transporters in drug development. Nat Rev Drug Discov 2010;9:215–236. (The initial white paper rom the International Transporter Consortium.) Giacomini KM, Huang SM. Transporters in drug development and clinical pharmacology. Clin Pharmacol Ther 2013;94:3–9. (Reviews roles o drug transporters in drug absorption, distribution, and elimination.) König J, Müller F, Fromm MF. Transporter and drug–drug interactions: important determinants of drug disposition and effects. Pharmacol Rev 2013;65: 944–966. (Reviews the role o transporters in drug–drug interactions.)

Morrissey KM, Wen CC, Johns SJ, Zhang L, Huang SM, Giacomini KM. The UCSF-FDA TransPortal: a public drug transporter database. Clin Pharmacol Ther 2012;92:545–546. (http://dbts.ucs .edu/ da transportal) Nigam SK. What do drug transporters really do? Nat Rev Drug Discov 2015;14:29–44. (Reviews endogenous unctions o drug transporters.) Palmeira A, Sousa E, Vasconcelos MH, Pinto MM. Three decades of P-gp inhibitors: skimming through several generations and scaffolds. Curr Med Chem 2012;19:1946–2025. (Overview o P-gp activity modulators or the reversal o multidrug resistance in cancer.) Roth M, Obaidat A, Hagenbuch B. OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. Br J Pharmacol 2012;165:1260–1287. ( Extensive review o the biology and pharmacology o organic anion and cation transporters.)

D

6

D

Drrugg m ta bo me bbolilis m

Drrug g m e ta bolis me b m

D-X X

D-X

Drug Toxicity M ich a e l W. Co n n e r, Ca t h e rin e Do ria n -Co n n e r, Vis h a l S. Va id ya , La u ra C. Gre e n , a n d David E. Go la n Uniinte Un in nde d Inte nde d Inte nde d re ce p pttor ree ce pt ptor or re cee pt ptor or INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700–711 On-ta On-targ a rg e tt"DR "On targ MECHANISMS DRUG UG TOXIC ICIT ITY Y"Off-targ . . . . . . . . . . .g. .e. t" . . . . . . . . . . . 71 fects . . . ve . . . rs . . . .c. .ts . . . . . . . . 72 ve Ta ras ge e ff fffe fe c ts . . . . . . . . .adve ad r. .s. e. . .e. ffe adOn Off-Target Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Dos os e too t hi high h . . . . . . . . . IIn corr c. t. .re ptor Idiosyncratic Toxicity .n . .co . . .rre . .e. .ct . . ce . . . .p . . . . . . . 75 CO TOXICITY . . is . . .a . .ctiva . . . . . .te . . .d. .or . . . . . . . . . . 75 Chro Ch h ro roni o nic niic a cct ctiv cti tiv tti ivvUG a ttion Overdose inhi in inhibite . . .hibi . b te t. . .d. . . . . . . . . . . . . . . . 75 orrDrug inhibition ib o . . . . . . . . . . . . . . inhib Drug–Drugg In nte tera ract ctio ions ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755 e ffe ffffePharmacokinetic ccts ts Drug–Drug Interacttio ions ns . . . . . . . . . . . . . . 75 ns Phar Ph arma rm maco cody dyna nami micc Drug–Drug Interactions ti . . . . . . . . . . . . . 75 Drug–Herb g Interactionss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Cellularr Mechanisms of Toxicity: Apoptosis i and Necrosis . . . 76 Organ and a Tissue Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Harmful Immune Responses and Immunotoxicityy . . . . . . . . 76 Drug-Induced Hepatotoxicityy . . . . . . . . . . . . . . . . . . . . . . . . . 79

INTRODUCTION Like many medical interventions, the use o drugs or therapeutic benef t is subject to the law o unintended consequences. These consequences—termed side effects , adverse effects , or toxic effects —are a unction o the mechanisms o drug action, the size o the drug dose, and the characteristics and health status o the patient. As such, the principles o pharmacology, presented in the preceding chapters, apply to drug toxicology as well. Many subsequent chapters contain Drug Summary Tables that list, among other properties, the specif c adverse e ects that can be caused by each drug. This chapter ocuses on the mechanisms underlying these adverse e ects. As a general matter, adverse e ects range rom those that are common and relatively benign to those that pose serious risk o organ damage or death. Even the ormer group o adverse e ects, however, can cause considerable discom ort and lead patients to avoid or reduce their use o medication. Also, in general, the type and risk o adverse e ects depend on the margin of safety between the dose required or e f cacy and the dose that causes adverse e ects. When the margin o sa ety is large, toxicity results primarily rom overdoses; when this margin is small or nonexistent, adverse e ects may be mani est at otherwise therapeutic doses. These principles apply both to prescription medications and to overthe-counter drugs such as acetaminophen and aspirin. Note 70

Un inte Unin te nd ndee d re ce ptor

Drug-Indduc uced Renal Toxicityy . . . . . . . . . . . . . . . . . . . . . . . . . . 80 y . . . . ."Off-targ .Off . . . . . tar . . . .g. .e. t" DO ugn-ta "O "Onrg eNeurotoxicity t" targ t. . . . . . . . 81 ttarced Musc scle lead Toxicity . . . c. .ts . . . . . 81 dDrug-Induced ve rs rs e e ffeSkel c tstall Mu adve adve d ve rrs.s. e. . .e .ff ffe Drug-Induced Cardiovascular Toxicity ty y. . . . . . . . . . . . . . . . . . 81 CDrug-Induced Corre ct re ce ptor ptor,, Toxxic In co corree cct ree ce pto ptorr82 Pulmonary icit ciiityyIn Carcinogenesis . .r. . . . . 82 but b ut incorre ctDttis is s ueug Theis a ctiva ctiv ct iva.a te te.d . oor Terato toge gene nesi ne siss Du si Duee to Dru r g Th Therapy . . . . . . . . . . . . 82 inhibite hibite d inh Dos Dos o s e too t hi high h PRINCIPLES FOR TREATING PATIENTS WITH DRUG-INDUCED RUChronic Chro Ch roni nicc aTOXICITY ccti ctiva tiv va tio tio ion o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 on TOWARD EARLYiDETECTION AND PREDICTION OF or inhibition inhibiti on DRUG TOXICITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 e ffe ffe cts CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . 85 Suggested S uggesttedd R Reading e adi ding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

that sa ety margins are a unction not only o the drug but also o the patient, in that genetic or other characteristics— such as polymorphisms in enzymes that detoxi y harm ul metabolites, comorbidities, or reduced unctional reserve in key organs—render patients more or less capable o de ending against toxicity. This is one reason why, all other things being equal, new medications should be initiated at the lowest doses likely to be therapeutic. Drug toxicity is critically important in drug development (see Chapter 51, Drug Discovery and Preclinical Development, and Chapter 52, Clinical Drug Evaluation and Regulatory Approval). Early in drug development, preclinical and clinical studies are used to evaluate compound potency, selectivity, pharmacokinetic and metabolic prof les, and toxicity. Prior to marketing, the regulatory agencies responsible or drug approval review the test data and decide whether the benef ts o the drug outweigh its risks. Once a drug is marketed and many more patients are exposed, the appearance o unexpected types or requencies o adverse e ects may cause a reevaluation o the drug, such that its use may be restricted to specif c patient populations or withdrawn entirely (as in the cases, or example, o the nonsteroidal anti-in ammatory drug rofecoxib and the antidiabetic drug troglitazone ). In this chapter, categories o drug toxicities that derive rom inappropriate activation or inhibition o the intended drug target (on-target adverse effects ) or unintended targets

C h a p t e r 6 Drug Toxicity 71

Ms . G is a n 80-ye a r-o ld p ia n o te a ch e r w ith p ro g re s s ive ly s e ve re rig h t le g p a in ove r a p e rio d o 5 to 10 ye a rs . S h e h a s co n tin u e d to te a ch in h e r s tu d io b u t a t th e co s t o in cre a s in g p a in a n d a tig u e . Im a g in g s tu d ie s re ve a l s e ve re o s te o a rth ritis o th e rig h t h ip . S h e is s ch e d u le d o r e le ctive re p la ce m e n t o th e rig h t h ip w ith a p ro s th e tic jo in t. Th e to ta l h ip re p la ce m e n t is p e r o rm e d w ith o u t im m e d ia te com p lica tio n s . Du rin g th e f rs t e w d a ys a te r th e o p e ra tio n , Ms . G is g ive n lo w-m o le cu la rw e ig h t h e p a rin a n d wa r a rin a s p ro p hyla xis a ga in s t d e e p ve in th ro m b o s is . S ix d a ys a te r th e o p e ra tio n , s h e d e ve lo p s e xcru cia tin g p a in in th e a re a o th e o p e ra tio n . Rig h t la te ra l h ip a n d b u tto ck s w e llin g is n o te d o n p hys ica l e xa m in a tio n . A co m p le te b lo o d co u n t re ve a ls s ig n if ca n t b lo o d lo s s (d ro p in h e m a to crit ro m 35% to 25%), a n d s h e is ta ke n b a ck to th e o p e ra tin g ro om o r e va cu a tion o a la rg e he m a to m a a ro u n d th e p ro s th e tic jo in t. Alth o u g h th e h e m a to m a does not appear to be gros sly in ected, cultures o the h e m a to m a a re pos itive or S ta phylococcus a u re us . Be ca u s e p ro s th e tic jo in t in e ctio n s a re d i f cu lt to tre a t s u cce s s u lly w ith o u t re m ova l o th e p ro s th e s is , Ms . G is s ta rte d o n a n a g g re s s ive 12-w e e k co u rs e o co m b in a tio n a n tib io tics in w h ich in tra ve n o u s va n co m ycin a n d o ra l ri a m p in a re a d m in is te re d o r 2 w e e ks o llo w e d b y o ra l cip ro o xa cin a n d ri a m p in o r 10 w e e ks . S h e to le ra te s th e f rs t 2 w e e ks o a n tib io tics w ith o u t co m p lica tio n s . Ho w e ve r, 36 h o u rs a te r s w itch in g h e r a n tib io tic ro m va n co m ycin to cip ro o xa cin , s h e d e ve lo p s a h ig h e ve r to 103°F

(off-target adverse effects ) are discussed f rst. The phenotypic e ects o these drug toxicities are then discussed at the physiologic, cellular, and molecular levels. General principles and specif c examples are also illustrated in this chapter and throughout the book. The development o rational therapeutic strategies o ten requires an understanding o the mechanisms o both drug action and drug toxicity.

MECHANISMS OF DRUG TOXICITY Whether a drug will do more harm than good in an individual patient depends on many actors, including the patient’s age, genetic makeup, and preexisting conditions; the dose o the drug administered; and other drugs that the patient may be taking. For example, the very old or very young may be more susceptible to the toxic e ects o a drug because o age-dependent di erences in pharmacokinetic prof les or drug-metabolizing enzymes. As discussed in Chapter 4, Drug Metabolism, genetic actors may determine individual characteristics o drug metabolism, receptor activity, or repair mechanisms. Adverse drug reactions may be more likely in patients with preexisting conditions, such as liver or kidney dys unction, and, o course, in patients allergic to specif c

a n d e xtre m e w e a kn e s s . As p ira tio n o th e h ip re ve a ls o n ly a s ca n t a m o u n t o s tra w-co lo re d (i.e ., n o n p u ru le n t) u id . Ms . G is th e re o re a d m itte d to th e h o s p ita l o r clo s e o b s e rva tio n . Tw e lve h o u rs a te r h e r a d m is s io n , Ms . G d e ve lo p s a n e xte n s ive m a cu lo p a p u la r ra s h o ve r h e r ch e s t, b a ck, a nd e xtre m itie s . He r cip ro oxa cin a n d ri a m p in a re d is co n tin u e d , a n d va n co m ycin is re s ta rte d . Gra d u a lly, ove r th e n e xt 72 h o u rs , h e r te m p e ra tu re re tu rn s to n o rm a l a n d h e r ra s h b e g in s to a d e . Th e re is n o g ro w th in th e cu ltu re o th e rig h t h ip a s p ira te . Ms . G is co n tin u e d o n va n co m ycin a s a s in g le a g e n t o r th e n e xt 4 w e e ks w ith o u t in cid e n t; ri a m p in is re s ta rte d , a ga in w ith o u t in cid e n t; a n d th e 12-w e e k a n tib io tic co u rs e is e ve n tu a lly co m p le te d u s in g a co m b in a tio n o trim e th o p rim -s u l a m e th oxa zo le a n d ri a m p in . Fo u r m o n th s a te r h e r h ip s u rg e ry, Ms . G is b a ck to te a ch in g h e r p ia n o s tu d e n ts a n d m a kin g s lo w b u t s te a d y p ro g re s s in h e r re h a b ilita tio n p ro g ra m .

Questions 1 . How likely was it that Ms. G’s high ever, weakness, and skin rash represented a drug reaction to ciprof oxacin? 2 . What was the rationale or co-administration o lowmolecular-weight heparin and war arin in the immediate postoperative period? 3 . Was there a cause-and-e ect relationship between administration o the prophylactic anticoagulants and Ms. G’s li e-threatening bleeding complication?

drugs. Concomitant medications can con ound both e f cacy and toxicity o drugs, particularly when these medications share or modulate the same metabolic pathways or transporters. Drug interactions with health supplements are also an important but o ten under-recognized cause o drug toxicity. Drug–drug and drug–herb interactions are discussed later in this chapter. The clinical determination o a drug’s toxicity may not always be straight orward: as in the case o Ms. G, or example, a patient being treated with an antibiotic to combat an in ection can develop a high ever, skin rash, and signif cant morbidity due either to recurrence o the in ection or, instead, to an adverse reaction to the antibiotic. Although a spectrum o adverse e ects may be associated with the use o any drug or drug class, it is help ul to conceptualize the mechanisms o drug toxicity based on several general paradigms: ■

■

“On-target” adverse e ects, which are the result o the drug binding to its intended receptor, but at an inappropriate concentration, with suboptimal kinetics, or in the incorrect tissue (Fig. 6-1) “O -target” adverse e ects, which are caused by the drug binding to a target or receptor or which it was not intended (Fig. 6-1)

72 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY Intended tissue

Unintended tissue

D

D

Drug metabolism

Drug metabolism

D-X

Inte nde d re ce ptor "On-target" adverse effects Dos e too high Chronic a ctiva tion or inhibition e ffe cts

D-X

Uninte nde d re ce ptor

Inte nde d re ce ptor

"Off-target" adverse effects

"On-target" adverse effects

Incorre ct re ce ptor is a ctiva te d or inhibite d

Corre ct re ce ptor, but incorre ct tis s ue Dos e too high Chronic a ctiva tion or inhibition e ffe cts

Uninte nde d re ce ptor "Off-target" adverse effects Incorre ct re ce ptor is a ctiva te d or inhibite d

Toxic cellular effects

FIGURE 6-1. On-target and off-target adverse drug effects. Drug D is intended to modulate the unction o a specif c receptor (intended receptor) in a particular tissue (intended tissue). On-target adverse e ects in the intended tissue could be caused by a supratherapeutic dose o the drug or by chronic activation or inhibition o the intended receptor by Drug D or its metabolite D–X. The same on-target e ects could occur in a second tissue (unintended tissue); in addition, the intended receptor could mediate an adverse e ect because the drug is acting in a tissue or which it was not designed. O -target e ects occur when the drug and/or its metabolites modulate the unction o a target (unintended receptor) or which it was not intended.

■ ■

Adverse e ects mediated by the immune system (Fig. 6-2) Idiosyncratic responses or which the mechanism is not known

These our mechanisms are discussed below. Note that many drugs can have both on-target and o -target e ects, and adverse e ects observed in patients can be due to multiple mechanisms.

On-Target Effects An important concept in drug toxicity is that an adverse e ect may be an exaggeration o the desired pharmacologic action as a result o changes in exposure or sensitivity to the drug (see Fig. 6-1). This can occur due to deliberate or accidental overdoses, alterations in the pharmacokinetics o the drug (e.g., due to liver or kidney disease or to interactions with other drugs), or changes in the pharmacodynamics o the drug–receptor interaction that alter the pharmacologic response (e.g., an increase in receptor number). All such changes can lead to an increase in the e ective concentration o the drug and thus to an increased biological response. Because on-target e ects are mediated via the desired mechanism o action o the drug, these e ects are o ten shared by every member o the therapeutic class and are thus also known as class effects . An important set o on-target adverse e ects may occur because the drug, or one o its metabolites, interacts with the

appropriate receptor but in tissues other than those a ected by the disease condition being treated (Fig. 6-1). Many drug targets are expressed in more than one cell type or tissue. For example, the antihistamine diphenhydramine is an H1 receptor antagonist used to ameliorate the e ects o histamine release in allergic conditions. This drug also crosses the blood–brain barrier to antagonize H1 receptors in the central nervous system, leading to somnolence. This adverse e ect led to the design o second-generation H1 receptor antagonists that do not cross the blood–brain barrier and thus do not induce drowsiness. Notably, the f rst o these second-generation H1 antagonists, terfenadine , produced an o -target e ect (interaction with cardiac potassium channels) that led to a di erent and serious adverse e ect—an increased risk o cardiac death. This example is discussed later in this chapter. Local anesthetics such as lidocaine and bupivacaine provide a second example o an on-target adverse e ect. These drugs are intended to prevent axonal impulse transmission by blocking sodium channels in neuronal membranes near the site o injection. Blockade o sodium channels in the central nervous system (CNS) ollowing overdose or inappropriate administration (e.g., intravascular administration) can lead to tremors, seizures, and death. These on-target e ects are discussed in greater detail in Chapter 12, Local Anesthetic Pharmacology. The antipsychotic agent haloperidol is thought to produce its benef cial e ect through blockade o mesolimbic and mesocortical D2 receptors. One consequence o blocking D2

C h a p t e r 6 Drug Toxicity 73 A Ma s t ce ll

Ha pte n 1

2 Ha pte n-bound prote in

P rote in

Comple me nt-me dia te d RBC lys is

B Cytotoxic T ce ll

Antige n 1

2

Antige n-bound RBC

RBC

3

RBC lys is

Antibodie s bind RBC

RBC re mova l by re ticuloe ndothe lia l s ys te m

C

Ma cropha ge Antige n

1

Antibodie s

2

Antige n–a ntibody comple xe s

3

Immune comple x de pos ition in tis s ue s

D Ha pte n 1

P rote in

2

3

Ha pte n-bound prote in Antige n pha gocytos is

Antige n pre s e nte d Activa te d T ce ll

FIGURE 6-2. Mechanisms of hypersensitivity reactions. A. Type I hypersensitivity reactions occur when a hapten binds to a protein (1). The antigen cross-links IgE antibodies on the sur ace o a mast cell, leading to mast cell degranulation (2). Mast cells release histamine and other inf ammatory mediators. B. Type II hypersensitivity reactions occur when an antigen binds to the sur ace o a circulating blood cell, usually a red blood cell (RBC) (1). Antibodies to the antigen then bind the sur ace o the RBC (2), attracting cytotoxic T cells (3), which release mediators that lyse the RBC. Binding o antibody to RBCs can also directly activate complement-mediated RBC lysis and RBC removal by the reticuloendothelial system. C. Type III hypersensitivity reactions occur when antibodies bind to a soluble toxin, acting as an antigen (1). The antigen–antibody complexes are then deposited in the tissues (2), attracting macrophages (3) and activating a complement-mediated reaction sequence (not shown). D. Type IV hypersensitivity reactions occur when a hapten binds to a protein (1) and the hapten-bound protein is phagocytosed by a Langerhans cell (2). The Langerhans cell migrates to a regional lymph node, where it presents the antigen to a T cell, thereby activating the T cell (3).

receptors in the pituitary gland is an increase in prolactin secretion, leading in some cases to amenorrhea, galactorrhea, sexual dys unction, and osteoporosis. These on-target e ects are discussed in Chapter 14, Pharmacology o Dopaminergic Neurotransmission. Sometimes, on-target adverse e ects unmask important unctions o the biological target. A prominent example o this phenomenon occurs with administration o hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (so-called statins ), which are used to decrease cholesterol levels. The intended target tissue o these drugs is the liver, where they inhibit HMG-CoA reductase, the rate-limiting enzyme o isoprenoid synthesis. A rare adverse e ect o statin therapy is muscle toxicity, including rhabdomyolysis and myositis; the act that this e ect occurs highlights the physiologic role o HMG-CoA reductase in regulating the post-translational modif cation o several

muscle proteins through a lipidation process called geranylgeranylation. Statins, as examples o drugs causing skeletal muscle injury, are also re erenced later in this chapter.

Off-Target Effects Very ew drugs are so selective that they interact with only one molecular target. O -target adverse e ects occur when a drug interacts with unintended targets (Fig. 6-1). A prominent example o an o -target e ect is the interaction o numerous compounds with cardiac IKr potassium channels. (Because the human ether-à-go-go-related gene [hERG] codes or one subunit o the human IKr channel, these channels are also called hERG channels.) Inhibition o potassium currents carried by IKr channels can lead to delayed repolarization o cardiac myocytes (see Chapter 24, Pharmacology o Cardiac Rhythm). In turn, delayed repolarization can lead

74 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

to an increase in the heart-rate corrected QT interval (QTc), cardiac arrhythmias including torsades de pointes, and sudden death. The antihistamine ter enadine was one o the earliest examples o compounds ound to inter ere with cardiac potassium channel currents, leading to potentially atal arrhythmias. This drug was designed to avoid drowsiness, an adverse e ect o the f rst-generation H1 antagonists (see earlier discussion). The observation o increased deaths due to cardiac arrhythmias in patients receiving ter enadine led to both withdrawal o this compound rom the market and vigorous e orts to understand how to prevent such events. It is now known that, although many compounds inhibit the hERG channel, compounds with a hal -maximal inhibitory concentration (IC50) more than 30- old greater than the maximum plasma concentration at the recommended therapeutic dose (Cmax, adjusted or protein binding) pose a low risk o causing QTc prolongation and cardiac arrhythmia. The active metabolite o ter enadine, exo enadine , inhibits the hERG channel only weakly, and exo enadine is now marketed as a sa er antihistamine. Many compounds can inter ere with cardiac potassium channels: accordingly, all new drug candidates are evaluated or the potential to interact with these promiscuous channels. In the hERG assay, the potential e ect o compounds on human cardiac potassium currents is measured in an in vitro system using cells trans ected with hERG. In addition to the hERG assay, the potential or altering cardiac electrophysiology is evaluated in a nonrodent animal model (see Chapter 51). As a condition o marketing approval, new drugs are also evaluated or their ability to prolong QTc in humans; this evaluation is generally per ormed in large, late-stage clinical trials. Compounds that increase QTc by more than a specif ed value at an exposure near that required or the therapeutic e ect are considered to pose a risk or producing arrhythmias. The positive control compound used in many o these “thorough QTc studies” is moxif oxacin, an antibiotic that increases QTc at clinical doses (but con ers a low risk o arrhythmogenesis). While the approach o relying most heavily on hERG inhibition in vitro and on thorough QTc clinical trials has prevented the recent introduction o “torsadogenic” drugs, this approach has been criticized or its low sensitivity, cost, and lack o assessment o the impact o other ion channel e ects that mitigate or enhance the e ects o compounds on QTc. Based on these concerns, alternative approaches are being considered, including preclinical evaluations that study a broader range o ion channel e ects and clinical studies at an earlier stage o drug development. Enantiomers (mirror-image isomers) o a drug can also cause o -target e ects. As described in Chapter 1, Drug– Receptor Interactions, drug receptors are exquisitely sensitive to the three-dimensional structure o the drug molecule; there ore, receptors can o ten distinguish between enantiomers o a drug. A tragic and well-known example o this phenomenon occurred with the administration o racemic thalidomide (mixture o [R]- and [S]-enantiomers) in the 1960s as a treatment or morning sickness in pregnant women. While the [R]-enantiomer o thalidomide was an e ective sedative, the [S]-enantiomer was a potent teratogen that caused severe birth de ects such as amelia (absence o limbs) and various degrees o phocomelia in an estimated 10,000 newborns in 46 countries (but not in the United States, thanks to Frances Kelsey at the Food

and Drug Administration [FDA], who doubted the sa ety o thalidomide and prevented its approval). The use o drugs in pregnant patients is discussed later in this chapter (see “Teratogenesis Due to Drug Therapy” and Box 6-1). The potential or signif cant pharmacologic di erences between drug enantiomers has led researchers and the FDA to evaluate such compounds as separate chemical entities. I a single enantiomeric preparation o a drug can be shown to have improved pharmacologic properties over a racemic version, then the purif ed enantiomer can be approved as a new drug. For example, the racemic proton pump inhibitor omeprazole and its [S]-enantiomer esomeprazole (as in [S]omeprazole) are marketed as separate drugs. Another common o -target e ect is the unintended activation o di erent receptor subtypes. For example, the 1-adrenergic receptor is expressed in the heart, and its activation increases heart rate and myocardial contractility. Closely related 2-adrenergic receptors are expressed in smooth muscle cells in the airways and in the vasculature, and activation o 2 receptors leads to smooth muscle relaxation and dilation o these tissues (see Chapter 11, Adrenergic Pharmacology). The clinical uses o -adrenergic receptor antagonists ( -blockers ) are o ten targeted to the 1 receptor to control heart rate and reduce myocardial oxygen demand in patients with angina or heart ailure. However, some 1 receptor antagonists are not entirely selective or the 1 receptor and can also antagonize the 2 receptor. -Adrenergic receptor antagonists with nonselective e ects are there ore contraindicated in patients with asthma, because such drugs could cause bronchoconstriction by antagonizing 2 receptors. Similarly, the use o inhaled 2 agonists in the treatment o asthma, particularly at high doses, may lead to increased heart rate. A second o -target e ect due to unintended activation o di erent receptor subtypes is the valvulopathy caused by the anorectic agent enf uramine . This drug’s primary mechanism o action appears to involve release o serotonin (5-hydroxytryptamine [5-HT]) and inhibition o 5-HT reuptake in brain areas that regulate eeding behavior. However, the compound also activates 5-HT2B receptors, leading to proli eration o myof broblasts in the atrioventricular valves. Pulmonary hypertension can develop and, in some cases, lead to death. Because o this adverse e ect, en uramine has been withdrawn rom the market (see “Drug-Induced Cardiovascular Toxicity”). The potential o -target e ects o some drugs can be explored by using genetically modif ed laboratory mice or rats in which the intended target receptor has been deleted (sometimes only in specif c tissues). I the drug nonetheless a ects the physiology o these rodents, then targets other than the intended target must be involved. O -target e ects o some drugs and drug metabolites can be determined only empirically, underscoring the importance o extensive drug testing both in preclinical experiments and in clinical trials. Despite such testing, some rare drug toxicities are discovered only when exposure occurs in a much larger population than that required or clinical trials. For example, f uoroquinolones , a class o broad-spectrum antibiotics derived rom nalidixic acid, displayed minimal toxicities in preclinical studies and clinical trials. Wider clinical use o these drugs, however, led to reports o anaphylaxis, QTc prolongation, and potential cardiotoxicity, resulting in the removal o two drugs o this class, temaf oxacin and

C h a p t e r 6 Drug Toxicity 75

grepaf oxacin, rom the market. Use o another uoroquinolone, trovaf oxacin, is signif cantly restricted due to hepatic toxicity. In comparison, ciprof oxacin and levof oxacin

are generally well tolerated and are requently used in the treatment o bacterial in ections. As seen in the introductory case, however, even these agents can occasionally cause a severe drug hypersensitivity reaction.

Idiosyncratic Toxicity Idiosyncratic drug reactions are adverse e ects that appear unpredictably, in a small raction o patients, or unknown reasons. These e ects are not typically mani est in premarketing testing in either laboratory animals or patients. The appearance o idiosyncratic injury leading to permanent organ dys unction and/or death, even i rare, o ten prompts withdrawal o the drug rom the market, precisely because susceptible patient populations cannot be identif ed. The systematic study o patient variations in response to di erent drugs may help to elucidate the genetic or other mechanisms that underlie idiosyncratic drug reactions.

CONTEXTS OF DRUG TOXICITY Drug Overdose The Swiss physician and alchemist Paracelsus noted nearly 500 years ago that “all substances are poison; there is none which is not a poison. The right dose di erentiates a poison and a remedy.” In some cases, such as a suicide or homicide, the overdose o a drug is intentional. Most cases o overdose, however, are accidents. Adverse drug events due to medication errors are estimated to a ect some 7 million people each year, with associated costs o $21 billion annually. This signif cant cost to both the patient and the health care system has led to systematic e orts intended to minimize errors in prescribing and dosing practices.

Drug–Drug Interactions As the population has aged and increasing numbers o patients have been prescribed multiple medications, the potential or drug–drug interactions has grown. Numerous adverse interactions have been identif ed, and the mechanisms o ten involve pharmacokinetic or pharmacodynamic e ects. Drug–herb interactions are also an important subset o drug–drug interactions.

Pharmacokinetic Drug–Drug Interactions

Pharmacokinetic interactions arise when one drug changes the absorption, distribution, metabolism, or excretion o another drug, thereby altering the concentration o active drug in the body. As discussed in Chapter 4, some drugs can inhibit or induce hepatic P450 enzymes. I two drugs are metabolized by the same P450 enzyme, competitive or irreversible inhibition o that P450 enzyme by one drug can result in an increase in the plasma concentration o the second drug. On the other hand, induction o a specif c P450 enzyme by one drug can lead to a decrease in the plasma concentrations o other drugs that are metabolized by the same enzyme. The anti ungal drug ketoconazole is a potent inhibitor o cytochrome P450 3A4 (CYP3A4). Co-administration o drugs that are also metabolized by CYP3A4 may result in reduced metabolism o these drugs and higher plasma drug

levels. I the co-administered drug has a low therapeutic index, toxicity may occur. Because o its potent inhibition o CYP3A4, ketoconazole is o ten used in clinical studies designed to assess the importance o pharmacokinetic drug– drug interactions. In addition to altering the activity o P450 enzymes, drugs can a ect the transport o other drugs into and out o tissues (see Chapter 4 and Chapter 5, Drug Transporters). For example, P-glycoprotein (P-gp), encoded by the multidrug resistance 1 (MDR1) gene, is an e ux pump that transports drugs into the intestinal lumen. Administration o a drug that inhibits or is a substrate or P-gp can lead to an increase in the plasma concentration o other drugs that are normally pumped out o the body by this mechanism. Since P-gp also plays a role in transport o drugs across the blood–brain barrier, compounds that inhibit P-gp can a ect drug transport into the CNS. Other transporters, such as the organic anion transporting polypeptide 1 (OATP1), mediate uptake o drugs into hepatocytes or metabolism and transport o drugs across the tubular epithelium o the kidney or excretion; both o these mechanisms promote clearance o drugs rom the body. Interactions o a drug or one o its metabolites with these classes o transporters can lead to inappropriately high plasma concentrations o other drugs that are handled by the same transporter. A pharmacokinetic interaction can sometimes be desirable. For example, because penicillin is cleared via tubular secretion in the kidney, the elimination hal -li e o this drug can be increased i the drug is given concomitantly with probenecid, an inhibitor o renal tubular transport. A second example is the combination o imipenem, a broad-spectrum antibiotic, with cilastatin, a selective inhibitor o a renal brush border dipeptidase (dehydropeptidase I). Because imipenem is rapidly inactivated by dehydropeptidase I, co-administration o imipenem with cilastatin is used to achieve therapeutic plasma concentrations o the antibiotic. A drug that binds to plasma proteins (such as albumin) may displace a second drug rom the same proteins to increase its ree plasma concentration and thereby increase its bioavailability to target and nontarget tissues. This e ect can be enhanced in a situation in which circulating albumin levels are low, such as liver ailure or malnutrition (decreased albumin synthesis) or nephrotic syndrome (increased albumin excretion).

Pharmacodynamic Drug–Drug Interactions

Pharmacodynamic interactions arise when one drug changes the response o target or nontarget tissues to another drug. Toxic pharmacodynamic interactions can occur when two drugs activate complementary pathways, leading to an exaggerated biological e ect. Such a drug interaction occurs upon co-administration o sildena l ( or erectile dys unction) and nitroglycerin ( or angina pectoris). Sildenaf l inhibits phosphodiesterase type 5 (PDE5) and thus prolongs the action o cyclic GMP (cGMP), and nitroglycerin stimulates guanylyl cyclase to increase cGMP levels in vascular smooth muscle. Co-exposure to the two drugs increases cGMP to an even greater degree, increasing the risk o severe hypotension (see Chapter 22, Pharmacology o Vascular Tone). A second example is the co-administration o antithrombotic drugs. A ter hip replacement surgery, patients are o ten treated with prophylactic war arin or a number o weeks to prevent the development o postoperative deep vein

76 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

thrombosis. Because plasma war arin concentrations may not reach a therapeutic level or several days, patients are sometimes co-administered low-molecular-weight heparin and war arin during this time. As seen in the case o Ms. G, however, signif cant bleeding may result i the e ects o the heparin and war arin synergize to produce supratherapeutic levels o anticoagulation. Newer antithrombotic drugs used or prophylaxis a ter orthopedic surgery (e.g., apixaban, rivaroxaban) may avoid this increased risk o bleeding because they reach therapeutic concentrations rapidly and do not require co-administration with another antithrombotic drug such as heparin.

Drug–Herb Interactions

The sa ety and e f cacy o a drug may also be altered by coexposure with various nonpharmaceuticals, such as oods, beverages, and herbal and other dietary supplements. Many herbal products are complex mixtures o biologically active compounds, and their sa ety and e ectiveness have rarely been tested in controlled studies. The wide use o unregulated herbal products among the public should lead clinicians to inquire about patient use o such products. The literature contains some reports o therapeutic ailure o drugs taken in conjunction with herbal products and some reports o toxicity. For example, the herbal preparation ginkgo biloba ( rom the tree o the same name) inhibits platelet aggregation. Concomitant use o ginkgo and nonsteroidal anti-inf ammatory drugs (NSAIDs), which also inhibit platelet aggregation, may increase the risk o bleeding. In combination with selective serotonin reuptake inhibitors , St. J ohn’s wort may cause serotonin syndrome.

Cellular Mechanisms o Toxicity: Apoptosis and Necrosis Cells are equipped with various mechanisms to avoid or repair damage: toxicity occurs i and when these de enses are overwhelmed. In some cases, toxicity can be minimized in the short term, but repeated insults (e.g., those leading to f brosis) can eventually compromise organ unction. The primary cellular responses to a potentially toxic drug are illustrated in Figure 6-3A and 6-3B, using the hepatocyte as an example. Depending on the severity o the toxic insult, a cell may undergo apoptosis (programmed cell death) or necrosis (uncontrolled cell death). Apoptosis allows the cell to undergo ordered sel -destruction by the coordinated activation o a number o dedicated proteins. Apoptosis can be benef cial when it eliminates damaged cells without damage to surrounding tissue. Inhibition o apoptosis is common in many cancer cells. I the toxic insult is so severe that ordered cell death cannot be accomplished, the cell may undergo necrosis . Necrosis is characterized by enzymatic digestion o cellular contents, denaturation o cellular proteins, and disruption o cellular membranes. While apoptotic cells undergo cell death with minimal in ammation and disruption o adjacent tissue, necrotic cells attract in ammatory cells that can damage nearby healthy cells.

Organ and Tissue Toxicity Most chapters in this book contain tables that list the serious and common adverse e ects o the drugs discussed in that chapter. Here, we consider common mechanisms o injury

and repair pertaining to the toxic e ects o drugs on the major organ systems. This chapter cannot catalogue every known or suspected injury to each organ or organ system, since the range o drug-associated organ and tissue toxicity is quite large. Instead, a ew specif c examples o injury are provided to demonstrate the general eatures o drug toxicity.

Harmful Immune Responses and Immunotoxicity

Stimulation o the immune system plays a role in the toxicity o several drugs and drug classes. Drugs can be responsible or immune reactions (the classic type I through type IV reactions), syndromes that mimic some eatures o immune responses (red man syndrome), and skin rashes (eruptions)— including severe and li e-threatening conditions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. Drugs can also compromise the normal unction o the immune system (immunotoxicity), leading to secondary e ects such as increased risk o in ection. Some drugs may be recognized by the immune system as oreign substances. Small-molecule drugs with a mass o less than 600 daltons are not direct immunogens but can act as haptens , such that the drug binds (o ten covalently) to a protein in the body and is then capable o triggering an immune response. I a drug is su f ciently large (e.g., a therapeutic peptide or protein), it may directly activate the immune system. The two principal immune mechanisms by which drugs can produce damage are hypersensitivity responses (allergic responses) and autoimmune reactions . The hypersensitivity responses are classically divided into our types (Fig. 6-2). Table 6-1 provides in ormation about the mediators and clinical mani estations o the our types o hypersensitivity reactions. Prior exposure to a substance is required or each o the our types o reactions. Type I hypersensitivity responses (immediate hypersensitivity or anaphylaxis ) result rom the production o IgE antibody a ter exposure to an antigen. The antigen may be a oreign protein, such as the bacterially derived thrombolytic drug streptokinase , or it may be an endogenous protein modif ed by a hapten to become immunogenic. Penicillin ragments—either in the administered drug ormulation or ormed in vivo—can act as haptens and activate the immune system. Subsequent exposure to the antigen causes mast cells to degranulate, releasing in ammatory mediators such as histamine, serotonin, and leukotrienes that promote bronchoconstriction, vasodilatation, and in ammation. Type I hypersensitivity responses mani est as a wheal-and-f are reaction in the skin. Symptoms o “hay ever” such as conjunctivitis and rhinitis may develop in the upper respiratory tract, while asthmatic bronchoconstriction may occur in the lower respiratory tract (see Chapter 48, Integrative In ammation Pharmacology: Asthma). Type II hypersensitivity responses (antibody-dependent cytotoxic hypersensitivity) occur when a drug binds to cells, usually red blood cells, and is recognized by an antibody, usually IgG or IgM. The antibody triggers cell lysis by complement f xation, phagocytosis by macrophages, or cytolysis by cytotoxic T cells. Type II responses are rare adverse responses to several drugs, including penicillin and quinidine . Type III hypersensitivity responses (immune complexmediated hypersensitivity) occur when antibodies, usually IgG or IgM, are ormed against soluble antigens. The antigen– antibody complexes are deposited in tissues such as kidneys, joints, and lung vascular endothelium. These complexes

C h a p t e r 6 Drug Toxicity 77 Drug

A

Quie s ce nt Kupffe r ce ll

He pa tocyte

S tre s s e d he pa tocyte

Cytotoxins (IL-1β, α -TNF)

Mitochondria l uncoupling Cons umption of ATP Cons umption of a ntioxida nts Lipid a nd prote in oxida tion Dis turba nce of Ca 2+ home os ta s is

Activa te d Kupffe r ce ll

ROS

RNI

IL-1 EC-GF

ROS

TGF-β

ROS

ROS TGF-β

Apoptotic he pa tocyte

Endothe lia l ce lls (a ctiva tion a nd prolife ra tion)

Ito ce ll Fibros is

B Drug

He pa tocyte

Circula ting monocyte s

Quie s ce nt Kupffe r ce ll

Che mota ctic a ctiva ting fa ctors (LTB4, LP O)

Injure d he pa tocyte

P la s ma me mbra ne ble bs Incre a s e d ce llula r volume Mitochondria l s we lling Dila te d e ndopla s mic re ticulum

Cytotoxins (RNI, ROS )

RNI

Ne crotic he pa tocyte

ROS

Activa te d Kupffe r ce ll IL-1

ROS

Endothe lia l ce lls (a ctiva tion)

FIGURE 6-3. Subtoxic and toxic damage to hepatocytes in response to moderate and high doses of drug. A. Subtoxic damage. Moderate doses o a potentially toxic drug activate Kup er cells and are metabolized by hepatocytes. The resulting hepatocyte stress may be exacerbated by the e ects o reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) elaborated by activated endothelial cells. Hepatocyte apoptosis and Ito cell activation may result, leading to f brosis. B. Toxic damage. High doses o a toxic drug are metabolized by hepatocytes to reactive metabolites that can induce cell injury. Chemotactic activating actors released by the injured hepatocytes activate Kup er cells and endothelial cells, which elaborate toxic ROS and RNI. The end result o this toxic cascade is hepatocyte necrosis. EC-GF, endothelial cell growth actor; IL-1, interleukin-1; IL-1 , interleukin-1 ; LPO, lipid peroxidation; LTB4, leukotriene B4; TGF- , trans orming growth actor ; -TNF, tumor necrosis actor .

78 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 6-1 Types o Hypersensitivity Reactions EXAMPLES OF SIGNS AND SYMPTOMS

EXAMPLES OF DRUGS

CLASSIFICATION

PRIMARY TRIGGERS

PRIMARY MEDIATORS

Type I or immediatetype hypersensitivity (humoral)

Antigen-binding IgE on mast cells

Histamine, serotonin

Hives and urticaria, bronchoconstriction, hypotension, shock

Penicillin

Type II or antibodydependent cellular cytotoxicity (humoral)

IgG, IgM, and cell-bound antigen

Activated complement; neutrophils, macrophages, natural killer cells

Hemolysis

Cefotetan

Type III or immunecomplex disease (humoral)

IgG, IgM, and soluble antigen

Activated complement; neutrophils, macrophages, natural killer cells; reactive oxygen species, chemokines

Cutaneous vasculitis

Mitomycin C

Type IV or delayed-type hypersensitivity (cell-mediated)

Antigen in association with major histocompatibility complex (MHC) protein on the surface of antigen-presenting cells

Cytotoxic T lymphocytes, macrophages, cytokines

Macular rash, organ failure

Sulfamethoxazole

Adapted from Table 2 in Bugelski PJ . Genetic aspects of immune-mediated adverse drug effects. Nat Rev Drug Discov 2005;4:59–69.

cause damage by initiating serum sickness , an in ammatory response in which leukocytes and complement are activated within the tissues. For example, type III hypersensitivity can be caused by the administration o antivenins , horse serum proteins obtained by inoculating a horse with the venom to be neutralized. Examples o other drugs that may pose a risk o serum sickness are bupropion and ce aclor. Type IV hypersensitivity responses (cell-mediated or delayed-type hypersensitivity) result rom the activation o TH1, TH17, and cytotoxic T cells. This type o hypersensitivity most commonly presents as contact dermatitis when a substance acts as a hapten and binds to host proteins. The f rst exposure does not normally produce a response, but subsequent dermal exposure can activate Langerhans cells, which migrate to local lymph nodes and activate T cells. The T cells then return to the skin and initiate an immune response. Wellknown type IV hypersensitivity responses include reactions to poison ivy and the development o latex allergies. Repeated exposure to a drug that is recognized as oreign by the immune system can trigger a massive immune response. This “cytokine storm” can lead to ever, hypotension, and even organ ailure. Thus, physicians should consider possible immune reactions to all administered drugs, even those that have appeared to be sa e in large populations. In the case presented at the beginning o the chapter, Ms. G’s ever and rash were likely caused by a T-cell-mediated hypersensitivity reaction to cipro oxacin. Once this was recognized and the cipro oxacin was stopped, her ever and rash resolved as well. Autoimmunity results when a person’s immune system attacks her or his own cells (see Chapter 46, Pharmacology o Immunosuppression). Several drugs and other chemicals can initiate autoimmune reactions. Methyldopa can cause hemolytic anemia by eliciting an autoimmune reaction against the Rhesus antigens (Rh actors) on red blood cells. Several other drugs, such as hydralazine , isoniazid, and procainamide , can cause a lupus-like syndrome by inducing antibodies to myeloperoxidase (hydralazine and isoniazid) or DNA (procainamide).

Red man syndrome occurs in a small percentage o patients receiving intravenous drugs such as the antibiotic vancomycin.

The reaction is caused by the direct e ect o the drugs on mast cells, causing these cells to degranulate. Unlike in type I hypersensitivity reactions, the mast cell degranulation in red man syndrome is independent o pre ormed IgE or complement. Red man syndrome is associated with the emergence o cutaneous wheals and urticaria (similar to type I reactions); however, it is o ten a relatively localized phenomenon a ecting the neck, arms, and upper trunk. Only rarely does red man syndrome progress to severe toxicity such as angioedema and hypotension. Red man syndrome has also been called an “anaphylactoid reaction” because o its resemblance to anaphylaxis (type I reaction). Because red man syndrome is initiated by the direct action o a drug on mast cells, it typically develops during the drug in usion (vancomycin in usions, or example, are o ten administered over a period o 60 minutes). The syndrome usually diminishes in severity or resolves a ter reducing the in usion rate or discontinuing the in usion; it can be reduced by the prophylactic use o antihistamines; and it may diminish in severity with repeated intravenous administrations. In addition to vancomycin, ciprof oxacin, amphotericin B, and ri ampin can cause this reaction. Red man syndrome is also associated with certain excipients used in intravenous ormulations; one such excipient is Cremophor (also known as Kolliphor® ), an excipient or paclitaxel, cyclosporine , and several other drugs. Many drugs can elicit the development o skin rashes , which are usually diagnosed as erythema multi orme. The more severe (sometimes li e-threatening) conditions known as Stevens-Johnson syndrome and toxic epidermal necrolysis have been reported with barbiturates, sul onamides, antiepileptics (phenytoin, carbamazepine ), nonsteroidal anti-in ammatory drugs (ibupro en, celecoxib, valdecoxib), allopurinol, and other drugs. The pathogenesis o Stevens-Johnson syndrome is not completely understood, but the morphologic appearance o mucous membrane and skin in ammation, with the development o blisters and separation o the epidermis rom the

C h a p t e r 6 Drug Toxicity 79

dermis, is consistent with an immune etiology. A temporal relationship may exist between the administration o a drug and the development o skin lesions, but some cases o StevensJohnson syndrome are idiopathic or related to in ection. Thus, not every case o Stevens-Johnson syndrome can be attributed to drug exposure. Immunotoxicity, or injury to the immune system, can occur either as an adverse e ect o therapy or as the specif c intent o therapy. Cytotoxic agents used in cancer chemotherapy are designed to kill proli erating neoplastic cells but also routinely damage proli erating normal cells in the bone marrow, lymphoid tissues, gastrointestinal tract, and hair ollicles at concentrations o drug that are required or e f cacy. For these agents, there is generally little sa ety margin or damage to normal tissues, and success ul therapy depends on a greater sensitivity o the cancer cells compared to normal tissues (see Chapter 33, Principles o Antimicrobial and Antineoplastic Pharmacology). An increased risk o in ection o ten accompanies therapy with agents that are cytotoxic to white blood cells. The margin between adverse e ects and therapeutic e ects may be increased by the use o agents that stimulate leukocyte production (e.g., f lgrastim). Targeting o the immune system may be appropriate when the disease is caused or exacerbated by a deleterious immune response (see Chapter 46). For example, inhaled corticosteroids may be used to control symptoms in patients with requent, severe exacerbations o chronic obstructive pulmonary disease (see Chapter 48). By inhibiting immune responses to pathogenic microorganisms, however, such treatment is also associated with an increased risk o pneumonia. Some immunotherapies target specif c cell types in the immune system and are associated with an increased risk o serious in ection. Rituximab is a monoclonal antibody (mAb) that targets CD20-positive B cells, which are involved in the pathogenesis o non-Hodgkin’s lymphoma (malignant CD20-positive B cells) and rheumatoid arthritis (antibodyproducing CD20-positive B cells). Two potentially serious adverse e ects that have been observed with the use o rituximab are progressive multi ocal leukoencephalopathy (PML), an in ection caused by a polyomavirus, the JC virus (JCV), and hepatitis B reactivation with the potential or ulminant hepatitis. These in ectious agents are generally present in latent orm in patients prior to treatment with rituximab, but the loss o immunocompetence as a result o treatment allows expression o these serious in ections. Similarly, e alizumab is a monoclonal antibody that targets CD11a, the subunit o leukocyte unction-associated antigen-1 (LFA1), which is expressed on all leukocytes. By decreasing the cell sur ace expression o CD11a and inhibiting the binding o LFA-1 to intercellular adhesion molecule-1 (ICAM-1), e alizumab inhibits leukocyte adhesion and is an e ective immunotherapy or psoriasis. However, because CD11a is also expressed on the sur ace o B cells, monocytes, neutrophils, natural killer cells, and other leukocytes, e alizumab can a ect the activation, adhesion, migration, and destruction o these cells as well. Like rituximab, e alizumab has been associated with PML; this serious adverse e ect led to its withdrawal rom the market in 2009. A similar increase in the requency o PML has been ound in patients receiving natalizumab or multiple sclerosis. Natalizumab binds to the 4 subunit o 4 1 and 4 7 integrins expressed on the sur ace o all leukocytes except neutrophils; by inhibiting

the 4-mediated adhesion o leukocytes to their target cells, leukocyte recruitment and activation are prevented. Cytotoxic antineoplastic agents o ten decrease the proli eration not only o the target cancer cells but also o proli erating cells o normal tissues (see earlier discussion). Targeted antineoplastic therapies may reduce the impact on normal proli erating cell populations (see Chapter 33), but immunosuppression and in ection remain risks. In recent years, numerous tyrosine kinase inhibitors have been developed or the treatment o malignant tumors. The mechanism o action o these compounds is competitive inhibition with ATP at the ATP-binding site o the kinases. The role o tyrosine kinases in cancer is presented in detail in Chapter 40, Pharmacology o Cancer: Signal Transduction. Sunitinib is an example o an anticancer tyrosine kinase inhibitor. This agent is approved or use in gastrointestinal stromal tumors, advanced renal cell carcinoma, and well-di erentiated pancreatic neuroendocrine tumors. Although sunitinib is selective or VEGF and other receptor tyrosine kinases expressed in these cancers, its adverse e ects include serious in ections o the perineum, respiratory tract, urinary tract, and skin as well as sepsis and septic shock. These in ections may occur with or without neutropenia, and necrotizing asciitis o the perineum may lead to death. Increased risk o in ections has also been observed with the tyrosine kinase inhibitors a atinib, bosutinib, ibrutinib, and ponatinib.

Drug-Induced Hepatotoxicity

Many drugs are metabolized in and/or excreted by the liver, and some o these metabolites can cause liver damage. A clinically signif cant example is acetaminophen, a widely used analgesic and antipyretic. In its therapeutic dose range, acetaminophen is metabolized predominantly by glucuronidation and sul ation, resulting in readily excreted metabolites; a small raction o the dose is also excreted unchanged. As shown in Figure 6-4, however, acetaminophen can also be oxidized to a reactive and potentially toxic species, N-acetyl-p-benzoquinoneimine (NAPQI). Glutathione can conjugate with and thereby detoxi y NAPQI, but overdosing with acetaminophen depletes glutathione reserves (as can other conditions), leaving NAPQI ree to attack cellular and mitochondrial proteins, resulting ultimately in the necrosis o hepatocytes. Timely (within about 10 hours o acetaminophen overdosing) administration o the antidote N-acetylcysteine (NAC) replenishes glutathione stores and can avert liver ailure and death. This example underscores the importance o dose : although acetaminophen is used sa ely by millions o individuals every day, the same drug, when taken in excess, is responsible or some 50% o the cases o acute liver ailure in the United States. The liver metabolizes and excretes many endogenous compounds as well as drugs. O particular interest with regard to drug-induced hepatotoxicity are the hepatocyte transporters and enzymes responsible or bile acid absorption rom the blood, bile acid processing within the hepatocyte, and bile acid secretion into the bile canaliculi (see Chapter 5). Some drugs share these pathways with the endogenous bile acids. Inhibition o bile acid transporters by a drug or its metabolites can lead to decreased bile acid secretion, with subsequent increases in intracellular and plasma concentrations o bile acids and accompanying hepatocellular injury. Inhibition o the bile salt export pump (BSEP) was a contributing actor in the hepatotoxicity that caused

80 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY O O

NH O

NH NH

-

O

O OH

O

O

Glucuronyl transferase

Sulfotransferase -

O

OH OH

O

OH

Acetaminophen

Ace ta minophe n glucuronide

O

S O

Ace ta minophe n s ulfa te

P450 enzyme or PHS O

O

NH

N

Gly S

Gluta thione

OH

O

N-a ce tyl-p-be nzoquinone imine (NAP QI)

O Glu

NH

Gluta thione conjuga te

Hepatotoxicity

Excretion

O H N O

OH

HS

N-acetylcysteine (NAC)

FIGURE 6-4. Mechanism of acetaminophen poisoning and treatment. Therapeutic doses of acetaminophen are nontoxic, but one metabolite formed at supratherapeutic doses can cause potentially lethal hepatotoxicity. Under ordinary circumstances, acetaminophen is metabolized primarily by glucuronidation ( 55–60%) and sulfation ( 30–35%); another 5% or less is excreted unchanged. The remaining 5–10% is oxidized to a reactive intermediate, N-acetyl-p-benzoquinoneimine (NAPQI). This oxidation is catalyzed by cytochrome P450 enzymes (CYP), primarily CYP2E1, as well as CYP3A4 and CYP1A2, and by prostaglandin H synthase (PHS). At therapeutic doses, NAPQI reacts rapidly with glutathione to form a nontoxic metabolite that is readily excreted. Under conditions of overdosing, however, NAPQI formation exceeds glutathione production, allowing free NAPQI to attack mitochondrial and cellular proteins. If this process is unchecked, hepatocyte necrosis and acute liver failure can result. Timely administration of the antidote N-acetylcysteine (NAC) can be lifesaving (within 10 hours of acetaminophen overdosing), in that NAC both reacts directly with NAPQI (not shown) and serves as a precursor to glutathione.

troglitazone , an insulin-sensitizing agent developed to treat

diabetes, to be withdrawn rom the market. Unexpected hepatotoxicity is the most requent reason or drug withdrawals in the United States. Many cases o ulminant hepatitis a ter drug therapy are idiosyncratic— that is, the mechanism by which the patient develops hepatic injury is not known—making it di f cult to identi y at-risk patients. In some cases, ailure to determine the mechanism(s) responsible or hepatic injury is due to the

inability to reproduce the injury in laboratory animals. A urther challenge is that hepatotoxicity may not be anticipated based on preclinical studies, because compounds exhibiting signif cant hepatotoxicity in animal studies at doses near the anticipated therapeutic exposures in humans are generally withdrawn rom development. Further conounding the prevention o hepatotoxicity is that clinical trials o a drug typically include a ew thousand patients, even though a risk o drug-induced hepatotoxicity in the range o 1 in 10,000 to 1 in 100,000 patients would be o su f cient concern to result in withdrawal rom the market. In other words, many clinical trials are too small, or have been designed with exclusion criteria that are not maintained once the drug is marketed, to detect unacceptable risks o hepatotoxicity. Withdrawal o troglitazone, or example, occurred only a ter it was noted that approximately 1 in 10,000 patients taking the drug died rom acute liver ailure. The serum levels o certain enzymes (alanine aminotrans erase [ALT], aspartate aminotrans erase [AST], and alkaline phosphatase [ALP]) and bilirubin are o ten used to monitor or potential hepatotoxicity in patients. The combination o hepatocellular injury (indicated by increased serum levels o ALT, AST, and ALP) and decreased hepatic unction (indicated by elevated bilirubin) is the best predictor o outcome or drug-induced hepatotoxicity. Elevation o serum ALT to 3 times the upper limit o normal, combined with elevation o serum bilirubin to 2 times the upper limit o normal, is associated with a mortality rate o at least 10%. This predictor has become known as Hy’s rule, named or the hepatologist Hyman Zimmerman.

Drug-Induced Renal Toxicity

The kidney is the major route o excretion o many drugs and their metabolites. Nephrotoxicity may mani est as alterations in renal hemodynamics, tubular damage and obstruction, glomerular nephropathy, and interstitial nephritis. Progressive renal ailure, characterized by progressive increases in serum creatinine, may result rom loss o unction o a su f cient number o nephrons. Examples o drug classes that can cause renal ailure include certain antibiotics, NSAIDs, antineoplastic agents, immunomodulators, and angiotensin converting enzyme (ACE) inhibitors. Here, we describe the mechanisms o nephrotoxicity caused by the aminoglycoside antibiotic gentamicin and the anti ungal agent amphotericin B. Renal injury is a common adverse e ect o treatment with both o these agents. Gentamicin causes renal injury in part through its inhibition o lysosomal hydrolases (sphingomyelinase, phospholipases) in proximal tubules o the kidney, leading to the lysosomal accumulation o electron-dense lamellar structures containing undegraded phospholipids. This process is called renal phospholipidosis. Lysosomal rupture leads to cell death in the orm o acute tubular necrosis. Renal tubular injury by gentamicin and other aminoglycoside antibiotics is reversible upon cessation o treatment, provided that the initial injury is not too severe. The polyene amphotericin B damages ungal cell membranes by interacting with ergosterol and orming membrane pores through which potassium leaks, leading to cell death. Amphotericin-induced renal injury appears to occur via a similar mechanism, with initial binding o drug to sterols in the membranes o renal tubular epithelial cells. Because the mechanism responsible or e f cacy is shared by the mechanism

C h a p t e r 6 Drug Toxicity 81

responsible or toxicity, the margin between the exposures required or anti ungal activity and those required or renal injury is small, leading to a high requency o renal injury in patients receiving amphotericin B. Liposomal ormulations o amphotericin B have been developed in an attempt to reduce this toxicity and to increase the plasma hal -li e o the drug. I the initial injury is not too severe, cessation o treatment with amphotericin o ten results in recovery o renal unction. Contrast media is administered intra-arterially or intravenously to provide radiographic delineation o the vasculature in organs such as the heart and the brain. These agents appear to cause renal injury both by direct toxicity to renal tubular epithelial cells and by constriction o the vasa recta, leading to reduced renal medullary blood ow. The nephrotoxicity o contrast media is dose-related, and patients with preexisting reductions in medullary blood ow—due, or example, to renal insu f ciency, intravascular volume depletion, heart ailure, diabetes, or diuretic or NSAID use—are at higher risk.

Drug-Induced Neurotoxicity

Drug-induced neurotoxicity is most o ten associated with the use o cytotoxic cancer chemotherapeutic agents. In most cases, neurotoxicity mani ests in the peripheral nerves, but the central nervous system may be a ected as well. Peripheral neuropathy has been associated with vinca alkaloids (e.g., vincristine , vinblastine ), taxanes (e.g., paclitaxel), and platinum compounds (e.g., cisplatin). The neuropathy caused by vinca alkaloids and taxanes is directly related to their primary mechanism o action, microtubule disruption (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance). In peripheral nerves, microtubule disruption is thought to result in altered axonal tra f cking and both sensory and motor neuropathy. Platinum-containing compounds may have direct toxic e ects on peripheral nerves. Methotrexate use has been associated with serious toxicity in the CNS (e.g., leukoencephalopathy, seizures).

Drug-Induced Skeletal Muscle Toxicity

Drugs and drug classes associated with skeletal muscle injury include HMG-CoA reductase inhibitors (statins ), corticosteroids (dexamethasone , betamethasone , prednisolone , hydrocortisone ), zidovudine , and daptomycin. Statin-induced myopathy appears to relate to the inhibition o geranyl-geranylation o several muscle proteins. Corticosteroid-induced muscle injury is complex, involving altered carbohydrate metabolism, decreased protein synthesis, and alterations in mitochondrial unction that reduce oxidative capacity. Patients treated with corticosteroids can mani est weakness, atrophy, myalgia, and microscopic decreases in muscle f ber size. Corticosteroid-associated muscle injury is reversible, albeit slowly. Understanding the pathogenesis o zidovudine-induced myopathy is complicated by the ability o HIV, the retroviral in ection or which zidovudine is administered, to induce myopathy in the absence o drug therapy. Nonetheless, the improvement in muscle unction upon withdrawal o zidovudine and the independent demonstration o zidovudine-induced myopathy in rodents suggest that the drug itsel causes myopathy, at least in some patients. The mechanism o zidovudine-associated myopathy is not well understood, but accumulation o the drug in skeletal muscle, disruption o mitochondrial cristae, and decreased oxidative phosphorylation are thought to play a role.

Daptomycin is a cyclic lipopeptide antibiotic active against Gram-positive bacteria. Its mechanism o action is a subject o active investigation. Daptomycin binds to the cell membrane o bacteria. It appears to induce the ormation o curved patches o membrane, leading to aberrant recruitment o proteins involved in cell division, membrane ion leaks, membrane depolarization, and bacterial cell death. Its spectrum o action is thought to result rom the higher content o negatively charged anionic phospholipids in the membranes o Gram-positive bacteria compared to Gramnegative bacteria. In preclinical studies o daptomycin, skeletal muscle was the main target organ o toxicity. The degree o skeletal myopathy was related to both the requency o dosing (i.e., the incidence and severity o toxicity increased i the once-daily dose was divided into several smaller doses) and the total exposure to drug (expressed as the area under the curve [AUC]). The mechanism o skeletal muscle injury by daptomycin is not ully understood. It is hypothesized that daptomycin disrupts muscle cell membranes, consistent with its lipophilic nature and its mechanism o action in bacteria. The membrane injury causes leakage o creatine kinase (CK), and increased CK activity in the serum is a clinical indicator o myotoxicity. Myopathy associated with an increase in CK has been observed in patients as well as in animals and is reversible upon discontinuation o daptomycin. Myopathy seldom occurs in patients administered once-daily doses.

Drug-Induced Cardiovascular Toxicity

Three major mechanisms o drug-induced cardiovascular toxicity have been recognized. First, as discussed earlier, many drugs interact with cardiac potassium channels to cause QTc prolongation, delayed repolarization, and cardiac arrhythmias. Second, some drugs are directly toxic to cardiac myocytes. The anthracycline antineoplastic agent doxorubicin avidly binds to iron; in the presence o oxygen, the iron can cycle between the iron(II) and iron(III) states, leading to the production o reactive oxygen species (ROS). These ROS promote cytotoxicity and death o cardiac myocytes; the mechanism o cell death may relate to the low activity o antioxidant enzyme systems in these cells. Cardiotoxicity, leading to heart ailure and arrhythmia, is o ten the dose-limiting toxicity in patients receiving this drug. Third, as noted earlier, some drugs are toxic to heart valves. The amphetamine analog fenfluramine exerts its desired anorectic e ect by increasing the release and decreasing the uptake o serotonin. Fen uramine and its metabolite nor en uramine also bind with high a f nity to 5-HT2B receptors. Drug binding to 5-HT2B receptors in heart valves activates mitogenic pathways, resulting in proli eration o valvular myof broblasts that orm myxoid plaques on the atrioventricular valves, leading to valvular insu f ciency and death in some patients. The increased serotonergic activity o en uramine can also increase vascular resistance and remodel the pulmonary arterial system, leading to pulmonary hypertension. As noted earlier, the severe adverse e ects o en uramine have caused the drug to be withdrawn rom the market. Because o the potential severity o all these cardiovascular toxicities, there is a concerted e ort to avoid selection o compounds or drug development that exhibit signif cant prolongation o the QTc interval or binding a f nity or 5-HT2B receptors.

82 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Drug-Induced Pulmonary Toxicity

Adverse e ects in the lungs range rom acute, reversible exacerbations o asthmatic symptoms to chronic injury characterized by remodeling and/or f brosis. Reversible airway obstruction can be associated with -adrenoceptor antagonist therapy, whereas chronic injury is observed in some patients receiving the chemotherapeutic agent bleomycin or the antiarrhythmic drug amiodarone . The response to injury a ter cellular damage is largely determined by the regenerative capacity o the target organ. Repeated insults to the lung, particularly to the epithelial cells lining conducting airways and alveoli, may be ollowed by regeneration. However, repeated cycles o epithelial injury can also lead to excessive deposition o collagen and extracellular matrix proteins in alveolar septa and the alveolar spaces, causing f brosis . Pulmonary f brosis is mani ested as loss o lung unction. Bleomycin and amiodarone are contraindicated in patients with existing disease o the lung parenchyma because both o these agents can cause pulmonary f brosis.

Carcinogenesis Due to Drug Therapy

Drugs (and other agents) that can cause cancer are termed carcinogens. More broadly, a carcinogen is a chemical, physical, or biological insult that acts by causing specif c types o DNA damage (these agents are termed initiators) or by acilitating proli eration o cells carrying precancerous mutations (these agents are promoters ). Initiators act by damaging DNA, inter ering with DNA replication, or inter ering with DNA repair mechanisms. Most initiators are reactive species that covalently modi y the structure o DNA, preventing accurate replication and, i unrepaired or misrepaired, leading to one or more mutations. I the mutation a ects a gene that controls cell cycle regulation, neoplastic trans ormation may be initiated. Carcinogenesis is a complex process, involving multiple genetic and epigenetic changes, that usually takes place over years or decades. For most therapeutic areas, compounds that cause direct DNA damage are avoided. Yet DNA damage and/or inter erence with DNA repair is the desired therapeutic e ect o many agents used to treat neoplasia. Damage to normal blood cell progenitors is an important on-target adverse e ect o cytotoxic alkylating agents used in cancer chemotherapy (chlorambucil, cyclophosphamide , melphalan, nitrogen mustards , and nitrosoureas ). These agents can cause myelodysplasia and/or acute myeloid leukemia (AML). Indeed, 10% to 20% o cases o AML in the United States arise secondary to treatment o other cancers with such anticancer drugs. Tamoxi en, a nongenotoxic estrogen receptor modulator, is an e ective treatment in patients with estrogen-sensitive breast cancer. However, this agent also increases the risk o some tumors. Although tamoxi en is an antagonist o estrogen receptors in the breast, it is a partial agonist in other tissues that express the estrogen receptor, most notably the uterus. There ore, an adverse e ect o breast cancer treatment with tamoxi en can be the development o endometrial cancer. Newer estrogen receptor modulators, such as raloxi ene , do not stimulate uterine estrogen receptors and may be used to treat or prevent breast cancer with a lower risk o endometrial cancer (see Chapter 30, Pharmacology o Reproduction). Product labels describe the preclinical assessment o each drug in the section o the label entitled “Carcinogenesis,

Mutagenesis, Impairment o Fertility.” In this section, it is not unusual to f nd descriptions o rodent studies that suggest carcinogenic potential or drugs. Since mutagens are not typically developed as drugs (with the exceptions noted above), the treatment-related tumors observed in these li etime studies in rodents administered high doses o drug are generally attributed to nongenotoxic (epigenetic) mechanisms. To assess whether the rodent f ndings represent a risk to the intended patient population, it is important to understand the mechanism by which these tumors occur. The proton pump inhibitor omeprazole , or example, causes tumors o the gastric enterochroma f n-like (ECL) cells in rodents. The development o these tumors results rom a dose-related and sustained increase in gastrin, which is secondary to the desired e ect o the compound (decreased acid secretion). However, the exposures required or sustained gastrin elevation and tumor ormation in rodents ar exceed the exposures required or e f cacy in patients. Further, the gastrin elevations noted in patients are o low magnitude and are not sustained. Thus, the carcinogenic f nding in the rodent studies is not considered to signal a risk or tumor development in patients treated with omeprazole.

Teratogenesis Due to Drug Therapy

Drugs given to pregnant patients may adversely a ect the etus. Teratogenesis is the induction o structural de ects in the etus, and a teratogen is a substance that can induce such de ects. The etus’s exposure to a drug is determined by maternal absorption, distribution, metabolism, and excretion o the drug and by the ability o the active teratogen to cross the placenta. These issues are urther discussed in Box 6-1. Drugs that might have ew adverse e ects on the mother may cause substantial damage to the etus. Because development o the etus is precisely timed, the teratogenic e ect o any substance is dependent on the developmental timing o the exposure. In humans, organogenesis generally occurs between the third and eighth weeks o gestation, and it is during this period that teratogens have the most pro ound e ects. Be ore the third week, most toxic compounds result in death to the embryo and spontaneous abortion, whereas a ter organogenesis, teratogenic compounds may a ect growth and unctional maturation o organs but do not a ect the basic developmental plan. For example, retinoic acid (vitamin A) possesses signif cant on-target teratogenic toxicity. Retinoic acid activates nuclear retinoid receptors (RARs) and retinoid X receptors (RXRs) that regulate a number o key transcriptional events during development. Given the severity o birth de ects that can occur, women who take RAR/ RXR agonists such as isotretinoin or acne must sign FDAmandated in ormed consent orms to demonstrate that they are aware o the risk o serious drug-related birth de ects. Another example o an on-target teratogenic e ect is in utero exposure o the etus to ACE inhibitors . Although ACE inhibitors were previously not contraindicated in the f rst trimester o pregnancy, recent data indicate that etal exposure during this period signif cantly increases the risks o cardiovascular and central nervous system mal ormations. ACE inhibitors can cause a group o conditions including oligohydramnios, intrauterine growth retardation, renal dysplasia, anuria, and renal ailure, re ecting the importance o the angiotensin pathway on renal development and unction.

C h a p t e r 6 Drug Toxicity 83

B O X 6 - 1 Ap p lica tio n to Th e ra p e u tic De cis io n Ma kin g : Dru g s in Pre g n a n cy Prescribing drugs to women who are, or might become, pregnant requires a risk–benef t evaluation or both the mother and the etus. However, many drugs have not been systematically studied in pregnant populations, so such risk–benef t evaluations may be uncertain. The FDA places drugs into f ve “pregnancy categories” based on data rom studies in laboratory animals, observations rom well-controlled epidemiologic studies (or lack thereo ), and/or case reports. These categories appear on drug labels and are listed below. Note that the categories are not strictly scaled according to risk; although Category A drugs are typically the sa est or use in pregnancy, and Category X drugs are, as the name suggests, contraindicated, drugs in Category B— or which, by def nition, human data are limited or inadequate—are not necessarily “almost as sa e” as those in Category A.

Category A Adequate and well-controlled studies have ailed to demonstrate a risk to the etus in the f rst trimester o pregnancy (and there is no evidence o risk in later trimesters).

Category B Animal reproduction studies have ailed to demonstrate a risk to the etus, and there are no adequate and well-controlled studies in pregnant women.

Category X Studies in animals or humans have demonstrated etal abnormalities and/or there is positive evidence o human etal risk based on adverse reaction data rom investigational or marketing experience, and the risks involved in use o the drug in pregnant women clearly outweigh potential benef ts. Category Xdrugs include not only teratogens but also drugs that have no proper use in pregnant patients. Statins are in this category, or example, because the normal physiologic increase in serum cholesterol that occurs during pregnancy should not be suppressed. Despite their long history o use, the label categories remain a source o con usion, even occasionally within the FDA itsel . For example, the antibiotic tigecycline is classif ed in Category D, but the absence o controlled data rom humans indicates that it should instead be placed in Category C. More generally, the FDA pregnancy categorization o drugs, like any such scheme, is not per ect and may ail to capture the nuances o some drug-specif c and patient-specif c circumstances. Thus, the physician should also rely on his or her own judgment, keeping in mind the ollowing issues: ■

■

Category C Animal reproduction studies have shown an adverse e ect on the etus, and there are no adequate and well-controlled studies in humans, but potential benef ts may warrant use o the drug in pregnant women despite potential risks.

Category D There is positive evidence o human etal risk based on adverse reaction data rom investigational or marketing experience or studies in humans, but potential benef ts may warrant use o the drug in pregnant women despite potential risks.

PRINCIPLES FOR TREATING PATIENTS WITH DRUG-INDUCED TOXICITY Treatment o drug-induced toxicity may include (1) reducing or eliminating exposure to the drug, (2) administering specif c treatments based on antagonizing the mechanism o action o the drug or altering its metabolism, and/or (3) providing supportive measures. Reduction o exposure to a therapeutic agent in a patient who experiences adverse e ects may seem intuitive, but it is not always the correct choice. The appearance o an adverse e ect during therapy does not necessarily indicate that the e ect is due to the drug, despite the temporal relationship between the initiation o therapy and the appearance o the adverse e ect. Even i the adverse e ect most likely occurred because o the drug, the risks o cessation must be weighed against the benef ts o continuing the drug. Cessation o therapy is more obviously a correct choice when the adverse e ects have been previously associated with the

■

What are the risks to both etus and mother o nott treating the illness or which the drug is being considered? Is the drug known to cross the placenta? Based on its molecular weight, charge, hydrophobicity, and/or potential or carrier-mediated transport, is it likely to cross the placenta? Is there a pharmacologic rationale or how the drug could a ect the etus (e.g., through e ects on organogenesis, organ development, organ unction, or a delivery complication) should the etus be exposed?

When appropriate, drugs that have proven e ective or treating a patient’s condition should be continued. To minimize etal risk, drugs should be prescribed at the lowest therapeutic dose, taking into account the metabolic and physiologic changes that occur during pregnancy. ■

drug and are li e-threatening, such as anaphylaxis due to a beta-lactam antibiotic. Needless to say, or such patients, uture therapy with this class o antibiotics would also be contraindicated. Adverse e ects that are irreversible and/or likely to increase in severity with continued treatment may also lead to the appropriate decision to terminate therapy. Many adverse e ects, however, are considered tolerable and reversible. Depending on the severity o the disease condition being treated, it may be that the overall benef t to the patient is greater with drug treatment than without. An example o such circumstances is the leukopenia that o ten occurs in patients receiving chemotherapy with cytotoxic drugs. Thus, the decision to withdraw or reduce therapy can be complex and o ten requires evaluation o many actors a ecting the patient’s immediate and long-term health. Therapies designed to counteract the adverse e ects produced by a drug are o ten based on antagonizing the pharmacologic (on-target) activity o the drug or inter ering with e ects related to metabolism o the drug. Antagonizing the

84 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

pharmacologic activity o a drug is a use ul approach in overdoses o opioids, benzodiazepines, and acetylcholinesterase (AChE) inhibitors. Inter erence with the toxic e ects o drug metabolites is a use ul approach in the treatment o acetaminophen toxicity. These examples are brie y discussed below. Conceptually, the simplest treatment or drug overdose is the administration o an antagonist that blocks the action o the drug (see Fig. 2-4). For example, an opioid overdose can be treated with naloxone , a competitive antagonist at the -opioid receptor. By competitively binding to opioid receptors, naloxone prevents or reverses the toxic e ects o natural or synthetic opioids, including respiratory depression, sedation, and hypotension. Naloxone has a rapid onset o action and is highly potent; indeed, i no clinical improvement is observed within 10 minutes a ter naloxone doses o up to 10 mg, a di erent diagnosis or multiple toxic entities should be considered. Naloxone has a relatively short hal -li e, so it must be given every 1 to 4 hours to provide adequate receptor antagonism while the opioid is being cleared. Flumazenil, a competitive antagonist at the GABAA (benzodiazepine) receptor, is used to treat benzodiazepine overdose. Flumazenil acts by competitive antagonism at benzodiazepine receptors in the central nervous system to completely or partially reverse the sedative e ects o benzodiazepines. Like naloxone, it has a rapid onset o action and is highly potent; its e ects should be seen within 5 minutes at a dose o not more than 3 mg. Flumazenil also has a short hal -li e (approximately 1 hour) and must be given requently to provide adequate receptor antagonism while the benzodiazepine is being cleared. Pharmacologic antagonism can also be used when the toxic agent is not a direct agonist but instead indirectly increases the concentration o the natural ligand or a receptor. AChE inhibitors produce a supraphysiologic concentration o acetylcholine at cholinergic synapses and a characteristic toxidrome o cholinergic excess—bradycardia, miosis, hypersalivation, sweating, diarrhea, vomiting, bronchoconstriction, weakness, respiratory paralysis, and convulsions. Although it is sometimes possible to restore AChE activity, the treatment o AChE inhibition generally depends on administering an anticholinergic agent such as atropine . By antagonizing the muscarinic acetylcholine receptor, atropine restores cholinergic balance and prevents bronchoconstriction, the most common cause o death in patients exposed to AChE inhibitors. As noted earlier, a consequence o acetaminophen overdose is depletion o intracellular glutathione by the drug’s metabolite N-acetyl-p-benzoquinoneimine (NAPQI). Glutathione stores can be replenished by administering N-acetylcysteine (NAC), a metabolic precursor o glutathione (see Fig. 6-4 or details). In addition to supportive therapy (gastric lavage and/or charcoal), NAC is given orally or intravenously within about 10 hours a ter ingestion o a potentially hepatotoxic dose o acetaminophen to prevent or lessen hepatic injury. Finally, supportive therapy can be provided in the ace o drug-induced toxicity. One example is the administration o intravenous uids to patients with renal injury in order to maintain adequate renal blood ow. In cases o severe renal injury, dialysis may be required until renal unction is regained. Another example is the treatment o bone marrow suppression resulting rom the administration o cytotoxic agents in cancer chemotherapy. Filgrastim, a recombinant

human granulocyte colony-stimulating actor (G-CSF), can be used to stimulate leukocyte production and provide supportive therapy until endogenous production o leukocytes resumes in the bone marrow.

TOWARD EARLY DETECTION AND PREDICTION OF DRUG TOXICITY A key element o the US FDA’s strategic plan o August 2011 states: “Modernizing toxicology and continually improving the ability o nonclinical tests, models, and measurements to predict product sa ety issues will increase the likelihood that toxicity risks will be identif ed earlier in product development, assuring patient sa ety, and mitigating the need to withdraw previously approved products.” One e ective approach toward early detection and prediction o drug toxicity is the use o sensitive, specif c, and qualif ed translational biomarkers. Many current approaches to detect and predict drug toxicity in animal studies use a combination o microscopic tissue examination and measurement o “traditional” biomarkers to assess organ injury. As described above, examples o traditional biomarkers include serum concentrations o urea nitrogen and creatinine to evaluate potential adverse renal e ects and serum activities o alanine aminotrans erase (ALT), aspartate aminotrans erase (AST), and gamma-glutamyltrans erase (GGT) and serum concentrations o bilirubin and bile acids to evaluate potential adverse hepatic e ects. However, these traditional markers are now viewed as relatively insensitive, particularly those or monitoring renal injury. Because o renal reserve, or instance, creatinine may not increase until there has been considerable (greater than 70%) loss o renal unction. O additional concern is drug-induced renal injury in patients with preexisting renal dys unction, since these patients have diminished reserve capacity. It is also important to note that the degree o loss o renal unction in drug studies cannot be equated with the potential or reversibility o the morphologic changes that may accompany the loss o unction. As noted above, nonclinical studies and clinical experience have demonstrated that drug-induced renal injury is o ten reversible, depending on the extent o injury. With these considerations in mind, the goal o recent e orts is to identi y sa ety biomarkers that may improve the detection and prediction o drug toxicity by (1) identi ying toxicity early in drug development, thereby reducing the rate o attrition o drug candidates during later stage clinical trials, and (2) providing markers to monitor toxicity in patients, with the goal o reducing the entry o drugs into the market that have unacceptable toxicity and acilitating the management o patients who su er organ damage or injury. Over the last decade, consortia such as the European Innovative Medicines Initiative (IMI), Predictive Sa ety Testing Consortium (PSTC), and Health and Environmental Sciences Institute (HESI) have dedicated resources to identiying translational biomarkers or early detection o toxicity. In 2008, the US FDA, European Medicines Agency (EMA), and Japanese Pharmaceuticals and Medical Devices Agency (PMDA) jointly announced the qualif cation o seven urinary biomarkers to monitor kidney toxicity in preclinical studies. This international e ort showed that, in many instances, kidney injury molecule-1 (KIM-1), clusterin (CLU), albumin, total protein, 2-microglobulin, cystatin C, and tre oil

C h a p t e r 6 Drug Toxicity 85

TABLE 6-2 Online Resources for Information on Drug Toxicity TYPE OF INFORMATION

SOURCE

WEBSITE

Product labels

Physician’s Desk Reference Drug manufacturer

http://csi.micromedex.com/Login.asp Various websites by manufacturer

Regulatory agencies

US Food and Drug Administration European Medicines Agency (EMA)

http://www.fda.gov/ http://www.ema.europa.eu/

Government databases

National Library of Medicine National Toxicology Program TOXNET International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)

http://www.ncbi.nlm.nih.gov/pubmed/ http://ntp.niehs.nih.gov/ http://toxnet.nlm.nih.gov/ http://www.ich.org/products/guidelines.html

Commercial databases

Pharmapendium Medscape DiscoveryGate INCHEM

https://www.pharmapendium.com/ http://www.medscape.com/ https://www.discoverygate.com http://www.inchem.org

actor 3 (TFF3) provide an earlier signal than traditional biomarkers and also add signif cant in ormation with regard to potential localization o the adverse renal e ects. Notably, these newer biomarkers correlated with the “gold standard” o kidney toxicity, quantitative histopathology. Thus, although the newer biomarkers generally do not o er higher sensitivity in nonclinical models, they do provide important perspective on which biomarkers would be use ul to monitor in both clinical studies (be ore drug approval) and in patients (a ter drug approval) in order to better understand potential risks to humans. As with traditional biomarkers, decisions on whether to withdraw therapy or individual patients or to continue clinical development o a drug in which increases in these biomarkers have been observed still require a risk–benef t analysis (see “Principles or Treating Patients with DrugInduced Toxicity” earlier in this chapter). The kidney biomarker qualif cation process has led some pharmaceutical companies to include an assessment o newer biomarkers in nonclinical and clinical data submitted or review to regulatory agencies in the United States, Europe, and Japan. Similar e orts are underway to identi y sa ety biomarkers or liver, heart, skeletal muscle, testicular, and vascular toxicity, including an evaluation o the per ormance o these biomarkers in diagnosis and prognosis o toxicity in clinical studies.

CONCLUSION AND FUTURE DIRECTIONS This chapter has presented a mechanism-based approach to understanding drug toxicity and provided examples to illustrate these principles in major organ systems. Drug development aims to discover compounds that are both e ective and highly selective and thus less likely to cause serious or otherwise undesirable o -target e ects. The challenges o the uture lie particularly with understanding the basis or variability o therapeutic and toxic responses to drugs. In an attempt to predict which patient populations will be

most susceptible to an adverse drug reaction, one approach under evaluation is to f nd correlations between individual single nucleotide polymorphisms (SNPs) and possible adverse reactions by comparing the SNPs o patients who have adverse reactions with those who do not. The identif cation o patients with genetic variants o the molecular target (and closely related targets) o a drug could provide use ul in ormation about patients who might be more likely to experience adverse e ects. Predicting e f cacy and sa ety in individual patients remains a challenge to the treating physician. The decision to use drug therapy requires knowledge o the potential benef ts and risks o the therapy. Moreover, physicians have the responsibility to communicate these risks and benef ts to the patient so that the ull range o therapeutic options can be considered. One challenge to the physician is where to f nd this in ormation. Sources include the scientif c literature, the product label, direct communications to prescribing physicians, and reviews o the preclinical and clinical data prepared by the FDA during its review o a New Drug Application (NDA; see Chapter 52). The key toxicity in ormation, both preclinical and clinical, is contained in the product label. Revisions o the label may occur as serious adverse events are attributed to drugs during postmarketing surveillance, and it is incumbent on the physician to consult the most recent version o the product label. Alerts o serious consequences may also be transmitted in the orm o direct communications to prescribing physicians, and the FDA website can be consulted or current regulatory actions related to a drug’s sa ety. The European Medicines Agency (EMA) website contains in ormation regarding regulatory actions or medicines marketed in Europe. Table 6-2 lists some o the online sources that can be consulted or in-depth in ormation about drug toxicity. Good sources o detailed in ormation on preclinical toxicity and clinical adverse events are the documents prepared by the FDA pharmacologist (preclinical) and medical reviewer (clinical) as part o their review o the NDA.

86 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Acknowledgment We thank Cullen Taniguchi, Sarah R. Armstrong, Vivian Gonzalez Le ebre, and Robert H. Rubin or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Agranat I, Caner H, Caldwell J. Putting chirality to work: the strategy o chiral switches. Nat Rev Drug Discov 2002;1:753–768. (An overview o enantiomeric-specif c properties o drugs and the strategies o switching drugs rom achiral to chiral preparations.) Bonventre JV, Vaidya VS, Schmouder R, Feig P, Dieterle F. Next-generation biomarkers or detecting kidney toxicity. Nat Biotechnol 2010;28:436–440. (Reviews potential new biomarkers or assessing renal unction in both nonclinical and clinical studies.) Bugelski PJ. Genetic aspects o immune-mediated adverse drug e ects. Nat Rev Drug Discov 2005;4:59–69. (Overview o immune-mediated adverse e ects, including detailed mechanistic in ormation.) Campion S, Aubrecht J, Boekelheide K, et al. The current status o biomarkers or predicting toxicity. Expert Opin Drug Metab Toxicol 2013;9:1391–1408. (Reviews sensitivity and utility o currently used biomarkers or assessment o toxicity during drug development.) Elangbam CS. Current strategies in the development o anti-obesity drugs and their sa ety concerns. Vet Pathol 2009;46:10–24. (Provides examples o drug development guided by knowledge o mechanisms o toxicity.) Fujimoto K, Kumagai K, Ito K, et al. Sensitivity o liver injury in heterozygous Sod2 knockout mice treated with troglitazone or acetaminophen. Toxicol Pathol 2009;37:193–200. (Demonstrates use o genetically engineered animals to study mechanisms o toxicity.) Hondeghem LM. QTc prolongation as a surrogate or drug-induced arrhythmias: act or allacy? Acta Cardiol 2011;66:685–689. (Commentary

on relative utility o drug-induced changes in QTc as a predictor o arrhythmogenic potential o drugs.) International Con erence on Harmonisation o Technical Requirements or Registration o Pharmaceuticals or Human Use. ICH harmonised tripartite guideline: immunotoxicity studies or human pharmaceuticals S8, 2005. http://www.ich.org/f leadmin/Public_Web_Site/ICH_Products/Guidelines/ Sa ety/S8/Step4/S8_Guideline.pd . (Summary o principles and guidelines or evaluating potential immunotoxicity o drug candidates.) Liebler DC, Guengerich FP. Elucidating mechanisms o drug-induced toxicity. Nat Rev Drug Discov 2005;4:410–420. (Introduces the concept o a mechanism-based approach to drug toxicity.) Morgan RE, Trauner M, van Staden CJ, et al. Inter erence with bile salt export pump unction is a susceptibility actor or human liver injury in drug development. Toxicol Sci 2010;118:485–500. (Correlation between drug-induced inhibition o bile salt export protein (BSEP) and hepatotoxicity in patients.) Morgan RE, van Staden CJ, Chen Y, et al. A multi actorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development. Toxicol Sci 2013;136:216–241. (Describes in vitro approaches or reducing risk o hepatotoxicity during selection o candidates or drug development.) Navarro VJ, Senior JR. Drug-related hepatotoxicity. N Engl J Med 2006;354:731–739. (Overview o pharmacogenomic approaches to understanding and predicting drug hepatotoxicity.) Owczarek J, Jasińska M, Orszulak-Michalak D. Drug-induced myopathies: an overview o the possible mechanisms. Pharmacol Rep 2005;57:23–34. (Overview o mechanisms leading to skeletal muscle toxicity.) Sager PT, Gintant G, Turner JR, Petit S, Stockbridge N. Rechanneling the cardiac proarrhythmia sa ety paradigm: a meeting report rom the Cardiac Sa ety Research Consortium. Am Heart J 2014;167:292–300. (A summary o new initiatives to better evaluate risk o arrhythmogenic potential o drugs and reduce the need or thorough QTc studies.) U.S. Food and Drug Administration. Pregnancy, lactation, and reproductive potential: labeling or human prescription drug and biological products—content and ormat. June 2015. Washington, DC: U.S. Department o Health. (Reviews proposals to revise product labels to better describe reproductive and developmental risks.)

OH H

Ta T a mo xife n (TAM)

4 -hydroxyTAM T

CYP 3 A4/5

C YP 3A4/55

(CYP 2C99 + othe r CYP is ofor f rms m )

Pharmacogenomics

N O

7

N

H

O

CYP 2D6

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 87 8 –88 88 N-de N de s me m e th thylTAM thylT ylTAM T AM PHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Genomic Variationn an a d Pharmacogenomics . . . . . . . . . . . . . . . 87 PHARMACOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Vari a riat atio ion in Enzymes of Drug ug Metabolism: Pha h rmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Variation in Drug Ta Targ rget ets: s: Pharmacodynamics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

H

Am b e r DaS h lin a n1dAKe ULT T 1 la n Ta n t is ira OH Pathway Basedd Ph Phar arma maco ma c oge g neticsEndo o x xife xif i fe f e n Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Role of Epigenetics in PharmacogeneticsPharm ha r acogenomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 9 Modern Pharm rmac acog ogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Pharmacogenomics and nd Regul egu atory Science . . . . . . . . . . . . . . 94 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . 95 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

B INTRODUCTION PHYSIOLOGY Modern pharmacologic100 agents are used to treat or control Genomic Variation and Pharmacogenomics %

)

diseases that range rom hypertension to human immunodef ciency virus (HIV) in ection. In many cases, the same drug regimens are used or populations o people. Large variations among individuals are o ten ound in response to drug therapy, however. These variations range rom potentially li e-threatening adverse drug reactions to equally serious lack o therapeutic e f cacy. Many actors in uence the drug response phenotype, including age, gender, and underlying disease, and genetic variation plays an important role. Interindividual di erences in the genes that encode drug targets, drug transporters, and enzymes that catalyze drug metabolism can pro oundly a ect the success or ailure o pharmacotherapy. Pharmacogenetics is the study o the role o inheritance in variation in drug response. The convergence o recent advances in genomic science and equally striking advances in molecular pharmacology has resulted in the evolution o pharmacogenetics into pharmacogenomics. The promise o pharmacogenetics-pharmacogenomics is the possibility that knowledge o a patient’s DNA sequence could be used to enhance pharmacotherapy, maximize drug e f cacy, and reduce the incidence o adverse drug reactions. There ore, pharmacogenetics and pharmacogenomics represent an important aspect o the aspiration to “personalize” or “individualize” medicine—in this case, drug therapy. This chapter describes the principles o pharmacogenetics and pharmacogenomics as well as recent developments in this discipline. Several key examples are cited in which knowledge o pharmacogenetics-pharmacogenomics may help to individualize drug therapy.

The human genome contains approximately 3 billion nucleotides. According to current estimates, the genome contains approximately 19,000–20,000 protein-coding genes that, through alternative splicing and post-translational modif cation, may encode 100,000 or more proteins. On average, any two people di er at about one nucleotide in every 1,000 in their genome, totaling an average interindividual di erence o 3 million base pairs throughout the genome. The majority o these di erences are so-called single nucleotide polymorphisms or SNPs (pronounced “snips”), in which one nucleotide is exchanged or another at a given position. SNPs and other di erences in DNA sequence can occur anywhere in the genome, in both coding regions and noncoding regions. I a SNP changes the encoded amino acid, it is called a nonsynonymous coding SNP (cSNP). The remaining di erences in DNA sequence involve insertions, deletions, duplications, and reshu ings, sometimes o just one or a ew nucleotides but occasionally o whole genes or larger DNA segments that include many genes. Functionally signif cant DNA sequence di erences that we currently understand tend to all within genes, either within their coding sequences or in the promoters, enhancers, splice sites, or other sequences that control gene transcription or mRNA stability. Heritable genetic regulation that does not occur through DNA sequence changes, termed epigenetics, also contributes to unctionally signif cant variation in genes and gene expression. Taken together, these di erences constitute each person’s genetic individuality. Some o that individuality a ects the way in which each person will respond to drug treatment.

87

88 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

Ro b e rt H, a 66-ye a r-o ld m a n , is s h o ve lin g s n o w o n e w in try m o rn in g in Min n e s o ta w h e n h e s lip s a n d fa lls o n a p a tch o f ice . He im m e d ia te ly fe e ls p a in in h is le ft h ip a n d is u n a b le to s ta n d . He is b ro u g h t to th e h o s p ita l, w h e re x-ra ys re ve a l th a t h e h a s fra ctu re d h is h ip . He u n d e rg o e s s u rg e ry th e n e xt d a y a n d is d is ch a rg e d to a re h a b ilita tio n h o s p ita l 3 d a ys la te r. Afte r le s s th a n 24 h o u rs a t th e re h a b ilita tio n h o s p ita l, Mr. H d e ve lo p s th e s u d d e n o n s e t o f p le u ritic ch e s t p a in . He is b ro u g h t to th e e m e rg e n cy d e p a rtm e n t, w h e re a co m p u te d to m o g ra p hy (CT) s ca n w ith in tra ve n o u s co n tra s t re ve a ls a p u lm on a ry e m b o lu s . He is tre a te d w ith h e p a rin a n d is a n tico a g u la te d w ith wa rfa rin a t a s ta rtin g d o s e o f 5 m g e a ch d a y, w ith a ta rg e t in te rn a tio n a l n o rm a lize d ra tio (INR) o f 2.0–3.0. Mr. H is d is ch a rg e d b a ck to h is re h a b ilita tio n h o s p ita l a n d re fe rre d to h is

PHARMACOLOGY The concept that inheritance might be an important determinant o individual variation in drug response emerged hal a century ago. It originally grew out o clinical observations o striking di erences among patients in their response to “standard” doses o a drug. Those observations, coupled with twin and amily studies that showed inherited variations in plasma drug concentrations and other pharmacokinetic parameters, led to the birth o pharmacogenetics. Many o those original examples o pharmacogenetic variation, and many o the most striking examples even today, involve pharmacokinetics— actors that in uence the concentration o drug reaching its target(s). However, examples o pharmacogenetic variation in the drug target, so-called pharmacodynamic actors, are also being reported with increasing requency. Pharmacogenetic variation related to unpredicted (idiosyncratic) adverse e ects rom drugs has also been described. While not detailed urther in this chapter, an example o an idiosyncratic pharmacogenetic e ect is provided by the serious hypersensitivity reaction associated with the HLA-B*5701 allele in patients taking abacavir, a reverse transcriptase inhibitor and anti-HIV agent (see Chapter 38, Pharmacology o Viral In ections). Predrug testing or HLA-B*5701 has been shown to nearly eliminate such hypersensitivity reactions.

Variation in Enzymes of Drug Metabolism: Pharmacokinetics Inherited variation in enzymes that catalyze drug metabolism is the most common actor responsible or pharmacogenetic variation in response to medications. The enzymes involved in drug metabolism are discussed in Chapter 4, Drug Metabolism. There are two broad categories o drug-metabolizing enzymes: those that catalyze phase I reactions ( unctionalization reactions that typically involve oxidation or reduction) and those that catalyze phase II reactions (typically, conjugation reactions that add groups, such as glucuronic acid, that enhance drug solubility and thus drug excretion).

lo ca l p hys icia n . Wh e n th e INR is s u b s e q u e n tly m e a s u re d , it is 6.2, a va lu e a s s o cia te d w ith a n in cre a s e d ris k o f h e m o rrh a g e . He is ta kin g n o o th e r m e d ica tio n th a t m ig h t in te rfe re w ith p la s m a le ve ls o f wa rfa rin . Th e p hys icia n a d vis e s Mr. H to s to p ta kin g wa rfa rin fo r 2 d a ys . Afte r m u ltip le a tte m p ts a t a d ju s tin g h is d o s e o f wa rfa rin , Mr. H e ve n tu a lly re a ch e s a s ta b le INR o f 2.5 w h e n ta kin g 1 m g o f wa rfa rin e a ch d a y.

Questions 1 . What molecular mechanisms could be responsible for the apparent sensitivity of Mr. H to warfarin? 2 . What additional laboratory information could assist in anticoagulating this patient? 3 . Would that information have helped in the selection of Mr. H’s initial warfarin dose?

Phase I and phase II reactions do not necessarily occur in that order, and metabolic intermediates resulting rom both types o reactions may be pharmacologically active. In act, some medications are administered as inactive prodrugs that must undergo phase I and/or phase II metabolism be ore they can exert their pharmacologic e ect. Genetic polymorphisms are common in enzymes that catalyze drug metabolism, and clinically signif cant polymorphisms have been ound in nearly all o the major enzymes involved in both phase I and phase II reactions (Table 7-1). Two “classic” examples are provided by the inherited variations in the enzymatic hydrolysis o the short-acting muscle relaxant succinylcholine by the enzyme butyrylcholinesterase (BChE; also known as serum cholinesterase) and the enzymatic acetylation o drugs such as the antituberculosis drug isoniazid (see Chapter 35, Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis). Patients with variations in BChE have a decreased rate o metabolism o succinylcholine, resulting in prolonged paralysis a ter drug exposure. A genetically polymorphic phase II enzyme, N-acetyltrans erase 2 (NAT2), catalyzes the acetylation o isoniazid. Patients treated with isoniazid can be classif ed as either “slow acetylators,” who metabolize isoniazid slowly and have high blood drug levels, or “ ast acetylators,” who metabolize isoniazid rapidly and have low blood drug levels. Family studies have shown that the rate o isoniazid biotrans ormation is inherited. The slow-acetylator phenotype is associated with drug toxicities that result rom excessive drug accumulation; examples include hydralazine- and procainamide-induced lupus and isoniazid-induced neurotoxicity and liver injury. Although the antihypertensive agent hydralazine is rarely used today in the treatment o hypertension, this drug has recently reemerged as one o the two active components in BiDil, a combination drug approved or the treatment o patients with symptomatic heart ailure. It is o interest that the US Food and Drug Administration (FDA) has approved BiDil or use only in patients o A rican ancestry, presumably because o an ethnically dependent genetic di erence in response to this drug.

C h a p t e r 7 Pharmacogenomics

89

TABLE 7-1 Examples of Genetic Polymorphisms and Drug Metabolism ENZYME

AFFECTED DRUG, CLASS, OR COMPOUND

Phase I (Oxidation/Reduction) Enzyme CYP1A2

Acetaminophen, ca eine, propranolol

CYP1B1

Estrogens

CYP2A6

Halothane, nicotine

CYP2B6

Cyclophosphamide

CYP2C8

Paclitaxel, retinoic acid

CYP2C9

Nonsteroidal anti-inf ammatory drugs, phenytoin, war arin

CYP2C19

Omeprazole, phenytoin, propranolol

CYP2D6

Antidepressants, -adrenergic antagonists, codeine, debrisoquine, dextromethorphan

CYP2E1

Acetaminophen, ethanol

CYP3A5

Calcium channel blockers, cyclosporine, dapsone, etoposide, lidocaine, lovastatin, macrolides, midazolam, quinidine, steroids, tacrolimus, tamoxi en

Phase II (Conjugation) Enzyme N-Acetyltrans erase 1

Sul amethoxazole

N-Acetyltrans erase 2

Dapsone, hydralazine, isoniazid, procainamide, sul onamides

Sul otrans erases (SULTs)

Acetaminophen, dopamine, epinephrine, estrogens

Catechol-O-methyltrans erase

Catecholamines, levodopa, methyldopa

Histamine N-methyltrans erase

Histamine

Thiopurine S-methyltrans erase

Azathioprine, mercaptopurine, thioguanine

UDP-glucuronosyltrans erases

Androgens, ibupro en, irinotecan, morphine, naproxen

Early examples o the importance o pharmacogenetic variation, such as those represented by BChE and NAT2, served as a stimulus to search or additional examples. Most o the second-generation examples continued to be associated with pharmacokinetics and continued to be recognized rom clinical observations—o ten rom adverse drug responses. They were most o ten studied either by administering a “probe drug” to a group o subjects and measuring plasma or urinary drug and/or metabolite concentrations or by directly assaying a drug-metabolizing enzyme in an easily accessible tissue such as the red blood cell (e.g., a series o methyltrans erase enzymes). Two prototypic examples that have become pharmacogenetic “icons” are the cytochrome P450 2D6 (CYP2D6) and thiopurine S-methyltransferase (TPMT) genetic polymorphisms. Because o the clinical implications o these polymorphisms, the FDA in its 2003 “Guidance on Pharmacogenomic Data” cited CYP2D6 and TPMT as examples o valid pharmacogenomic biomarkers. CYP2D6 is a member o the cytochrome P450 (CYP) amily o microsomal, phase I drug-metabolizing enzymes. CYP2D6 contributes to the metabolism o a large number o medications, including antidepressants, antipsychotics, antiarrhythmics, and analgesics. The CYP2D6 polymorphism was originally described by two di erent laboratories studying two di erent probe drugs: the antihypertensive

debrisoquine and the oxytocic agent sparteine . The requency

distribution o the debrisoquine urinary metabolic ratio, the ratio o the parent drug to its oxidized metabolite, is shown in Figure 7-1A or a Northern European population. Shown at the ar right o the f gure is a group o “poor metabolizers” o debrisoquine, subjects homozygous or recessive alleles (genes) coding or enzymes with decreased activity or or deletion o the CYP2D6 gene; shown in the middle is the large group o “extensive metabolizers,” subjects heterozygous or homozygous or the “wild-type” allele; and shown at the ar le t is a small subset o “ultrarapid metabolizers,” some o whom have multiple copies o the CYP2D6 gene. Several molecular genetic mechanisms are responsible or variation in CYP2D6 enzyme activity, including nonsynonymous cSNPs, gene deletion, and gene duplication; some ultrarapid metabolizers can have up to 13 copies o the gene. It has been estimated that 6% to 10% o Caucasians are CYP2D6 poor metabolizers. Among East Asians, in contrast, the poor-metabolizer phenotype is present at a requency o just 1% to 2%. The ultrarapid-metabolizer phenotype, rare in most Caucasian populations, has a requency o 3% in Spaniards and up to 13% in Ethiopians. These ethnic di erences have potentially important medical implications because CYP2D6 metabolizes many commonly prescribed medications, including the -adrenergic blocker metoprolol,

90 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY A CYP2D6 pharmacogenetics Exte ns ive me ta bolize rs

P oor me ta bolize rs

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Ultra ra pid me ta bolize rs

40

0 0.01

0.10

1

10

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De bris oquine : 4-Hydroxyde bris oquine me ta bolic ra tio

B

AmpliChip CYP450 array

FIGURE 7-1. CYP2D6 pharmacogenetics. A. Frequency distribution o the metabolic ratio or the cytochrome P450 2D6 (CYP2D6)-catalyzed metabolism o debrisoquine to orm its 4-hydroxy metabolite. Data or 1,011 Swedish subjects are plotted as the ratio o metabolites in the urine. Most subjects metabolize debrisoquine extensively, while some subjects metabolize the compound ultrarapidly and others metabolize the compound poorly. B. The AmpliChip CYP450 array can be used to determine variant genotypes or cytochrome P450 genes that inf uence drug metabolism.

the neuroleptic haloperidol, the opioids codeine and dextromethorphan, and the antidepressants f uoxetine , imipramine , and desipramine , among many others (Table 7-1). There ore, poor metabolizers or CYP2D6 can potentially experience an adverse drug e ect when treated with standard doses o agents such as metoprolol that are inactivated by CYP2D6, whereas codeine is relatively ine ective in poor metabolizers because it requires CYP2D6-catalyzed metabolism to orm the more potent opioid morphine. Conversely, ultrarapid metabolizers may require unusually high doses o drugs that are inactivated by CYP2D6, but those same subjects can be “overdosed” with codeine, su ering respiratory depression or even respiratory arrest in response to “standard” doses. In one tragic case, a nursing in ant whose mother was an ultrarapid CYP2D6 metabolizer died when the mother was prescribed a standard dose o codeine and the baby was overdosed on morphine present in the breast milk. For ultrarapid

and poor metabolizers, alternative analgesics are now recommended as substitutes or codeine. CYP2D6 genetic polymorphisms are also important or the e f cacy o the breast cancer drug tamoxi en. Tamoxi en is used to block the estrogen receptor (ER) in approximately 60% o breast cancer patients with ER-positive tumors. However, tamoxi en is a prodrug that requires metabolic activation to orm 4-hydroxytamoxi en and 4-hydroxy-N-desmethyltamoxi en (endoxi en) (Fig. 7-2A). These metabolites are approximately 100 times more potent as antagonists o the ER than the parent drug. As a result, patients who are CYP2D6 poor metabolizers (Fig. 7-1) are relatively unable to orm the active 4-hydroxy metabolites o tamoxi en. Poor-metabolizer patients have worse outcomes with respect to breast cancer recurrence than do CYP2D6 extensive metabolizers (EMs) (Fig. 7-2B). Furthermore, i EM patients are co-administered other drugs such as antidepressants that are good CYP2D6 substrates, they may receive less benef t rom tamoxi en therapy than do CYP2D6 EMs who are not co-administered drugs that compete with tamoxi en or CYP2D6-catalyzed metabolism. In the past, an individual’s genotype or CYP2D6 and many other genes encoding drug-metabolizing enzymes was in erred rom phenotype (e.g., the urinary metabolic ratio that can be measured by assaying the urinary excretion o a specif c metabolite a ter the administration o a probe drug) (Fig. 7-1A). As discussed below, genotype assignment is now increasingly dependent on DNA-based tests per ormed with commercially available, direct-to-consumer testing devices such as the genotyping “chip” shown in Figure 7-1B. TPMT represents another example o an important and clinically relevant genetic polymorphism or drug metabolism. TPMT catalyzes the S-methylation o thiopurine drugs such as 6-mercaptopurine and azathioprine (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance). Among other indications, these cytotoxic and immunosuppressive agents are used to treat acute lymphoblastic leukemia and in ammatory bowel disease. Although thiopurines are use ul drugs, they have a narrow therapeutic index (i.e., the ratio between the toxic dose and the therapeutic dose is small), with occasional patients su ering rom li e-threatening thiopurine-induced myelosuppression. In Caucasians, the most common variant allele or TPMT is TPMT*3A; the requency o this allele is approximately 5%, so 1 in 300 subjects carries two copies o the TPMT*3A allele. TPMT*3A is predominantly responsible or the trimodal requency distribution o the level o red blood cell TPMT activity shown in Figure 7-3. TPMT*3A has two nonsynonymous cSNPs, one in exon 7 and another in exon 10 (Fig. 7-3). The presence o TPMT*3A results in a striking decrease in tissue levels o TPMT protein. Mechanisms responsible or the observed decrease in TPMT*3A protein level include both accelerated TPMT*3A degradation and intracellular TPMT*3A aggregation, probably as a result o protein mis olding. As a result, drugs such as 6-mercaptopurine are poorly metabolized and may reach toxic levels. Subjects homozygous for TPMT*3A are at greatly increased risk for life-threatening myelosuppression when treated with standard doses of thiopurine drugs. These patients should be treated with approximately one-tenth to one-f teenth the standard dose. There are striking ethnic di erences in the requency o variant alleles or TPMT. For example, TPMT*3A is rarely observed in East Asian populations, whereas TPMT*3C, which has only the exon 10 SNP, is the most common variant

C h a p t e r 7 Pharmacogenomics

91

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TP MT a ctivity (units /ml RBC) CYP2D6

SULT1A1 OH

N-de s me thylTAM

1

TPMT*1 (wild type )

Endoxife n

2

3

4

5

6

7

8

9

10

VNTR

TPMT*3A VNTR

B

FIGURE 7-3. TPMT pharmacogenetics. Frequency distribution o red blood

80

cell (RBC) thiopurine S-methyltrans erase (TPMT) activity or 298 unrelated Caucasian subjects. TPMTL indicates an allele or alleles or the trait o low activity, while TPMTH re ers to the “wild type” (TMPT*1) allele or the trait o high activity. The observed trimodal requency distribution or RBC TPMT activity is due mainly to the e ect o TPMT*3A, the most common variant allele or low activity in a Caucasian population. TMPT*1 and TPMT*3A di er by two nonsynonymous single nucleotide polymorphisms (SNPs), one in exon 7 and one in exon 10. VNTR, variable number tandem repeat.

EM

60

s

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Ye a rs a fte r ra ndomiza tion FIGURE 7-2. Tamoxifen pharmacogenetics. A. Tamoxi en is metabolized by two cytochrome P450 pathways to orm the active metabolites 4-hydroxytamoxi en (4-hydroxyTAM) and endoxi en, which are urther metabolized by sul otrans erase (SULT) 1A1 (not shown). Genetic variations in CYP2D6 can inf uence the extent o tamoxi en metabolism. B. Kaplan-Meier curves showing the inf uence o CYP2D6 “metabolizer” status on the survival o women with estrogen receptor-positive (ER[ ]) breast cancer who were treated with tamoxi en. Patients who were extensive metabolizers (EM) o tamoxien had improved relapse- ree survival and disease- ree survival relative to intermediate metabolizers (IM) and poor metabolizers (PM).

allele in those populations. For these populations, alternative agents or dose reductions are recommended or thiopurine drugs. Because o its clinical signif cance, TPMT was the f rst example selected by the FDA or public hearings on the inclusion o pharmacogenetic in ormation in drug labeling. For the same reason, clinical testing or TPMT genetic polymorphisms is widely available. The phenomenon o marked changes in the level o a protein as a result o the alteration o only one or two amino acids in the protein has been observed repeatedly or multiple other genes o pharmacogenetic signif cance and is a common explanation or the unctional e ects o nonsynonymous cSNPs within these genes. The BChE, NAT2, CYP2D6, and TPMT genetic polymorphisms all behave as monogenic (single-gene) Mendelian traits, as do many other early examples rom pharmacogenetics. However, pharmacogenetics-pharmacogenomics has now moved beyond monogenic traits with pharmacokinetic phenotypes, and the ocus increasingly involves unctionally and clinically signif cant variation in drug targets as well as drug-metabolizing enzymes. Variation can also involve multiple genes and pathways that in uence both pharmacokinetics and pharmacodynamics.

Variation in Drug Targets: Pharmacodynamics Drugs generally exert their e ects by interacting with specif c target proteins. There ore, genetic variations in these target proteins, or in signaling pathways downstream rom

92 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

TABLE 7-2 Examples of Genetic Polymorphisms and Drug Targets PROTEIN

AFFECTED DRUG CLASS (EXAMPLE)

5-Lipoxygenase

Zileuton

Angiotensin converting enzyme (ACE)

ACE inhibitors (lisinopril)

Apolipoprotein E

Statins (pravastatin)

2-Adrenergic

receptor

-Adrenergic agonists (albuterol)

Epidermal growth actor receptor

Gef tinib

Sul onylurea receptor

Tolbutamide

Vitamin K epoxide reductase complex 1

War arin

the target proteins, can in uence the outcome o pharmacotherapy (Table 7-2). One example o genetic variation or a drug target in germline DNA involves a class o drugs used to treat asthma. As noted in Chapter 48, Integrative In ammation Pharmacology: Asthma, the anti-asthma medication zileuton decreases airway in ammation by inhibiting the enzyme 5-lipoxygenase , an enzyme encoded by the gene ALOX5. Variations in 5-lipoxygenase illustrate the point that variation in many areas o a gene can a ect protein unction. Polymorphisms in gene regulatory regions, such as the gene promoter, can in uence transcription and thereby alter protein expression. The promoter o the ALOX5 gene displays variation in the number o tandem repeats o the sequence GGGCGG. These repeat sequences bind the transcription actor complex Sp1, which up-regulates ALOX5 transcription. The most common ALOX5 allele contains f ve repeats and is present in about 77% o ALOX5 transcripts. As a result, approximately 94% o the population has at least one copy o the f ve-repeat allele. The most common variant alleles contain our and three repeats and are present at requencies o about 17% and 4%, respectively. Because o increased Sp1 binding, people who carry the f ve-repeat allele are thought to express more 5-lipoxygenase than those who lack it. Interestingly, there seems to be no relationship between the presence or absence o the f ve-repeat allele and the severity o asthma in the population; that is, this ALOX5 promoter polymorphism does not seem to a ect the disease process itsel . However, in trials o zileuton and closely related 5-lipoxygenase inhibitors, only subjects who had at least one copy o the f ve-repeat allele responded to the drug. This result suggests that zileuton-like compounds are unlikely to help the 6% o the population who lack the f ve-repeat allele and that identi ying this subgroup would allow the use o alternative, more e ective medications. The ALOX5 example demonstrates that pharmacodynamic-pharmacogenetic variation (i.e., variation in genes encoding drug targets) can be just as important, i not more important, than the pharmacokinetic-pharmacogenetic variation represented by CYP2D6 and TPMT. Table 7-2 lists several drug target proteins with genetic polymorphisms that have been associated with variation in drug response. This example also

illustrates an important principle, that a polymorphism in a drug target need not cause a disease in order to inf uence the treatment o that disease.

Pathway-Based PharmacogeneticsPharmacogenomics Each o the preceding examples o CYP2D6, TPMT, and ALOX5 involves clinically signif cant pharmacogenetic variation as a result o sequence variation in a single gene (i.e., monogenic inheritance). However, it is also possible or multiple genes encoding proteins that in uence both pharmacokinetics and pharmacodynamics to alter the drug response phenotype. One o the best examples o this type is provided by the anticoagulant warfarin. War arin (see Chapter 23, Pharmacology o Hemostasis and Thrombosis) is one o the most widely prescribed oral anticoagulants in both North America and Europe. However, despite the existence o a laboratory test that is used universally to ollow war arin’s e ect on coagulation (INR), serious adverse reactions—involving both hemorrhage and undesired thrombosis—continue to complicate war arin therapy. These complications are illustrated by the case o Mr. H at the beginning o this chapter: a ter a “standard” dose o war arin, his INR was elevated to 6.2, a level associated with increased risk o hemorrhage. Why might that have occurred? First, we need to remember that war arin is a racemic mixture. S-war arin is three to f ve times more potent than R-war arin, and S-war arin is metabolized predominantly by the cytochrome P450 isoorm CYP2C9. CYP2C9 is a highly polymorphic gene, and the variant alleles CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile358Leu) are associated with only 12% and 5%, respectively, o the level o enzyme activity observed with the wild-type allele (CYP2C9*1). Patients who carry these variant alleles require decreased doses o war arin to achieve an anticoagulant e ect, and these same subjects have increased risk o hemorrhage during war arin therapy. However, this pharmacokinetic-pharmacogenetic variation ails to explain most o the variance in the therapeutic war arin dose in patients who are anticoagulated with this power ul, but potentially dangerous, drug. The molecular target o war arin was not identif ed until 2004. The gene encoding that target, vitamin K epoxide reductase complex 1 (VKORC1), was also cloned that year. When the VKORC1 gene was sequenced in a number o patients, although no nonsynonymous cSNPs were ound, a series o haplotypes (combinations o SNPs on a single chromosome) was observed that were associated with the war arin dose requirement. In one study, patients with VKORC1 haplotypes that were associated with a low dose requirement had an average war arin maintenance dose approximately hal o that required by subjects with haplotypes associated with a high dose requirement. Several subsequent studies conf rmed that the VKORC1 haplotype is associated with approximately 25% to 30% o the variance in the war arin maintenance dose, while 5% to 15% can be explained by the CYP2C9 genotype. The roles o CYP2C9 and VKORC1 in war arin pharmacokinetics and pharmacodynamics are shown schematically in Figure 7-4. Because the genes encoding both o these proteins contribute to variation in drug response, genotyping or CYP2C9 and haplotyping or VKORC1 represent potentially use ul strategies or the determination o an initial war arin dose or Mr. H. An initial analysis o combined data or over 5,000 patients worldwide who were anticoagulated with war arin

C h a p t e r 7 Pharmacogenomics P re curs ors of clotting fa ctors

Active clotting fa ctors

O2

CO 2

Vitamin K-dependent γ-glutamyl carboxylase

Vita min K re duce d

Vita min K e poxide

Vitamin K epoxide reductase

6-Hydroxywa rfa rin 7-Hydroxywa rfa rin CYP2C9

S-Warfarin

FIGURE 7-4. Warfarin pharmacokinetics and pharmacodynamics. Vitamin K is a required co actor or the post-translational -carboxylation o glutamate residues in certain clotting actor precursors (see Chapter 23). Vitamin Kis oxidized to the inactive epoxide as a consequence o the carboxylation reaction. The enzyme vitamin Kepoxide reductase (VKORC1) converts the inactive epoxide into the active, reduced orm o vitamin K. War arin acts as an anticoagulant by inhibiting VKORC1 and thereby preventing the regeneration o reduced vitamin K. S-war arin is metabolized to 6-hydroxywar arin and 7-hydroxywar arin by cytochrome P450 2C9 (CYP2C9).

and were genotyped or both CYP2C9 and VKORC1 showed that adding genotype data to clinical variables provided a superior prediction o the war arin dose requirement than an algorithm that used only clinical data such as age, diet, and weight. Due to the increasing evidence that genetic as well

93

as clinical variables are important or anticoagulant therapy, in 2010, the FDA guidelines or war arin recommended that patient genotypes (i known) should be considered in wararin dose selection and monitoring. However, the question o whether genotype-guided dosing regimens are superior to dosing regimens that utilize clinical variables alone is not yet answered. In particular, two o three recent multicenter randomized controlled clinical studies to evaluate the e f cacy o including patient genotype in ormation in clinical dosing regimens demonstrated little improvement over clinical dosing protocols or algorithms in terms o time spent within the therapeutic range. It remains uncertain whether genotypeguided war arin dosing improves clinical outcomes and prevents the bleeding complications associated with supratherapeutic war arin dosing. To date, such e f cacy measures have been associated with genotype guidance, but only in a study comparing a large cohort o subjects with historical controls. War arin provides an important example o a situation in which pharmacokinetic-pharmacogenetic data may prove inadequate or clinical translation because those data explain too little o the variation in therapeutic drug dose. Thus, wararin may represent, probably in a simplif ed orm, the type o polygenic, pathway-based pharmacogenetic-pharmacogenomic model (i.e., one that combines pharmacokinetic and pharmacodynamic variants) that may become increasingly common in the uture.

Role of Epigenetics in PharmacogeneticsPharmacogenomics Epigenetics is an emerging area o pharmacogenomics (Fig. 7-5). Epigenetics re ers to heritable changes in gene unction and expression that are not a result o DNA sequence changes. Several recent studies show that, in addition to DNA sequence changes, epigenetic changes including DNA

Compone nts of the e pige ne tic ma chine ry

DNA methylation proteins DNMT1 DNMT3A DNMT3B

TET1-3 MBD1-4 MECP 2

Histone modifying proteins P CAF p300/CBP S IRT1-7 HDAC1-11 LS D1 J ARID1A-C

EZH1/2 DOT1L S ET7/9 MLL2 G9a

miRNAs miR-24 miR-27b miR-125b miR-126* miR-378 miR-631

P TGS

mRNA

miRNA miRNA miRNA

Ribos ome

Me t CG TGS

ADME ge ne

GC Me t

Ge nomic DNA Nucle os ome

Drug tra ns porte r or me ta bolizing e nzyme

FIGURE 7-5. Epigenetic control of absorption, distribution, metabolism, and excretion (ADME) genes. The molecules that regulate epigenetic e ects include a host o proteins or DNA methylation (DNMT1, DNMT3A, DNMT3B, TET1-3, MBD1-4, MECP2), histone modif cation (HDAC1-11, p300/CBP, PCAF, SIRT1-7, EZH1/2, DOT1L, MLL2, and others), and miRNAs (miR-24, miR-27b, miR-125b, miR-126*, miR-378, and miR-631). Collectively, these proteins and miRNAs contribute to transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) o drug transporters and metabolizing enzymes. Down-regulation o expression o ADME genes can lead to variations in the pharmacokinetic and pharmacodynamic prof les o the drugs that interact with their gene products.

94 FUNDAMENTAL PRINCIPLES OF PHARMACOLOGY

methylation, histone modif cation, and post-transcriptional regulation via noncoding RNAs (including micro RNAs [miRNAs]) can regulate the expression o genes involved in pharmacokinetic and pharmacodynamic responses. Furthermore, since DNA variation alone cannot entirely explain interindividual di erences in drug response and adverse drug event phenotypes, investigation o epigenetic mechanisms is likely to have clinical benef t. The most common orm o epigenetic change is DNA methylation. DNA methyltrans erases (DNMTs) add a methyl group rom S-adenosylmethionine to cytosine in specif c promoter sites called CpG sites. In the human genome, up to 80% o the CpG sites are methylated. Large regions o unmethylated CpG sites, called CpG islands, are present near promoters and are associated with increased transcription, whereas methylation o CpG sites within promoters is associated with gene silencing. Examples o pharmacoepigenetic mechanisms related to variation in drug responses may include altered methylation o specif c CpG sites in promoters o genes involved in drug absorption, distribution, metabolism, and excretion (ADME). For instance, di erential methylation o the CYP3A4 promoter correlates with its hepatic gene expression. Drug- and toxin-related changes in CYP1A1 and CYP1B1 expression are also directly related to altered methylation patterns o these genes. In one investigation, treatment o HepG2 liver cells with the anticancer agent decitabine (a DNA methyltrans erase inhibitor) led to partial demethylation o the CYP1B1 promoter and restored CYP1B1 inducibility. Evidence also exists or the role o tissue-specif c miRNAs in regulating the expression o multiple pharmacokinetic enzymes and transporters, including CYP3A4, CYP1B1, ABCB1, ABCG2, and ABCC1. Clari ying the role o miRNAs in the control o ADME gene expression will improve mechanistic understanding o variation in drug levels and pharmacodynamic e ects and help in identi ying new potential therapeutic targets.

Modern Pharmacogenomics Completion o the Human Genome Project and the ongoing 1,000 Genomes Project points the way to uture developments in pharmacogenetics and pharmacogenomics in the “postgenomic” era. Application o modern genomic assay techniques such as genome-wide association studies (GWAS), combined with an increasing ocus on pathways— pathways that encompass genes encoding all o the drugmetabolizing enzymes and transporters that could in uence the f nal concentration o drug reaching the target (i.e., pharmacokinetics), together with genes encoding the drug target and signaling pathways downstream rom that target (i.e., pharmacodynamics)—represent the uture or this aspect o “individualized medicine.” Attaining the goal o truly personalized drug therapy and translating genomic knowledge into clinical practice rapidly will require the application o high-throughput genotyping technologies. Simvastatin was one o the f rst drugs to be identif ed through GWAS as a genomic biomarker. Cholesterol-lowering HMG-CoA reductase inhibitors, such as simvastatin and atorvastatin (see Chapter 20, Pharmacology o Cholesterol and Lipoprotein Metabolism), are among the most widely prescribed drugs worldwide. Although these drugs are generally very sa e, statins can rarely cause serious myopathy with rhabdomyolysis and renal ailure. In an attempt to predict and prevent this serious adverse drug

reaction, the SEARCH collaborative group per ormed a GWAS in which approximately 300,000 SNPs across the genome were genotyped using DNA rom 85 patients who had developed severe statin-induced myopathy and 90 control subjects who had not developed this adverse drug reaction. A single SNP located within the SLCO1B1 gene, which encodes an organic anion transporter that mediates statin uptake by the liver, had a strong association with myopathy. The odds ratio or myopathy risk in subjects homozygous or the variant nucleotide at the SNP was 16.9, and it was estimated that more than 60% o the cases o myopathy in this 12,064-patient trial were associated with this one SNP. Patients homozygous or the variant SNP may have higher plasma levels o statins and there ore be more likely to develop rhabdomyolysis at any given dose o drug. Over the last decade, a growing number o GWASs have identif ed additional genes and SNPs that can predict treatment response to an increasing list o medications. For example, a pharmacogenomic GWAS was conducted to evaluate the symptomatic response to inhaled corticosteroids in more than 900 patients with asthma. That study identif ed a unctional SNP in the promoter region o GLCCI1, a suspected mediator o glucocorticoid-induced apoptosis. Patients who carried the SNP had worse therapeutic responses to inhaled glucocorticoids a ter 4–8 weeks o therapy and were there ore at higher risk o developing treatment-resistant asthma and asthma exacerbations. While GWAS has success ully identif ed multiple genes that may explain a portion o the variability observed in therapeutic responses to various drugs, these genes alone ail to account or the majority o the heritability in therapeutic response. The variability observed in drug responses is likely due to the coordinated e ects o unctional SNP genotypes, gene pathways, epigenetic e ects, and environmental actors. In recent years, integrative pharmacogenomics and systems biology approaches have been utilized to integrate diverse data types and to per orm predictive modeling o the most important interactions that contribute to clinical response phenotypes. These models not only provide insight into the pharmacogenetic mechanisms but can also be evaluated and tested in cell lines, animal models, and clinical trials. In addition, these models may help to identi y new potential drug targets or therapeutic intervention. As an example, investigators interested in pharmacogenetic variation in response to statin medications per ormed an expression quantitative trait loci (QTL) study in which global gene expression was measured in immortalized patient-derived B cells treated with and without simvastatin. Six genes related to statin levels were identif ed in these cellular models; glycine amidinotrans erase (GATM) was the most signif cant gene. (GATM is essential or the synthesis o creatine.) Subsequent association analyses demonstrated that variation in this gene was strongly associated with the presence o statin-induced myopathy in multiple independent cohorts. Thus, in this evolving f eld, downstream approaches involving integration o GWAS in ormation with gene expression and computational modeling o gene interactions can in er mechanistic in ormation to clari y the roles o pharmacogenomic candidates.

Pharmacogenomics and Regulatory Science To achieve individualized drug therapy, we need not only to understand the science underlying pharmacogenetics and pharmacogenomics and to develop state-o -the-art technologies to detect and assay DNA sequence data but also to translate that

C h a p t e r 7 Pharmacogenomics

knowledge into the clinic. That translation process will require the active involvement o the FDA and the pharmaceutical industry, which develops virtually all new drugs. Early e orts by the FDA began with thiopurine drugs and TPMT and were ollowed by hearings on a genetic polymorphism in UGT1A1, a gene encoding a phase II enzyme involved in biotrans ormation o the antineoplastic agent irinotecan. Public hearings have also been held on CYP2C9, VKORC1, and war arin— resulting in relabeling—and on tamoxi en and CYP2D6. Incorporating in ormation rom pharmacogenetics could also contribute to postmarketing surveillance, not only to help avoid adverse reactions but also to “rescue” drugs that might be o benef t to groups o patients selected on the basis o genetic variation in drug response. The latter situation was highlighted by reports that a polymorphism in the 1-adrenoceptor in uences response to the 1-adrenergic antagonist bucindolol—both in vitro and in patients with heart ailure. This -antagonist had initially ailed in a clinical trial that did not include genotyping, perhaps because only patients with the wild-type 1-adrenoceptor genotype displayed the desired clinical response.

CONCLUSION AND FUTURE DIRECTIONS Pharmacogenetics and pharmacogenomics involve the study o ways in which DNA sequence variation a ects the response o individual patients to medications. The goal o pharmacogenetics and pharmacogenomics is to maximize e f cacy and minimize toxicity based on knowledge o an individual’s genetic composition. Although many actors other than inheritance in uence di erences among patients in their response to drugs, the past hal -century has demonstrated that genetics is an important actor responsible or variation in the occurrence o adverse drug reactions or the ailure o individual patients to achieve the desired therapeutic response. Pharmacogenetics has evolved during that hal -century rom classical examples, such as CYP2D6 and TPMT, to include more complex situations such as that represented by the pharmacogenetics o war arin, a drug that displays both pharmacokinetic and pharmacodynamic pharmacogenetic variation, and that represented by new loci and SNPs related to pharmacogenomic phenotypes identif ed through GWAS. The areas o genomic medical science, epigenetics, and systems biology also present unique challenges

95

in translation into the clinic. However, there can no longer be any doubt that pharmacogenetics and pharmacogenomics will be applied to clinical medicine with increasing breadth and depth and that, ultimately, they will enhance our ability to individualize drug therapy.

Acknowledgment The authors would like to acknowledge Liewei Wang and Richard M. Weinshilboum, the authors o this chapter in the Second and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy, who provided the template or and insight into the construction o this chapter.

Suggested Reading Caudle KE, Klein TE, Ho man JM, et al. Incorporation o pharmagenomics into routine clinical practice: the Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline development process. Curr Drug Metab 2014;15:209–217. (Overview o metrics used to determine which pharmacogenetic variants are suitable or clinical implementation.) Drazen JM, Yandava CN, Dube L, et al. Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment. Nat Med 1999;22:168–171. (Original study that showed di erent pharmacologic responses in people with di erent polymorphisms o the ALOX5 gene.) Ingelman-Sundberg M, Zhong XB, Hankinson O, et al. Potential role o epigenetic mechanisms in the regulation o drug metabolism and transport. Drug Metab Dispos 2013;41:1725–1731. (Review o emerging evidence or the role o epigenetics in drug transport and metabolism.) Mallal S, Phillips E, Carosi G, et al. HLA-B*5701 screening or hypersensitivity to abacavir. N Engl J Med 2008;358:568–579. (A double-blind randomized study o a genetic biomarker or an idiosyncratic adverse drug response.) Mangravite LM, Engelhardt BE, Medina MW, et al. A statin-dependent QTL or GATM expression is associated with statin-induced myopathy. Nature 2013;502:377–380. (Investigation o pharmacogenetic variation in response to statin medications.) SEARCH Collaborative Group. SLCO1B1 variants and statin-induced myopathy—a genomewide study. N Engl J Med 2008;359:789–799. (The f rst genome-wide association study o a drug response.) Tantisira KG, Lasky-Su J, Harada M, et al. Genomewide association between GLCCI1 and response to glucocorticoid therapy in asthma. N Engl J Med 2011;365:1173–1183. (The f rst genome-wide association study to evaluate lung unction changes in response to corticosteroid treatment in asthma.) Zineh I, Pacanowski M, Woodcock J. Pharmacogenetics and coumarin dosing—recalibrating expectations. N Engl J Med 2013;369:2273–2275. (Perspective on caveats or adopting genotype-guided dosing regimens or anticoagulants.)

C e n tra l p e rc e ptionn

II

Thh a la mus

R e lay a n d d e s c e n d in g m o d u la tion

Principles of Neuropharmacology Braa ins te m

Tra ns m is s ion S pinn a l cord

C onduu c tion

P e rip h e ra l s timuluss

S igna l tra nss d u c tio n

Corr tex

IIA Fundamental Principles of Neuropharmacology

P re s ynapti ticc ne uro n Acc ti tio ion o n pote ntia l Ne urotra ns mitte r tra n s port p te r

2

8

1 P ree curs c or

6b Na +

Na +

N urotra ns m itte r Ne

Principles of Cellular Excitability and Electrochemical Transmission C a 2+

3

4

S y na napt ptiic cle ft

Eliza b e t h M ayn e , La u re n K. Bu h l,5 a a n dK+ Ga ry R. SAde t rich a rtcycla z se n yl ylyl 5b

Ca 2+

6a

Na +

α

β

γ

α

E

GTPCHANN INTRO INTR ODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 98– 98–99 88–9 –9 –99 ON . . . . . . . . . . . . . . . . . . . . . 105 PHARMA ARMA ARM AGDP CO COL LO LOGY L OG OF ION C CHANNELS HA A CELLULAR EXCITABILITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 9 ELECTROCHEMICAL TRANSMISSION ANSMISSIO ON . . . . . . . . . . . . . . . . . . . . . 105 Ohm’s Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999 Oh Synaptic Vesicle Regulation . . .c.AMP . . P. . . . . . . . . . . . . . . . . . . . . 107 ATP AT P A. MP Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Postsynaptic Recepttors . . . . . . . . . . . . .7. . . . . . . . . . . . . . . . . . 10 1077 Channel Selectivity, the Nernst Equation, Ch upP hos phodie p s te ra s e. . . . . . . 108 Po o s ts ynaptic p c e ll Transmitter Metabolism and Reuptake and the Resting Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 CONCLUSION AND FUTURE DIRECTIONS DIRE EC E C . . . . . . . . . . . . . . . . . . 109 AMP The Goldman Equation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Su Sugg gges este tedd Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 1099 The Action Potential. Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

INTRODUCTION Cellular communication is essential or the e ective unctioning o any complex multicellular organism. The major mode o intercellular communication is the transmission o chemical signals, such as neurotransmitters, neuropeptides, and hormones. In excitable tissues, such as nerves and muscles, rapid intracellular communication relies on the propagation o electrical signals—action potentials—along the plasma membrane o the cell. Both chemical and electrical transmission commonly involve the movement o ions across the plasma membrane or across the membranes o internal organelles such as the endoplasmic reticulum. Ionic movements can directly change the cytoplasmic concentration o ions, such as Ca2 , that are key regulators o biochemical and physiologic processes like phosphorylation, secretion, and contraction. Ionic movements also change the electrical potential across the membrane through which the ions f ow, thus regulating various voltage-dependent unctions including the opening o other ion channels. Some o these events are brie , with durations and actions o several milliseconds (0.001 sec). Others can take many seconds, with biochemical consequences— or example, phosphorylation o proteins— that can persist or minutes or hours. Even gene expression can be regulated by changes in ion concentrations, resulting in long-term changes in cellular physiology, growth, di erentiation, and death. 98

Many drugs modi y chemical or electrical signaling to increase or decrease cellular excitability and electrochemical transmission. To appreciate how such drugs act, the present chapter explains the electrochemical oundations that underlie signaling within and between electrically active cells. These general principles are applicable to many areas o pharmacology, including those discussed in Chapters 10 to 12 (Section IIB, Principles o Autonomic and Peripheral Nervous System Pharmacology), Chapters 13 to 19 (Section IIC, Principles o Central Nervous System Pharmacology), and Chapter 24, Pharmacology o Cardiac Rhythm.

CELLULAR EXCITABILITY Excitability re ers to the ability o a cell to generate and propagate electrical action potentials . Neuronal, cardiac,

smooth muscle, skeletal muscle, and many endocrine cells have an excitable character. Action potentials may propagate over large distances, as in peripheral nerve axons that conduct over several meters, or they may stimulate activity in cells o much smaller size such as the 30- to 50- m-diameter interneurons that are contained within a single autonomic ganglion. The unction o action potentials di ers depending on the cell in which they occur. Propagating waves o action potentials rapidly conduct encoded in ormation with delity over long distances along axons. Within a small cell, action potentials excite the whole cell at once, causing an increase

C h a p t e r 8 Principles o Cellular Excitability and Electrochemical Transmission

Du rin g a b u s in e s s trip to J a p a n , Ka rl G a tte n d s a d in n e r in h is h o n o r a t a re s ta u ra n t th a t s p e cia lize s in u g u f s h . Ka rl is im p re s s e d b e ca u s e h e h a s h e a rd th a t th is s p e cia l d is h is n o t a va ila b le in th e Un ite d S ta te s a n d is a n e xp e n s ive d e lica cy in J a p a n . Be o re th e m e a l is ove r, Ka rl n o te s a n u n u s u a l a n d d e lig h t u l s e n s a tio n o tin g lin g a n d n u m b n e s s in h is m o u th a n d a ro u n d h is lip s . His h o s ts a re p le a s e d th a t h e is e xp e rie n cin g th e d e s ire d e e ct o u g u f s h in g e s tio n . Ka rl is a s cin a te d a n d s o m e w h a t e a r u l o th e p o te n tia l toxic e e cts o th e u g u n e u ro toxin (te tro d o to xin ) a s th e y a re d e s crib e d to h im b y h is kn o w le d g e a b le h o s ts . Ho w e ve r, h is h o s ts a s s u re h im th a t

in intracellular ions (e.g., Ca2 ) ollowed by a rapid release o chemical transmitter molecules or hormones. These chemicals then travel to speci c receptors, near or ar rom the releasing cell, to e ect chemical transmission, which is discussed in the second part o this chapter. Cellular excitability is fundamentally an electrical event. There ore, an understanding o basic electricity is necessary to explain the biological processes o excitability and synaptic transmission. The ollowing sections present basic principles o electricity as applied to two important cellular components— the plasma membrane and ion-selective channels.

Ohm’s Law The magnitude o a current (I , measured in amperes) f owing between two points is determined by the potential di erence (V, measured in volts) between those two points and the resistance to current f ow (R, measured in Ohms): I

V/R

gV

th e s u s h i ch e a t th e re s ta u ra n t is u lly lice n s e d to p re p a re u g u f s h a n d is ce rtif e d b y th e g ove rn m e n t. Ka rl is re lie ve d w h e n h e a wa ke n s th e n e xt m o rn in g w ith o u t a n y s ig n s o w e a kn e s s o r p a ra lys is . Ho w e ve r, h e d e cid e s th a t h e w ill p o lite ly o rg o s e a o o d o r th e re s t o th e trip a n d a s k o r Ko b e b e e in s te a d .

Questions 1 . What is the molecular mechanism o action o tetrodotoxin? 2 . What is the e ect o tetrodotoxin on the action potential?

inside and outside o the cell, which in turn avors an increased rate o charge movement across the membrane. The convention used in most texts and in this chapter is that the voltage across a membrane, the “membrane potential,” is expressed as the di erence between the intracellular and extracellular potentials (Vm Vin Vout). For most normal cells, Vm is negative when the cell is at rest (Vin Vout). The membrane is termed hyperpolarized when Vm is more negative than at rest, and it is described as depolarized when Vm is more positive than at rest. Current is conventionally de ned with respect to the direction in which positive charge f ows. Positive charge moving rom inside to outside is called outward current and is represented graphically by positive values. Positive charge moving rom outside to inside is called inward current and is represented graphically by negative values. Movement o negative charge is de ned in the exact opposite way. Note that an e f ux o K cations is electrically equivalent to an inf ux o Cl anions; both are outward currents.

Equation 8-1a

For example, current may f ow rom the extracellular to the intracellular compartment in response to an electrical potential di erence (also known as a voltage difference) across the plasma membrane. Voltage can be thought o as a potential energy or as the propensity or charged particles to f ow rom one area to another. Resistance is the obstacle to this f ow. Decreased resistance allows greater ion f ow and there ore increased current (current has units o charge/time). When this relationship, known as Ohm’s law, is applied to biological membranes such as the plasma membrane, the electrical resistance is o ten replaced by its reciprocal, the conductance (g, measured in reciprocal ohms, or siemens [S]): I

99

Outwa rd curre nt I I=gV

Ne ga tive pote ntia l

V

P os itive pote ntia l

Equation 8-1b

For simplicity, assume that all resistive elements in the cell membrane behave in an “ohmic way”; that is, their current– voltage (I-V) relationship is described by Equations 8-1a, b. In this case, the I-V relationship is linear, with a slope given by the conductance, g. Figure 8-1 represents the transmembrane current (I ) measured at di erent transmembrane potentials (V ) in a hypothetical cell. The slope o the I-V curve equals the conductance. From a conceptual perspective, current increases as voltage increases because a higher voltage results in a greater potential energy di erence between the

Inwa rd curre nt

FIGURE 8-1. Ohm’s law. Ohm’s law states that there is a linear relationship between current (I) and voltage (V) and that the slope o the I versus V plot yields the conductance (g). By convention, outward current is the f ow o positive charge rom inside the cell to outside the cell. Transmembrane potential is de ned by the di erence in electric potential (voltage) between the inside and outside o the cell. For most cells, the resting potential inside the cell is negative relative to that outside the cell. Conductance, g, is the reciprocal o resistance.

100 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

Ion Channels How does current actually f ow across a cell membrane? Biological membranes are composed o a lipid bilayer within which some proteins are embedded and to which other proteins are attached (schematically represented in Fig. 8-2). Pure lipid membranes are virtually impermeable to most polar or charged substances, thus having a very high intrinsic resistance. From an electrical perspective, the lipid bilayer also acts as a capacitor by separating the extracellular and intracellular ions. To enable the passage o ions that carry electrical current, ion channels span the membrane. Most ion channels are gated—that is, they remain closed until speci c signals dictate their opening. Once open, they exhibit ion selectivity, allowing only certain ions or types o ions to pass. From an electrical perspective, a set o gated ion channels is a variable conductor: it provides many individual conductances or di erent ions to f ow between the extracellular and intracellular environments. The magnitude o the overall conductance depends on the raction o channels in the open state and the conductance o the individual open channels.

Channel Selectivity, the Nernst Equation, and the Resting Potential By itsel , the hypothetical I-V relation in Figure 8-1 does not explain the electrical behavior o most real cells. I a cell behaved according to Equation 8-1, then the potential di erence across the membrane would be zero in the absence o an externally applied current. Instead, most cells maintain a negative potential di erence across their plasma membrane.

Ca pa citor (pla s ma me mbra ne )

Re s is tor (ion cha nne l)

Ii

Ic

IT = Ii + Ic

FIGURE 8-2. Electric circuit model of the cell membrane. The cell membrane can be modeled as a simple electric circuit containing a resistor and a capacitor. Ion-selective channels unction as resistors (identical to conductors), through which ions can f ow down their electrochemical gradient. The lipid bilayer acts as a capacitor by maintaining a separation o charges between the extracellular and intracellular spaces. This circuit (re erred to as an RC, or resistor-capacitor, circuit) changes the timing between the f ow o charges across the membrane (current) and changes in transmembrane potential (voltage), because the lipid bilayer, acting as a capacitor, stores some o the charge that passes across the membrane. Time is required to store this charge; there ore, the initial change in voltage associated with a step o current is slow. As the capacitor (lipid bilayer) lls with charges and the voltage change grows, more o the charge passes through the resistor, until a new steady state is reached and the current– voltage relationship becomes more linear. (Ic , capacitor current; Ii, ionic current; IT, total current.)

This voltage di erence is most pronounced in neuronal and cardiac ventricular cells, where a resting potential (the voltage di erence across the membrane in the absence o external stimuli) o 60 to 90 mV can be recorded. The resting potential results rom three actors: (1) an unequal distribution o positive and negative charges on each side o the plasma membrane, (2) a di erence in selective permeabilities o the membrane to the various cations and anions, and (3) the current-generating action o active (energy-requiring) and passive pumps that help to maintain the ion gradients. The e ects o these interrelated actors can be explained best with an example. Consider the case when there are only potassium ions (K ) and protein-bound anions (A ) inside the cell and no other ions outside the cell (Fig. 8-3). I this cell’s membrane is permeable to potassium alone, due to the presence o channels that are open at rest and pass only K ions, then K will f ow outward, while A will remain inside. The K ions f ow outward because o a chemical gradient; that is, K e f ux is energetically avorable because the K concentration inside the cell is greater than that outside the cell. A potential e f ux o the anion, A , is also avored by its chemical gradient, but the absence o transmembrane channels permeable to A prevents this anion rom f owing across the membrane. Because o this selective permeability or K , every K ion that exits the cell leaves one net negative charge (an A ion) on the inside o the cell and adds one net positive charge (a K ion) on the outside o the cell. This separation o charges across the membrane creates a negative membrane potential. I a negative membrane potential were not established as K leaves the cell, then K ions would continue to exit until the extracellular concentration o K was equal to the intracellular concentration o K . However, the establishment o a voltage di erence creates an electrostatic force that eventually prevents net K e f ux (Fig. 8-3B). Thus, the electrical gradient (Vm) and the chemical gradient “pull” the K ions in opposite directions: the electrical gradient avors an inward f ow o K ions, while the chemical gradient avors an outward f ow o K ions. These orces combine to create an electrochemical gradient, which is equal to the sum o the electrical gradient and the chemical gradient. The transmembrane electrochemical gradient is the net driving force for passive ion movement across biological membranes. As a result o the electrochemical gradient, the extracellular concentration o K does not equalize with the intracellular concentration. Instead, an equilibrium is established in which the electrostatic orce “pulling” K back into the cell is balanced exactly by the chemical gradient driving K e f ux. The electrical potential at which this equilibrium occurs, or any permeant ion X, is a unction o the charge o the ion (z), the temperature (T), and the intracellular and extracellular concentrations o the ion. This relationship is expressed as the Nernst equation: [X]out RT ln Vx Vin Vout Equation 8-2 [X]in zF where Vx is the transmembrane potential that a membrane selectively permeable to ion X would reach at equilibrium (i.e., the Nernst potential or that ion), Vin Vout is the transmembrane voltage di erence, RT/zF is a constant or a given temperature and charge (this number simpli es to 26.7 mV or a charge o 1 at a temperature o 37°C), and [X]out and [X]in are the extracellular and intracellular

C h a p t e r 8 Principles o Cellular Excitability and Electrochemical Transmission

A

K+ s e le ctive cha nne l

B

+

K

+

K

C

+

K

101

+

K

+

K +

+

K

K

-

+

A

K

-

A

+

K

A

A

+

-

-

A

A -

-

A

A

A

+

A

-

A

K

+

K -

-

A

K

A

+

-

A

-

-

K

-

+

A

-

A

A

K

+

K

+

K

-

-

-

K

+

K

+

+

K -

+

A

A

A

A

K

-

-

-

+

K

Che mica l force Ele ctrica l force

ZERO

Ele ctroche mica l gra die nt = che mica l force + e le ctrica l force

ZERO

FIGURE 8-3. Electrochemical basis of the resting membrane potential. A. Consider a prototypical cell that initially contains equal concentrations o intracellular potassium ions (K ) and impermeant anions (A ). Assume urther that ions can exit the cell only via a single K -selective channel. In this case, there is a strong chemical gradient or K to exit the cell, but there is no electrical orce avoring ion f ow because the electrical sum o the intracellular charges is zero. B. K begins to exit the cell through the K -selective channel, but A remains inside the cell because it has no exit route. There ore, the K chemical gradient across the membrane becomes smaller. As K exits the cell, the net negative charge rom the A remaining inside the cell produces a negative membrane potential that exerts an electrical orce opposing K e f ux. This orce is opposite in direction to that o the chemical gradient; as a result, the total electrochemical gradient (the sum o the chemical orce and the electrical orce) is less than the chemical gradient alone. C. When the electrical gradient is equal and opposite to the chemical gradient, the system is in equilibrium and no net ion f ow occurs. The voltage resulting rom the separation o charges at equilibrium is re erred to as the Nernst potential.

concentrations, respectively, of ion X. The electrochemical driving force on ion X is equal to the difference between the actual membrane potential and the Nernst potential for that ion, Vm Vx. The third determinant of the resting membrane potential is the active and passive ion pumps that move ions across the membrane. These pumps govern the concentration of ions inside and outside the cell and act as generators of net current by moving net charge across the membrane, termed electrogenic transport. Numerous pumps play an important physiologic role in maintaining ion gradients; these include the ATP-dependent Na /K pump (which uses the energy of ATP hydrolysis to extrude three Na ions for every two K ions that enter the cell) and the Na /Ca2 exchanger (which extrudes one Ca2 for every three Na ions that enter the cell). The coordinated action of these pumps closely regulates the intracellular and extracellular concentrations of all biologically important cations and anions. By knowing the values of these ion concentrations, it is possible to calculate

the Nernst potentials for these cations and anions at physiologic temperature and, hence, the value of the transmembrane potential at which the net driving force for each ion is equal to zero (Table 8-1). Variations in the magnitude and direction of transport for each ion (mediated by pumps and exchangers in the plasma membrane) and differences in the membrane permeability for each ion (mediated by channels selective for each ionic species) generate the distinct intracellular and extracellular concentrations for each of the four key ions. The relative ionic permeabilities of the neuronal membrane at rest are K Cl Na Ca2 . Because the plasma membrane contains K -selective channels that are open under resting conditions while most other channels are closed, the resting membrane potential most closely approximates the Nernst potential for K (about 90 mV). In reality, the additional, weak permeabilities of other ionic species raise the resting membrane potential above that for K . Thus, although K is the most permeant ion, the permeability of the other ions and

TABLE 8-1 Nernst Equilibrium Potentials for Major Ions ION

EXTRACELLULAR CONCENTRATION

INTRACELLULAR CONCENTRATION

NERNST EQUATION FOR ION

NERNST POTENTIAL FOR ION

Na

145 mM

15 mM

26.7 ln (145/15)

VNa

K

4 mM

140 mM

26.7 ln (4/140)

VK

95 mV

Cl

122 mM

4.2 mM

VCl

90 mV

Ca 2

1.5 mM

1

10 5 mM

26.7 ln (122/4.2) 26.7/2 ln (1.5/1

10 5)

VCa 2

61 mV

159 mV

The calculated values o the Nernst potential are typical o mammalian skeletal muscle. Many human cells have similar transmembrane ion gradients.

102 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

the membrane to that ion. Quantitatively, this relationship is expressed as the Goldman-Hodgkin-Katz equation:

I(nA)

Vm

IK

INe t = IK + INa

VK

VNa VR

V(mV) g K : g Na = 5:1

INa -92

-70

+40 IK VK gK INa VNa g Na VR INe t

K+ curre nt K+ Ne rns t pote ntia l K+ conducta nce Na + curre nt Na + Ne rns t pote ntia l Na + conducta nce Re s ting me mbra ne pote ntia l Ne t curre nt

FIGURE 8-4. Relative contribution of K and Na to the resting membrane potential. The relative membrane permeabilities o K , Na , and other ions, and the Nernst (electrochemical equilibrium) potentials o these ions, together determine the resting membrane potential. In the example shown, the conductance o K is f ve times greater than the conductance o Na (shown by the slopes o the I versus V lines or IK and INa , respectively). That is, the membrane is f ve times more permeable to K than to Na . The K current is described by IK [IK gK(V VK)], while the Na current is described by INa [INa gNa (V VNa )]. (In this example, gK and gNa are constant conductances over all voltages.) INet, the net membrane current, is the sum o these two currents (INet IK INa ). The “resting” membrane potential (VR) is the value o V at which INet equals zero. In this example, note that VR is close to, but greater than, VK. This is because, although K is the primary determinant o the resting potential, the minor Na current depolarizes VR to a value more positive than VK.

the action o the “electrogenic” pumps also contribute to the overall resting potential. At the steady state that describes the true resting membrane potential (Fig. 8-4), Vm does not equal the Nernst potential or any o the individual ions, and each ionic species experiences a net electrochemical orce. In other words, (Vm Vion ) is nonzero, and small ion f uxes occur. The algebraic sum o these inward and outward currents is small and is balanced by currents rom active, electrogenic pumps, so there is no net current across the resting membrane. It has been estimated that up to 25% o all cellular energy in excitable tissues is expended in maintaining ion gradients across cellular membranes.

The Goldman Equation The example shown in Figure 8-3 addresses a situation where only one ionic species f ows across the plasma membrane. In reality, many cells possess a number o di erent channels that are selective or di erent ions, all o which contribute to the overall resting membrane potential. When the resting potential is determined by two or more species o ions, the inf uence o each species is governed by its concentrations inside and outside the cell and by the relative permeability o

P [K ]o RT ln K F PK[K ]i

PNa [Na ]o PNa [Na ]i

PCl[Cl ]i PCl [Cl ]o

Equation 8-3

where P x is the membrane permeability o ion x. (P x is expressed as a raction, with a value o 1 indicating maximum permeability.) Essentially, this expression states that the larger the concentration gradient o a particular ion and the higher its membrane permeability, the greater its role is in determining the membrane potential. In the extreme case, when the permeability o one ion is exclusively dominant, the Goldman equation reverts to the Nernst equation or that ion. For example, i P K P Cl, P Na , the equation becomes Vm

[K ]o RT ln F [K ]i

Alternatively, i P Na greatly exceeds P K, P Cl, then Vm VNa , and the membrane is strongly depolarized. This important concept links changes in ion channel permeability to changes in membrane potential. Whenever an ion-selective channel opens, the membrane potential shifts toward the Nernst potential for that ion. The relative contribution o a given channel to the overall membrane potential depends on the extent o ion f ow through that channel. Time-dependent changes in the membrane permeabilities o Na and K (and, in cardiac cells, Ca2 ) account or the major distinguishing eature o electrically excitable tissues—the action potential.

The Action Potential According to Ohm’s law, passage o current across a cell membrane causes the voltage across the membrane to change, reaching a new steady-state value that is determined by the membrane’s resistance (see above). The time course o this voltage change is exponential, determined by the product o the resistance r m and the capacitance cm o the membrane, with a rate constant equal to [r m cm] 1. (The membrane’s capacitance results rom having an insulator, the hydrocarbon core o the phospholipids in the membrane, between two conductors, the ionic solutions on either side o the membrane [see Fig. 8-2]. Capacitors store charge at both sur aces and require time to change the magnitude o this charge.) I the stimulated potential change is less than the threshold value or triggering an action potential (see below), then the membrane voltage changes smoothly and returns to its resting value when the stimulating current is turned o (Fig. 8-5A). On the other hand, i the membrane voltage changes positively by more than the threshold value, then a dramatic event occurs: the membrane voltage rises much more rapidly, to a value o approximately 50 mV, and then drops to its resting value o approximately 80 mV (Fig. 8-5B). This “suprathreshold” event is known as the action potential (AP). Importantly, hyperpolarizing stimuli cannot trigger an AP (Fig. 8-5C). In most neurons, the balance between voltage-gated Na and K channels regulates the AP. (In cardiac cells and many secretory cells, voltage-gated Ca2 channels are also involved in AP regulation; see Chapter 24.) Voltage-gated Na channels conduct an inward current that depolarizes the cell at the beginning o the AP. Voltage-gated K channels conduct an outward current that repolarizes the cell at the end o the AP, in preparation or the next excitatory event.

C h a p t e r 8 Principles o Cellular Excitability and Electrochemical Transmission

0

m

V

)

A

V

o

l

t

a

g

e

(

Thre s hold volta ge

-50 -90

S ma ll de pola rizing s timulus

0

are open; as the membrane is rapidly depolarized through 0 mV, most or all Na channels open; and ast depolarizations to 25 mV open about hal o the Na channels. These are the relations that occur when a constant depolarization is imposed on the membrane (in a process called voltageclamping); when the brie depolarization o an AP stimulates the membrane, ewer Na channels have time to reach the open state, and a large reserve o unopened channels provides a “margin o sa ety” or impulse transmission. Recall that ionic current is the product o the ionic conductance (g) and a potential di erence. For ions, the potential di erence is the same as the electrochemical driving orce, Vm Vx, where Vx is the Nernst potential or the speci ed ion. For example, or Na current: I Na

-50 -90

I Na

0

La rge hype rpola rizing s timulus Thre s hold volta ge

-50

V

o

l

t

a

g

e

(

m

V

)

C

La rge de pola rizing s timulus

-90

Time FIGURE 8-5. The action potential. A. In the example shown, a resting cell has a membrane potential o approximately 80 mV. I a small depolarizing stimulus is applied to the cell (e.g., a stimulus that opens a ew voltagegated Ca 2 channels), the membrane slowly depolarizes in response to the inf ux o Ca 2 ions. Once the stimulus ends and the Ca 2 channels close, the membrane returns to its resting potential. The time course o the voltage change is determined by the membrane capacitance (see Fig. 8-2). B. I a larger depolarizing stimulus is applied to the cell, such that the membrane potential exceeds its “threshold” voltage, the membrane rapidly depolarizes to about 50 mVand then returns to its resting potential. This event is known as an action potential; its magnitude, time course, and shape are determined by voltage-gated Na and K channels that open in response to membrane depolarization. C. In comparison, application o a hyperpolarizing stimulus to a cell does not generate an action potential, regardless o the magnitude o hyperpolarization.

Figure 8-6 shows the current–voltage (I-V) relationships or the voltage-gated Na channel and the “resting” K channel. The total Na conductance o the membrane is the product o the constant conductance o a single open Na channel, the total number o Na channels, and the probability that an individual Na channel is open, P o. Po depends on the membrane potential and increases with depolarization (shown in Fig. 8-6A), and it is this voltage dependence that enables a cell to overcome the resting K conductance to generate and propagate action potentials in response to depolarizing inputs. The open channel probability represents the raction o all Na channels that open (albeit transiently, see below) in response to a single voltage step. For example, at very negative potentials (e.g., 85 mV), essentially no Na channels

gNa (Vm

VNa )

or

Thre s hold volta ge

V

o

l

t

a

g

e

(

m

V

)

B

103

gNa Po (Vm

Equation 8-4 VNa )

Here, gNa is the Na conductance o the membrane when all Na channels are open, and P o is, as above, the probability that any individual Na channel is open. The graphic illustration o this equation is shown in Figure 8-6B, where the Na current or a “ ully activated” membrane is described by the straight line that passes with positive slope through VNa. I there were no voltage dependence to the Na conductance (i.e., i gNa were always equal to gNa ), this line would extend throughout the negative voltage range, as shown by its dashed-line extrapolation. However, the voltage dependence o P o (Fig. 8-6A) causes the actual Na conductance gNa to be voltage-dependent, resulting in deviation o the actual INa rom this theoretical “ ully activated” condition. Thus, increasing depolarizations rom rest (caused, or example, by an applied stimulus) result in inward Na currents that rst become larger as more channels open and then become smaller as Vm approaches VNa , reducing the driving orce through open channels (Fig. 8-6B). Potassium channels conduct outward currents that oppose the depolarizing actions o inward Na currents. Although there are many types o K channels with diverse “gating” properties, only two types need to be considered in order to appreciate the role o K channels in excitability. These two K channel types include the voltage-independent “leak” channels and the voltage-gated “delayed recti er” channels. Leak channels are the K channels that contribute to the resting membrane potential by remaining open throughout the negative range o membrane potentials. The K current that f ows through these channels is shown by the dashed line labeled IK in Figure 8-6B; or these channels, K current will f ow or all Vm not equal to VK. The summation o INa and IK(leak) is represented by the dashed blue line in Figure 8-6C. Three important points on this line de ne three critical aspects o the AP. The net ionic current (INet) is zero at all three o these points. First, at rest, Vm VK. Under this condition, small, transient membrane depolarizations caused by “external” stimuli increase the driving orce or K ions, resulting in net outward currents rom ion conductances through leak channels that repolarize the membrane back to rest when the external stimulus ends. Second, at Vm VT, the outward potassium currents are matched by inward sodium currents, and the net current is also zero. Under this condition, however, even a small further depolarization results in a net inward current that

104 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY A

FIGURE 8-6. Voltage dependence o channel activity. A. Po, the probability that

1

P0 0 -50

0

B Outwa rd Curre nt

50

V (mV)

INa , IK INa Na + cha nne ls be gin to ope n

-90

IK

-50

50

Inwa rd Curre nt

V (mV)

VNa

All Na + cha nne ls ope n

C

INa , IK, INe t IK INa

Outwa rd Curre nt

VK -90

VT

VP

-50

50

V (mV)

Inwa rd Curre nt

INe t

urther depolarizes the membrane, which leads to a larger inward current and urther membrane depolarization. This positive feedback loop constitutes the rising phase of the AP. Thus, the AP occurs in response to any rapid depolarization beyond VT, which is de ned as the threshold potential. Third, Vp is the potential at the peak o the AP. As the membrane approaches Vp, it moves urther rom VK and closer to VNa, so the driving orce or sodium inf ux decreases while that or potassium e f ux grows. Once Vm reaches this maximum depolarization, the net current switches sign rom inward to outward, and consequently, the membrane begins to be repolarized. Voltage-gated (delayed rectif er) K channels contribute to the rapid repolarization phase o the AP. Although membrane depolarization opens these channels, they open and close more slowly than do Na channels in response to depolarization. There ore, inward Na current dominates the early (depolarization) phase o the AP, and outward K current dominates the later (repolarization) phase (Fig. 8-7). This is why the AP is characterized by an initial rapid depolarization (caused by ast inward Na current) ollowed by a prolonged repolarization (caused by slower and more sustained outward K current). The nal eature determining membrane excitability is the limited duration o Na channel opening in response to membrane depolarization. A ter opening in response to rapid membrane depolarization, most Na channels enter a closed state in which they are inactivated (i.e., prevented rom subsequent opening). Recovery rom inactivation

an individual voltage-gated Na channel will open, is a unction o the membrane voltage (V). At voltages more negative than 50 mV, there is a very low probability that a voltage-gated sodium channel will open. At voltages more positive than 50 mV, this probability begins to increase and approaches 1.0 (i.e., a 100% chance o opening) at 0 mV. These probabilities are also generalizable to a population o voltage-gated Na channels, so that virtually 100% o voltage-gated Na channels in the membrane will open at 0 mV. B. The Na current across a membrane (INa) is a unction o the voltage dependence o the Na channels that carry the current and the driving orce (that is, how ar Vm is rom VNa , the reversal potential or Na ). At voltages more negative than 50 mV, the Na current is zero. As the voltage increases above 50 mV, Na channels begin to open, and there is an increasing inward (negative) Na current. The maximum inward Na f ux is reached at 0 mV, when all the channels are open. As the voltage continues to increase above 0 mV, the Na current is still inward, but decreasing, because inward f ow o the positively charged Na ions is opposed by the increasingly positive intracellular potential. The Na current is zero at VNa (the Nernst potential or Na ) because, at this voltage, the electrical and chemical gradients or Na ion f ow are balanced. At voltages more positive than VNa, the Na current is outward (positive). The dashed line indicates the relationship that would exist between Na current and voltage i the open probability o the Na channels were not voltage-dependent. The potassium current that f ows through voltage-independent K “leak channels” is shown by the dashed line labeled IK. C. The summation o plasma membrane Na currents (INa ) and K currents (IK ) demonstrates three key transition points in the I-V graph (denoted by blue circles) at which the net current is zero. The rst o these points occurs at a membrane potential o 90 mV, where V VK. At this voltage, a small increase in potential (i.e., a small depolarization) results in an outward (positive) K current that brings the membrane potential back toward VK. The second point occurs at VThreshold, the threshold voltage (VT ). At this voltage, INa IK; urther depolarization results in the opening o more voltage-dependent Na channels and a net negative (inward) current, which initiates the action potential. The third point occurs at VPeak, the peak voltage (VP). At this voltage, the transition occurs rom a net negative current to a net positive (outward) current. As the Na channels inactivate, the net positive current is dominated by IK, and the membrane potential returns toward VK (i.e., the membrane is repolarized).

occurs only when the membrane is repolarized, whereupon the Na channels return relatively slowly to the closed, resting state rom which they can then open in response to a stimulus. This inactivation o Na conductance, combined with the slowly decaying voltage-gated K conductance, produces dynamic changes in membrane excitability. Following just one AP, ewer Na channels are available to open (i.e., gNa is temporarily smaller), more K channels are open (i.e., gK is larger), the corresponding ionic currents are changed, and VT is therefore more positive than it was before the AP. An excitable membrane is in its socalled re ractory state during this period, which lasts rom just a ter the AP until the conditions o ast gNa inactivation and slow gK activation have returned to their resting values. Very slow depolarizing stimuli will ail to induce an AP, even when the membrane reaches the threshold potential de ned by a rapid depolarizing stimulus, because o the accumulation o inactivated Na channels during the slow depolarizing stimulus. The inactivation property o Na channels is important in the concept o use-dependent block, as discussed in Chapter 12, Local Anesthetic Pharmacology, and Chapter 24. Also, under pathologic conditions, cells may express Na channels that inactivate incompletely and there ore continue to carry an inward current a ter termination o the AP. Such currents may be adequate to raise the membrane potential above VT and thus induce repetitive ring. Diseases such as myotonia and certain types o neuropathic pain appear to arise rom expression o this type o altered Na channel.

C h a p t e r 8 Principles o Cellular Excitability and Electrochemical Transmission

lowering systemic vascular resistance. Some cardiovascular diseases are also treated by selective blockers o cardiac Ca2 channels (see Chapter 22, Pharmacology o Vascular Tone). Highly potent and selective blockers o a certain class o neuronal Ca2 channel have been puri ed rom the venom o a marine snail (Conus sp.) and administered into the spinal f uid to treat severe cases o neuropathic pain. Tetrodotoxin, the ugu neurotoxin rom the introductory case, blocks most neuronal voltage-gated Na channels with high a nity. As a result, tetrodotoxin can inhibit AP propagation in the nervous system, leading to atal paralysis i ingested in su cient amounts. Ion channel unction can also be modi ed through pharmacologic modulation o the receptors that regulate the channels, as described below.

VNa

V

o

l

t

a

g

e

Vm

VT Vr VK 0

105

1

2

3

4

ELECTROCHEMICAL TRANSMISSION g Na

C

o

n

d

u

c

t

a

n

c

e

gK

0

0 De pola riza ting s timulus

1

2

3

4

Time (ms )

FIGURE 8-7. Time course of voltage-dependent Na and K conductances. During the course o an action potential, the transmembrane voltage (Vm) f rst increases rapidly rom VT toward VNa , then decreases below VT, and more slowly approaches VK. The shape and duration o the action potential can be explained by the di erential time courses o the voltagedependent Na and K currents. In response to a depolarizing stimulus, the Na conductance (gNa ) increases rapidly because o the rapid opening o voltage-gated Na channels, then decreases because o Na channel inactivation. The K conductance (gK) increases concurrently with gNa but takes a longer time to reach its maximum conductance because there is a slower rate constant or opening o voltage-dependent K channels. Eventually, gK is greater than gNa , and the membrane repolarizes. (VNa , VK, Nernst potentials or Na and K , respectively; Vr, resting membrane potential; VT, threshold potential or action potential f ring.)

PHARMACOLOGY OF ION CHANNELS Many drugs act directly on ion channels to produce changes in membrane excitability. For example, local anesthetics are injected locally at high concentrations to block Na channels in peripheral and spinal neurons; this Na channel block inhibits AP propagation and prevents sensory transmission (e.g., pain) and motor nerve impulses by these nerves (see Chapter 12). At much lower concentrations, these and structurally similar antiarrhythmic drugs act systemically to suppress abnormal APs in the heart and to treat neuropathic pain and some orms o myotonia (see Chapter 24). Drugs that block K channels are used to treat certain types o cardiac arrhythmias and may be used in the uture to overcome nerve conduction de cits secondary to demyelinating conditions such as multiple sclerosis and spinal cord injury. Calcium channels are blocked directly by some drugs used in the treatment o hypertension; such drugs act by relaxing vascular smooth muscle and

Neurons communicate with one another and with other cell types through the regulated release o small molecules or peptides known as neurotransmitters . Neurotransmitters may be released into the circulation, rom which they can act on distant organs, or they may di use only a short distance to act on juxtaposed target cells at specialized connections called synapses . Synaptic transmission thus integrates electrical signals (voltage changes in the plasma membrane o the presynaptic cell) with chemical signals (release o neurotransmitter by the presynaptic cell and subsequent binding o the transmitter to receptors in the membrane o the postsynaptic cell). For this reason, synaptic transmission is o ten re erred to as electrochemical transmission. The general sequence o processes essential or electrochemical transmission is as ollows (Fig. 8-8): 1. Neurotransmitters are synthesized by cytoplasmic enzymes and stored in the neuron. Common neurotransmitters include acetylcholine, norepinephrine, aminobutyric acid (GABA), glutamate, dopamine, and serotonin. Most neurons are specialized to release only one type o neurotransmitter, and this specialization is determined largely by the synthetic enzymes expressed in that neuron. A ter synthesis, neurotransmitters are actively transported rom the cytoplasm into intracellular vesicles (o ten called synaptic vesicles ) in which they reach high concentrations. Loading o these vesicles is accomplished by the coordinated activity o a number o vesicular membrane proteins. In most cases, an ATP-dependent transporter pumps protons rom the cytoplasm into the vesicle, thereby creating a proton gradient across the vesicle membrane. The electrochemical energy in this proton gradient is used to provide specialized neurotransmitter transporters with the uel or active transport o neurotransmitter molecules rom the cytoplasm into the vesicle. Neurotransmitter- lled vesicles undergo a docking process and are primed at the “active zone” inside the plasma membrane o the presynaptic terminal, a cellular structure that is specialized or neurotransmitter release. 2. When the threshold condition is reached in the neuron, an AP is initiated and propagated along the axonal membrane to the presynaptic nerve terminal. 3. Depolarization o the nerve terminal membrane causes opening o voltage-dependent Ca2 channels and inf ux o Ca2 through these open channels into the presynaptic nerve terminal. In many neurons, this Ca2 inf ux

106 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

4. Presynaptic neuron Action pote ntia l Ne urotra ns mitte r tra ns porte r

2 1 P re curs or

5. 6b

Na +

Na +

Ne urotra ns mitte r

Ca 2+

3 4

Synaptic cleft 5a Ca 2+

K+

5b

Ade nylyl cycla s e

6a

Na +

α GDP

β

γ

E

α GTP

ATP

cAMP

Postsynaptic cell

7

Phosphodiesterase AMP

6.

FIGURE 8-8. Steps in synaptic transmission. Synaptic transmission can be divided into a series of steps that couple electrical depolarization of the presynaptic neuron to chemical signaling between the presynaptic and postsynaptic cells. 1. Neuron synthesizes neurotransmitter from precursors and stores the transmitter in vesicles. 2. An action potential traveling down the neuron depolarizes the presynaptic nerve terminal. 3. Membrane depolarization activates voltage-dependent Ca 2 channels, allowing Ca 2 entry into the presynaptic nerve terminal. 4. The increased cytosolic Ca 2 stimulates vesicle fusion with the plasma membrane of the presynaptic neuron, with subsequent release of neurotransmitter into the synaptic cleft. 5. Neurotransmitter diffuses across the synaptic cleft and binds to one of two types of postsynaptic receptors. 5a. Neurotransmitter binding to ionotropic receptors causes channel opening and changes the permeability of the postsynaptic membrane to ions. This may also result in a change in the postsynaptic membrane potential. 5b. Neurotransmitter binding to metabotropic receptors on the postsynaptic cell activates intracellular signaling cascades; the example shows G protein activation leading to the formation of cAMP by adenylyl cyclase. In turn, such a signaling cascade can activate other ion-selective channels (not shown). 6. Signal termination is accomplished by removal of transmitter from the synaptic cleft. 6a. Transmitter can be degraded by enzymes (E) in the synaptic cleft. 6b. Alternatively, transmitter can be recycled into the presynaptic cell by reuptake transporters. 7. Signal termination can also be accomplished by enzymes (such as phosphodiesterase) that degrade postsynaptic intracellular signaling molecules (such as cAMP).

7.

8.

is mediated by P/Q-type (Cav 2.1) or N-type (Cav 2.2) Ca2 channels. In the presynaptic terminal, the rapid rise in cytosolic ree Ca2 concentration is sensed by specialized protein machinery, causing neurotransmitter- lled vesicles to use with the presynaptic plasma membrane (see the next section, “Synaptic Vesicle Regulation”). A ter vesicle usion, neurotransmitter is released into the synaptic cle t. The rate o neurotransmitter release di ers depending on the substance released. Released neurotransmitter di uses across the synaptic cle t, where it can bind to two broad classes o receptors localized in the nearby postsynaptic membrane and present more sparsely at distant sites: a. Binding o neurotransmitter to ligand-gated ionotropic receptors directly opens channels that mediate ion f ux across the postsynaptic membrane. Within milliseconds, this ion f ux leads to excitatory or inhibitory postsynaptic potentials . b. Binding o neurotransmitter to metabotropic receptors (e.g., G protein-coupled receptors) causes activation o intracellular second messenger signaling cascades. These signaling events (e.g., phosphorylation by speci c kinases) can then modulate ion channel unction, leading to changes in channel gating and thus altering the postsynaptic potential. The time course o changes mediated by metabotropic receptors is generally seconds to minutes, which is slower than the time course o changes mediated by ionotropic receptors. Both ionotropic and metabotropic receptors are commonly present on the same cell, accounting or ast and slow changes, respectively, in the postsynaptic membrane potential. Some neurotransmitters may also bind to a third class o receptors on the presynaptic membrane. These receptors are called autoreceptors because they regulate neurotransmitter release. Excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) propagate passively (i.e., without generating an AP) along the membrane o the postsynaptic cell. A large number o EPSPs can summate to raise the postsynaptic membrane potential beyond threshold voltage (VT). I this occurs, a single or multiple APs can be generated in the postsynaptic cell. (This process is not shown in Fig. 8-8.) Stimulation o the postsynaptic cell is terminated by removal o the neurotransmitter rom the synaptic cle t, desensitization o the postsynaptic receptor, or a combination o both. Neurotransmitter removal occurs by two mechanisms: a. Degradation o the neurotransmitter by enzymes in the synaptic cle t; and b. Uptake o the neurotransmitter by speci c transporters into the presynaptic terminal (or the closely surrounding glial cells), which terminates synaptic action and allows the neurotransmitter to be recycled into synaptic vesicles in preparation or a new release event. For G protein-coupled metabotropic receptors in the postsynaptic cell, termination o the response to a transmitter stimulus is also dependent on intracellular enzymes that inactivate second messengers (e.g., phosphodiesterases that convert cAMP to its inactive metabolite AMP) or that reverse phosphorylation o target proteins.

C h a p t e r 8 Principles of Cellular Excitability and Electrochemical Transmission

The prototypic chemical synapse is that o the neuromuscular junction (see Fig. 10-4 or more detail). At this junction, terminal branches o the motor axon lie in synaptic troughs on the sur ace o the muscle cells. When the neuron res, acetylcholine (ACh) is released rom the motor neuron terminals. The released ACh di uses across the synaptic cleft to bind to ligand-gated ionotropic receptors located on the postsynaptic muscle membrane. This binding o ACh to its receptors causes a transient increase in the probability o opening o receptor-associated ion channels. The channel pore is equally permeable to Na and K , so these channels have a reversal potential (i.e., a potential at which there is no net current f owing through the channel) o approximately 10 mV (the average o the individual Na and K Nernst potentials; see Eq. 8-3). The net inward current passing through these open channels depolarizes the muscle cell membrane. Although this particular end-plate potential, resulting rom one presynaptic AP, is su ciently large to stimulate an AP in the muscle, such a magnitude is exceptional. Because most neuronal excitatory postsynaptic potentials are too small to stimulate an AP, several neuronal excitatory postsynaptic potentials must occur together, within a short time ( 10 ms) and at closely spaced synapses (allowing temporal and spatial integration), in order or the postsynaptic depolarization to reach the threshold value or ring o an AP. Many neurons synthesize and release neuropeptides in addition to neurotransmitters. Neuropeptides, which are short chains o amino acids, exert e ects on other neurons and play key roles in processes as diverse as energy homeostasis and cellular excitability. In contrast to the ast neurotransmission mediated by synaptic vesicle usion and neurotransmitter release, neuropeptides signal more slowly and are typically released via the regulated secretory pathway. Indeed, the synthesis, storage, and release o neuropeptides are similar to hormone production and secretion, and many neuropeptides were initially identi ed as hormones acting outside the central nervous system. Like hormones, neuropeptides are synthesized as precursor polypeptides (preproneuropeptides) on ribosomes at the endoplasmic reticulum and subsequently processed enzymatically into the propeptide, sorted, and packaged along with specialized proteases into dense-core secretory vesicles in the Golgi apparatus (see below). As with hormones, the proneuropeptide may include multiple distinct neuropeptides, and a neuron can there ore release more than one neuropeptide. Proteases within the vesicle cleave the proneuropeptide into the individual neuropeptides during ast axonal transport o the vesicle toward the synapse. Unlike classical neurotransmitter vesicles, neuropeptide vesicles do not undergo docking at the synapse, and release may occur at sites other than the synaptic terminal. Exocytosis o neuropeptide vesicles thus tends to occur only in response to sustained Ca2 elevation, which typically requires repeated or prolonged stimuli. Once released, neuropeptides act almost exclusively at metabotropic receptors. Because they are o ten released extrasynaptically, neuropeptides typically di use over longer distances than neurotransmitters to bind to receptors on numerous cells surrounding the release site, a process known as volume transmission. The ollowing discussion highlights steps in the basic processes o neurotransmission that can be modi ed by pharmacologic agents.

107

Synaptic Vesicle Regulation Nerve terminals contain two types o secretory vesicles: small, clear-core synaptic vesicles and large, dense-core synaptic vesicles . The clear-core vesicles store and secrete small organic neurotransmitters such as acetylcholine, GABA, glycine, and glutamate. Dense-core vesicles are more likely to contain neuropeptide or amine neurotransmitters. As described above, the larger dense-core vesicles are similar to the secretory granules o endocrine cells because their release is not limited to “active zones” on the presynaptic cell. Densecore vesicle release is also more likely to ollow a train o impulses (continuous or rhythmic stimulation) than a single AP. Hence, the smaller clear-core vesicles are involved in rapid chemical transmission, while the larger dense-core vesicles are implicated in slow, modulatory, or distant signaling. Recently, many o the proteins that control synaptic vesicle tra cking have been identi ed. Synaptic vesicles interact with a amily o proteins called synapsins that bind to the actin cytoskeleton in a phosphorylation-dependent manner. Activity at the synapse drives phosphorylation and dephosphorylation o synapsins via a variety o protein kinases and phosphatases, and synapsins are thus thought to regulate the availability o vesicles or Ca2 -dependent exocytosis. Vesicle docking and priming at the active zone are mediated by interactions between proteins in the synaptic vesicle membrane and the plasma membrane. Once docked and primed, Ca2 -sensing proteins called synaptotagmins play a key role in exocytosis. For both Ca2 -regulated and Ca2 -independent vesicle exocytosis, usion between the synaptic vesicle and plasma membranes is mediated by the SNARE/SM protein complex present in both the vesicle membrane (synaptobrevin/ VAMP) and the plasma membrane (syntaxin-1, SNAP-25) (Fig. 8-9). Certain neurotoxins, such as tetanus toxin and botulinum toxin (see Chapter 10, Cholinergic Pharmacology), appear to act by selectively cleaving SNAREs and thereby inhibiting synaptic vesicle exocytosis. Conversely, a toxin in the venom o the black widow spider binds to speci c receptors in presynaptic nerve terminals and oligomerizes to orm pores in the presynaptic plasma membrane, thereby bypassing the physiologic regulation o synaptic vesicle usion to stimulate spontaneous release o neurotransmitters. SNAREs and associated proteins may provide uture targets or pharmacologic control o synaptic transmission.

Postsynaptic Receptors A large number o neuropharmacologic drugs act on neurotransmitter or neuropeptide receptors. These integral membrane proteins all into two classes: ionotropic and metabotropic . Ionotropic receptors, such as nicotinic acetylcholine receptors, AMPA and NMDA glutamatergic receptors, and “A” type GABA receptors, are almost always composed o our to ve subunits that oligomerize in the membrane to orm a ligandgated channel. Relatively rapid binding o one or sometimes two ligand molecules to the receptor leads to a slower allosteric con ormational change that opens the channel pore. The subunits composing the same unctional receptor o ten di er among di erent tissues and, as a consequence, the detailed molecular pharmacology o the receptors is tissue-dependent. For example, although acetylcholine is the endogenous transmitter or all nicotinic cholinergic receptors, di erent synthetic agonists (or antagonists) selectively activate (or inhibit)

108 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

these receptors in skeletal muscle, autonomic ganglia, or the central nervous system (see Chapter 10). Ionotropic receptors are also ound presynaptically, where they modulate neurotransmitter release rom the presynaptic terminal. Metabotropic receptors, which exert their e ects through activation o intracellular signaling cascades, are similarly diverse in ligands, location, and e ect. They are ound both pre- and postsynaptically and commonly coexist with ionotropic receptors. Most metabotropic receptors are G proteincoupled receptors (GPCRs). Classical ast neurotransmitters (glutamate, ACh, and GABA), monoamines (dopamine, serotonin, and norepinephrine), and neuropeptides can all act via metabotropic receptors. A neurotransmitter or peptide may have multiple distinct metabotropic receptors, each with di erent or even opposing intracellular signaling pathways. For example, dopamine increases cAMP levels via the D1 amily o dopamine receptors but decreases cAMP levels via the D2 amily o dopamine receptors (see Chapter 14, Pharmacology o Dopaminergic Neurotransmission). The intracellular e ects o metabotropic receptor activation are numerous and diverse. Postsynaptically, metabotropic receptor signaling can open ion channels to create slow excitatory or inhibitory postsynaptic currents or modulate channel properties to change cellular excitability. Second messenger cascades regulate the activity o a host o intracellular targets and processes (see Chapter 1, Drug–Receptor Interactions, as well as chapters in Sections IIB and IIC). Activation o presynaptic receptors can regulate the probability o neurotransmitter release at the synapse. The diversity o structure and unction o metabotropic receptors makes them prime candidates or development o selective agonists or antagonists that activate or inhibit specif c subtypes o metabotropic receptors.

The transporters that acilitate neurotransmitter reuptake rom the synaptic cle t into the presynaptic cell are o even greater importance. Because these reuptake transporters are crucial or the termination o synaptic transmission, their inhibition has pro ound e ects. For example, the psychotropic e ects o cocaine derive rom this drug’s ability to inhibit dopamine and norepinephrine reuptake in the brain, and the therapeutic benef t o antidepressants such as f uoxetine likely results rom inhibition o serotonin-selective reuptake Ne urotra ns mitte r

A S yna ptic ve s icle me mbra ne Cytopla s m S NARE comple x P re s yna ptic pla s ma me mbra ne Volta ge -ga te d ca lcium cha nne l (clos e d)

B

S yna ptota gmin-1 (Ca 2+ s e ns or)

Ca 2+

Transmitter Metabolism and Reuptake Altering the metabolism o the neurotransmitter provides an important mechanism or pharmacologic intervention at the synapse. The two major types o intervention involve inhibition o neurotransmitter degradation and antagonism o neurotransmitter reuptake. Acetylcholinesterase, the enzyme responsible or degrading acetylcholine, is an example o the f rst type o drug target. Acetylcholinesterase inhibitors are the mainstays o treatment or myasthenia gravis (see Chapter 10).

Ca 2+

C

FIGURE 8-9. Current model o neurotransmitter release. A. Synaptic vesicles are tethered close to the plasma membrane of the presynaptic neuron by several protein–protein interactions. The most important of these interactions involve SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and SM (Sec1/Munc18-like) proteins present in both the vesicle membrane and the plasma membrane. The SNARE proteins include synaptobrevin (red), syntaxin-1 (yellow), and SNAP-25 (green). The SM proteins include Munc18-1 and others (not shown). Voltage-gated Ca 2 channels are located in the plasma membrane in close proximity to these SNARE/SM complexes; this facilitates the sensing of Ca 2 entry by Ca 2 -binding proteins (synaptotagmin-1, in blue) localized to the presynaptic plasma membrane and/or the synaptic vesicle membrane. B–D. Voltagegated calcium channels open in response to an action potential, allowing entry of extracellular Ca 2 into the cell. The increase in intracellular Ca 2 triggers binding of synaptotagmin-1 to the SNARE/SM complex and fusion of the vesicle membrane with the plasma membrane, releasing neurotransmitter molecules into the synaptic cleft. Several additional proteins (Munc13-1, complexin-1, and others) are also involved in the regulation of synaptic vesicle fusion (not shown).

Action pote ntia l

D

C h a p t e r 8 Principles of Cellular Excitability and Electrochemical Transmission

(see Chapter 15, Pharmacology o Serotonergic and Central Adrenergic Neurotransmission). Because reuptake transporters tend to be substrate-specif c, it is anticipated that new drugs can be designed to selectively target other specif c transporter subtypes as well.

CONCLUSION AND FUTURE DIRECTIONS Cellular excitability is a crucial component o intercellular communication. The undamental basis or cellular excitability lies in the electrochemical gradients that are established by ion pumps across the lipid bilayer o the plasma membrane and in the ion-selective channels that regulate the permeability o the membrane selectively or di erent ionic species, allowing a change in membrane voltage to be coupled to a chemical stimulus or response. The action potential, a special type o stereotyped response ound in excitable cells, is made possible by the voltage-dependent properties o Na and K channels. The basic processes o electrochemical transmission provide the substrate or pharmacologic modulation o cellular excitation and communication, topics that are addressed in more detail throughout this book.

109

Acknowledgment We thank Michael Ty or his valuable contributions to this chapter in the First and Second Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Catterall WA, Raman IM, Robinson HP, Sejnowski TJ, Paulsen O. The Hodgkin-Huxley heritage: rom channels to circuits. J Neurosci 2012;32:14064–14073. (Historical overview of our understanding of the action potential.) Choquet D, Triller A. The dynamic synapse. Neuron 2013;80:691–703. (Conceptual overview of synaptic physiology, with an emphasis on recent research advances.) Kullmann DM, Waxman SG. Neurological channelopathies: new insights into disease mechanisms and ion channel unction. J Physiol (London) 2010;588:1823–1827. (Review of neurologic diseases resulting from alterations in ion channel physiology.) Nestler EJ, Hyman SE, Holtzman DM, Malenka RC. Molecular neuropharmacology: a foundation for clinical neuroscience. 3rd ed. New York: McGraw-Hill Pro essional; 2015. (An overview of neuropharmacology.) Südho TC. Neurotransmitter release: the last millisecond in the li e o a synaptic vesicle. Neuron 2013;80:675–690. (Review of events and mechanisms of synaptic vesicle fusion.) van den Pol AN. Neuropeptide transmission in brain circuits. Neuron 2012;76:98–115. (Review of neuropeptide functions.)

9

B rai Br a in c a pillary

Pe ric icyt yte te

Principles of Nervous System Physiology and Pharmacology J o s h u a M . Ga la n t e r, S u s a n n a h B. Co rn e s , a n d Da n ie l H. Low e n s t e in

As trro g li liaa l p ro r ce c ss Baa s e me nt me mb ra ne

Mito Mi toch c on n dria INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110–111 INT NEUROANATOMY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Anatomy of the Peripphee ra An rall Ne Nervous System . . . . . . . . . . . . . . 110 Auto Au tono nomi micc Nerv mi Ne rvou o s System m . . . . . . . . . . . . . . . . . . . . . . . . . 111 Peripheral Motor and Sensoory Sys yste tems mss . . . . . . . . . . . . . . . 11 1122 Anat An atom omyy of the Cen entr tral al Nervous System. . . . . . . . . . . . . . . . . 11 1144 Cerebrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Dien Di ence ceph phal alon onn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Cerebellum m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Brainstem m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Spinal Cordd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Cellular Organization of the Nervous System . . . . . . . . . . . . . 116 Long-Tract Neuronal Organizationn . . . . . . . . . . . . . . . . . . . . 117

INTRODUCTION The nervous system contains more than 10 billion neurons. Most neurons orm thousands o synaptic connections, giving the nervous system complexity unlike that seen in any other organ system. Interactions among neuronal circuits mediate unctions ranging rom primitive ref exes to language, mood, and memory. To per orm these unctions, the individual neurons that comprise the nervous system must be organized into unctional networks, which, in turn, are organized into larger anatomical units. The previous chapter reviewed the physiology o individual neurons by describing electrical transmission within a neuron and chemical transmission rom one neuron to another. This chapter discusses neuronal systems by examining two levels o organization. First, the gross anatomical organization o the nervous system is presented to place in context the sites o action o pharmacologic agents that act on this system. Second, the major patterns o neuronal connectivity (so-called neuronal tracts) are presented, because knowledge o the ways in which neuronal cells are organized to transmit, process, and modulate signals acilitates a deeper understanding o the actions o drugs on these tracts. This chapter also discusses the major types o neurotransmitters and the blood–brain barrier; these unctional and metabolic concepts have important pharmacologic consequences or drugs that act on the nervous system. 110

j. . . . . . . . . . . . 117 Local Circuit Neuronal Org rganizattioonT.ight .g. . . . junction Single-Source Divergent Neur uroonal Organization. . . . . . . . 11 1188 NEUROPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Neurotransmittter erss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Amino Ami ino Acid Neurotransmitterss . . . . . . . . . . . . . . . . . . . . . . . 120 Biog Bi ogen e ic Aminess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Other Small Moleculle Neurottran ansm smit itte ters rs . . . . . . . . . . . . . . 123 Neuropeptidess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 The Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 124 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125

NEUROANATOMY The nervous system can be divided structurally and unctionally into peripheral and central components. The peripheral nervous system includes all nerves traveling between the central nervous system and somatic and visceral sites. It is divided unctionally into the autonomic (involuntary) nervous system and the sensory and somatic (voluntary) nervous system. The central nervous system (CNS) includes the cerebrum, diencephalon, cerebellum, brainstem, and spinal cord. The CNS relays and processes signals received rom the peripheral nervous system; the processing results in responses that are ormulated and relayed back to the periphery. The CNS is responsible or important unctions such as perception—including sensory, auditory, and visual processing—wake ulness, language, and consciousness.

Anatomy of the Peripheral Nervous System The autonomic nervous system regulates involuntary responses o smooth muscle and glandular tissue. For example, it controls vascular tone, heart rate and contractility, pupillary constriction, sweating, salivation, piloerection (“goose bumps”), uterine contraction, gastrointestinal (GI) motility, and bladder unction. The autonomic nervous system is

C h a p t e r 9 Principles o Nervous System Physiology and Pharmacology 111

Ma rth a P is a 66-ye a r-o ld w o m a n w ith a 4-ye a r h is to ry o w o r s e n in g Pa rkin s o n ’s d is e a s e , a n e u ro lo g ica l d is o rd e r re s u ltin g ro m th e p ro g re s s ive d e g e n e ra tio n o n ig ro s tria ta l n e u ro n s th a t u s e d o p a m in e a s a n e u ro tra n s m itte r. Th e d is e a s e ca u s e s a re s tin g tre m o r, rig id ity, d i f cu lty in itia tin g m o ve m e n t, a n d p o s tu ra l in s ta b ility. Wh ile vis itin g h e r p h ys icia n , Ms . P re g is te rs a n u n u s u a l co m p la in t: “ It s e e m s th a t m y S in e m e t d o e s n’t w o rk a s w e ll w h e n I ta ke it w ith m e a ls .” Ms . P e xp la in s th a t s h e h a s re ce n tly s ta rte d o n a n e w “ lo w-ca rb ” d ie t th a t h a s in cre a s e d h e r p ro te in in ta ke a t th e e xp e n s e o h ig h -ca rb o h yd ra te o o d s . Co n ce rn e d , Ms . P a s ks , “ Co u ld m y d ie t h a ve a nyth in g to d o w ith th is ?” He r p hys icia n e xp la in s th a t le vo d o p a , a co m p o n e n t o h e r S in e m e t, h e lp s re p la ce a ch e m ica l in h e r b ra in th a t is p ro d u ce d in in s u f cie n t q u a n titie s b e ca u s e o th e lo s s o ce rta in n e u ro n s in h e r b ra in . Alth o u g h m a ny a cto rs co u ld le a d to th e d e cre a s e d e e ctive n e s s o h e r m e d ica tio n , Ms . P’s d o cto r co n f rm s h e r s u s p icio n th a t h e r h ig h -p ro te in d ie t co u ld

divided into the sympathetic nervous system, responsible or “ ght or f ight” responses, and the parasympathetic nervous system, responsible or “rest and digest” responses. The sensory and somatic peripheral nervous system carries sensory signals rom the periphery to the CNS and motor signals rom the CNS to striated muscle; these signals regulate voluntary movement (Fig. 9-1).

Autonomic Nervous System

Autonomic nerve f bers interact with their target organs by a two-neuron pathway. The rst neuron originates in the brainstem or spinal cord and is termed a preganglionic neuron. The preganglionic neuron synapses outside the spinal cord with a postganglionic neuron that innervates the target organ. As discussed below, the anatomical location o these connections di ers or neurons o the sympathetic and parasympathetic divisions o the autonomic nervous system. Anatomy of the Sympathetic Nervous System

The sympathetic nervous system is also known as the thoracolumbar system, because its preganglionic bers arise rom the rst thoracic segment to the second or third lumbar segment o the spinal cord (Fig. 9-2). Speci cally, the preganglionic nerve cell bodies arise rom the intermediolateral columns in the spinal cord. Preganglionic nerves exit the spinal cord at the ventral roots o each vertebral level and make synaptic connections with postganglionic neurons in sympathetic ganglia. Most sympathetic ganglia are located in the sympathetic chain, which consists o 25 pairs o interconnected ganglia that lie on either side o the vertebral column. The rst three ganglia, termed the superior cervical ganglion, middle cervical ganglion, and inferior cervical ganglion, send their postganglionic bers via the cranial and cervical spinal nerves. The superior cervical ganglion innervates the pupil, salivary glands, and lacrimal glands, as well as blood vessels

in d e e d b e in te r e rin g w ith th e m e d ica tio n ’s a b ility to re a ch h e r b ra in . He re co m m e n d s th a t s h e m o d e ra te h e r p ro te in in ta ke , a n d , i n e ce s s a ry, ta ke a h ig h e r d o s e o S in e m e t a te r a h ig h -p ro te in m e a l. At h e r o llo w-u p vis it, Ms . P is h a p p y to re p o rt th a t h e r m e d ica tio n is m o re e e ctive n o w th a t s h e is e a tin g le s s p ro te in .

Questions 1 . Where is the nigrostriatal tract located? How does the degeneration o a specif c group o neurons result in specif c symptoms such as those seen in Parkinson’s disease? 2 . Why is levodopa used in the treatment o Parkinson’s disease, and what is the relationship o this compound to dopamine? 3 . Why does protein consumption inter ere with the action o levodopa? 4 . Why does Sinemet contain both levodopa and carbidopa?

and sweat glands in the head and ace (Fig. 9-2). Postganglionic neurons arising in the middle and in erior cervical ganglia, as well as the thoracic ganglia, innervate the heart and lungs. Fibers arising rom the remaining paravertebral ganglia innervate sweat glands, pilomotor muscles, and blood vessels o skeletal muscle and skin throughout the body. Postganglionic neurons that innervate the GI tract down to the sigmoid colon, including the liver and pancreas, arise rom ganglia that are located anterior to the aorta, at the origins o the celiac, superior mesenteric, and in erior mesenteric blood vessels (Fig. 9-2). Hence, these ganglia, collectively known as prevertebral ganglia , are named the celiac ganglion, superior mesenteric ganglion, and inferior mesenteric ganglion, respectively. In contrast to the paravertebral ganglia, the prevertebral ganglia have long preganglionic bers and short postganglionic bers. The adrenal medulla is contained within the adrenal glands that lie on the superior sur ace o the kidneys. The adrenal medulla contains postsynaptic neuroendocrine cells (Fig. 9-2). Unlike sympathetic postganglionic neurons, which synthesize and release norepinephrine, neuroendocrine cells o the adrenal medulla synthesize primarily epinephrine (85%) and release this neurotransmitter into the bloodstream rather than at synapses on a speci c target organ (see Chapter 11, Adrenergic Pharmacology). Many pharmacologic agents modulate sympathetic nervous system activity. As discussed in Chapter 11, the sympathetic nervous system has an organ-speci c distribution o adrenergic receptor types. This organ-speci c receptor expression allows drugs to modulate sympathetic activity selectively. For example, certain sympathetic agonists, such as albuterol, can dilate bronchioles selectively, while certain sympathetic antagonists, such as metoprolol, can selectively decrease heart rate and contractility.

112 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY Dors a l root ga nglion Dors a l root Gra y ma tte r White ma tte r

S ympa the tic ne rve trunk S pina l cord S kin Ve ntra l root P re ve rte bra l ga nglion S mooth mus cle S e ns ory ne uron

P a ra ve rte bra l cha in ga nglion

S oma tic motor ne uron

S ke le ta l mus cle

P re ga nglionic ne uron P os tga nglionic ne uron

Adre na l me dulla

FIGURE 9-1. Organization of the peripheral nervous system. The peripheral nervous system contains sensory, somatic motor, and autonomic components. Sensory neurons (solid blue line) arise principally in the skin or joints, have cell bodies and nuclei in the dorsal root ganglia, and project onto neurons located in the dorsal horn o the spinal cord. Somatic motor neurons (solid black line) arise in the ventral horn o the spinal cord, exit through the ventral roots, and join f bers o sensory neurons to orm spinal nerves, which then innervate skeletal muscle. The autonomic component o the peripheral nervous system consists o a two-nerve system; the two nerves are called preganglionic and postganglionic neurons, respectively. Sympathetic preganglionic neurons (dashed gray line) arise in the ventral horn o the thoracic and lumbar segments o the spinal cord and project onto postganglionic neurons in the paravertebral and prevertebral ganglia. Sympathetic postganglionic neurons (dashed blue line) innervate many organs, including smooth muscle. The adrenal medulla is also innervated by preganglionic neurons o the sympathetic nervous system (see Fig. 9-2). Parasympathetic preganglionic neurons (not shown) arise in nuclei in the brainstem and the sacral segments o the spinal cord and project onto postganglionic neurons in ganglia located near the innervated organs.

Anatomy of the Parasympathetic Nervous System

Nearly all o the parasympathetic ganglia lie in or near the organs they innervate. The preganglionic bers o the parasympathetic nervous system arise in the brainstem or in sacral segments o the spinal cord; thus, the parasympathetic system is also called the craniosacral system (Fig. 9-2). In some cases, parasympathetic preganglionic neurons can travel almost 1 meter be ore synapsing with their postganglionic targets. Preganglionic nerve bers o cranial nerve (CN) III, the oculomotor nerve , arise rom a region o the midbrain termed the Edinger-Westphal nucleus and innervate the pupil, stimulating it to constrict. The medulla o the brain contains nuclei or parasympathetic nerve bers in CNs VII, IX, and X. Parasympathetic bers in the acial nerve (CN VII) stimulate salivary secretion by the submaxillary and sublingual glands as well as tear production by the lacrimal gland. Parasympathetic bers in the ninth cranial nerve, the glossopharyngeal nerve , stimulate the parotid gland. The 10th cranial nerve, termed the vagus nerve , provides parasympathetic innervation to the major organs in the chest and abdomen, including the heart, tracheobronchial tree, kidneys, and GI system down to the proximal colon. Parasympathetic nerves originating in the sacral region o the spinal cord innervate the remainder o the colon, urinary bladder, and genitalia. Many pharmacologic agents modulate parasympathetic nervous system activity. For example, bethanechol is a parasympathomimetic that promotes GI and urinary tract motility.

Antagonists o parasympathetic activity include atropine , a drug used locally to dilate the pupils or systematically to increase heart rate, and ipratropium, a drug used to dilate bronchioles. These agents and others are discussed in Chapter 10, Cholinergic Pharmacology.

Peripheral Motor and Sensory Systems

Fibers o the somatic nervous system innervate their target striated muscles directly (Fig. 9-1). The rst-order neurons rom the motor cortex send projections that cross in the lower medulla and descend through the spinal cord in the lateral corticospinal tract be ore synapsing on the secondorder neurons in the ventral horns o the spinal cord. Projections rom the second-order neurons exit through the ventral roots and join the dorsal roots , carrying sensory nerve bers, to orm the spinal nerves . Spinal nerves exit the vertebral column through the intervertebral oramina, a ter which they separate into peripheral nerves. Somatic components o the peripheral nerves innervate muscles directly. Muscles are innervated in a myotomal distribution. That is, neurons originating rom a particular ventral root level o the spinal cord (e.g., C6) innervate speci c muscles (e.g., f exor muscles o the orearm). Sensory neurons have cell bodies in the dorsal root ganglia . The endings o sensory nerves lie in the skin and joints and enter the spinal cord through the dorsal roots . Neurons or vibration and position sense (proprioception) ascend through the ipsilateral dorsal columns in the spinal cord and synapse with secondary neurons in the contralateral

C h a p t e r 9 Principles of Nervous System Physiology and Pharmacology 113

Sympathetic nervous system

Parasympathetic nervous system Eye s Oculomotor ne rve (CN III)

S a liva ry gla nds Fa cia l ne rve (CN VII)

Re s pira tory tra ct

Glos s opha rynge a l ne rve (CN IX)

Cra nia l Va gus ne rve (CN X)

C1 C2 C3 C4 C5 C6 C7 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 L1 L2

He a rt Ce rvica l

S kin

Live r

S toma ch

Thora cic Ce lia c ga nglion Adre na l gla nd

P a ncre a s

Lumba r

L3

Bla dde r

L4

S upe rior me s e nte ric ga nglion

L5 S1 S2 S3 S4 S5

Inte s tine s

Kidne y

P e riphe ra l blood ve s s e l

S a cra l

S ympa the tic trunk Infe rior me s e nte ric ga nglion P a ra ve rte bra l ga nglia

P reve rte bra l ga nglia

Exte rna l ge nita lia S ympa the tic pre ga nglionic fibe rs S ympa the tic pos tga nglionic fibe rs P a ra s ympa the tic pre ga nglionic fibe rs P a ra s ympa the tic pos tga nglionic fibe rs

FIGURE 9-2. Patterns of sympathetic and parasympathetic innervation. Sympathetic preganglionic neurons arise in the thoracic and lumbar segments of the spinal cord. Sympathetic preganglionic neurons project onto postganglionic neurons in ganglia that lie close to the spinal cord, most notably the paravertebral ganglia, and in the prevertebral ganglia located near the aorta. Parasympathetic ganglia generally lie close to the organs they innervate. Thus, parasympathetic preganglionic neurons, which arise in nuclei in the brainstem and the sacral segments of the spinal cord, are generally long and project onto short postganglionic neurons.

lower medulla. Sensory neurons that carry sensations of pain and temperature synapse with secondary neurons in the posterior horn of the spinal cord and then cross within the spinal cord to ascend in the contralateral spinothalamic tract. Both the spinothalamic tract and the dorsal column tracts connect with third-order neurons in the thalamus, part of the

diencephalon (see below), before ultimately reaching the somatosensory cortex. Sensory information is encoded in a dermatomal distribution. That is, neurons originating from a particular dorsal root level of the spinal cord (e.g., C6) carry sensory information corresponding to a particular area of the skin (e.g., the lateral aspects of the forearm and hand).

114 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

A number o pharmacologic agents modulate the activity o the somatic nervous system. For example, antagonists o neuromuscular junction activity, such as pancuronium, are used to induce paralysis during surgery. In contrast, drugs that increase neuromuscular junction activity, such as edrophonium and neostigmine , are used in the diagnosis and treatment o myasthenia gravis, an autoimmune disease characterized by decreased skeletal muscle stimulation at the neuromuscular junction. These agents and others are discussed in Chapter 10.

Ce re bra l he mis phe re : Ce re bra l corte x Ba s a l ga nglia

Bra ins te m

Midbra in P ons Me dulla

Anatomy of the Central Nervous System

The cerebral hemispheres constitute the largest division o the human brain. These structures contain several subdivisions, including the cerebral cortex, its underlying white matter, and the basal ganglia (Fig. 9-4). The cerebral hemispheres are divided into le t and right sides that are connected by the corpus callosum. The cerebral cortex is responsible or high-level unctions, including sensory perception, planning and ordering motor unctions, cognitive unctions, such as abstract reasoning, and language. The cortex is divided anatomically and unctionally into the frontal, temporal, parietal, and occipital lobes (Fig. 9-4A). Subregions o the cortex have speci c unctions. For example, stimulation o part o the precentral gyrus, which lies in the rontal cortex, induces peripheral motor unction (movement), and ablation o this structure inhibits movement. From a pharmacologic perspective, the cerebral cortex is a site o action o many drugs, sometimes as part o their intended mechanism o action and sometimes as an adverse e ect. Barbiturates and benzodiazepines (see Chapter 13, Pharmacology o GABAergic and Glutamatergic Neurotransmission) are commonly prescribed hypnotics and sedatives that potentiate the action o inhibitory neurotransmitters in the cortex. General anesthetics (see Chapter 17, General Anesthetic Pharmacology) are also thought to have e ects on the cerebral cortex. The cerebral white matter, which includes the corpus callosum (Fig. 9-4B), transmits signals between the cortex and other areas o the central nervous system and rom one area o the cortex to another. The white matter consists primarily o myelinated axons that, as in other areas o the brain, have an associated vascular network o small arteries, veins, and capillaries. It is around these small vessels that inf ammatory cells collect in diseases such as multiple sclerosis, and it is the small arterioles that are especially a ected by systemic hypertension. The basal ganglia consist o three deep nuclei o gray matter (Fig. 9-4C), including the caudate and putamen— together known as the striatum—and the globus pallidus . In a general sense, these nuclei help initiate and control cortical actions. These actions include not only intended movement but also behavior and certain rudimentary aspects o cognition. Regions o the basal ganglia responsible or movement ensure that intended actions are carried out and irrelevant movements are inhibited. As seen in the

Ce re be llum

Ce rvica l

The CNS is divided anatomically into seven major divisions, namely, the cerebral hemispheres , diencephalon, cerebellum, midbrain, pons , medulla , and spinal cord (Fig. 9-3). The midbrain, pons, and medulla are collectively known as the brainstem and together connect the spinal cord with the cerebrum, diencephalon, and cerebellum.

Cerebrum

Die nce pha lon

S pina l cord Thora cic

Lumba r

S a cra l

FIGURE 9-3. Anatomic organization of the central nervous system. The central nervous system is divided into seven major regions: the cerebral hemispheres, diencephalon (thalamus), cerebellum, midbrain, pons, medulla, and spinal cord. The cerebral hemispheres include the cerebral cortex, underlying white matter (not shown), and basal ganglia. The midbrain, pons, and medulla together make up the brainstem. The spinal cord is further divided into cervical, thoracic, lumbar, and sacral segments.

case o Ms. P, Parkinson’s disease is caused by degeneration o a dopaminergic pathway that arises in the substantia nigra in the midbrain (see below) and terminates in the striatum (hence its name, the nigrostriatal tract or pathway). This degeneration prevents the basal ganglia rom properly initiating motor activity—resulting in decreased intended movement and an unintended tremor—and causes the decreased (“f at”) a ect characteristic o Parkinson’s disease. Levodopa , a component o Ms. P’s Sinemet medication, acts on the striatum to ameliorate these clinical mani estations o the disease (see Chapter 14, Pharmacology o Dopaminergic Neurotransmission).

C h a p t e r 9 Principles o Nervous System Physiology and Pharmacology 115 A Fronta l lobe

P a rie ta l lobe

Te mpora l lobe

available, although many agents a ecting this region o the brain are in development. It should be noted that many drugs o abuse (see Chapter 19, Pharmacology o Drugs o Abuse) stimulate the brain reward pathway, which includes the nucleus accumbens and its projections to the limbic system.

Diencephalon

Occipita l lobe

B Cingula te gyrus

C Ca uda te

Corpus ca llos um

e In t

r n a l c a p s u le

Tha la mus P uta me n

FIGURE 9-4. Anatomy o the cerebral hemispheres. A. In this lateral view, the cerebral hemispheres are divided into our lobes— rontal, parietal, occipital, and temporal—which are structurally and unctionally distinct rom each other. B. A sagittal view o the cerebral hemispheres shows the corpus callosum and cingulate gyrus. The corpus callosum connects the le t and right hemispheres and coordinates their actions. The cingulate gyrus is part o the limbic system; it lies immediately superior to the corpus callosum. C. The basal ganglia include the caudate and putamen, which are together known as the striatum, and the globus pallidus (medial to the putamen, not shown). The thalamus lies medial to the basal ganglia. Arrows indicate the trajectory o neurons in the internal capsule, a bundle o white matter that carries motor commands rom the cortex to the spinal cord.

A rim or “limbus” around the cortex has “older,” more basic unctions and is loosely termed the limbic system. This system consists o the cingulate gyrus (Fig. 9-4B), the hippocampal ormation (including the hippocampus and surrounding structures), and the amygdala . These structures are responsible or emotion, social behavior, autonomic control, the perception o pain, and memory. For example, the memory loss associated with Alzheimer’s disease is caused by degeneration o the hippocampal ormation. Only a ew drugs that specif cally a ect the limbic system are currently

The diencephalon is divided into the thalamus and hypothalamus . The thalamus, which has several distinct nuclei, is located medially in the brain and in erior to the cerebral cortex. Some thalamic nuclei link sensory pathways rom the periphery to the cerebral cortex. Other nuclei act as connections between the basal ganglia and the cortex. The thalamus is not a simple signal relay; rather, it f lters and modulates sensory in ormation, in part dictating which signals reach conscious awareness. The hypothalamus lies ventral to the thalamus. It controls the autonomic nervous system, the pituitary gland, and essential behaviors such as hunger and thermoregulation. Descending pathways rom the medial hypothalamus regulate autonomic preganglionic neurons in the medulla and spinal cord. It is generally believed that the antihypertensive e ect o clonidine is mediated by its action at receptors on brainstem neurons controlled by the hypothalamus (see Chapter 11). Other neurons originating in the medial hypothalamus secrete hormones either directly into the systemic circulation (e.g., vasopressin rom axon terminals in the posterior pituitary gland) or into a portal system that, in turn, controls hormone secretion by the anterior pituitary gland (see Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland). The hypothalamus also initiates complex behaviors in response to hunger, extremes in temperature, thirst, and time o day.

Cerebellum

The cerebellum lies in erior to the posterior end o the cerebrum and dorsal to the brainstem. It has three unctionally distinct regions: the central cerebellar vermis , the lateral cerebellar hemispheres , and the small f occulonodular lobe (Fig. 9-5). The cerebellum has a relatively well-def ned pattern o neural connections, receiving inputs rom a wide

Flocculonodula r lobe

Ce re be lla r ve rmis Ce re be lla r he mis phe re s

FIGURE 9-5. Anatomy o the cerebellum. The cerebellum is divided into the cerebellar hemispheres (laterally), the vermis (medially), and the small f occulonodular node. The area immediately above the f occulonodular lobe in this drawing is a cross section o the cerebellar peduncles.

116 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

variety o sources and sending output primarily to the motor areas o the cerebral cortex via the thalamus. The cerebellum coordinates voluntary movement in space and time, maintains balance, controls eye movement, and has roles in motor learning ( or example, hand–eye coordination) and certain cognitive unctions such as the timing o repetitive events and language. Few drugs are designed primarily to a ect the cerebellum. However, several agents, notably alcohol and certain antiepileptic drugs, are toxic to the cerebellum. These agents especially a ect the vermis, which controls balance.

Gra y ma tte r White ma tte r

Ve ntra l horn Dors a l horn

Dors a l root

Brainstem

The midbrain, pons, and medulla are collectively known as the brainstem. The brainstem connects the spinal cord to the thalamus and cerebral cortex. It is arranged with the midbrain superior, the medulla in erior, and the pons bridging the midbrain and medulla (Fig. 9-3). White matter pathways interconnecting the spinal cord, cerebellum, thalamus, basal ganglia, and cerebral cortex course through this small region o the brain. In addition, the brainstem gives rise to most o the cranial nerves . Some o these nerves are conduits or sensation rom the head and ace, including hearing, balance, and taste. The cranial nerves also control the motor output to the skeletal muscles o mastication, acial expression, swallowing, and eye movement. The brainstem also regulates parasympathetic output to the salivary glands and the iris. The medulla contains several control centers that are essential or li e, including centers that direct the output o the autonomic nuclei, pacemakers that regulate heart rate and breathing, and centers that control ref ex actions such as coughing and vomiting. Several relay structures in the pons also play a role (in conjunction with the midbrain) in regulating vital unctions such as respiration. The base o the pons contains white matter tracts connecting the cerebral cortex and the cerebellum. Neurons in the periaqueductal gray, especially in the midbrain, send descending projections to the spinal cord that modulate pain perception (see Chapter 18, Pharmacology o Analgesia). Clusters o di usely projecting neurons lie throughout the brainstem, hypothalamus, and the surrounding base o the brain. These nuclei, which include the locus ceruleus , raphe nucleus , and several others, comprise the reticular activating system, which is responsible or consciousness and sleep regulation. The nuclei each use a di erent neurotransmitter system (see below), and thus a variety o classes o medications can have e ects on this system. For example, it is via these nuclei that rst-generation antihistamines cause sedation (see Chapter 44, Histamine Pharmacology) and stimulants such as cocaine cause heightened alertness.

Spinal Cord

The spinal cord is the most caudal division o the central nervous system. It runs rom the base o the brainstem (medulla) at the level o the rst cervical vertebra down to the rst lumbar vertebra. Like the cerebrum, the spinal cord is organized into white matter tracts and regions o gray matter. The white matter tracts connect the periphery and spinal cord to more rostral divisions o the CNS, while the gray matter orms the nuclear columns that lie in an H-shaped pattern in the center o the spinal cord (Fig. 9-6). Neurons in the spinal cord can be de ned by their spatial location relative to the gray matter “H.” These neurons include sensory neurons located in the dorsal horns o the “H,”

Dors a l root ga nglion

Dura

FIGURE 9-6. Anatomy of the spinal cord. The spinal cord has an H-shaped wedge of gray matter that includes the dorsal and ventral horns. The dorsal horn is responsible for sensory relays to the brain, and the ventral horn is responsible for motor relays to skeletal muscle. The white matter carries signals to and from more rostral divisions of the CNS.

motor neurons located in the ventral horns o the “H,” and spinal interneurons. The sensory neurons relay in ormation rom the periphery to more rostral divisions o the CNS via the dorsal columns or spinothalamic tracts (see above). The motor neurons relay commands arising in the central motor areas and descend in the corticospinal tract to peripheral muscles. Interneurons connect sensory and motor neurons and are responsible or mediating ref exes, such as the deep tendon ref exes, by coordinating the action o opposing muscle groups. Because the spinal cord carries sensory signals— including sensations o pain—to the central nervous system, it is an important target or analgesic drugs such as opioids (see Chapter 18).

Cellular Organization of the Nervous System Cellular organization in the autonomic and peripheral nervous system involves a limited number o neurons that make ew connections. For example, somatic and sensory in ormation is carried directly between the spinal cord and the periphery. Autonomic nerves are slightly more complex, in that the signal must undergo synaptic transmission between a preganglionic and a postganglionic neuron. In both cases, however, ew ancillary neuronal connections are made, and little or no modi cation o in ormation occurs. In contrast, cellular organization in the central nervous system is ar more complex. In ormation is not simply relayed rom one area to another; instead, central neurons receive signals rom numerous sources and distribute their own axons widely. Some neurons synapse with hundreds o thousands o other neurons. Moreover, not every synaptic connection is excitatory (i.e., designed to depolarize the postsynaptic neuron). Some connections are inhibitory (i.e., designed to hyperpolarize the postsynaptic neuron). Other neurons projecting onto a target neuron can modulate the relative excitability o the cell, a ecting the response o

C h a p t e r 9 Principles of Nervous System Physiology and Pharmacology 117

A Long-tra ct

B Loca l circuit

C S ingle -s ource dive rge nt

Conve rge nt s igna ling

Dive rge nt s igna ling

FIGURE 9-7. Cellular organization of the central nervous system. The CNS has three main organizational motifs. A. Long-tract neurons act as relays between the periphery and higher sites in the CNS. Long-tract neurons receive signals from many different neurons (convergent signaling) and synapse with many downstream neurons (divergent signaling). B. Local circuit neurons show a complicated structural motif, arranged in layers, which includes both excitatory and inhibitory neurons. These circuits are used to process information. C. Single-source divergent neurons typically originate in a nucleus in the brainstem and have axonal terminals that innervate thousands of neurons, usually in the cerebral cortex.

the postsynaptic neuron to other signals. The complexity generated by this variability is needed to carry out the many intricate processes per ormed by the brain. Although the CNS possesses immense complexity at the level o neuronal connectivity, three major moti s are used to organize neurons into unctional units in the nervous system: the long-tract neuronal systems , local circuits , and singlesource divergent systems (Fig. 9-7). The peripheral nervous system is organized exclusively as a long-tract system, while the central nervous system uses all three moti s.

Long-Tract Neuronal Organization

Long-tract neuronal organization involves neural pathways that connect distant areas of the nervous system to one another (Fig. 9-7A). It is the organization used by the peripheral nervous system, and it is important or the transmission o signals rom one region to another within the central nervous system. In the peripheral nervous system, signals are transmitted with little modi cation. Sensory neurons respond to stimuli such as touch, temperature, pressure, vibration, and noxious chemicals and, i the initial membrane depolarization is strong enough, transmit an action potential directly to the spinal cord. There, sensory neurons synapse directly with somatic motor neurons, orming ref ex arcs, and with ascending spinal neurons that transmit the in ormation to higher levels. Motor neurons carry in ormation directly rom the spinal cord out through the ventral roots and project directly on the motor end plates o the muscles they innervate. The long axon tracts o the peripheral sensory and motor neurons are bundled together and travel as peripheral nerves. As described above, preganglionic neurons o the autonomic nervous system orm synaptic connections with postganglionic neurons at ganglia that are located prevertebrally, paravertebrally, or near the innervated visceral organs. One preganglionic neuron typically makes synaptic connections with up to several thousand postganglionic neurons,

an organization that is termed divergent signaling. Although divergent signaling does result in some processing and modi cation o in ormation, the autonomic nervous system does not generally modi y neural signals appreciably. In contrast to neurons in the peripheral pathways, neurons in long-tract systems o the central nervous system not only relay but also integrate and modi y signals. CNS long-tract neurons display divergent signaling like autonomic neurons but also receive synaptic connections rom many upstream neurons (convergent signaling). The CNS uses both excitatory and inhibitory neurotransmitters to localize a signal, a strategy that is known as center-surround signaling. For example, sensory perception in the CNS can precisely localize a signal by activating cortical neurons that map to one area o the body and inhibiting neurons that map to surrounding areas o the body.

Local Circuit Neuronal Organization

Local circuit neurons maintain connectivity primarily within

the immediate area. These neurons are generally responsible or modulating signal transmission (Fig. 9-7B). For example, neurons in the cerebral cortex are organized in layers, usually six in number. While in ormation f ows into one layer and out o a di erent layer through long-tract connections, links between the layers process the signals and interpret the inputs. Local synaptic connections can be both excitatory and inhibitory, ensuring that only certain patterns o inputs are passed along. For example, in ormation originating in the lateral geniculate neurons enters the primary visual cortex through a long-tract connection called the optic tract. In an area o the cortex designed to perceive lines, the outgoing neurons will be excited only i the incoming neurons re in a particular pattern, in this case designating a line in a particular orientation. The outgoing signal might then serve as the input to another area o the brain that recognizes shapes. I this area receives an appropriate pattern o lines rom the appropriate sources, it might recognize a particular object, such as the grid on a tic-tac-toe board.

118 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

Single-Source Divergent Neuronal Organization

Nuclei in the brainstem, hypothalamus, and basal orebrain ollow single-source divergent circuit organization (Fig. 9-7C), in which neurons originating in one nucleus innervate many target cells. Because single-source divergent neuronal organization involves the action o signals on a wide variety o neurons, it is also commonly re erred to as a diffuse system of organization. Instead o stimulating their targets directly, divergent neurons typically exert a modulatory inf uence by using neurotransmitters—generally, biogenic amines (see below)—that act on G protein-coupled receptors. These receptors alter the resting potential and ion channel conductance o the neuronal membranes in which they are located, thereby altering the ease o depolarization o these neurons. Neurons constituting single-source divergent circuits do not generally have myelin sheaths, because their modulatory inf uences vary over the course o minutes or hours rather than ractions o a second. In addition, their axons are highly branched, enabling synaptic connections with a large number o target neurons. The principal single-source divergent neuronal systems are summarized in Table 9-1. They include pigmented dopaminergic neurons that originate in the substantia nigra , widely innervate the striatum, and are responsible or regulating the activity o neurons that control intended actions (Fig. 9-8A). Speci cally, neurons in the nigrostriatal tract excite downstream pathways that initiate movement and inhibit pathways that suppress movement. The nigrostriatal tract degenerates in Parkinson’s disease, which is why Ms. P displayed a paucity o movement. Other dopaminergic neurons medial to the substantia nigra project to the pre rontal cortex and inf uence thought processes. Another example o a single-source divergent circuit involves the noradrenergic nucleus in the pons termed the locus ceruleus (Fig. 9-8B). Neurons originating in this nucleus widely innervate the cerebral cortex and cerebellum

and maintain vigilance and responsiveness to unexpected stimuli. Thus, drugs such as cocaine , which inhibits the reuptake o catecholamines such as norepinephrine, can activate this system and cause hypervigilance (see Chapter 19). Neurons that originate in the raphe nuclei in the caudal brainstem use the neurotransmitter serotonin and are responsible or modulating pain signals in the spinal cord and locus ceruleus (Fig. 9-8B). Other neurons originating in the raphe

A Dopa mine rgic a nd choline rgic pa thwa ys Me dia l s e pta l nucle i

S tria tum

Nucle us ba s a lis Ve ntra l te gme nta l a re a S ubs ta ntia nigra Dopa mine rgic ne urons

P e dunculopontine nucle us Choline rgic ne urons

B Nora dre ne rgic a nd s e rotone rgic pa thwa ys

TABLE 9-1 Single-Source Divergent Neuronal Systems ORIGIN

NEUROTRANSMITTER FUNCTIONS

Substantia nigra (midbrain)

Dopamine

Enable intended movement; executive function; emotional regulation; memory

Locus ceruleus (pons)

Norepinephrine

Vigilance; responsiveness to unexpected stimuli

Raphe nuclei (medulla, pons, and midbrain)

Serotonin

Basal nucleus of Meynert

Acetylcholine

Alertness

Pedunculopontine nucleus

Acetylcholine

Sleep–wake cycles

Tuberomamillary nucleus (hypothalamus)

Histamine

Forebrain arousal

Perception of pain; responsiveness of cortical neurons; mood (?)

Locus ce rule us Ra phe nucle i S pina l cord Nora dre ne rgic ne urons

S e rotone rgic ne urons

FIGURE 9-8. Diffuse neuronal systems. A. Dopaminergic neurons (blue) arise in the substantia nigra and the ventral tegmental area and project to the striatum and the cerebral cortex, respectively. These neurons are associated with the initiation of movement and the brain reward pathways. Cholinergic neurons (red) arise in the nucleus basalis, pedunculopontine nucleus, and medial septal nuclei. These neurons, which project widely throughout the brain, are responsible for maintaining sleep–wake cycles and regulating sensory transmission. B. Noradrenergic neurons (blue) arise in the locus ceruleus and innervate the entire brain. These neurons are responsible for maintaining alertness. Serotonergic neurons (red) arise in the raphe nuclei and project to the diencephalon, to the basal ganglia, and, via the basal forebrain, to the cerebral hemispheres as well as the cerebellum and spinal cord. Serotonergic neurons are believed to have a role in modulating affect and pain.

C h a p t e r 9 Principles of Nervous System Physiology and Pharmacology 119

nuclei widely innervate the orebrain, modulating the responsiveness o neurons in the cortex. Serotonergic neurons regulate wake ulness and sleep, and dys unction o the serotonergic system is hypothesized to be a cause o depression. Because antidepressants block the reuptake o serotonin, this class o drugs may activate the serotonergic raphe pathway (see Chapter 15, Pharmacology o Serotonergic and Central Adrenergic Neurotransmission). Three other important nuclei that widely innervate the cortex are the basal nucleus of Meynert, the pedunculopontine nucleus , and the tuberomamillary nucleus . The basal nucleus and the pedunculopontine nucleus use acetylcholine as a neurotransmitter (Fig. 9-8A). The ormer nucleus projects to the cortex and regulates alertness, while the latter nucleus controls sleep–wake cycles and arousal. Cells in the basal orebrain that receive inputs rom the pedunculopontine nucleus degenerate in several diseases, including Alzheimer’s disease. The tuberomamillary nucleus uses the neurotransmitter histamine (see below) and may help maintain arousal through its actions on the orebrain. The somnolence induced by f rst-generation antihistamines—histamine H1 receptor

A Sympathetic

P re ga nglionic ne uron

antagonists used to treat allergies (see Chapter 44)—may be caused by inhibition o transmission involving tuberomamillary nucleus neurons.

NEUROPHYSIOLOGY Neurotransmitters The peripheral nervous system uses only two neurotransmitters: acetylcholine and norepinephrine (Fig. 9-9). In contrast, the CNS uses not only a wide variety o small molecule neurotransmitters, including acetylcholine and norepinephrine (Table 9-2), but also many neuroactive peptides . These peptides may be transmitted concurrently with the small molecule neurotransmitters, and they generally have a neuromodulatory role. The small molecule neurotransmitters can be organized into several broad categories based on both their structure and unction (Fig. 9-10). The f rst category, the amino acid neurotransmitters, includes glutamate , aspartate , GABA, and glycine . The biogenic amine neurotransmitters, which are

B Parasympathetic

C Somatic

Ace tylcholine Nicotinic re ce ptors

Ace tylcholine Nicotinic re ce ptors P os tga nglionic ne uron

Nore pine phrine or Ace tylcholine Tis s ue re ce ptor

Adre ne rgic Mus ca rinic (s we a t gla nds )

Ace tylcholine

Mus ca rinic

Ace tylcholine

Nicotinic

FIGURE 9-9. Neurotransmitters in the peripheral nervous system (A-C). Only two neurotransmitters are required to mediate neurotransmission in the peripheral nervous system. Acetylcholine is released by sympathetic and parasympathetic preganglionic neurons, parasympathetic postganglionic neurons, somatic motor neurons, and sympathetic postganglionic neurons that innervate sweat glands. All other sympathetic postganglionic neurons release norepinephrine. Acetylcholine stimulates nicotinic acetylcholine receptors on sympathetic and parasympathetic postganglionic neurons and at the neuromuscular junction. Acetylcholine stimulates muscarinic acetylcholine receptors on sweat glands and on tissues innervated by parasympathetic postganglionic neurons. Norepinephrine stimulates - and -adrenergic receptors on tissues (except for sweat glands) innervated by sympathetic postganglionic neurons.

120 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

TABLE 9-2 Small Molecule Neurotransmitters in the Central Nervous System NEUROTRANSMITTER

RECEPTOR SUBTYPE

RECEPTOR MOTIF

MECHANISM

GABA

GABAA GABAB

Ionotropic Metabotropic

↓ cAMP ↑ Cl conductance ↑ K , Cl conductance

Ionotropic

↑ Cl conductance

Glycine

,

Subunits

Glutamate, Aspartate

AMPA Kainate NMDA mGlu (1–7)

Ionotropic Ionotropic Ionotropic Metabotropic

↑ ↑ ↑ ↓ ↑

Dopamine

D1, D5 D2, D3, D4

Metabotropic Metabotropic

↑ cAMP ↓ cAMP; ↑ K , ↓ Ca 2 conductance

Metabotropic Metabotropic Metabotropic

↑ IP3/DAG/Ca 2 ↓ cAMP; ↑ K , ↓ Ca 2 conductance ↑ cAMP

Norepinephrine

1 2 1,

2,

3

Na , K conductance Na , K conductance Na , K , Ca 2 conductance cAMP IP3/DAG/Ca 2

Serotonin

5-HT1 5-HT2 5-HT3 5-HT4–7

Metabotropic Metabotropic Ionotropic Metabotropic

↓ ↑ ↑ ↑

Histamine

H1 H2 H3

Metabotropic Metabotropic Metabotropic

↑ IP3/DAG/Ca 2 ↑ cAMP ↓ cAMP

Acetylcholine

Nicotinic Muscarinic

Ionotropic Metabotropic

↑ Na , K , Ca 2 conductance ↑ IP3/DAG/Ca 2 ↓ cAMP; ↑ K conductance

Adenosine

P1 P2X P2Y

Metabotropic Ionotropic Metabotropic

↓ cAMP; ↓ Ca 2 , ↑ K conductance ↑ Ca 2 , K , Na conductance ↑ IP3/DAG/Ca 2

cAMP; ↑ K conductance IP3/DAG/Ca 2 Na , K conductance cAMP

Neurotransmitters can be organized into several categories, including the amino acids, biogenic amines, acetylcholine, adenosine, and nitric oxide (NO). Each neurotransmitter can bind to one or more receptors. Except or the NO receptor, which is intracellular (not shown), the other small molecule receptors are all at the cell sur ace. These cell sur ace receptors may be ionotropic or metabotropic. The mechanism o action o each receptor is indicated. In addition to the small molecule neurotransmitters, more than 50 neuroactive peptides have been identif ed. AMPA, kainate, and NMDA receptors are named a ter agonists that selectively activate them. AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate; cAMP, cyclic adenosine-3′,5′-monophosphate; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate.

derived from decarboxylated amino acids, include dopamine , norepinephrine , epinephrine , serotonin, and histamine . Acetylcholine , which is neither an amino acid nor a biogenic amine, is used as a neurotransmitter in both the CNS and the peripheral nervous system. The purines adenosine and adenosine triphosphate (ATP) are also used in central neurotransmission, although their roles have not been studied in as much detail as those of other neurotransmitters. The lipid-soluble gas nitric oxide (NO), which has many effects in peripheral tissues, has recently been shown to act as a diffusible neurotransmitter in the CNS.

Amino Acid Neurotransmitters

The amino acid neurotransmitters are the primary excitatory and inhibitory neurotransmitters in the CNS. Two types of amino acid neurotransmitters are used: the acidic amino acids glutamate and aspartate, which are primarily excitatory, and the neutral amino acids GABA and glycine, which are primarily inhibitory. Glutamate, aspartate, and glycine are all alpha-amino acids that are also building blocks for

protein synthesis. Glutamate is the primary excitatory neurotransmitter; it acts on both ionotropic (ligand-gated ion channels) and metabotropic (G protein-coupled) receptors (see Chapter 13). Excessive excitation of certain glutamate receptors is one of the mechanisms by which ischemic injury causes neuronal death. For this reason, glutamate receptors are a major target for pharmaceutical research. To date, however, there are few therapeutic agents in clinical use that bind selectively to glutamate receptors. Felbamate , used in the treatment of refractory epilepsy, inhibits the NMDA glutamate receptor, thereby reducing the excessive neuronal activity associated with seizures. Unfortunately, its use is limited by adverse effects such as bone marrow suppression and liver failure (see Chapter 16, Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System). GABA, which is also discussed in Chapter 13, is the primary inhibitory neurotransmitter in the CNS. Several classes of therapeutic agents, most notably the barbiturates and benzodiazepines, bind to GABA receptors and, by allosteric mechanisms, potentiate the effect of endogenous GABA.

C h a p t e r 9 Principles of Nervous System Physiology and Pharmacology 121

FIGURE 9-10. Structures of the small molecule neurotransmitters. The

Amino Acid Neurotransmitters

principal small molecule neurotransmitters can be divided into two broad categories. Amino acids are the primary excitatory (glutamate and aspartate) and inhibitory (glycine and -aminobutyric acid) neurotransmitters in the CNS. Their amino and carboxylic acid groups are shown in blue. Biogenic amines are the primary modulatory neurotransmitters in the CNS. The amine moiety is shown in blue. Dopamine, norepinephrine, and epinephrine share a common catechol group; histamine has an imidazole group; and serotonin has an indole group. Acetylcholine (a neurotransmitter in diffuse modulatory systems in the CNS), adenosine, and nitric oxide (NO) do not fall into either structural category. The bond order is 2.5 for the nitrogen–oxygen bond in NO, intermediate in strength between a double bond and a triple bond.

O

O

H2 N

H2 N

OH

OH

HO O

HO

As pa rtic a cid

O Gluta mic a cid

O

O H2 N

H2 N

OH Glycine

Biogenic Amines

OH

γ-Aminobutyric a cid (GABA)

Biogenic Amine Neurotransmitters OH HO

NH2

HO

H N

HO

HO Dopa mine

Epine phrine

OH HO

NH2

HO Nore pine phrine

NH2

HN

NH2

N

OH

HN His ta mine

S e rotonin

Other Neurotransmitters O

N H2 N

N N

N

N+

O Ace tylcholine

O OH

HO Ade nos ine

OH

NO Nitric oxide

The biogenic amines (along with acetylcholine) are used by the di use neuronal systems to modulate complex central nervous system unctions such as alertness and consciousness. In the peripheral nervous system, norepinephrine is released by sympathetic postganglionic neurons to e ect the sympathetic response. The adrenal medulla is a neuroendocrine tissue that releases the biogenic amine epinephrine into the circulation in response to stress. The biogenic amines are all synthesized rom amino acid precursors. Based on these precursors, the biogenic amines can be divided into three categories. The catecholamines (dopamine, norepinephrine, and epinephrine) are derivatives o tyrosine. The indoleamine serotonin is synthesized rom tryptophan. Histamine is ormed rom histidine. These three categories are described brief y below. The catecholamines are all derived rom tyrosine in a series o biochemical reactions (Fig. 9-11). First, tyrosine is oxidized to L-dihydroxyphenylalanine (L-DOPA). L-DOPA is then decarboxylated to dopamine. In the case o Ms. P, L-DOPA (levodopa) is one o the components o the medication used to compensate or the loss o dopaminergic neurons in the substantia nigra. (Dopamine is not an e ective therapeutic or Parkinson’s disease because it does not cross the blood–brain barrier; see below.) Central dopaminergic receptors have been the target o a wide variety o therapeutics. For example, both dopamine precursors and direct dopamine receptor agonists are used in the treatment o Parkinson’s disease, as discussed in Chapter 14. Dopamine receptor antagonists have been used with success in treating the psychotic symptoms o schizophrenia; this topic is also discussed in Chapter 14. Certain drugs o abuse, such as cocaine and the amphetamines, can activate brain reward pathways that depend on dopaminergic neurotransmission, as discussed in Chapter 19. Dopamine is synthesized rom tyrosine and L-DOPA in the cytoplasm but is then transported into synaptic vesicles. In dopaminergic neurons, the dopamine contained in synaptic vesicles is released as the neurotransmitter. In adrenergic and noradrenergic neurons, dopamine is converted to norepinephrine within the synaptic vesicles by the enzyme dopamine- hydroxylase . In a small number o neurons and in the adrenal medulla, norepinephrine is then transported back into the cytoplasm, where it is methylated to orm epinephrine. Chapter 11 discusses the pharmacology o drugs that target peripheral adrenergic receptors, including both agonists, such as bronchodilators and vasopressors, and antagonists, such as antihypertensives. Several classes o therapeutic agents act on central adrenergic receptors. Clonidine is a partial agonist that acts on presynaptic 2-receptors. Some antidepressants increase the synaptic concentration o norepinephrine by

122 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

O OH NH2

HO Tyros ine Te tra hydrobiopte rin O 2 , Fe 2+

Tyrosine hydroxylase (TH)

O HO

OH NH2

HO L-DOPA

Aromatic L-amino acid decarboxylase

P yridoxa l phos pha te

HO

blocking its reuptake (tricyclic antidepressants [TCAs] and serotonin-norepinephrine reuptake inhibitors [SNRIs]), while others increase the intracellular pool of norepinephrine available for synaptic release by inhibiting its chemical degradation (monoamine oxidase inhibitors [MAOIs]). 5-Hydroxytryptamine (5-HT, also known as serotonin) is formed from the amino acid tryptophan by enzymatic oxidation at the 5 position followed by enzymatic decarboxylation. This sequence of reactions is similar to that used in the synthesis of dopamine, although the enzymes that carry out the reactions are different (Fig. 9-12). Several classes of drugs target serotonergic neurotransmission. TCAs and SNRIs, which block norepinephrine reuptake, also block serotonin reuptake. Selective serotonin reuptake inhibitors (SSRIs), which act more selectively on serotonin reuptake transporters, are also used to treat depression. The role of serotonergic neurons in depression and the various therapies for depression that target serotonergic neurotransmission are discussed in more detail in Chapter 15. Histamine is formed by decarboxylation of the amino acid histidine. Histamine functions as a diffuse neurotransmitter in

NH2

HO

O HN

OH

Dopa mine As corbic a cid O 2 , Cu 2+

NH2

Dopamine ß-hydroxylase Tryptopha n

OH HO

Tryptophan hydroxylase (TPH)

NH2

O

HO HN

Nore pine phrine

OH NH2

Phenylethanolamine N-methyltransferase

S -a de nos ylme thionine

OH

OH H N

HO

5-Hydroxytryptopha n

Aromatic L-amino acid decarboxylase

HO Epine phrine

FIGURE 9-11. Synthesis of catecholamines. Catecholamines are all synthesized rom tyrosine. Sequential enzymatic reactions result in hydroxylation o tyrosine to orm L-DOPA, decarboxylation o L-DOPA to orm dopamine, hydroxylation o dopamine to orm norepinephrine, and methylation o norepinephrine to orm epinephrine. Depending on the enzymes (shown in blue lettering) expressed in a particular type o presynaptic neuron, the reaction sequence may stop at any o the last three steps, so that dopamine, norepinephrine, or epinephrine can be the f nal product that is synthesized and used as a neurotransmitter.

NH2

HN

OH 5-Hydroxytrypta mine (S e rotonin)

FIGURE 9-12. Synthesis of 5-hydroxytryptamine (serotonin). Tryptophan is f rst oxidized by tryptophan hydroxylase (TPH) and then decarboxylated by aromatic L-amino acid decarboxylase to yield serotonin.

C h a p t e r 9 Principles of Nervous System Physiology and Pharmacology 123

the CNS; it also has a particular role in the maintenance o arousal via the tuberomamillary nucleus o the hypothalamus and in the sensation o nausea via the area postrema in the f oor o the ourth ventricle. Few therapeutics intentionally target central histaminergic neurotransmission. Instead, most drugs in this class act on peripheral histamine H1 receptors, at which histamine mediates the inf ammatory response to allergic stimuli, or on H2 receptors in the treatment o peptic ulcer disease (see Chapters 44 and 47, Integrative Inf ammation Pharmacology: Peptic Ulcer Disease). Peripherally acting antihistamines are sometimes used to e ect sedation or as antiemetics, acting via the central neuroanatomic substrates noted above.

insulins , and gastrins . Neuropeptides also include the pituitary hormone release and inhibiting actors, including corticotropin-releasing hormone (CRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), growth hormone-releasing hormone (GRH), and somatostatin. The opioid peptide amily includes the enkephalins , dynorphins , and endorphins . Opioid receptors, which are distributed

Other Small Molecule Neurotransmitters

In the case o Ms. P, L-DOPA, the immediate precursor o dopamine, is administered rather than the neurotransmitter itsel . Unlike L-DOPA, which is able to cross rom the blood to the brain tissue where it acts to treat Ms. P’s Parkinson’s disease, dopamine is unable to cross that boundary. The reason or this exclusion is the existence o a selective lter, termed the blood–brain barrier, which regulates the transport o many molecules rom the blood into the brain (Fig. 9-13). The blood–brain barrier protects the brain tissue both rom toxic substances that circulate in the blood and rom neurotransmitters such as epinephrine, norepinephrine, glutamate, and dopamine that have systemic e ects in body tissues but that would bind receptors in the CNS and cause undesirable e ects i access were permitted. The structural basis or the blood–brain barrier resides in the unique design o the cerebral microcirculation. In most tissues, there are small gaps, called fenestrae , between the endothelial cells that line the microvasculature. These gaps allow water and small molecules to di use across the lining without resistance but lter out large proteins and cells. In the CNS, the endothelial cells orm tight junctions that prevent di usion o small molecules across the vessel wall. Also, unlike peripheral endothelial cells, CNS endothelial cells do not generally have pinocytotic vesicles that transport f uid rom the blood vessel lumen to the extracellular space. In addition, blood vessels in the CNS are covered by cellular processes derived rom astroglia . These processes play an important role in selectively transporting certain nutrients rom the blood to central neurons. In the absence o a selective transport mechanism, the blood–brain barrier generally excludes water-soluble substances. In contrast, lipophilic substances, including important lipid-soluble gases such as oxygen and carbon dioxide, can usually di use across the endothelial membranes. The oil/water partition coe cient is a good indicator o the ease with which a small molecule can enter the CNS. Lipophilic substances with high oil/water partition coe cients can generally di use across the blood–brain barrier, whereas hydrophilic substances with low oil/water partition coe cients are typically excluded (Fig. 9-14). Many important hydrophilic nutrients, such as glucose and a number o amino acids, would not be able to cross the blood–brain barrier without the existence o speci c transporters. Glucose, or example, is transported across the barrier by a hexose transporter that allows this nutrient to move down its concentration gradient in a process called facilitated diffusion. Amino acids are transported by three di erent transporters: one or large neutral amino acids such as valine and phenylalanine; one or smaller neutral amino acids and polar amino acids, such as glycine and

Acetylcholine plays a major role in peripheral neurotransmission. At the neuromuscular junction, this molecule is used by somatic motor neurons to depolarize striated muscle. In the autonomic nervous system, acetylcholine is the neurotransmitter used by all preganglionic neurons and by parasympathetic postganglionic neurons. The multiple unctions o acetylcholine in the peripheral nervous system have spurred the development o a wide range o drugs that target peripheral cholinergic neurotransmission. These include muscle paralytics, which inter ere with neurotransmission at the motor end plate, acetylcholinesterase inhibitors, which increase local acetylcholine concentration by inter ering with the metabolic breakdown o the neurotransmitter, and receptor-speci c agonists and antagonists. In the CNS, acetylcholine acts as a di use-system neurotransmitter. Like the biogenic amines, it is thought to regulate sleep and wake ulness. Donepezil, a reversible acetylcholinesterase inhibitor that acts at central cholinergic synapses, helps to “brighten” patients with dementia (see Chapter 10). Peripheral anticholinergic agents may cause central cholinergic blockade and thereby result in major adverse e ects. For example, the antimuscarinic drug scopolamine can cause drowsiness, amnesia, atigue, and dreamless sleep. In contrast, cholinergic agonists such as pilocarpine can induce adverse e ects o cortical arousal and alertness. The purinergic neurotransmitters adenosine and adenosine triphosphate have a role in central neurotransmission. This role is most evident in the e ects o caffeine , which is a competitive antagonist at adenosine receptors and causes a mild stimulant e ect. In this case, the adenosine receptors, which are located on presynaptic noradrenergic neurons, act to inhibit the release o norepinephrine. Antagonism o these adenosine receptors by ca eine causes the release o norepinephrine to be disinhibited, which results in the characteristic stimulatory e ects o the drug. Nitric oxide (NO), which has generated signi cant interest as a peripheral vasodilator, acts in the brain as a neurotransmitter. Unlike the other small molecule neurotransmitters, NO di uses through the neuronal membrane and binds to its receptors within the target cell. Receptors or NO are thought to reside in presynaptic neurons, allowing NO to act as a retrograde messenger. While many therapeutics target the peripheral vasodilatory e ects o NO, none as o yet target its actions as a central neurotransmitter.

Neuropeptides

The neuroactive peptides are the last major class o neurotransmitters. Many neuropeptides also have endocrine, autocrine, and paracrine actions. Major examples o neuroactive peptide amilies are the opioids , tachykinins , secretins ,

widely in areas o the spinal cord and brain that are involved in pain sensation, are the principal pharmacologic targets o opioid analgesics such as morphine (see Chapter 18) and o some drugs o abuse such as heroin (see Chapter 19).

The Blood–Brain Barrier

124 FUNDAMENTAL PRINCIPLES OF NEUROPHARMACOLOGY

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Dia ze pa m

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o

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Peripheral capillary

Chlora mphe nicol

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o

n

Glucos e

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L-DOPA

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P he nytoin

p

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P he noba rbita l

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Fe ne s tra

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i

v

Dopa mine

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Me thotre xa te Ma nnitol

0.01

P inocytotic ve s icle s

Endothe lia l ce ll

S odium

0.001

Morphine P e nicillin

0.01

0.1

1.0

10

100

Oil/wa te r pa rtition coe fficie nt

FIGURE 9-14. Relative ability of compounds to enter the brain from the Brain capillary

P e ricyte

As troglia l proce s s Ba s e me nt me mbra ne Mitochondria Tight junction

FIGURE 9-13. Features of capillaries in the central nervous system compared to the peripheral vasculature. In the periphery, capillary endothelial cells have gaps (termed fenestrae) between them and use intracellular pinocytotic vesicles to acilitate the transcapillary transport o uid and soluble molecules. In contrast, CNS vessels are sealed by tight junctions between the endothelial cells. The cells have ewer pinocytotic vesicles and are surrounded by pericytes and astroglial processes. In addition, capillary endothelial cells in the CNS have more mitochondria than those in systemic vessels; these mitochondria may re ect the energy requirements necessary or CNS endothelial cells to transport certain molecules into the CNS and transport other molecules out o the CNS.

glutamate, respectively; and one or alanine, serine, and cysteine. L-DOPA is transported by the large neutral amino acid transporter, but dopamine itsel is excluded by the blood– brain barrier. For this reason, L-DOPA is administered in lieu o dopamine to patients with Parkinson’s disease. A ter meals with a high protein content, however, the transporter can become overwhelmed, and its transport o L-DOPA can become ine ective. This explains Ms. P’s complaint that her medication was less e ective a ter she began a diet high in protein. The blood–brain barrier also contains a number o ion channels, which ensure that ion concentrations in the brain are maintained at homeostatic levels. Just as certain vital hydrophilic nutrients are allowed access to the brain tissue via particular transporters, many potentially toxic lipophilic compounds can be excluded rom the

blood. In general, there is a correlation between the oil/water partition coe f cient o a compound and its ability to enter the brain rom the systemic circulation. Specif c transporters acilitate the entry into the brain o certain compounds (squares), such as glucose (glucose transporter) and L-DOPA (large neutral L-amino acid transporter). Transporters also pump certain compounds out o the CNS (diamonds), such as phenobarbital and phenytoin. The metabolic blood–brain barrier, consisting o a number o drugmetabolizing enzymes, also limits the CNS concentration o certain drugs.

brain by a class o proteins known as multiple drug resistance (MDR) transporters . These transporters pump hydrophobic compounds out o the brain and back into the blood vessel lumen. (Note that MDR transporters are present in many cell types, playing an important role in processes such as the resistance o tumor cells to chemotherapeutic agents; see Chapter 5, Drug Transporters.) A metabolic blood–brain barrier adds a layer o protection against toxic compounds; this barrier is maintained by enzymes that metabolize compounds transported into CNS endothelial cells. One such enzyme, aromatic L-amino acid decarboxylase (sometimes called DOPA decarboxylase ), metabolizes peripheral L-DOPA to dopamine, which is unable to cross the blood–brain barrier. For this reason, Ms. P’s medication includes a second component, carbidopa , which is an inhibitor o DOPA decarboxylase. Carbidopa ensures that L-DOPA is not metabolized to dopamine peripherally be ore crossing the blood–brain barrier. Importantly, carbidopa itsel is unable to cross the blood–brain barrier and, there ore, does not inter ere with the conversion o L-DOPA to dopamine in the CNS.

CONCLUSION AND FUTURE DIRECTIONS This chapter discusses the anatomical organization o the peripheral and central nervous systems, the transmission and processing o electrical and chemical signals by neurons, the principal neurotransmitters used by CNS neurons, and the structure and unction o the blood–brain barrier. Although this chapter introduces some specif c drugs as examples, the ocus is on the general principles o anatomy and neurotransmission that are important or understanding the action o all pharmacologic agents a ecting the nervous system. The remaining chapters in this section discuss specif c

C h a p t e r 9 Principles of Nervous System Physiology and Pharmacology 125

neurotransmitter systems and specif c agents that act on the peripheral and central nervous systems. Thus, Chapters 10 and 11 describe peripheral cholinergic and adrenergic systems, and Chapter 12, Local Anesthetic Pharmacology, discusses the production o local anesthesia by inhibition o electrical transmission through peripheral and spinal neurons. Chapter 13 describes central excitatory and inhibitory neurotransmission. Although ew therapeutics currently take advantage o glutamatergic neurotransmission, two major classes o drugs, the benzodiazepines and the barbiturates, a ect GABAergic neurotransmission by potentiating the e ect o GABA at the GABAA receptor. Chapter 14 discusses dopaminergic systems, describing in more detail the concept, introduced in the present chapter, that some o the symptoms o Parkinson’s disease can be alleviated by drugs that increase dopaminergic transmission. Chapter 14 also explains how inhibiting dopaminergic transmission can alleviate some o the symptoms o schizophrenia, implying that dopamine may play a role in this disease. Chapter 15 discusses drugs that modi y a ect, the outward mani estations o mood. These agents include antidepressants, which block reuptake or inhibit metabolism

o the biogenic amines norepinephrine and serotonin, as well as the “mood stabilizer” lithium, which is thought to a ect a signal transduction pathway. Chapter 16 explores the pharmacology o abnormal electrical neurotransmission, including the action o channel blockers, such as phenytoin, which block the propagation o action potentials and thereby inhibit many types o seizures. Chapter 17 describes the pharmacology o general anesthetics, agents whose mechanism o action remains an area o active investigation. Chapter 18 discusses the pharmacology o analgesia, including opioid receptor agonists and nonopioid analgesics. To conclude, Chapter 19 ocuses on the pharmacology o drugs o abuse.

Suggested Reading Blumen eld H. Neuroanatomy through clinical cases. 2nd ed. Sunderland, MA: Sinauer Associates, Inc.; 2010. (Thorough review of human neuroanatomy with an emphasis on clinical correlation; includes many exemplary clinical cases.) Squire LR, Berg D, Bloom F, du Lac S, Ghosh A, Spitzer NC, eds. Fundamental neuroscience. 4th ed. Waltham, MA: Academic Press; 2013. (Comprehensive textbook containing detailed information on human neuroanatomy and neurophysiology.)

IIB Principles of Autonomic and Peripheral Nervous System Pharmacology

Naa + Choliine

Ch o line rg ic n e uro n Cho AcCoA + C hol holine in nnee

He micc ho h o liiniu nium m

Cho line Cholin acetyl tyltra trans ns ffeeeras r e

Ve s amic o l V

10

ACh h

Ca lciu lcium m cha nne l ch Ca 2+

LEMS (a utoo a ntibodd y))

Ca lciuu m cha nne l

Cholinergic Pharmacology Ca 2+ C

A h H+ AC

Alire za

Bo tulinum toxin At ri, M ich a e l

S. Ch a n g , a n d Ga ry R. S t rich a rt z

ACh AC

INTRODUCTION CASE 127– 7 12 1288 PH HARMA H MANicotinic CACh HARMACOLOGIC CLASSES S AN ND AGENTS . . . . . . . . . . . . . . . . 137 Mus ca rinic r& inic ini c ACh ACh re ce c e pto p tor. . . . . . . . . . . . . . . . . . . . . 12 pt ptor ACh re ce ptor (MD MHYSIOLOGY 2, P 4) Inhibitors of Acetylcholine Syntthesi In he sis, s, ACh AC h BIOCHEMISTRY AND OF F Storage, o Choline and+Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1377 CHOLINERGIC NEUROT TRA RANSMISSION . . . . . . . . . . . . . . . . . . . . 127 a ce c taa ttee S ynaptic c cle ft Acet cet etyl ylch c olinesterase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 13 137 Synthesis of Acetylcholiine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 12 Structur St Stru truc urA allChE Class Stor St orag agee a ndd Release of Acee ty tylcholine oline oli liinnee . . . . . . . . . . . . . . . . . . . 1288 AChE hEssess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 hE AChE Clinical Cli i Applications Inhibito rs rs s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Cholinergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Ac etylcholineste ras A rase Rec R Re eceptor eecep cep Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 139 Muscarinic Rec ecep epto tors rs.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Musca uscar usca carinic Receptor Agonistss . . . . . . . . . . . . . . . . . . . . . . . 13 car 1399 Nicotinic Receptors. . . . . . . . . . . . . . . . . . . . . . . . . .N Nicoti Nic Ni icotinic cot otinic nic 130 Nico Ni coti tini niic Re nic Rece cept ptor pt or Ago goni nist stss . . . . . . . . . . . . . . . . . . . . . . . . . 140 Degradation of Acetylcholine nticc . . .Mus . . . .ca. .rinic . . . . . . . .A .Ch re ce ppt 1331 rinic ACh ptor or P Po o s ts ynap ynaptic pin pt Rece Re cept ptor pt orr Annt ntag agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1411 A ACh Ch re ce pt p tor ptor Physiologic Effects Transmission . . . . . . . . . . 13 1322 c e llllof Cholinergic riinnic Receptor Anttago goni nist stss . . . . . . . . . . . . . . . . . . . . 141 Neuromuscular J unction onn M . .1., .M. 3. ,. M . .5. . M . .2., .M . .4 . . . . . . . . . . . 132 + + Muscari nne l Ope ns N Na /K chaaNicotini nic ni icc Receptor Antagonistss . . . . . . . . . . . . . . . . . . . . . . 143 Autonomic Effectss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Gq Gi CNS Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 CONCLUSIO IO ON AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 144 ON Ex Excita tor tory y The Non-Neuronal Cholinerg erg rgi giic ggic System ((NNCS) N . . . . . . . . . 136 Su P LC AC, AC , Sugg gges este tted te e d Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 1444 K+ h

l

INTRODUCTION Cholinergic pharmacology is centered on the properties o the f rst identif ed neurotransmitter, acetylcholine (ACh). The unctions o cholinergic pathways are diverse; generally, they involve the neuromuscular junction (NMJ), the autonomic nervous system, the central nervous system (CNS), and the non-neuronal cholinergic system (NNCS). In the neuronal cholinergic systems, ACh acts as a neurotransmitter at the NMJ, at autonomic ganglia, at terminal synapses o parasympathetic postganglionic f bers and a ew sympathetic postganglionic f bers, and in the CNS. Many non-neuronal cells also express ACh receptors and can thereby serve as e ector cells or both neuronally and non-neuronally released ACh. Despite the many physiologic actions o ACh, the current therapeutic uses or cholinergic and anticholinergic drugs are limited by the ubiquitous nature o cholinergic pathways, and thus, by the inherent di f culty o e ecting a specif c pharmacologic intervention without inducing adverse e ects. Nonetheless, medications with somewhat targeted cholinomimetic and anticholinergic actions are in widespread clinical use or their e ects on the brain (especially cognition and behavior), neuromuscular junction, heart, eyes, lungs, and genitourinary and gastrointestinal tracts. Other chapters that discuss applications o cholinergic pharmacology are Chapter 18, Pharmacology o Analgesia;

Chapter 47, Integrative In ammation Pharmacology: Peptic Ulcer Disease; and Chapter 48, Integrative In ammation Pharmacology: Asthma.

BIOCHEMISTRY AND PHYSIOLOGY OF CHOLINERGIC NEUROTRANSMISSION Acetylcholine synthesis, storage, and release ollow a similar set o steps in all cholinergic neurons. The specif c e ects o ACh at a particular cholinergic synapse are largely determined by the ACh receptor type at that synapse. Cholinergic receptors are divided into two broad classes. Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors that are expressed at the terminal synapses o all parasympathetic postganglionic f bers and a ew sympathetic postganglionic f bers, at autonomic ganglia, and in the CNS. Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels that are concentrated postsynaptically at many excitatory autonomic synapses and presynaptically in the CNS. Acetylcholinesterase (AChE), the enzyme responsible or acetylcholine degradation, is also an important pharmacologic target. In this section, the biochemistry o each o these pharmacologic targets is described and the physiologic e ects o acetylcholine at the neuromuscular junction, in the autonomic nervous system, in the CNS, and in the nonneuronal cholinergic system are discussed.

127

128 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

Th e ye a r is 1744. Virg in ia n s e ttle rs ca p tu re Ch ie Op e ch a n ca n o u g h , w a rrio r ch ie o th e Po w h a ta n s a n d u n cle to Po ca h o n ta s . Op e ch a n ca n o u g h is co n s id e re d a m a s te r ta cticia n a n d h a s a re p u ta tio n a s a b ru ta l wa rrio r. On e co lo n ia l co rre s p o n d e n t p o rtra ys a d i e re n t p ictu re o th e ca p tu re d ch ie , h o w e ve r: “ Th e e xce s s ive a tig u e s h e e n co u n te re d w re cke d h is co n s titu tio n ; h is f e s h b e ca m e m a ce ra te d ; h is s in e w s lo s t th e ir to n e a n d e la s ticity a n d h is e ye lid s w e re s o h e a vy th a t h e co u ld n o t s e e u n le s s th e y w e re li te d u p b y h is a tte n d a n ts . . . h e wa s u n a b le to wa lk; b u t h is s p irit, ris in g a b ove th e ru in s o h is b o d y, d ire cte d [h is o llo w e rs ] ro m th e litte r o n w h ich h e wa s ca rrie d b y h is In d ia n s .” Du rin g th e co n n e m e n t o Op e ch a n ca n o u g h to a p ris o n in J a m e s to w n , it is d is co ve re d th a t, a te r a p e rio d o in a ctivity, h e is a b le to ra is e h im s e l ro m th e g ro u n d to a s ta n d in g p o s itio n . It is th o u g h t th a t th e s to ry o Op e ch a n ca n o u g h p ro vid e s th e e a rlie s t re co rd e d d e s crip tio n o m ya s th e n ia g ra vis , a n e u ro m u s cu la r d is e a s e re s u ltin g ro m th e a u to im m u n e p ro d u ctio n o a n tib o d ie s d ire cte d a g a in s t ch o lin e rg ic re ce p to rs a t th e n e u ro m u s cu la r ju n ctio n . In 1934, a lm o s t 2 ce n tu rie s la te r, th e En g lis h p hys icia n Ma ry Bro a d o o t Wa lke r e n co u n te rs s e ve ra l p a tie n ts w ith s im ila r s ym p to m s o m u s cle w e a kn e s s , w h ich re m in d h e r o th e

Synthesis of Acetylcholine Acetylcholine is synthesized in a single step rom choline and acetyl coenzyme A (acetyl CoA) by the enzyme choline acetyltransferase (ChAT): Acetyl Coenzyme A Acetylcholine

Choline

Coenzyme A

æ ChAT æ æ æÆ

H2O

Equation 10-1

In the CNS, choline used or the synthesis o acetylcholine arises rom one o three sources. Approximately 35% to 50% o the choline generated by acetylcholinesterase in the synaptic cle t (see Fig. 10-1 and below) is transported back into the axon terminal, where it comprises about hal o the choline used in ACh synthesis. Plasma-based stores o choline may also be transported to the brain as part o phosphatidylcholine (a phospholipid), which is then metabolized to ree choline. (The incorporation o choline into phosphatidylcholine is essential, because choline itsel cannot cross the blood–brain barrier.) Choline is also stored in phospholipids as phosphorylcholine, where it can be used when needed. Acetyl CoA or the synthesis reaction is derived mainly rom glycolysis and is ultimately produced by the enzyme pyruvate dehydrogenase. Although the synthesis o acetyl CoA occurs at the inner membrane o mitochondria, choline acetyltrans erase is located in the cytoplasm. It is hypothesized that citrate serves as the carrier or acetyl CoA rom the mitochondrion to the cytoplasm, where the citrate is reed by citrate lyase.

s ym p to m s o p a tie n ts w ith tu b o cu ra re p o is o n in g . Give n h e r n d in g s , Dr. Wa lke r a d m in is te rs a n a n tid o te , p hys o s tig m in e , to h e r im m o b ile p a tie n ts . Th e re s u lts a re s ta rtlin g —w ith in m in u te s , h e r p a tie n ts a re a b le to ris e a n d wa lk a cro s s th e ro o m . Dr. Wa lke r h a s d is cove re d th e rs t tru ly e e ctive m e d ica tio n o r m ya s th e n ia g ra vis . De s p ite th e s ig n i ca n ce o h e r a cco m p lis h m e n t, it is la rg e ly rid icu le d b y th e s cie n ti c co m m u n ity b e ca u s e th e tre a tm e n t im p rove s th e s ym p to m s o m ya s th e n ia g ra vis to o ra p id ly a n d e e ctive ly to b e b e lie va b le . It is n o t u n til m a ny ye a rs la te r th a t th e s cie n ti c co m m u n ity co m e s to a cce p t h e r n d in g s .

Questions 1 . Why do tubocurare poisoning and myasthenia gravis produce similar symptoms? 2 . How does physostigmine improve the symptoms of myasthenia gravis? Why is it dangerous to administer physostigmine to every patient presenting with muscle weakness? 3 . What are the therapeutic uses of anticholinergic drugs in other diseases such as Alzheimer’s dementia? 4 . What are the advantages and disadvantages of using medications with anticholinergic effects in older individuals and individuals with cognitive impairments?

The choline acetyltransferase reaction is not the ratelimiting step in ACh synthesis. Rather, ACh synthesis is limited by the availability of the choline substrate, which depends on uptake of choline into the neuron. Two processes are responsible or choline transport. The f rst is low-a f nity 10–100 M) acilitated di usion. This transport (Km system is not saturable and is ound in cells that synthesize choline-containing phospholipids, such as the corneal epithelium. Far more important is a sodium-dependent, higha f nity transport system (Km 1–5 M) ound specif cally in cholinergic nerve terminals. Because the high-a f nity transporter is saturated at concentrations o choline 10 M, it sets an upper limit on the supply o choline or ACh synthesis. As the rate-limiting component in ACh synthesis, this transporter is a target or several anticholinergic drugs (e.g., hemicholinium-3, see Fig. 10-1).

Storage and Release of Acetylcholine A ter its synthesis in the cytoplasm, ACh is transported into synaptic vesicles or storage. An ATPase that pumps protons into the vesicle provides the energy necessary or this process. Transport o protons out o the vesicle (i.e., down the H concentration gradient) is coupled to uptake o ACh into the vesicle (i.e., against the ACh concentration gradient) via an ACh-H antiport channel. This antiporter is a target or some anticholinergic drugs, such as vesamicol, and its inhibition results in a def cit o ACh storage and subsequent release (Fig. 10-1). Cholinergic synaptic vesicles contain not only ACh but also ATP and heparan sul ate proteoglycans,

C h a p t e r 1 0 Cholinergic Pharmacology 129

Na + Choline

Cholinergic neuron AcCoA + Choline

Hemicholinium

Choline acetyltransferase

Vesamicol

ACh

Ca lcium cha nne l

Ca lcium cha nne l

Ca 2+

Ca 2+ ACh H+

LEMS (a utoa ntibody)

Botulinum toxin ACh

Mus ca rinic ACh re ce ptor (M2 , M4 )

Nicotinic ACh re ce ptor ACh

Choline + a ce ta te

Synaptic cleft AChE

AChE Inhibitors

Acetylcholinesterase

Postsynaptic Mus ca rinic ACh re ce ptor cell M1 , M3 , M5 M2 , M4 Gq P LC

Excita tory

Nicotinic ACh re ce ptor

Gi

Ope ns Na +/K+ cha nne l Excita tory

AC,

K+ cha nne l Inhibitory

FIGURE 10-1. Acetylcholine synthesis, storage, release, and degradation pathways and pharmacologic agents that act on these pathways. Choline is transported into the presynaptic cholinergic nerve terminal by a high-a f nity Na -choline co-transporter. This transporter is inhibited by hemicholinium. The cytosolic enzyme choline acetyltrans erase catalyzes the ormation o acetylcholine (ACh) rom acetyl coenzyme A (AcCoA) and choline. Newly synthesized ACh is packaged (together with ATP and proteoglycans) into vesicles or storage. Transport o ACh into the vesicle is mediated by a H -ACh antiporter, which is inhibited by vesamicol. The ACh-containing vesicles use with the plasma membrane when intracellular calcium levels increase in response to a presynaptic action potential, releasing the neurotransmitter into the synaptic cle t. Lambert-Eaton myasthenic syndrome (LEMS) results rom an autoantibody that blocks the presynaptic Ca 2 channel. Botulinum toxin prevents the exocytosis o presynaptic vesicles, thereby blocking ACh release. Acetylcholine di uses in the synaptic cle t and binds to postsynaptic and presynaptic receptors. Acetylcholine receptors are divided into nicotinic and muscarinic receptors. Nicotinic receptors are ligand-gated ion channels that are permeable to cations, while muscarinic receptors are G protein-coupled receptors that alter cell signaling pathways, including activation o phospholipase C (PLC), inhibition o adenylyl cyclase (AC), and opening o K channels. Postsynaptic nicotinic receptors and M1, M3, and M5 muscarinic receptors are excitatory; postsynaptic M2 and M4 muscarinic receptors are inhibitory. Presynaptic nicotinic receptors enhance Ca 2 entry into the presynaptic neuron, thereby increasing vesicle usion and release o ACh; presynaptic M2 and M4 muscarinic receptors inhibit Ca 2 entry into the presynaptic neuron, thereby decreasing vesicle usion and release o ACh. Acetylcholine in the synaptic cle t is degraded by membrane-bound acetylcholinesterase (AChE) into choline and acetate. Numerous inhibitors o AChE exist; most clinically relevant anticholinesterases are competitive inhibitors o the enzyme.

both of which serve as counter-ions for ACh. By neutralizing the positive charge of ACh, these molecules disperse electrostatic forces that would otherwise prevent dense packing of ACh within the vesicle. (Released ATP also acts as a neurotransmitter, through purinergic receptors, to inhibit the release of ACh and norepinephrine from autonomic nerve endings.) Release of ACh into the synaptic cleft occurs via fusion of the synaptic vesicle with the plasma membrane. This process

depends on axon terminal depolarization and the opening of voltage-dependent calcium channels. The increase in intracellular Ca2 facilitates the binding of synaptotagmin to the SNARE-complex proteins, which together mediate vesicle– membrane attachment and fusion. The result is that the contents of the vesicle are released as discrete “quanta” into the synaptic cleft. (See Chapter 8, Principles of Cellular Excitability and Electrochemical Transmission, for additional details on electrochemical transmission.)

130 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

Two stores o ACh have distinct roles during the process o ACh release. One store, known as the depot pool, consists o vesicles positioned near the plasma membrane o the axon terminal. Axonal depolarization causes these vesicles to release ACh rapidly. The reserve pool serves to ref ll the depot pool as it is being used. An adequate rate o reserve pool mobilization is required to sustain ACh release or an extended period o time. O the two stores, the depot pool is replenished f rst by vesicles loaded with newly synthesized ACh; this process displaces some o the older depot pool vesicles into the reserve pool.

Cholinergic Receptors A ter ACh has been released into the synaptic cle t, it binds to one o two classes o receptors, usually on the membrane sur ace o the postsynaptic cell. Muscarinic acetylcholine receptors (mAChR) are seven-transmembrane-helix G proteincoupled receptors (GPCRs), and nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels. Although muscarinic receptors and nicotinic receptors are sensitive to the same neurotransmitter, these two classes of cholinergic receptors share little structural similarity.

Muscarinic Receptors

Muscarinic cholinergic transmission occurs mainly at autonomic ganglia, at end organs innervated by the parasympathetic division o the autonomic nervous system, and in the CNS. As G protein-coupled receptors, muscarinic receptors transduce signals across the cell membrane and interact with GTP-binding proteins. Because the e ects o muscarinic receptor activation occur through the actions o these G proteins, there is a latency o at least 100–250 ms associated with muscarinic responses to receptor activation. (In contrast, nicotinic receptor channels have latencies on the order o 5 ms.) Activation o G proteins by agonist binding to muscarinic receptors may have several di erent e ects on cells. These include inhibition o adenylyl cyclase (via Gi) and stimulation o phospholipase C (via Gq/11), both mediated by an subunit o the G protein. (See Chapter 1, Drug–Receptor Interactions, or a discussion o these signaling mechanisms.) Muscarinic activation also modulates ion channels via the subunit o a G protein. The predominant e ect o such mAChR stimulation is to increase the opening o specif c potassium channels (G protein-modulated inwardly recti ying K channels, or GIRKs), thereby hyperpolarizing the cell. The subunit o the Gi protein binds to the channel and enhances its probability o being open. Five distinct cDNAs or muscarinic receptors, denoted M1–M5, have been isolated and detected in human cells. These receptor types orm two unctionally distinct groups. M1, M3, and M5 are coupled to G proteins responsible or the stimulation o phospholipase C. M2 and M4, on the other hand, are coupled to G proteins responsible or adenylyl cyclase inhibition and K channel activation. The receptors o each unctional group can be distinguished based on their responses to pharmacologic antagonists (Table 10-1). Generally, M1 is expressed in cortical neurons and autonomic ganglia, M2 in cardiac muscle, and M3 in smooth muscle and glandular tissue. Because stimulation o M1, M3, and M5 receptors acilitates excitation o the cell, while stimulation o M2 and M4 receptors suppresses cellular excitability, there is

a predictable correlation between the receptor subtype and the e ect o ACh on the cell. The various muscarinic receptor subtypes account or much o the diversity in cellular responses to mAChR agonists.

Nicotinic Receptors

Nicotinic acetylcholine receptors (nAChRs) mediate nicotinic cholinergic transmission via a process known as direct ligand-gated conductance (Fig. 10-2). The binding o two ACh molecules to one nAChR elicits a con ormational change in the receptor that creates a monovalent cationselective pore through the cell membrane. Open channels o the activated nAChR are equally permeable to K and Na ions. (Since the resting membrane potential is close to the Nernst potential or K and ar below the Nernst potential or Na , the predominant ion passing through the open nACR is Na .) A relatively small permeability to Ca2 ions also results in important elevations o intracellular [Ca2 ]. There ore, when open, these channels produce a net inward current that depolarizes the postsynaptic cell. Stimulation o multiple nAChRs may depolarize the cell su f ciently to generate action potentials and to open voltage-dependent calcium channels. The latter action, and the direct entry o Ca2 through the nAChR pore, can lead to activation o several intracellular signaling pathways. Because ACh dissociates rapidly rom active-state receptor molecules and acetylcholinesterase rapidly degrades ree (unbound) ACh in the synaptic cle t (see below), the depolarization mediated by nAChRs is brie ( 10 ms). Although the simultaneous binding o two ACh molecules is required or channel opening, it is not necessary or both molecules to dissociate rom the receptor in order or the channel to open again; the binding o a second ACh molecule to a receptor that still has one ACh bound may, once again, result in channel opening. The kinetics o nAChR binding and channel opening are detailed in Figure 10-3. Structurally, the nicotinic acetylcholine receptor comprises f ve subunits, each o which has a mass o approximately 40 kilodaltons (Fig. 10-2A). Several types o nAChR subunits have been identif ed; these are designated , , , , and . All o these subunits share 35–50% homology with one another. Each receptor at the NMJ is composed o two subunits, one and one subunit, and either one or one subunit. (The 2 orm dominates at the neuromuscular junction in mature skeletal muscle, while the 2 orm is expressed in embryonic muscle.) Agonist molecules bind at a hydrophobic pocket that is ormed between each subunit and the adjacent, complementary subunit—this is the structural basis or the binding o two ACh molecules to each receptor. The con ormational change in the subunits induced by the binding o ACh initiates the overall changes in the pore that permit ion ow through the receptor (i.e., that open the channel). Besides simply opening and closing in response to ACh binding, nicotinic receptors also modulate their responses to various concentration prof les o ACh. The receptors react di erently to discrete, brie pulses o ACh than to neurotransmitter that is present continuously. As noted above, under normal conditions, a closed, resting-state channel responds to dual ACh binding by opening transiently, and the low a f nity o the receptor or ACh causes rapid dissociation o ACh rom the receptor and return o the receptor to its resting con ormation. In contrast, continuous exposure

C h a p t e r 1 0 Cholinergic Pharmacology 131

TABLE 10-1 Characteristics of Cholinergic Receptor Subtypes RECEPTOR

TYPICAL LOCATIONS

Muscarinic M1

Autonomic ganglia

CNS Muscarinic M2

Heart: SA node

RESPONSES

MECHANISM

PROTOTYPE AGONIST

PROTOTYPE ANTAGONIST

Late excitatory postsynaptic potential (EPSP) Complex: at least arousal, attention, analgesia

Gq/11 → PLC → ↑ IP3 ↑ DAG → ↑ Ca 2 ↑ PKC

Oxotremorine

Pirenzepine

Heart: ventricle

Slowed spontaneous depolarization; hyperpolarization ↓ Conduction velocity ↓ Re ractory period; ↓ contractile orce Slight ↓ in contractility

Muscarinic M3

Smooth muscle

Contraction

Muscarinic M4

Heart: AV node Heart: atrium

AF-DX 117

subunit o Gi → inhibits AC; subunit o Gi → ↑ K channel (GIRK) opening

As M1

Hexahydrosiladi enidol

CNS

As M2; presynaptic autoreceptors, negative eedback to suppress ACh release

Himbacine

Muscarinic M5

CNS

As M1

Nicotinic NM

Skeletal muscle at neuromuscular junction (NMJ )

End-plate depolarization; skeletal muscle contraction

Opening o nAChR Na /K channels

Phenyltrimethylammonium

Tubocurare

Nicotinic NN

Autonomic ganglia

Depolarization and f ring o postganglionic neuron Secretion o catecholamines Complex: at least arousal, attention, analgesia

Opening o nAChR Na /K channels; postsynaptic depolarization and presynaptic interactions with other receptors, calcium channels

Dimethylphenylpiperazinium

Trimethaphan

Adrenal medulla CNS

Cholinergic receptors are divided into nicotinic and muscarinic receptors. All nicotinic receptors are ligand-gated cation-selective channels, while muscarinic receptors are G protein-coupled receptors. Specif c pharmacologic agonists and antagonists exist or most subclasses, although the majority o these agents are currently used only or experimental purposes.

o the receptor to ACh causes it to undergo a change to a “desensitized” con ormation in which the channel is locked closed. The desensitized state is also characterized by a greatly increased a f nity o the receptor or ACh, so that ACh remains bound to the receptor or a relatively long period o time. This prolonged binding o ACh to the desensitized con ormation delays the conversion o the receptor to its resting state and hence prolongs the time during which the receptor is incapable o being activated by agonist. Nicotinic cholinergic receptors at autonomic ganglia and in the central nervous system (termed N2 or NN) are similar to receptors at the NMJ (N1 or NM), with the exception that the subunits in NN receptors are composed solely o and subunits. To complicate matters, however, nine di erent subunit types ( 2– 10) and three subunit types ( 2– 4) have been detected in neuronal tissues. ( 1 and 1 re er to the distinct subunit types ound at the NMJ.) This diversity o and subunit combinations is responsible or the variable responses o CNS and autonomic nAChRs to pharmacologic agents. Presynaptic nAChRs in the CNS modulate the release both o ACh itsel and o other excitatory and inhibitory neurotransmitters. This e ect may involve prolonged elevations

o [Ca2 ] in the presynaptic nerve terminals, which lead to inactivation o neuronal calcium channels.

Degradation of Acetylcholine In order or acetylcholine to be use ul or rapid, repeated neurotransmission, there must be a mechanism to limit its duration o action. Degradation o ACh is essential not only to prevent unwanted activation o neighboring neurons or muscle cells but also to ensure proper timing o signaling at the postsynaptic cell. A single receptor molecule is typically capable o distinguishing between two sequential presynaptic release events because degradation o ACh in the synaptic cle t occurs aster than the time course o nAChR activation. Enzymes collectively known as cholinesterases are responsible or degrading acetylcholine. The two types o cholinesterase, AChE and butyrylcholinesterase (BuChE, also known as pseudocholinesterase or nonspecif c cholinesterase), are distributed widely throughout the body. AChE is indispensable or the degradation o ACh and is capable o hydrolyzing about 4 105 molecules o ACh per enzyme molecule per minute; its turnover time o 150 s makes it one

132 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

A Ove ra ll S tructure

α

δ

β

α

N

M4

ε

M1

C

M3 M2

o the most e f cient hydrolytic enzymes known. AChE is concentrated on the postsynaptic membrane, and the choline that it rees there is e f ciently transported back into the presynaptic terminal. BuChE has a secondary role in ACh degradation; the enzyme can hydrolyze ACh but at rates much slower than that o AChE. Evidence suggests that BuChE may be involved in early neural development as a co-regulator o ACh and may also be involved in the pathogenesis o Alzheimer’s disease. Because o its central importance to cholinergic transmission, a class o drugs known as acetylcholinesterase inhibitors has been designed to target AChE.

Physiologic Effects of Cholinergic Transmission B Ace tylcholine Binding S ite

δ

β

β α

α

Neuromuscular J unction Amino a cids

α

Y W W

ACh binding s ite s

ε

Y N Y

Ace tylcholine binding s ite

C C

Y

ε

C Ion Cha nne l M2 M2

M2

M2

M2

M2

M2

Amino a cids

Le ucine ring

FIGURE 10-2. Structural biology of the nicotinic acetylcholine receptor. A. Overall structure o the nicotinic acetylcholine receptor (NM type) and its f ve subunits ( 2 ). Each subunit is composed o a transmembrane protein that has our membrane-spanning (hydrophobic) alpha-helical regions (M1, M2, M3, M4). The large hydrophilic N-terminal domains o the two subunits contain the binding sites or acetylcholine. B. Acetylcholine binding site viewed rom above (inset: lower magnif cation). The labeled amino acids o the subunit hydrophilic domain are particularly important in binding acetylcholine. The con ormational change that results rom the binding o two acetylcholine molecules opens the channel. C. The M2 domains o the f ve subunits all ace the interior o the protein and together orm the transmembrane channel (inset). Three negatively charged rings o f ve amino acids (one rom each M2 subunit) draw positively charged ions through the channel. At the center, an uncharged leucine ring (purple) participates in closing the ion channel when the receptor becomes desensitized to acetylcholine.

Acetylcholine is the principal neurotransmitter at the neuromuscular junction (Fig. 10-4). ACh is released by motor neurons and it binds to nicotinic receptors in the muscle cell membrane, resulting in motor end-plate depolarization. The extent o depolarization depends on the quantity o ACh released into the synaptic cle t. Release o ACh is quantal in nature; that is, ACh is released in discrete quantities by the presynaptic motor neuron. Each quantum o ACh corresponds to the contents o a single synaptic vesicle and elicits a small depolarization in the motor end-plate termed a miniature end-plate potential (MEPP). Under resting conditions, sporadic MEPPs are detected at the motor end-plate, corresponding to a low baseline level o unstimulated ACh release that arises rom spontaneous synaptic vesicle usion with the motor axon’s presynaptic membrane. In contrast, the arrival o an action potential at the motor axon terminal causes many more vesicles (up to thousands) to use with the neuronal membrane and release their ACh. At the motor endplate, the result is a relatively large depolarization termed the end-plate potential (EPP) (Fig. 10-5). The magnitude o an EPP is more than su f cient to trigger an action potential that propagates rom the end-plate throughout the muscle f ber and, hence, produces a single contraction or “twitch.” Acetylcholine not only triggers muscle contraction as its primary effect at the NMJ, but also modulates its own action at this site. Presynaptic cholinergic receptors, located on the axon terminal o the motor neuron, respond to ACh binding by facilitating the mobilization o synaptic vesicles rom the reserve pool to the depot pool. This positive eedback loop, in which the release o ACh stimulates additional ACh release, is necessary to ensure su f cient ACh release when the nerve is stimulated with high requency ( 100 Hz). Despite this mechanism, the ACh output per nerve impulse wanes rapidly during prolonged high- requency stimulation. Fortunately, because an excess o ACh is released and an excess o ACh receptors is present, there is a large sa ety margin. Only when 50% or more o the postsynaptic receptors are desensitized is a decline in muscle tension observed during tetanic stimulation (a phenomenon known as tetanic fade ). Importantly, selective blockade o the modulatory presynaptic cholinergic receptors by antagonists such as hexamethonium prevents acilitation and causes rapid tetanic ade to occur under otherwise normal conditions (Fig. 10-6).

Autonomic Effects

Autonomic activity can be classif ed as either tonic activity, which accounts or end organ stimulation at rest, or phasic activity, which triggers an elevated response to changing

C h a p t e r 1 0 Cholinergic Pharmacology 133 ACh ACh

ACh binding s ite s

ACh

ACh

Re ce ptor ga te (clos e d)

ACh

kon 2A + R

ACh

koff

k'off

ACh

β

k'on A + AR

ACh

A2 R

α

A2 R

*

Re ce ptor ga te (ope n)

FIGURE 10-3. Kinetics of nicotinic acetylcholine receptor binding and channel opening. Each transition between states o receptor binding and channel opening is completely reversible, and it is not necessary to go through all o the possible con ormations be ore returning to a given state. For example, a receptor with two associated ligands may lose one and then gain another to return to its initial state, without the need or both ligands to dissociate. A, agonist ligand (ACh); R, nicotinic ACh receptor (closed); R*, nicotinic ACh receptor (open); kon, rate constant or association (binding) o the f rst ACh molecule to the receptor; k′on, rate constant or association o the second ACh molecule to the receptor; ko , rate constant or dissociation o the f rst ACh molecule rom the receptor; k′o , rate constant or dissociation o the second ACh molecule rom the receptor; , rate constant o channel opening a ter both ACh molecules have bound; , rate constant o channel closure. Note that channel opening and closing are much aster events than binding and dissociation o ACh to the receptor, so that a receptor with two bound ACh molecules can open and close multiple times be ore one o the agonists dissociates.

conditions. Neurotransmission through autonomic ganglia is complicated because several distinct receptor types contribute to the complex changes observed in postganglionic neurons. The generalized postsynaptic response to presynaptic impulses can be separated into our distinct components (Fig. 10-7). The primary event in the postsynaptic ganglionic response is a rapid depolarization mediated by nicotinic ACh receptors in the cell membrane of postganglionic neurons. The mechanism is similar to that in the NMJ, in that an inward current elicits a near-immediate excitatory postsynaptic potential (EPSP) o 10–50 ms in duration. Typically, the amplitude o such an EPSP is only a ew millivolts, and many such events must sum or the postsynaptic cell membrane to reach the threshold or f ring an action potential (Fig. 10-7A). The three remaining events o ganglionic transmission modulate this primary signal and are known as the slow EPSP, the IPSP (inhibitory postsynaptic potential), and the late, slow EPSP. The slow EPSP occurs a ter a latency o 1 second and is mediated by M1 muscarinic ACh receptors. The duration o this e ect is 10–30 seconds (Fig. 10-7C). The IPSP is largely a product o catecholamine (i.e., dopamine and norepinephrine) stimulation o dopaminergic and -adrenergic receptors (see Chapter 11, Adrenergic Pharmacology), although some IPSPs in a ew ganglia are mediated by M2 muscarinic receptors. The latency and duration o the IPSPs generally vary between those o the ast and slow EPSPs. The late, slow EPSP is mediated by a decrease in potassium conductance induced by stimulation o receptors or peptide transmitters (i.e., angiotensin, substance P, and luteinizing hormone-releasing hormone). Lasting or several minutes, the late, slow EPSP is thought to have a role in the long-term regulation o postsynaptic neuron sensitivity to repetitive depolarization. One pharmacologic consequence o such a complex pattern o depolarization in autonomic ganglia is that drugs selective or the IPSP, slow EPSP, and late, slow EPSP are generally not capable o eliminating ganglionic transmission.

Instead, such agents alter only the e f ciency o transmission. For example, methacholine , a muscarinic receptor agonist, has modulatory e ects on autonomic ganglia that resemble the stimulation o slow EPSPs (see below). Blockade o excitatory transmission through autonomic ganglia relies on inhibition o the nAChRs that mediate ast EPSPs. The overall effect of ganglionic blockade is complex and depends on the relative predominance of sympathetic and parasympathetic tone at the various end organs (Table 10-2). For example, the heart is in uenced at rest primarily by the parasympathetic system, whose tonic e ect is a slowing o the heart rate. Thus, blockade o autonomic ganglia that innervate the heart by moderate to high doses o the antimuscarinic agent atropine results in blockade o vagal slowing o the sinoatrial node and hence in relative tachycardia. (It should be noted that in low doses, the central parasympathetic stimulating e ects o atropine predominate, initially resulting in bradycardia prior to its peripheral vagolytic action.) Blood vessels, in contrast, are innervated only by the sympathetic system. Because the normal e ect o sympathetic stimulation is to cause vasoconstriction, ganglionic blockade results in vasodilation. It is important to realize, however, that the responses described above ignore the presence o muscarinic ACh receptors at many o the end organs. When stimulated directly by cholinergic agents, such receptors o ten mediate a response that overrides the response produced by ganglionic blockade. In general, the expected net cardiovascular e ects o muscarinic blockade produced by clinical doses o atropine in a healthy adult with a normal hemodynamic state are mild tachycardia, with or without ushing o the skin, and no pro ound e ect on blood pressure. The muscarinic receptor subtypes expressed in visceral smooth muscle, cardiac muscle, secretory glands, and endothelial cells mediate highly diverse responses to cholinergic stimulation. These e ects are detailed in Table 10-3. In general, these end-organ e ects tend to predominate over

134 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

ganglionic in uences. That is, or systemically administered cholinergic agonists, the overall response is generally similar to that caused by direct stimulation o the postganglionic e ector sites and o ten di erent rom that caused by ganglionic stimulation alone.

Ne uron

CNS Effects Ne uromus cula r junction Mus cle fibe r

Mye lin Axon

S chwa nn ce ll s he a th

P re s yna ptic boutons

End-pla te re gion

S yna ptic cle fts

Mitochondria S yna ptic ve s icle (ACh) De ns e ba r (a ctive zone ) P re s yna ptic me mbra ne S yna ptic cle ft P os ts yna ptic me mbra ne J unctiona l fold

ACh re ce ptors

Ace tylcholine s te ra s e s

FIGURE 10-4. The neuromuscular junction (NMJ ). At the neuromuscular junction, motor neurons innervate a group o muscle f bers. The area o muscle f bers innervated by an individual motor neuron is re erred to as the end-plate region. Multiple presynaptic terminals extend rom the axon o the motor neuron. When the motor neuron is depolarized, its synaptic vesicles use with the presynaptic membrane, releasing ACh into the synaptic cle t. ACh receptors o the neuromuscular junction are exclusively nicotinic, and stimulation o these receptors results in depolarization o the muscle cell membrane and generation o an end-plate potential.

CNS unctions o ACh include modulation o sleep, wake ulness, learning, and memory; suppression o pain at the spinal cord level; and essential roles in neural plasticity, early neural development, immunosuppression, and epilepsy. Both nicotinic and muscarinic receptors are expressed in central neurons. Nicotinic receptors are primarily involved as presynaptic heteroreceptors that modulate the release o other neurotransmitters, such as glutamate, whereas muscarinic presynaptic receptors are primarily autoreceptors that modulate the release o ACh. While the past two decades have improved understanding o the diversity o subunits and molecular properties o neuronal nicotinic receptors, important questions remain about the anatomical distributions and unctional roles o di erent neuronal receptor subtypes in the CNS and o their changes in disease states and during nicotine abuse (as occurs with smoking). As part o the ascending reticular activating system, cholinergic neurons play an important role in arousal and attention (see Fig. 9-8). Levels o ACh throughout the brain increase during wake ulness and REM sleep and decrease during inattentive states and non-REM/slow-wave sleep (SWS). During an awake or aroused state, cholinergic projections rom the pedunculopontine nucleus, the lateral tegmental nucleus, and the nucleus basalis o Meynert (NBM) are all active. Because the NBM projects di usely throughout the cortex and hippocampus (see Fig. 9-8), activation o the NBM causes a global increase in ACh levels. Acetylcholine markedly potentiates the excitatory e ects o other inputs to its cortical target cells without a ecting the baseline activity o these neurons, an e ect that likely derives rom its modulation o excitatory neurotransmitter release. This primed state is thought to improve the ability o such neurons to process incoming inputs. For the brain as a whole, the result is a heightened state o responsiveness. The cholinergic link to memory processes is supported by evidence rom diverse experimental models. Whereas elevated ACh levels during wake ulness appear to benef t memory encoding processes, consolidation o hippocampusmediated, episodic, explicit memories benef t rom SWS, when ACh levels are at their minimum. By artif cially keeping ACh levels elevated during SWS (e.g., by administration o an AChE inhibitor), consolidation o newly acquired explicit learning and episodic memories can be disrupted. Current understanding o the interplay among ACh, sleep, and memory is as ollows. During awake states, ACh prevents inter erence with initial learning in the hippocampus by suppressing retrieval o previously stored memories (to prevent them rom inter ering with new encoding), but release o this suppression is necessary to allow consolidation o new memories. During sleep (in particular, during SWS), lower ACh levels are required or proper consolidation o newly acquired memories because stronger excitatory eedback transmission is needed to reactivate memories or consolidation within neocortical brain areas. There ore, it may be use ul to remember to sleep, as sleep is needed to remember, or at least to remember better.

Action pote ntia l

Action pote ntia l

0

t

i

a

l

(

m

V

)

Thre s hold pote ntia l of pos ts yna ptic ce ll

-55 -70

M

e

m

b

r

a

n

e

p

o

t

e

n

Re s ting pote ntia l

Q

A

Q

Q

S ubthre s hold, no s umma tion

B

Q

2Q

Te mpora l s umma tion

C

S pa tia l s umma tion

FIGURE 10-5. Quantal release of acetylcholine and muscle contraction. Muscle contraction relies on the accumulation o a su f cient concentration o acetylcholine at the motor end-plate to depolarize the muscle beyond the threshold potential (typically, about 55 mV). A ter local depolarization occurs, a sel -propagating action potential is generated that can spread along the muscle f ber and result in muscle contraction. A. As a single cholinergic vesicle releases its contents into the NMJ , a small depolarization (Q), otherwise known as a miniature end-plate potential (MEPP), occurs in the local region o the muscle. This MEPP is insu f cient to generate an action potential. When a su f cient number o individual cholinergic vesicles empty their contents into the NMJ , either in quick succession (B) or simultaneously (C), su f cient depolarization occurs (termed the end-plate potential, or EPP) that the motor end-plate threshold or action potential generation is exceeded, and muscle contraction occurs. An isolated action potential produces a twitch, while a train o action potentials may produce sustained contraction o the muscle. Note that although this example uses two MEPPs or simplicity, many more than two MEPPs are actually required to achieve threshold-level depolarization. In this f gure, the x-axis is time.

A

A Fa s t EP S P

0.1 Hz

e

50 Hz

g

2 Hz

a

0.1 Hz

(

m

V

)

B IP S P

V

o

l

t

C S low EP S P

B D La te , s low EP S P

S timulus a pplie d

0.1 Hz

2 Hz

50 Hz

0.1 Hz

15 s e c

30 s e c

5 min

Time

FIGURE 10-7. Four types of synaptic signals in an autonomic ganglion. FIGURE 10-6. Tetanic fade and the effects of hexamethonium. A. Control stimulation. Rapid stimulation o muscle contraction relies on presynaptic acetylcholine autoreceptors that provide positive eedback and thereby increase the amount o acetylcholine released with each depolarization. The diagram shows control muscle responses to single-shock stimulation (0.1 Hz), a train o our stimulations (2 Hz), or tetanic stimulation (50 Hz). Positive eedback increases the amount o ACh released with each depolarization during tetanic stimulation, providing enhanced muscle contraction that gradually ades back to baseline during subsequent single-shock stimulation. B. Stimulation a ter the administration o hexamethonium. Note that although the response to isolated (0.1 Hz) stimuli is unchanged in the presence o hexamethonium, the drug prevents the increased e ect that normally occurs with higher requency (50 Hz) stimulation. This is a result o hexamethonium’s antagonism o the acetylcholine autoreceptor on the presynaptic terminal that is normally responsible or positive eedback o ACh release.

The response o autonomic ganglia to neurotransmission is a complex event mediated by a number o di erent neurotransmitters and receptor types and occurring on several distinct time scales. A. The primary mode o neurotransmission is the action potential, which is produced by a su f ciently strong (suprathreshold) excitatory postsynaptic potential (EPSP). The ast EPSP is mediated by acetylcholine acting on postsynaptic nicotinic ACh receptors. B. The inhibitory postsynaptic potential (IPSP) is a membrane hyperpolarization response. This response is thought to be mediated by several di erent postsynaptic receptor types, including modulatory dopamine receptors and -adrenergic receptors as well as M2 muscarinic ACh receptors. C. The slow EPSP is mediated by M1 muscarinic receptors, has a latency o about 1 second a ter an initial depolarization, and lasts or 10–30 seconds. D. The late, slow EPSP occurs on the order o minutes a ter a depolarization event. This excitatory response may be mediated by peptides that are co-released with acetylcholine. 135

136 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

PREDOMINANT TONE

EFFECTS OF GANGLIONIC BLOCKADE

Arterioles

Sympathetic (adrenergic)

Vasodilation; ↑ peripheral blood f ow; hypotension

Veins

Sympathetic (adrenergic)

Vasodilation; pooling o blood; ↓ venous return; ↓ cardiac output

gross cognitive de ects. These abnormal f ndings are also observed in etal alcohol syndrome and Rett syndrome, both o which demonstrate dramatically reduced numbers o cholinergic neurons in the brain. There is also some evidence or an immunomodulatory role or ACh, as many cells o the immune system both release ACh and express ACh receptors. Finally, mutations have been identif ed in nicotinic ACh receptor genes that are responsible or autosomal dominant nocturnal rontal lobe epilepsy (ADNFLE); this milestone in epilepsy research is the f rst demonstration that alterations in a ligand-gated ion channel can cause epilepsy.

Heart

Parasympathetic (cholinergic)

Tachycardia

The Non-Neuronal Cholinergic System (NNCS)

Iris

Parasympathetic (cholinergic)

Mydriasis (pupil dilation)

Ciliary muscle

Parasympathetic (cholinergic)

Cycloplegia ( ocused to ar vision)

Gastrointestinal tract

Parasympathetic (cholinergic)

↓ Tone and motility; constipation; ↓ secretions

Urinary bladder

Parasympathetic (cholinergic)

Urinary retention

Salivary glands

Parasympathetic (cholinergic)

Xerostomia (dry mouth)

Sweat glands

Sympathetic (cholinergic)

Anhidrosis (absence o sweating)

TABLE 10-2 Effects of Autonomic Ganglionic Blockade on Tissues SITE

The clinical importance o ACh or cognitive unction is illustrated by the pathophysiology and treatment o Alzheimer’s disease (AD) and other neurodegenerative dementias, including di use Lewy body dementia (DLB) and Parkinson’s disease with dementia (PDD). Neurodegenerative dementias and brain injury produce central cholinergic dys unction. Patients with these conditions mani est cognitive, unctional, and behavioral def cits that are at least partially related to cholinergic def cits and amenable to symptomatic treatment with procholinergic medications. An example is the symptomatic treatment o AD with acetylcholinesterase inhibitors. Acetylcholine also plays a role in pain modulation through inhibition o spinal nociceptive transmission. Cholinergic neurons located in the rostral ventromedial medulla extend processes to the superf cial lamina o the dorsal horn at all levels o the spinal cord, where secondary neurons in a erent sensory pathways are located. ACh released by the cholinergic neurons is believed to bind to muscarinic ACh receptors located on secondary sensory neurons specif c or pain transmission, resulting in suppression o action potential f ring in these cells and consequently in analgesia (see Chapter 18). Clinically, the analgesic properties o ACh can be demonstrated by injecting AChE inhibitors into the spinal uid. Acetylcholine has CNS e ects unrelated to its role as a neurotransmitter. ACh has been observed to inhibit neurite growth. During the early phases o neural development, when such growth is essential, AChE levels are increased. The presence o ACh in chick limb buds and myotomes suggests other, morphogenetic roles or this compound. Lesioning o rat cholinergic neurons during development results in cortical abnormalities, including aberrant growth and positioning o pyramidal cell dendrites, altered cortical connectivity, and

Cells o various non-neuronal tissues and organs produce ACh (non-neuronal ACh [NN-ACh]). NN-ACh acts in an autocrine and paracrine ashion on nicotinic and muscarinic ACh receptors that are expressed on neighboring ACh-producing or e ector cells. The NNCS regulates physiologic processes including cell growth, adhesion, migration, and di erentiation. Dys unction o the NNCS may contribute to the pathogenesis o disease in several organ systems, including the skin (e.g., atopic dermatitis, pemphigus, psoriasis, vitiligo) and the urinary (e.g., overactive bladder syndrome), gastrointestinal (e.g., gastroesophageal re ux disease, peptic ulcer disease, pancreatitis), immune (e.g., Sjogren’s syndrome, rheumatoid arthritis, sepsis), pulmonary (e.g., asthma, chronic obstructive pulmonary disease, cystic f brosis), musculoskeletal (e.g., osteoporosis, tendinosis), reproductive (e.g., sperm dysmotility), and cardiovascular systems (e.g., atherosclerosis). For some conditions, anticholinergic drugs are already standard therapeutic choices. However, or most organ systems, the role o the NNCS remains to be urther delineated, and specif c therapeutic interventions targeted at the NNCS remain to be developed. TABLE 10-3 Effects of Acetylcholine on Muscarinic Receptors in Peripheral Tissues TISSUE

EFFECTS OF ACETYLCHOLINE

Vasculature (endothelial cells)

Release o nitric oxide and vasodilation

Eye iris (pupillae sphincter muscle)

Contraction and miosis

Ciliary muscle

Contraction and accommodation o lens to near vision

Salivary and lacrimal glands

Thin and watery secretions

Bronchi

Constriction; ↑ secretions

Heart

Bradycardia, ↓ conduction velocity, AV block at high doses, slight ↓ in contractility

Gastrointestinal tract

↑ Tone, secretions; relaxation o sphincters

Urinary bladder

Contraction o detrusor muscle; relaxation o sphincter

Sweat glands

Diaphoresis

Reproductive tract, male

Erection

Uterus

Variable

C h a p t e r 1 0 Cholinergic Pharmacology 137

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic manipulation o cholinergic transmission has met with only limited success because the complex actions o ACh make it di f cult to obtain selective e ects. For example, many cholinergic agents are capable o both stimulating and blocking cholinergic receptors through a mechanism known as depolarizing blockade (see below). There ore, only a relatively small raction o the many cholinergic and anticholinergic agents discovered over the past century are used in clinical practice. These drugs are used primarily or (1) modulation o gastrointestinal motility, (2) xerostomia (dry mouth), (3) glaucoma, (4) motion sickness and antiemesis, (5) neuromuscular diseases such as myasthenia gravis and Eaton-Lambert syndrome, (6) acute neuromuscular blockade and reversal during surgery, (7) ganglionic blockade during aortic dissection, (8) dystonias (e.g., torticollis), headache, and pain syndromes, (9) reversal o vagal-mediated bradycardia, (10) mydriasis, (11) bronchodilation in chronic obstructive pulmonary disease, (12) bladder spasms and urinary incontinence, (13) cosmetic e ects on skin lines and wrinkles, and (14) treatment o Alzheimer’s disease and other neurodegenerative dementias. Slight variations in the pharmacologic properties o individual cholinergic and anticholinergic agents are responsible or their large di erences in therapeutic utility. The relative selectivity o action o the most use ul agents depends on both pharmacodynamic and pharmacokinetic actors, including inherent di erences in receptor binding a f nity, bioavailability, tissue localization, and resistance to degradation. These variations, in turn, derive rom the molecular structure and charge o the drug. The structure o pirenzepine , or example, allows the drug to bind M1 muscarinic receptors (located in autonomic ganglia) with higher a f nity than M2 and M3 receptors (located at parasympathetic end organs). As a result, the drug’s predominant e ect at clinically used doses is ganglionic blockade (see Table 10-1). Similarly, the addition o a methyl group to acetylcholine yields methacholine , which is more resistant to degradation by AChE and, hence, possesses a longer duration o action. Charged agents such as muscarine generally do not cross membrane barriers. The absorption o such drugs through both the gastrointestinal (GI) mucosa and the blood–brain barrier is signif cantly impaired, unless specif c carriers are available to transport the drug; there ore, such drugs typically have little or no e ect on the CNS. In contrast, lipophilic agents have excellent CNS penetration. As one example, the high CNS penetration o physostigmine makes this drug the agent o choice or treating the CNS e ects o anticholinergic overdose. The ollowing discussion is ordered mechanistically. For each class o drugs, the selectivity o individual agents within the class is used as a basis to explain the therapeutic uses o each agent.

Inhibitors o Acetylcholine Synthesis, Storage, and Release Drugs that inhibit the synthesis, storage, or release o ACh have only recently begun to have clinical use (Fig. 10-1). Hemicholinium-3 blocks the high-a f nity transporter or choline and thus prevents the uptake o choline required or ACh synthesis. Vesamicol blocks the ACh-H antiporter

that transports ACh into vesicles, thereby preventing the storage o ACh. Both o these compounds are utilized only in research settings, however. Botulinum toxin A, produced by Clostridium botulinum, degrades SNAP-25 and thus prevents synaptic vesicle usion with the axon terminal (presynaptic) membrane. This paralysis-inducing property is currently used in the treatment o several diseases associated with increased muscle tone, such as torticollis, achalasia, strabismus, blepharospasm, and other ocal dystonias. Botulinum toxin is also approved or cosmetic treatment o acial lines or wrinkles and is used to treat various headache and pain syndromes (e.g., by intrathecal delivery into the spinal uid). Because it degrades a protein common to the synaptic vesicle usion machinery in multiple types o nerve terminals, botulinum toxin has a general e ect on the release o many di erent neurotransmitters, not just ACh.

Acetylcholinesterase Inhibitors Agents in this class bind to and inhibit AChE, thereby increasing the concentration o endogenously released ACh in the synaptic cle t. The accumulated ACh subsequently activates nearby cholinergic receptors. Agents in this class are also re erred to as indirectly acting ACh receptor agonists because they generally do not activate receptors directly. It is important to note that a ew AChE inhibitors have a direct action as well. For example, neostigmine , a quaternary carbamate, not only blocks AChE but also binds to and activates nAChRs at the neuromuscular junction.

Structural Classes

All indirectly acting cholinergic agonists inter ere with the unction o AChE by binding to the active site o the enzyme. There are three chemical classes o such agents, including (1) simple alcohols with a quaternary ammonium group, (2) carbamic acid esters o alcohols bearing either quaternary or tertiary ammonium groups, and (3) organic derivatives o phosphoric acid (Fig. 10-8). The most important unctional di erence among these classes is pharmacokinetic. Edrophonium is a simple alcohol that inhibits AChE by reversibly associating with the active site o the enzyme. Because o the noncovalent nature o the interaction between the alcohol and AChE, the enzyme–inhibitor complex lasts or only 2–10 minutes, resulting in a relatively rapid but completely reversible block. The carbamic acid esters neostigmine and physostigmine are hydrolyzed by AChE, so a labile covalent bond is ormed between the drug and the enzyme. However, the rate at which this reaction occurs is many orders of magnitude lower than for ACh. The resulting enzyme–inhibitor complex has a hal li e o approximately 15–30 minutes, corresponding to an e ective inhibition lasting 3–8 hours. Organophosphates such as diisopropyl f uorophosphate have a molecular structure that resembles the transition state ormed in carboxyl ester hydrolysis. These compounds are hydrolyzed by AChE, but the resulting phosphorylated enzyme complex is extremely stable and dissociates with a hal -li e o hundreds o hours. Furthermore, the enzyme– organophosphate complex is subject to a process known as aging, in which oxygen–phosphorus bonds within the inhibitor are broken spontaneously in avor o stronger bonds between the enzyme and the inhibitor. Once aging occurs, the duration o AChE inhibition is increased even urther. Thus, organophosphate inhibition is essentially irreversible,

138 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

A

Simple Alcohols

Carbamic Acid Esters

B

N+

HO

N

C

O

N+

O

O

O Edrophonium

Neostigmine

H N

Organophosphates

P F

O

Isoflurophate

O N O

N Physostigmine

FIGURE 10-8. Structural classes of acetylcholinesterase inhibitors. Acetylcholinesterase (AChE) inhibitors are divided into three structural classes. A. Simple alcohols such as edrophonium have a short half-time of AChE inhibition. Edrophonium is used in the diagnosis of myasthenia gravis and other diseases of the neuromuscular junction. B. Carbamic acid esters are hydrolyzed by AChE. This results in the formation of a covalent bond between the carbamic acid ester (boxed) and AChE and a consequently long half-time of AChE inhibition. Neostigmine is used to treat myasthenia gravis and, during or after surgery, to reverse paralysis induced by nicotinic acetylcholine receptor antagonists. Physostigmine, because it has good CNS penetration, is the agent of choice for treating anticholinergic poisoning. C. Organophosphates form an extremely stable phosphorus–carbon bond with AChE. This results in irreversible inactivation of AChE. As a result, many organophosphates are extremely toxic.

and the body must synthesize new AChE molecules to restore AChE activity. However, i strong nucleophiles (such as pralidoxime ) are administered be ore aging has occurred, it is possible to displace the organophosphate rom the inhibited AChE and recover enzymatic unction.

Clinical Applications

Acetylcholinesterase inhibitors have a number o clinical applications, including (1) increasing transmission at the neuromuscular junction, (2) increasing parasympathetic tone, and (3) increasing central cholinergic activity (e.g., to treat symptoms o AD). Because o their ability to increase the activity o endogenous ACh, AChE inhibitors are especially use ul in diseases o the neuromuscular junction, where the primary de ect is an insu f cient quantity o either ACh or AChR. In myasthenia gravis, autoantibodies are generated against NM receptors. These antibodies both induce NM receptor internalization and block the ability o ACh to activate the receptors. As a result, patients with myasthenia gravis present with signif cant weakness (recall the description o Chie Opechancanough in the introductory case). Eaton-Lambert syndrome is also characterized by muscle weakness, but this disorder is caused by autoantibodies generated against Ca2 channels; both presynaptic Ca2 entry and the subsequent release o ACh in response to axon terminal depolarization are attenuated. Certain anticholinergic drugs, such as tubocurare, also cause weakness or paralysis by acting as competitive antagonists at the nAChR, preventing ACh rom binding to the receptor and causing nondepolarizing blockade o cholinergic transmission. Acetylcholinesterase inhibitors (such as the physostigmine used in the introductory case) improve all three o these conditions by increasing the concentration o endogenously released ACh at the neuromuscular junction and thereby increasing ACh signaling. Because ACh binding to NM receptors results in muscle cell depolarization, AChE inhibitors are ineffective at

reversing the action of agents that cause paralysis by inducing sustained depolarization, such as succinylcholine (see below). In act, AChE inhibitors in su f ciently high doses can exacerbate existing weakness and paralysis caused by depolarizing blockade. Thus, it is o undamental importance that the cause o the muscle weakness should be determined be ore treatment is initiated. Short-acting AChE inhibitors such as edrophonium are ideal or such diagnostic purposes. Edrophonium mitigates weakness i the blockade is attributable to competitive AChR antagonists or to diseases such as myasthenia gravis or Eaton-Lambert syndrome. In contrast, i muscle strength decreases urther with edrophonium administration, then depolarizing blockade may be suspected. The short hal -li e o edrophonium ensures that exacerbation o the latter condition will last or a minimal amount o time. For chronic treatment o myasthenia gravis, longer acting AChE inhibitors such as pyridostigmine , neostigmine , and ambenonium are the pre erred agents. AChE inhibitors exert other therapeutic e ects by potentiating parasympathetic actions in target tissues. Topical application o AChE inhibitors to the cornea o the eye decreases intraocular pressure by acilitating the out ow o aqueous humor. The main e ect o AChE inhibitors on the gastrointestinal system is an increase in smooth muscle motility because o enhancement o ganglionic transmission at Auerbach’s plexus, although these agents also cause increased secretion o gastric acid and saliva. Neostigmine, the most popular drug or this application, is typically used or relie o abdominal distention. The use o anticholinesterases in reversing anticholinergic drug poisoning is also well established. The agent o choice or this indication is typically physostigmine ; its tertiary amine structure allows it ready access to the brain and spinal cord, where it can counteract the CNS e ects o anticholinergic toxicity. Acetylcholinesterase inhibitors are also used to treat the symptoms o AD dementia and other conditions causing

C h a p t e r 1 0 Cholinergic Pharmacology 139

TABLE 10-4 Pharmacokinetic and Mechanistic Characteristics of Donepezil, Rivastigmine, Rivastigmine Transdermal Patch, Galantamine, and Galantamine ER

TMAX (H)

ELIMINATION HALF-LIFE (H)

HEPATIC METABOLISM

REVERSIBLE INHIBITION OF AChE

OTHER CHOLINOMIMETIC EFFECTS

100

3–5

60–90

Yes

Yes

—

Rivastigmine

40

0.8–1.8

2

No

No*

BuChEI

Rivastigmine Patch

55–65

3.4

8–12

No

No*

BuChEI

Galantamine

85–100

0.5–1.5

5–8

Yes

Yes

nAChR agonist

Galantamine ER

85–100

4.5–5

25–35

Yes

Yes

nAChR agonist

DRUG

BIOAVAILABILITY (%)

Donepezil

*Rivastigmine is a “pseudo-irreversible” inhibitor of AChE and BuChE. Tmax, time to peak plasma concentration; AChE, acetylcholinesterase; BuChEI, butyrylcholinesterase inhibitor; nAChR agonist, allosteric (potentiating) ligand at some nicotinic acetylcholine receptors in the CNS.

dementia (e.g., Parkinson’s disease with dementia, di use Lewy body dementia, vascular-ischemic dementia), brain injury (e.g., traumatic brain injury), and cognitive impairment (e.g., cognitive impairment associated with multiple sclerosis and schizophrenia). Donepezil and rivastigmine are secondgeneration AChE inhibitors indicated or the treatment o AD dementia in the mild, moderate, and severe stages; galantamine is a second-generation AChE inhibitor indicated or the treatment o AD dementia in the mild and moderate stages. Rivastigmine is also approved by the US Food and Drug Administration (FDA) or the treatment o Parkinson’s disease with dementia. Tacrine is a f rst-generation AChE inhibitor that is no longer in clinical use—it had the disadvantage o our-times-daily dosing and it had the potential to cause hepatic toxicity. In both short-term (24–52 weeks) e f cacy trials and long-term clinical e ectiveness studies, these AChE inhibitors have demonstrated benef cial e ects in producing improvement or stabilization o symptoms and in slowing the progression o cognitive, unctional, and behavioral decline in AD dementia. Although there are mechanistic and pharmacokinetic di erences among these drugs (Table 10-4), there are no signif cant di erences in their e f cacy in the treatment o AD. For example, rivastigmine is a “pseudoirreversible” cholinesterase inhibitor because it orms a labile carbamoylate complex with AChE (and BuChE), inactivating the enzyme until the covalent bond is broken. While rivastigmine is available as a twice-daily oral preparation, it is now used mostly as a once-daily transdermal patch ormulation. Galantamine is both a reversible AChE inhibitor and an allosteric (potentiating) nicotinic receptor ligand. All o these drugs exhibit linear pharmacokinetics, and their time to peak plasma concentration (Tmax) values and elimination hal -lives are prolonged in elderly patients. With appropriately slow titration, these medications are generally well tolerated and have a avorable adverse e ect prof le (with the exception o tacrine, which is no longer used clinically; see above). While these medications are relatively selective or AChE in the CNS, the most common adverse e ects—including nausea, vomiting, anorexia, atulence, loose stools, diarrhea, and abdominal cramping—are related to peripheral cholinomimetic e ects

on the GI tract. The rivastigmine transdermal patch can also cause skin irritation, redness, and rash at the site o application. The adverse e ects o AChE inhibitors may occur in 5–20% o patients, are usually mild and transient, and are related to the dose and rate o dose escalation. For the oral preparations, the adverse GI e ects o AChE inhibitors can be minimized by administering the drug a ter a meal or in combination with memantine, an NMDA channel blocker that is indicated or the treatment o moderate to severe AD. For transdermal rivastigmine, adverse e ects can be minimized by applying the patch to a di erent site each day. These medications may also increase the risk o syncope, particularly in susceptible individuals and with overdose. Use o these agents is contraindicated in patients with unstable or severe cardiac disease (particularly cardiac arrhythmias), uncontrolled epilepsy, unexplained or recurrent syncope, or active peptic ulcer disease.

Receptor Agonists All cholinergic receptor agonists bind to the ACh binding site o cholinergic receptors. Receptor agonists can be divided into muscarinic and nicotinic receptor-selective agents, although some cross-reactivity exists with virtually all o these agents. Muscarinic receptor agonists are used clinically in the diagnosis o asthma and as miotics (agents that cause pupil constriction). Nicotinic receptor agonists are used clinically or induction o muscle paralysis.

Muscarinic Receptor Agonists

Agents in this class are divided structurally into choline esters and alkaloids (Fig. 10-9). The choline esters are charged, highly hydrophilic molecules that are poorly absorbed by the oral route and ine f ciently distributed to the CNS. Choline esters include acetylcholine, methacholine, carbachol, and bethanechol (Table 10-5). Acetylcholine is not administered in clinical settings because o its broad actions and its extremely rapid hydrolysis by AChE and pseudocholinesterase. Methacholine is at least three times more resistant to hydrolysis by AChE than is ACh. This agent is relatively selective or cardiovascular muscarinic cholinergic receptors, and it has relatively little a f nity or nicotinic

140 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

Choline Esters

A

B

Alkaloids

HO

O N+

O

N+ O

Ace tylcholine

Mus ca rine

O N+

O

Methacholine

N

O

O H2 N

N

O

Pilocarpine N+

O

Carbachol

O H2 N

N+

O

Bethanechol

FIGURE 10-9. Structural classes of muscarinic receptor agonists. Muscarinic receptor agonists are divided into choline esters and alkaloids. A. Choline esters are charged molecules and therefore have little CNS penetration. Methacholine is highly resistant to AChE and is used in the diagnosis of asthma. Carbachol has both nicotinic and muscarinic receptor activity; it is only used topically for treatment of glaucoma. Bethanechol is highly selective for muscarinic receptors; it is used to promote GI and bladder motility. Shown in blue are groups in the drug molecules that differ from acetylcholine. B. Alkaloids have highly variable structures; some have excellent CNS penetration. Muscarine, the prototypical muscarinic receptor agonist, is an alkaloid that is structurally similar to acetylcholine (boxed areas). Until recently, pilocarpine was the only alkaloid muscarinic receptor agonist used clinically. It is used to treat xerostomia (dry mouth) in patients with Sjögren’s syndrome and postradiation syndromes. Cevimeline, an M1 and M3 agonist, is also effective in xerostomia related to Sjögren’s syndrome (not shown).

cholinergic receptors. Although methacholine can stimulate receptors expressed on cardiovascular tissues, the magnitude o its response is unpredictable. This act has limited its use as a vasodilator or cardiac vagomimetic (i.e., a drug that mimics the cardiac response to vagus nerve

[parasympathetic] stimulation, which typically includes bradycardia, decreased contractility, and compensatory sympathetic re exes). Currently, methacholine is used only in the diagnosis o asthma; in this application, the bronchial hyperreactivity that is characteristic o asthma causes an exaggerated bronchoconstriction response to parasympathomimetics (see Chapter 48). Both carbachol and bethanechol are resistant to cholinesterases because, in these drugs, a carbamoyl group is substituted or the acetyl ester group o ACh (Fig. 10-9). This resistance to AChE extends their duration o action and allows time or distribution o the intact drug to areas o lower blood ow. Carbachol has enhanced nicotinic activity relative to other choline esters. This agent cannot be used systemically because its nicotinic action at autonomic ganglia leads to unpredictable responses. Instead, the agent is used principally as a topical miotic agent, typically in the treatment o glaucoma. Local application o the drug to the cornea o the eye results in both pupillary constriction (miosis) and decreased intraocular pressure. Bethanechol is almost completely selective or muscarinic receptors. It is an agent o choice or promoting GI and urinary tract motility, particularly or postoperative, postpartum, and drug-related urinary retention and or hypotonic neurogenic bladder. In contrast to the choline esters, the alkaloids vary greatly in structure. Some are amphipathic, while others are highly charged. Most o these agents are tertiary amines, although a ew are quaternary amines with protonated or permanently charged nitrogens substituting or the choline-centered N o ACh. The amphipathic nature o the tertiary amine alkaloids permits absorption through the GI mucosa and penetration into the CNS. Muscarine is an example o a quaternary amine alkaloid that has low bioavailability because o its permanently charged nature. Most alkaloids are primarily o value in pharmacologic research. The most clinically used alkaloid is pilocarpine , a miotic agent and a sialagogue (saliva-inducing agent) used to treat xerostomia (dryness o the mouth secondary to reduced salivary secretion). Cevimeline , an M1 and M3 agonist, is used to treat xerostomia in Sjögren’s syndrome.

Nicotinic Receptor Agonists

Succinylcholine is a choline ester that has high a f nity or

nicotinic receptors and is resistant to AChE. It is used to induce paralysis during surgery by means o depolarizing blockade . This e ect can be caused by any direct nAChR agonist because such drugs activate receptor-associated channels and produce depolarization o the cell membrane.

TABLE 10-5 Relative Pharmacologic Properties of Choline Esters ESTER

SUSCEPTIBILITY TO AChE

CARDIAC ACTIVITY

GI ACTIVITY

URINARY ACTIVITY

EYE ACTIVITY (TOPICAL)

ATROPINE ANTAGONISM

NICOTINIC ACTIVITY

Acetylcholine Methacholine Carbachol Bethanechol Note that all actions are mediated by muscarinic receptors, with the exception of nicotinic activity. “ ” indicates negligible activity. “ ” indicates unpredictable.

C h a p t e r 1 0 Cholinergic Pharmacology 141

TABLE 10-6 Comparison of Nondepolarizing and Depolarizing NMJ -Blocking Agents EFFECT

NONDEPOLARIZING

DEPOLARIZING

Effect on motor end-plate

Increased activation threshold to ACh; no depolarization

Partial; persisting depolarization

Initial excitatory effect on muscle

None

Transient fasciculations

Muscle response to tetanic stimulation during partial block

Poorly sustained contraction

Well-sustained contraction

Effect of previous administration of a competitive NMJ blocking agent

Additive effect

Antagonistic effect

Effect of previous administration of a depolarizing NMJ blocking agent

No effect or antagonistic effect

No effect or additive effect

In order to produce depolarizing blockade, the agent must persist at the neuromuscular junction and activate the nicotinic receptor channels continuously. Note that this e ect is unlike the depolarization pattern seen in the generation o a standard action potential or end-plate potential, in which ACh is present at the neuromuscular junction or only a brie period o time. The overall pattern is a brie period o excitation, maniested by widespread asciculations in muscle cells, ollowed by f accid paralysis. The paralysis occurs or two reasons. First, the open cholinergic channels maintain the cell membrane in a depolarized state, e ecting inactivation o voltagegated sodium channels so that they cannot open to support urther action potentials. Second, the agonist-bound nAChRs spontaneously desensitize, preventing their opening and response to any subsequently delivered, additional agonist. Because o this mechanism, any nAChR agonist, including ACh, is capable o producing depolarizing blockade at su f ciently high concentrations. Generally, depolarizing blockade with succinylcholine is used or only short durations because prolonged depolarization can lead to li e-threatening electrolyte imbalances (caused by prolonged Na inf ux and K e f ux). Table 10-6 compares the e ects o depolarizing and nondepolarizing NMJ-blocking agents. The concept o depolarizing blockade pertains to all cholinergic receptors and is not strictly limited to the NMJ. For example, this mechanism accounts or the paradoxical suppression o parasympathomimetic activity at autonomic ganglia by high levels o agonists, such as nicotine, that are selective or nicotinic receptors. The potential or inducing depolarizing blockade is partially responsible or the unpredictable e ects o nAChR agonists. Although muscarinic receptor agonists can also cause depolarizing blockade at autonomic ganglia, this e ect is obscured by the overwhelmingly parasympathomimetic responses seen at other neuroe ector sites. The toxic e ects o cholinergic agents and poisons are described in Box 10-1.

Receptor Antagonists Antagonists o AChRs act by binding directly to the agonist site and competitively blocking stimulation o the receptor by endogenous ACh or exogenously administered receptor agonists.

Muscarinic Receptor Antagonists

Anticholinergic compounds that act on muscarinic receptors are used to produce a parasympatholytic e ect in target organs. By blocking normal cholinergic tone, these compounds allow sympathetic responses to predominate (Table 10-2). The most commonly used anticholinergics are either naturally occurring alkaloids or synthetic quaternary ammonium compounds. The alkaloids are relatively selective or antagonist activity at muscarinic receptors, whereas the synthetic compounds also demonstrate substantial antagonism at nicotinic receptors. The prototypical muscarinic receptor antagonist is atropine , a naturally occurring alkaloid ound in the plant Atropa belladonna, or deadly nightshade. Belladonna derived its name rom Italian or “beauti ul woman”—during the Renaissance, women in Italy ingested or applied to their eyes extracts and juices o berries rom the plant to cause dilation o the pupils, which was considered a mark o beauty. Atropine is used clinically to induce mydriasis (pupil dilation) or ophthalmologic examinations, to reverse symptomatic sinus bradycardia, to inhibit excessive salivation and mucus secretion during surgery, to prevent vagal ref exes induced by surgical trauma o visceral organs, and to counteract the e ects o muscarine poisoning rom certain mushrooms (see Box 10-1). Because o its marginal activity at nicotinic receptors, extremely high doses o atropine are required or any e ects to be seen at the NMJ. Similarly, because nicotinic receptors are primarily responsible or excitatory transmission at autonomic ganglia, atropine produces only partial block at these sites and only at relatively high doses. Scopolamine (hyoscine hydrobromide), a tertiary amine, di ers rom atropine by virtue o its substantial CNS e ects. Scopolamine is requently used or the prevention and treatment o motion sickness, as an antiemetic, and, in the hospice setting, as an adjunct to end-o -li e com ort care medications to e ect mild sedation and management o oral secretions. A transdermal patch system has been developed to e ect slow absorption and long duration o the anti-motion sickness e ect while avoiding a rapid rise in plasma levels and adverse CNS e ects (e.g., anterograde disruption o novel learning and memory encoding, inattention, and slowing o psychomotor speed). Scopolamine can also be used to ameliorate nausea, particularly that associated with chemotherapy, and can be administered intravenously during procedures in which minimizing oral secretions is desirable. Methscopolamine and glycopyrrolate are quaternary amine antimuscarinics with low CNS penetration that are used or their peripheral e ects to decrease oral secretions, decrease GI spasms, and, in the case o glycopyrrolate, prevent bradycardia during surgical procedures. Both drugs have delayed but measurable CNS and cognitive anticholinergic e ects. Pirenzepine , which is selective or M1 and M4 receptors, was a potential alternative to H2 receptor antagonists in the treatment o peptic ulcer disease, but its use has been supplanted by the advent o the proton pump inhibitors (see Chapter 47). Ipratropium, a synthetic quaternary ammonium compound, is more e ective than -adrenergic agonists in the treatment o chronic obstructive pulmonary disease (COPD) but less e ective in treating asthma. Tiotropium has

142 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

B O X 1 0 - 1 Ch o lin e rg ic Toxicity The toxic e ects o cholinergic agents are a unction o their mechanism o action (e.g., muscarinic versus nicotinic stimulation), dose and duration o exposure, route o absorption, CNS penetration, and metabolism.

Muscarinic Cholinergic Toxicity Acute toxicity with direct muscarinic agents is o ten due to ingestion o toxic mushrooms (e.g., mushrooms in the genus Inocybe) and drugs such as pilocarpine. Adverse e ects o muscarinic overstimulation typically mani est within 15–30 minutes and include nausea, vomiting, diarrhea, sweating, hypersalivation, cutaneous f ushing, ref ex tachycardia (sometimes bradycardia), and bronchoconstriction. Intoxication with these agents can be treated by competitive blockade using atropine.

Nicotinic Cholinergic Toxicity Acute toxicity with nicotine, o ten due to ingestion o cigarettes and insecticides, produces adverse e ects on the CNS, skeletal muscle end-plate, and cardiovascular system. Acute nicotinic toxicity can cause CNS hyperexcitation (seizures progressing to coma and respiratory arrest), skeletal muscle depolarization blockade (respiratory arrest), and cardiovascular abnormalities (hypertension and arrhythmias). As little as 40 mg o nicotine (equivalent to 1 mg o pure liquid nicotine, or the amount o nicotine ound in two regular cigarettes) can be atal, especially in in ants. Treatment, including antiepileptic drugs and mechanical ventilation, is dictated by symptoms. Atropine may be used to counteract parasympathetic stimulation.

Cholinesterase Inhibitor Poisoning Acute cholinesterase inhibitor toxicity is o ten due to exposure to organophosphate pesticides. Such exposures remain an important threat to children and those in the developing world. Initially,

similar, and possibly superior, e f cacy to ipratropium as a bronchodilator in the treatment o COPD. The superior e f cacy o ipratropium and tiotropium in COPD is likely due to the act that the major reversible bronchoconstrictive component in COPD is mediated by cholinergic neural tone (see Chapter 48). In poorly controlled asthma, the addition o tiotropium to inhaled glucocorticoids and long-acting betaagonists may signif cantly increase the time to f rst severe exacerbation and provide modest sustained bronchodilation. Some antimuscarinic drugs are used in the treatment o urinary incontinence and overactive bladder syndrome. Muscarinic stimulation promotes voiding by causing (1) detrusor muscle contraction and (2) bladder trigone and sphincter muscle relaxation. Antimuscarinics produce the opposite e ects by promoting detrusor relaxation and tightening the bladder sphincter. Antimuscarinics currently approved or the treatment o overactive bladder include oxybutynin, propantheline , terodiline , tolterodine , fesoterodine , trospium, darifenacin, and solifenacin. Among these agents, oxybutynin, propantheline, tolterodine, esoterodine, and trospium are nonspecif c muscarinic receptor antagonists, whereas dari enacin and soli enacin are selective M3 receptor

signs o muscarinic toxicity predominate, including vomiting, diarrhea, pro use sweating, hypersalivation, miosis, and bronchoconstriction. Signs o nicotinic toxicity o ten ollow rapidly, including con usion and seizures due to CNS hyperexcitation and respiratory compromise due to depolarizing neuromuscular blockade. Treatment includes emergency management o vital signs (particularly maintaining respiratory integrity), decontamination, symptomatic treatment with atropine, and pralidoxime (PAM) administration to regenerate active enzyme rom the organophosphorus–cholinesterase complex (mostly at skeletal muscle neuromuscular junctions; PAM does not readily penetrate the CNS). Time is o the essence to maximize the potential or recovery. Large doses o atropine may be necessary in some cases (e.g., when toxicity is due to potent agents such as parathion and chemical nerve agents); 1–2 mg o intravenous atropine is administered every 5–15 minutes until signs o e ect (such as reversal o miosis and dry mouth) are noted and maintained. Repeated administration o atropine may be required or hours or days, depending on the elimination hal -li e o the organophosphate. Examples o misuse o cholinergic chemical agents include the use o Sarin nerve gas in the 1980s by Iraq against Kurdish civilians and Iranian troops and in 1995 by a J apanese terrorist in an attack on Tokyo subway passengers. Sarin, one o a class o nerve agents known as “G” agents that also includes tabun and soman, is a colorless and odorless gas with high toxic potency; as little as 0.5 mg o Sarin is lethal or adults. Time is o the utmost importance in recognizing an exposure, providing rapid decontamination in accordance with hazmat protocols, and administering atropine and pralidoxime. When exposure to nerve agents can be expected, prophylaxis may be achieved with pyridostigmine or physostigmine (e.g., as prophylactically administered to some US troops in the Gul War). ■

antagonists. These agents appear to have similar clinical e f cacy. Clinical trials suggest that tolterodine may cause less dry mouth than oxybutynin and that the newer M3-selective agents dari enacin and soli enacin may cause less dry mouth and constipation than the nonselective agents. Atropine, rom belladonna extract, was one o the drugs f rst used to treat symptoms o Parkinson’s disease (PD). Antimuscarinics are still used at times to ameliorate tremor and rigidity in patients with PD. These medications include amantadine , biperiden, benztropine , procyclidine , and trihexyphenidyl. Although antimuscarinics may be help ul in the treatment o PD-related tremor and rigidity, the use of antimuscarinics in elderly and cognitively susceptible patients should be avoided because o the high risk o potential adverse e ects (see Box 10-2). Benztropine and trihexyphenidyl are commonly used to treat extrapyramidal symptoms, dystonias, and akathisia associated with neuroleptics; these adverse e ects are thought to be due to an imbalance between dopaminergic and cholinergic pathways secondary to excessive neuroleptic-induced dopamine antagonism. Trihexyphenidyl is also used to treat neuroleptic-induced hypersalivation syndrome.

C h a p t e r 1 0 Cholinergic Pharmacology 143

B O X 1 0 - 2 Po te n tia l Ad ve rs e Effe cts o f Dru g s w ith An tich o lin e rg ic Pro p e rtie s in Ge ria tric a n d Co g n itive ly Im p a ire d Pa tie n ts Drug-related anticholinergic adverse e ects are potentially hazardous to elderly patients, especially those with cognitive impairment, and cause signif cant morbidity in this population. Additive anticholinergic e ects rom medications can compromise the sa ety o geriatric patients because (1) many common drugs possess at least a small measure o anticholinergic activity, (2) the elderly, and especially the cognitively impaired elderly, are exquisitely sensitive to cholinergic blockade (due to central cholinergic hypo unction and dys unction in aging and dementia, respectively), and (3) polypharmacy is a common practice in the geriatric population. Adverse e ects rom anticholinergic drugs in the elderly may include acute encephalopathy (delirium, con usional state), alls, urinary retention, constipation, and exacerbation and decompensation o underlying cognitive, unctional, and behavioral def cits (particularly in patients with dementia) and may necessitate increased care and hospitalization. It should be noted that many over-the-counter medications have anticholinergic e ects. For example, a common o ender in causing conusion and cognitive dys unction in the elderly and cognitively impaired individuals is diphenhydramine, an antihistamine with anticholinergic properties that is o ten used as a hypnotic either alone or in combination with acetaminophen. Clinicians and pharmacists should be vigilant to minimize polypharmacy in the geriatric population and to monitor and prevent medication-related anticholinergic adverse events. The updated Beers Criteria o potentially inappropriate drugs or elderly patients identif es medications (many with anticholinergic properties) and classes o medications, and specif c medications in patients with certain conditions, that may pose greater potential risks than benef ts in persons older than 65 years o age. Particular caution must be used with medications with strong anticholinergic e ects; these include diphenhydramine, scopolamine, antimuscarinic agents used or urinary incontinence, antispasmodics, skeletal muscle relaxants, tricyclic antidepressants, and some antipsychotics (Table 10-7). ■

Antimuscarinic toxicity causes substantial morbidity and unctional impairment in the geriatric population (see Box 10-2). Depending on the dose, antimuscarinic agents such as atropine and scopolamine may cause bradycardia and sedation at low to medium levels o muscarinic blockade, and tachycardia and CNS hyperexcitation (with delirium, hallucinations, and seizures) at higher levels. Other adverse e ects may include blurred vision (cycloplegia and mydriasis), dry mouth, ileus, urinary retention, f ushing and ever, agitation, and tachycardia. Antimuscarinic medications are contraindicated in patients with glaucoma. Patients with angle-closure glaucoma, which may be precipitated in individuals with shallow anterior chambers, are especially at risk. Antimuscarinics should also be used with caution in patients with prostatic hypertrophy and in patients with dementia or cognitive impairment. Antimuscarinic toxicity is considered dangerous in in ants and children, who are exquisitely sensitive to the hyperthermic adverse e ects caused by an overdose. Symptomatic treatment may include controlled cooling and

TABLE 10-7 Medications with Anticholinergic Properties That Can Be Inappropriate for Use by Cognitively Impaired Older Adults (2012 AGS Beers Criteria) Antihistamines Brompheniramine Carbinoxamine Chlorpheniramine Clemastine Cyproheptadine Dimenhydrinate Diphenhydramine Hydroxyzine Loratadine Meclizine

Antiparkinson Agents Benztropine Trihexyphenidyl

Antispasmodics Atropine products Belladonna alkaloids Dicyclomine Homatropine Hyoscyamine products Loperamide Propantheline Scopolamine

Antipsychotics Chlorpromazine Clozapine Fluphenazine Loxapine Olanzapine Perphenazine Pimozide Prochlorperazine Promethazine Thioridazine Thiothixene Tri uoperazine

Skeletal Muscle Relaxants Carisoprodol Cyclobenzaprine Orphenadrine Tizanidine

Antidepressants Amitriptyline Amoxapine Clomipramine Desipramine Doxepin Imipramine Nortriptyline Paroxetine Protriptyline Trimipramine Antimuscarinics (urinary incontinence) Dari enacin Fesoterodine Flavoxate Oxybutynin Soli enacin Tolterodine Trospium

Adapted with permission rom The American Geriatrics Society. American Geriatrics Society updated Beers Criteria or potentially inappropriate medication use in older adults. J Am Geriatr Soc 2012;60:616–631. doi:10.1111 /j.1532-5415.2012.03923.x.

antiepileptic drugs, but slow administration o low doses o intravenous physostigmine may also be required. High doses o quaternary antimuscarinics and shortacting ganglionic blockers (such as trimethaphan) can cause parasympathetic ganglionic toxicity, mani ested as autonomic blockade and severe orthostatic hypotension. The antimuscarinic e ects may be treated with neostigmine, and the hypotension may require treatment with sympathomimetics such as phenylephrine.

Nicotinic Receptor Antagonists

Selective nicotinic receptor antagonists are used primarily to produce nondepolarizing (competitive) neuromuscular blockade during surgical procedures. Nondepolarizing neuromuscular junction (NMJ) blockers, such as tubocurare , act by antagonizing nicotinic ACh receptors directly, thus preventing binding o endogenously released ACh and subsequent muscle cell depolarization. This leads to f accid paralysis that is similar in presentation to the paralysis

144 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

in myasthenia gravis. In selecting a specif c agent, the primary consideration is its duration o action—ranging rom very long-lasting agents (d-tubocurarine, pancuronium) to intermediate-duration agents (vecuronium, rocuronium) to rapidly degraded compounds (mivacurium). Because nicotinic receptors are expressed in autonomic ganglia as well as the NMJ, nondepolarizing blocking agents o ten have variable adverse e ects associated with ganglionic blockade. Both the muscular paralysis and the autonomic blockade can be reversed by administration o AChE inhibitors. A new class o agents, epitomized by sugammadex, can also be used to accelerate the recovery o blockade by vecuronium and rocuronium. These agents act by chelating vecuronium or rocuronium in an inactive complex, which is then slowly cleared rom the circulation. Sugammadex is investigational in the United States. In special cases, compounds with relatively selective antagonist activity at nAChRs can be used to induce autonomic blockade. The e ects o autonomic ganglionic blockade are discussed above and are listed in detail in Table 10-2. Most commonly, mecamylamine and trimethaphan are administered when ganglionic blockade is desired. The only current use or these agents is to treat hypertension in patients with acute aortic dissection, because the drugs lower blood pressure while simultaneously blunting the sympathetic re exes that would normally cause a deleterious rise in pressure at the site o the tear.

CONCLUSION AND FUTURE DIRECTIONS There are two major classes o cholinergic receptors: nicotinic and muscarinic. Nicotinic receptors are ligand-gated channels that require the direct binding o two acetylcholine molecules to open. These receptors comprise all o the cholinergic receptors at the neuromuscular junction (NM), and they predominate at autonomic ganglia (NN). Thus, the primary cholinergic unctions mediated by nAChRs include skeletal muscle contraction and autonomic activity. The predominant applications o pharmacologic agents directed at nAChRs are (1) neuromuscular blockade, through competitive antagonists and depolarizing blockers, and (2) ganglionic blockade, which results in e ector organ responses that are opposite to those produced by physiologic autonomic tone. Muscarinic receptors are G protein-coupled receptors that bind acetylcholine and initiate signaling through several intracellular pathways. These receptors are expressed in the autonomic ganglia and e ector organs, where they mediate a parasympathetic response. The primary use o muscarinic receptor agonists and antagonists is to modulate autonomic responses o e ector organs. Both nicotinic and muscarinic receptors are ubiquitous in the CNS, where the e ects o acetylcholine include analgesia, arousal, and attention. The relative roles o mAChRs and nAChRs in the brain and spinal cord are not ully understood, and the most e ective currently available CNS drugs increase endogenous cholinergic transmission by inhibiting the action o acetylcholinesterase, the enzyme that hydrolyzes ACh. Although cholinergic pharmacology is a relatively mature f eld with several receptor-selective agents, the specif city o action o the various agents continues to be ref ned. The discovery o muscarinic receptor subtype diversity may lead to the development o agents selective or subtypes that are expressed in a tissue-specif c pattern. Similarly, elucidation

o the role o nicotinic receptor subunit diversity in the CNS has spurred development o more selective agents that modulate the activity o these receptor subtypes. For example, a selective partial agonist at the 7 nicotinic ACh receptor is in late-stage clinical trial testing in AD dementia. Another avenue or uture investigation involves positive allosteric modulators o nicotinic receptors; these agents may augment endogenous cholinergic tone in a manner that is more spatially and temporally specif c, thus potentially providing di erential e f cacy and improved sa ety. Acetylcholinesterase inhibitors are widely used in clinical practice and are standard o care in the treatment o AD and other dementias. They may provide short-term (6–12 month) symptomatic benef ts in AD and, when used chronically, slow clinical decline. Several nicotinic and muscarinic agonists and receptor modulators are in clinical development or the treatment o cognitive impairment, AD dementia, neuropathic pain syndromes, and neuroprotection. Nicotinic receptors may also provide targets or uture treatment approaches in epilepsy. Finally, the physiologic and pathophysiologic roles o the non-neuronal cholinergic system remain to be ully delineated, and specif c therapies targeted at this system remain to be developed.

Suggested Reading Abirishami A, Ho J, Wong J, Yin L, Chung F. Sugammadex, a selective reversal medication or preventing postoperative residual neuromuscular blockade. Cochrane Database Syst Rev 2009;4:CD007362. (Reviews clinical trials on the e ectiveness o sugammadex in postoperative recovery.) Albuquerque EX, Pereira EFR, Alkondon M, Rogers SW. Mammalian nicotinic acetylcholine receptors: rom structure to unction. Physiol Rev 2009;89:73–120. (Excellent review o nAChR structure, gating, and physiologic roles.) Atri A, Shaughnessy LW, Locascio JJ, Growdon JH. Long-term course and e ectiveness o combination therapy in Alzheimer disease. Alzheimer Dis Assoc Disord 2008;22:209–221. (Assesses long-term clinical e ectiveness o cholinergic and glutamatergic anti-AD medications in slowing the course o AD dementia.) Beckmann J, Lips KS. The non-neuronal cholinergic system in health and disease. Pharmacology 2013;92:286–302. (Reviews the non-neuronal cholinergic system and its role in normal and pathologic processes and conditions.) Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms o the central nervous system. Ann Rev Pharmacol Toxicol 2007;47:699–729. (A thorough review o the nicotinic cholinergic system, with many citations.) Kerstjens HA, Engel M, Dahl R, et al. Tiotropium in asthma poorly controlled with standard combination therapy. N Engl J Med 2012;367: 1198–1207. (Replicate clinical trials demonstrating that the addition o tiotropium to inhaled glucocorticoids and long-acting beta-agonists signif cantly increases time to f rst severe exacerbation and provides sustained bronchodilation in poorly controlled asthma.) Marchi M, Grilli M. Presynaptic nicotinic receptors modulating neurotransmitter release in the central nervous system: unctional interactions with other coexisting receptors. Prog Neurobiol 2010;92:105–111. (A brie and readable review.) Rountree SD, Atri A, Lopez OL, Doody RS. E ectiveness o antidementia drugs in delaying Alzheimer’s disease progression. Alzheimers Dement 2013;9:338–345. (Reviews the evidence base or cholinergic and other medications in AD dementia.) Sher E, Chen Y, Sharples TJW, Broad LM. Physiological roles o neuronal nicotinic receptor subtypes: new insights on the nicotinic modulation o neurotransmitter release, synaptic transmission and plasticity. Curr Topics Med Chem 2004;4:283–297. (A thorough treatise on this important topic.) Uteshev VV. The therapeutic promise o positive allosteric modulation o nicotinic receptors. Eur J Pharmacol 2014;727:181–185. (Reviews theoretical and practical considerations or the role o positive allosteric modulators o nicotinic ACh receptors.)

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C h a p t e r 1 0 Cholinergic Pharmacology 149

Tyros ine

Na +

Tyross iinne ne h oxy hydro xyl yla yl s e

Tyros ro o s in e

11

Adre A d ne rg iic ne uro n

Dihydr D Dih y oxyyphe p nyl y a laa nninn e yl (L-DOPA P A)

Acttion Act on pote ntia l

Aromaa ti ttic L-a min n o a cid no de ca rbo booxxyla la s e

Dop mine Dopa VMAT VMA AT

Adrenergic Pharmacology Ca 2+

Na + H+ Dopa mine Dopa mi m inne β− −hyd ydr droxyla dro x se NE NE

a ddr dree ne rgic i ree ce pt pto toorr

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150–151 BIOCHEMISTRY AND PHYS HYSIOLOGY Y OF ADRENERG RG GIC FUNCT UNCTIO ION N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 150 Cate Ca te cholamine h Synthesis, Storage, and Release . . . . . . . . . . 150 Reuptake and Metabolism of Catecholami mine ness . . . . . . . . . . . . 153 Catecholamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 andd 2-A Adr dree noceptorss . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 1- an -Adrenoceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Regulation of Recept p orr Res espo pons nsee . . . . . . . . . . . . . . . . . . . . . . . 1555 Physiologic and Pharmacologic Effects of Endoggen enou ouss Ca Cate tech chol olam amiines . . . . . . . . . . . . . . . . . . . . . . . . 155 Epinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Norepinephrinee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Dopaminee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

INTRODUCTION Adrenergic pharmacology involves the study o agents that act on pathways mediated by the endogenous catecholamines norepinephrine, epinephrine, and dopamine. The sympathetic nervous system is the major source o endogenous catecholamine production and release. Signaling through catecholamine receptors mediates diverse physiologic e ects, including increasing the rate and orce o cardiac contraction, modi ying the peripheral resistance o the arterial system, inhibiting the release o insulin, stimulating hepatic release o glucose, and increasing adipocyte release o ree atty acids. Drugs that target the synthesis, storage, reuptake, and metabolism o norepinephrine and epinephrine or that directly target the postsynaptic receptors or these transmitters are requent therapies or many major diseases, including hypertension, shock, asthma, and angina. This chapter examines the biochemical and physiologic basis or adrenergic action and then discusses the action o the di erent classes o adrenergic drugs.

BIOCHEMISTRY AND PHYSIOLOGY OF ADRENERGIC FUNCTION The autonomic nervous system contributes to homeostasis through the concerted action o its sympathetic and parasympathetic branches. Catecholamines are the major e ectors o sympathetic signaling. The ollowing discussion 150

N NE

N NE

Nid h i Ge ra , Eh rin J . Arm s t ro n g , a n d David E. Go la n α 2 (a uto u re ce pt p or)

NE tra ns p o rte r

MAO NE

DOP GAL

PHARMA PHAR MACOLOGIC OLO OLOG LOGIC OG C CLASSES CLAS SSES S AN AND A ND A AGEN AGENTS AGE E . . . . . . . . . . . . . . . . 156 In nhi hibbitors off C Catecholamine chhol olaa ine Synthesis ynthe . . . . . . . . . . . . . . . . . . . 156 Inhibiitors rs of Ca C techolamine min Storage torage . . . . . . . . . . . . . . . . . . . . 156 S ynaptic naptic cle Inhibitors f ft of Catecholamin aminee Re Reup uupta t kee . . . . . . . . . . . . . . . . . . . 158 Inhibitors of Catecholamine ate chh atech e Me Meta M Met et e ta bo bolism m . . . . . . . . . . . . . . . . . 158 Receptor Agonists iist stss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 -Adrenergi renergic ergic Ago Aggon Agon onis ists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 -A -Adre -Adr Adren ner ergi gic Agon nis ists ts . . . .β.1. . . . . . . β . . . . . . . . . . . . . . . . . . 159 αgon 1 2 Receptor Re ec Antagonists . . . . . . . . . . . . . . . 159 P os ts t yna ptic p ti a ddre ne rgic i -Adrenergic Antagonists re ccee ptors ptors . . . . . . . . . . . . . . . . . . . . . . 159 -Adrenergic Antagoniists ts . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1600 Poo s tss yynaptic ap p tic c e lll CONCLUSION C CL AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 161 Suggested e est es Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161

presents the biochemistry o catecholamine action, rom synthesis to metabolism to receptor activation. The physiologic roles o the endogenous catecholamines epinephrine (adrenaline), norepinephrine (noradrenaline), and dopamine are then discussed, with emphasis on the specif city o receptor expression in di erent organ systems.

Catecholamine Synthesis, Storage, and Release Catecholamines are synthesized by sequential chemical modif cations o the amino acid tyrosine. This synthesis occurs primarily at sympathetic nerve endings and in chroma f n cells. Epinephrine is predominantly synthesized in chroma f n cells o the adrenal medulla; sympathetic neurons produce norepinephrine as their primary neurotransmitter (Fig. 11-1). Tyrosine, the precursor or catecholamine synthesis, is transported into neurons via an aromatic amino acid transporter that uses the Na gradient across the neuronal membrane to concentrate tyrosine (as well as phenylalanine, tryptophan, and histidine). The f rst step in catecholamine synthesis, the oxidation o tyrosine to dihydroxyphenylalanine (DOPA), is mediated by the enzyme tyrosine hydroxylase (TH). TH is the rate-limiting enzyme in catecholamine synthesis, and it is regulated by eedback (end-product) inhibition and by kinase-mediated phosphorylation o the enzyme. DOPA is converted to dopamine by a relatively nonspecif c aromatic amino acid decarboxylase. Dopamine is then hydroxylated by dopamine- -hydroxylase to yield

C h a p t e r 1 1 Adrenergic Pharmacology 151

Th e ye a r is 1960. Ms . S h a s e lt d e p re s s e d o r a n u m b e r o ye a rs . S h e h a s trie d s e ve ra l d i e re n t m e d ica tio n s to a lle via te h e r e e lin g s o h o p e le s s n e s s a n d la ck o m o tiva tio n , b u t n o th in g s e e m s to h e lp . Re ce n tly, h o w e ve r, h e r d o cto r h a s p re s crib e d ip ro n ia zid , a n e w m e d ica tio n re p o rte d to b e o b e n e f t in m a ny ca s e s o d e p re s s io n . He te lls h e r th a t s cie n tis ts b e lie ve th a t th e d ru g h a s b e n e f cia l e e cts in d e p re s s io n b y in h ib itin g a n e n zym e in th e b ra in ca lle d m o n o a m in e o xid a s e (MAO). MAO is o n e o th e e n zym e s re s p o n s ib le o r ca te ch o la m in e d e g ra d a tio n , a n d its in h ib itio n s ig n if ca n tly in cre a s e s th e a va ila b le co n ce n tra tio n s o ca te ch o la m in e s . Be ca u s e ip ro n ia zid is a n e w d ru g , its p o te n tia l a d ve rs e e e cts a re n o t w e ll d e f n e d , s o h e r d o cto r a d vis e s Ms . S to re p o rt a ny u n u s u a l e e cts o th e m e d ica tio n . Ho p e u l, b u t n o t e xp e ctin g s ig n if ca n t ch a n g e s , Ms . S ta ke s th e m e d ica tio n . With in a e w w e e ks , s h e b e g in s to e e l m o tiva te d a n d e n e rg e tic o r th e f r s t tim e in 20 ye a r s . Exu b e ra n t a t h e r n e w s e n s e o e n e rg y, Ms . S re cla im s h e r p a s t li e a s a s o cia lite b y h o s tin g a g a la w in e a n d ch e e s e re ce p tio n . Th e b e s t a n d b rig h te s t o th e city tu rn u p , e xp e ctin g a f n e e ve n in g . As s h e s ta n d s u p to g ive th a n ks to h e r a tte n d e e s , Ms . S ce le b ra te s w ith

norepinephrine. In tissues that produce epinephrine, norepinephrine is then methylated on its amino group by phenylethanolamine N-methyltransferase (PNMT). Expression o PNMT in the adrenal medulla is largely dependent on the high concentrations o cortisol that ow into the medulla via veins draining the adrenal cortex. The conversions o tyrosine to DOPA and o DOPA to dopamine occur within the cytoplasm. Dopamine is transported into synaptic vesicles by a 12-helix membrane-spanning proton antiporter called the vesicular monoamine transporter (VMAT). Unlike all the other enzymes in the catecholamine biosynthesis pathways, dopamine- -hydroxylase is associated with the inner sur ace o secretory vesicles, and it catalyzes the conversion o dopamine to norepinephrine inside these vesicles. There are three distinct vesicular transporters that di er in substrate specif city and localization. VMAT1 and VMAT2 (also known as Uptake 2 [Fig. 11-2]) both transport serotonin (5-HT), histamine, and all catecholamines. The tissue-specif c expression o VMAT1 and VMAT2 is mutually exclusive: VMAT1 expression is restricted mainly to nonneuronal cells (adrenal gland, gastric mucosa, intestine, and sympathetic ganglia) and VMAT2 is expressed primarily in the central nervous system (CNS). In addition to these expression di erences, the a f nity o VMAT2 or histamine (Km, 3 M) is signif cantly higher than that o VMAT1 or histamine (Km, 436 M). The vesicular acetylcholine transporter (VAChT) is expressed in cholinergic neurons, including motor nerves (see Chapter 10, Cholinergic Pharmacology). These antiporters use the proton gradient generated

a la rg e s w ig o h e r a vo rite 1954 Ch ia n ti. By th e e n d o th e p a rty, Ms . S h a s a s e ve re h e a d a ch e a n d n a u s e a . Re ca llin g h e r d o cto r ’s w a rn in g , Ms . S h a s a rie n d ru s h h e r to th e n e a re s t h o s p ita l. In th e e m e rg e n cy d e p a rtm e n t, th e a tte n d in g p h ys icia n re co rd s a b lo o d p re s s u re o 230/160 m m Hg . Re co g n izin g th a t Ms . S is e xp e rie n cin g a h yp e rte n s ive e m e rg e n cy, th e d o cto r a d m in is te rs p h e n to la m in e (a n -a d re n o ce p to r a n ta g o n is t). Ms . S ’s b lo o d p re s s u re q u ickly n o rm a lize s , a n d th e d o cto r ’s s u b s e q u e n t clin ica l in ve s tig a tio n id e n tif e s a n e w , a n d n o w a m o u s , d ru g – o o d in te ra ctio n in vo lvin g MAO in h ib ito rs . Th is p o te n tia l a d ve rs e in te ra ctio n is s h a re d b y s o m e o th e r MAO in h ib ito r s ; m o re re ce n t w o rk o n s u b typ e -s e le ctive a n d re ve r s ib le MAO in h ib ito r s h a s m in im ize d th e in cid e n ce o th is in te ra ctio n .

Questions 1 . Which enzymes metabolize catecholamines? What are the specif cities o iso orms o these enzymes or the various catecholamines? 2 . What is the mechanistic explanation or the interaction o MAO inhibitors with red wine and aged cheese? 3 . How did phentolamine lower Ms. S’s blood pressure?

by a H -ATPase in the vesicular membrane to concentrate dopamine (or, in the case o VAChT, acetylcholine) inside the vesicle. Norepinephrine concentrations within the vesicle can reach 100 mM. To stabilize the osmotic pressure resulting rom the high concentration gradient or norepinephrine across the vesicle membrane, norepinephrine is thought to condense with ATP. Consequently, ATP and norepinephrine are co-released upon vesicle exocytosis. In adrenal medullary cells, norepinephrine is transported or di uses rom vesicles back into the cytoplasm, where PNMT converts it to epinephrine. Epinephrine is then transported back into vesicles or storage until its eventual release by exocytosis. The nonselective nature o VMAT1 and VMAT2 has important pharmacologic consequences, as discussed below. Activation o the sympathetic nervous system and subsequent catecholamine release are initiated by signals originating in an array o processing areas in the CNS, especially the limbic system. These CNS neurons project axons that synapse on sympathetic preganglionic neurons in the intermediolateral columns o the spinal cord. The preganglionic axons project to the sympathetic ganglia. The preganglionic neurons use acetylcholine as the neurotransmitter to activate nicotinic acetylcholine (ACh) receptors, which are cationselective channels that depolarize the neuronal membrane and thereby generate postsynaptic potentials in postganglionic neurons. Ganglionic blockers such as hexamethonium and mecamylamine block the ganglionic nicotinic ACh receptor, without signif cant e ects on skeletal muscle ACh receptors (see Chapter 10). The sympathetic postganglionic axons

152 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

A Norma l upta ke of nore pine phrine from s yna ptic cle ft a nd

Aroma tic L-a mino a cid tra ns porte r

Na

conce ntra tion of NE in s yna ptic ve s icle

H+

ATP

Tyrosine hydroxylase

Tyros ine

Adrenergic neuron

Cytopla s m

Tyros ine

+

Dihydroxyphe nyla la nine (L-DOPA)

Action pote ntia l

ADP

VMAT

Aromatic L-amino acid decarboxylase

NE

VMAT

NE

NE NE

Dopa mine

NE tra ns porte r (NET)

H+

NE

S yna ptic cle ft

Na +

NE

NE tra ns porte r

NE

NE

Na + Ca 2+

H+ Dopa mine Dopamine β−hydroxylase NE

NE

B Coca ine inhibits NE tra ns porte r NE

α 2 (a utore ce ptor)

a dre ne rgic re ce ptor

H+

ATP

ADP

MAO NE

Cocaine

DOP GAL H+

NE

NE

NE NE

Synaptic cleft

α1

β1

β2

P os ts yna ptic a dre ne rgic re ce ptors Postsynaptic cell

NET

VMAT

NE NE

NE

NE

C Re s e rpine inhibits VMAT

ATP

H+

ADP

VMAT

NET

Na +

NE

FIGURE 11-1. Catecholamine synthesis, storage, release, and reuptake pathways. The endogenous catecholamines dopamine, norepinephrine, and epinephrine are all synthesized rom tyrosine. The rate-limiting step in catecholamine synthesis, the oxidation o cytoplasmic tyrosine to dihydroxyphenylalanine (L-DOPA), is catalyzed by the enzyme tyrosine hydroxylase. Aromatic L-amino acid decarboxylase then converts L-DOPA to dopamine. Vesicular monoamine transporter (VMAT) translocates dopamine (and other monoamines) into synaptic vesicles. In adrenergic neurons, intravesicular dopamine- -hydroxylase converts dopamine to norepinephrine (NE). Norepinephrine is then stored in the vesicle until release. In adrenal medullary cells, norepinephrine returns to the cytosol, where phenylethanolamine N-methyltrans erase (PNMT) converts norepinephrine to epinephrine. The epinephrine is then transported back into the vesicle or storage (not shown). -Methyltyrosine inhibits tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis (not shown). Released norepinephrine can stimulate postsynaptic 1-, 1-, or 2-adrenergic receptors or presynaptic 2-adrenergic autoreceptors. Released norepinephrine can also be taken up into presynaptic terminals by the selective NE transporter. NE in the cytoplasm o the presynaptic neuron can be urther taken up into synaptic vesicles by VMAT (not shown) or degraded to 3,4-dihydroxyphenylglycoaldehyde (DOPGAL; see Fig. 11-3) by mitochondrion-associated monoamine oxidase (MAO).

NE NE

NE

Reserpine

FIGURE 11-2. Mechanisms of action of cocaine and reserpine. A. Norepinephrine (NE) that has been released into the synaptic cle t can be taken up into the cytoplasm o the presynaptic neuron by the selective NE transporter (NET), an Na -NE co-transporter. Cytoplasmic NE is concentrated in synaptic vesicles by the nonselective vesicular monoamine transporter (VMAT), an H -monoamine antiporter. An H -ATPase uses the energy o ATP hydrolysis to concentrate protons in synaptic vesicles and thereby generates a transmembrane H gradient. This H gradient is used by VMAT to drive monoamine transport into the synaptic vesicle. B. Cocaine inhibits the NE transporter, allowing released NE to remain in the synaptic cle t or a longer period o time. By this mechanism, cocaine potentiates neurotransmission at adrenergic synapses. C. Reserpine inhibits the vesicular monoamine transporter, preventing the ref lling o synaptic vesicles with NE and eventually depleting the adrenergic terminal o neurotransmitter. By this mechanism, reserpine inhibits neurotransmission at adrenergic synapses.

C h a p t e r 1 1 Adrenergic Pharmacology 153

orm varicosities or en passant connections with target organs. The arrival o an action potential at these endings opens voltage-gated neuronal Ca2 channels, and the ensuing Ca2 in ux triggers exocytosis o the catecholamine-containing synaptic vesicles. Various novel substances—including peptides rom sea snails—block these Ca2 channels; ziconotide is an example o a drug in this class that has e f cacy in the treatment o severe pain (see Chapter 18, Pharmacology o Analgesia). Norepinephrine rapidly di uses away rom the sympathetic nerve endings and locally regulates target tissue responses (e.g., smooth muscle tone) by activating adrenergic receptors expressed on target tissues. (An exception is that ACh is the transmitter used at sympathetic nerve endings in sweat glands.) Importantly, adrenergic receptors are also expressed at sympathetic nerve endings; these receptors may serve as an autoregulatory mechanism or modulating the extent o neurotransmitter release.

Ne urotra ns mitte r OH HO

Catechol-O-methyl transferase (COMT)

The action o a catecholamine molecule at its postsynaptic receptor is terminated by one o three mechanisms: (1) reuptake o catecholamine into the presynaptic neuron, (2) metabolism o catecholamine to an inactive metabolite, and (3) di usion o the catecholamine away rom the synaptic cle t. The f rst two o these mechanisms require specif c transport proteins or enzymes and, there ore, are targets or pharmacologic intervention. Reuptake o catecholamine into the neuronal cytoplasm is mediated by a selective catecholamine transporter (e.g., norepinephrine transporter, or NET) that is also known as Uptake 1 (Fig. 11-2). Approximately 90% o the released norepinephrine is taken up by this process (recycled); the remainder is either metabolized locally or di uses into the blood. Uptake 1 is a symporter that uses the inward Na gradient to concentrate catecholamines in the cytoplasm o sympathetic nerve endings, thus limiting the postsynaptic response and allowing neurons to recycle the transmitter or subsequent release. Inside the nerve terminal, catecholamines can be urther concentrated in synaptic vesicles via VMAT, the same transporter used to transport dopamine into the vesicle or catecholamine synthesis. Thus, the pool of catecholamines available for release comes from two sources: molecules that are synthesized de novo and molecules that are recycled via neuronal reuptake. Catecholamine metabolism involves the two enzymes MAO and catechol-O-methyltransferase (COMT) (Fig. 11-3). MAO is a mitochondrial outer-membrane enzyme that is expressed in most neurons. It exists in two iso orms: MAO-A and MAOB. The two iso orms have some degree o ligand specif city: MAO-A pre erentially degrades serotonin, norepinephrine, and dopamine, while MAO-B degrades dopamine more rapidly than serotonin and norepinephrine. As indicated in the clinical case, MAO inhibitors are e f cacious in the treatment o depression. The MAO-A iso orm is responsible or detoxiying substances in cheese and wine be ore they reach the general circulation. COMT is a cytosolic enzyme that is relatively nonspecif c and is expressed primarily in the liver.

Catecholamine Receptors Adrenergic receptors (also called adrenoceptors ) are selective or norepinephrine and epinephrine. Supraphysiologic concentrations o dopamine can also activate some adrenoceptors. These receptors are divided into three main classes,

Monoamine oxidase (MAO)

HO

Nore pine phrine

OH HO

H

OH O

NH2

Aldehyde reductase

O

HO

DOP GAL

HO

Norme ta ne phrine

Aldehyde dehydrogenase

OH HO

OH OH

MAO

HO

O

HO

DOP EG

OH

OH O

HO

H

DOMA O

HO

Reuptake and Metabolism of Catecholamines

NH2

MOP GAL

COMT Aldehyde reductase

COMT

OH O

OH

OH O

HO

MOP EG HO

Aldehyde dehydrogenase

OH O

Va nillylma nde lic a cid (VMA) Ma jor me ta bolite (e xcre te d in urine )

FIGURE 11-3. Norepinephrine metabolism. Norepinephrine is degraded to metabolites by two main enzymes. Catechol-O-methyltransferase (COMT) is a widely distributed cytosolic enzyme; COMT in the liver is particularly important in the metabolism of circulating catecholamines. Monoamine oxidase (MAO), which is localized to the outer surface of mitochondria, is found in many monoaminergic (including adrenergic) neurons. COMT, MAO, aldehyde reductase, and aldehyde dehydrogenase metabolize catecholamines to multiple intermediates (abbreviated as DOPGAL, MOPGAL, DOPEG, DOMA, and MOPEG) that are eventually excreted. Vanillylmandelic acid (VMA) is the major metabolite excreted in urine.

termed 1, 2, and (Table 11-1). Each o these major classes has three subtypes: 1A, 1B, and 1D; 2A, 2B, and 2C; and 1, 2, and 3. Each o the adrenergic receptor subtypes is a member o the G protein-coupled receptor (GPCR) super amily (also known as seven-transmembrane helix receptors). GPCRs regulate complex intracellular signaling networks through intermediate transducing molecules, which are called G proteins because o their GTP binding and hydrolysis activity. G proteins are heterotrimeric, with , , and subunits. In the resting (inactive) state, G binds guanosine 5 -diphosphate (GDP) and is associated with G . Binding o agonist to the GPCR triggers the dissociation o GDP and the binding o guanosine 5′-triphosphate (GTP) to the G subunit. GTP binding initiates a con ormational change that leads to the dissociation o G and to the activation o G . Both G and G can activate downstream e ectors. In mammals, at least 27 G , 5 G , and 13 G subtypes are present, and downstream GPCR signaling depends on the specif c G combination. On the basis o the primary sequence o the G subunit, G proteins can be divided into our major amilies—Gs, Gi, Gq/11, and G12—and each amily o G subunit activates specif c downstream signaling pathways (see Chapter 1, Drug–Receptor Interactions).

154 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 11-1 Adrenoceptor Actions RECEPTOR SUBTYPE

SIGNALING MEDIATORS

TISSUE

EFFECTS

1

Gq / Gi / Go

Vascular smooth muscle Genitourinary smooth muscle Intestinal smooth muscle Heart Liver

Contraction Contraction Relaxation ↑ Inotropy and excitability Glycogenolysis and gluconeogenesis

2

Gi / Go

Pancreatic -cells Platelets Nerve Vascular smooth muscle

↓ Insulin secretion Aggregation ↓ Norepinephrine release Contraction

1

Gs

Heart Heart Renal juxtaglomerular cells

↑ Chronotropy and inotropy ↑ AV node conduction velocity ↑ Renin secretion

2

Gs

Smooth muscle Liver Skeletal muscle

Relaxation Glycogenolysis and gluconeogenesis Glycogenolysis and K uptake

3

Gs

Adipose

Lipolysis

1-

and

2-Adrenoceptors

1-Receptors

are expressed in vascular smooth muscle, genitourinary tract smooth muscle, intestinal smooth muscle, prostate, brain, heart, liver, and other cell types. The prototypical signaling mechanism o 1-receptors involves Gq/11, which is generally a stimulatory protein that activates various e ectors including phospholipase C, phospholipase D, phospholipase A2, Ca2 channels, K channels, Na / H exchangers, several members o the mitogen-activated protein (MAP) kinase pathways, and a variety o other kinases including phosphatidylinositol 3-kinase. Phospholipase C cleaves phosphatidylinositol-4,5-bisphosphate, generating the two second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts to increase intracellular [Ca2 ] via both release o endogenous Ca2 stores and in ux o Ca2 rom extracellular uid. Increased intracellular [Ca2 ] activates various regulatory proteins that mediate physiologic responses in various tissues. DAG activates protein kinase C, which urther activates a variety o protein substrates including ion channels such as Na /H exchangers, Ca2 channels, and K channels. Phospholipase D catalyses the hydrolysis o phosphatidylcholine to phosphatidic acid and choline. Phosphatidic acid may act directly as a signaling molecule or be urther metabolized to DAG by phosphatidic acid hydrolase. Gq/11-stimulated activation o phospholipase A2 is mediated by increased intracellular [Ca2 ] or by activation o protein kinase C and MAP kinase pathways. The various 1-receptor subtypes likely di er in their tissue-specif c localization and their capacity to activate downstream signaling pathways. The downstream signaling pathways activated by 1receptors can be complex. Stimulation o 1-receptors in vascular smooth muscle cells increases intracellular [Ca2 ], leading to activation o calmodulin, phosphorylation o myosin light chain, increased actin–myosin interaction, and muscle contraction (see Chapter 22, Pharmacology o Vascular Tone). There ore, 1-receptors are important in mediating increases in peripheral vascular resistance, which can

increase blood pressure and redistribute blood ow. While 1-receptor antagonists would seem to be attractive in the therapy o hypertension, their clinical e f cacy in preventing the complications o hypertension is uncertain. 1-Receptor activation also causes contraction o genitourinary smooth muscle, and 1-receptor antagonists are clinically e f cacious in the symptomatic treatment o benign prostatic hyperplasia (BPH) (see below). 2-Adrenoceptors activate Gi, an inhibitory G protein. Gi has multiple signaling actions, including inhibition o adenylyl cyclase (thus decreasing cAMP levels), activation o G protein-coupled inward rectif er K channels (causing membrane hyperpolarization), and inhibition o neuronal Ca2 channels. These e ects tend to decrease neurotransmitter release rom the target neuron. 2-Receptors are ound on both presynaptic neurons and postsynaptic cells. Presynaptic 2-receptors function as autoreceptors to mediate feedback inhibition of sympathetic transmission. 2-Receptors are also expressed on platelets and pancreatic -cells, where they mediate platelet aggregation and inhibit insulin release, respectively. The latter observations have led to the development o agents that are selective inhibitors o 2-receptors. The main pharmacologic approach to 2-receptors, however, has been in the treatment o hypertension. 2-Receptor agonists act at CNS sites to decrease sympathetic out ow to the periphery, resulting in decreased norepinephrine release at sympathetic nerve terminals and, there ore, decreased vascular smooth muscle contraction.

-Adrenoceptors

-Adrenoceptors are divided into three subclasses, termed 1, 2, and 3 (Table 11-1). All three subclasses activate a stimulatory G protein, Gs. Gs activates adenylyl cyclase, which catalyzes the ormation o intracellular cAMP rom adenosine triphosphate (ATP). Increased intracellular cAMP activates protein kinases, especially protein kinase A (PKA), by binding to the regulatory subunit o the enzyme. This results in the release and activation o the catalytic subunit o PKA, which

C h a p t e r 1 1 Adrenergic Pharmacology 155

phosphorylates and activates a variety o intracellular proteins including ion channels and transcription actors. The nature o the signaling di erences among the -adrenoceptor subtypes is unclear since they all appear to couple e f ciently to Gs, and stimulation o 1- and 2-adrenoceptors causes increased intracellular cAMP. It has been suggested that specif city may be con erred by di erences in the composition o the G protein subunits associated with the two receptors. 1-Adrenoceptors couple exclusively to Gs, but 2-adrenoceptors can also activate e ectors via coupling to Gi. Thus, 2-adrenoceptors can limit and spatially restrict cAMP production by switching between Gs- and Gi-mediated signaling, which, in turn, a ects PKA-dependent regulation o target proteins. Pharmacologic selectivity among the -adrenoceptors appears to reside in the tissue-selective distribution o each -adrenoceptor subtype. 1-Adrenoceptors are localized primarily in the kidney and heart. In the kidney, they are present mainly on renal juxtaglomerular cells, where receptor activation causes renin release (see Chapter 21, Pharmacology o Volume Regulation). Stimulation o cardiac 1-receptors (which represent 70–80% o all cardiac -adrenergic receptors) causes an increase in both inotropy ( orce o contraction) and chronotropy (heart rate). The inotropic e ect is mediated by increased phosphorylation o Ca2 channels by protein kinase A, including calcium channels in the sarcolemma and phospholamban in the sarcoplasmic reticulum, and by phosphorylation o troponin I and troponin C, which reduces myof lament sensitivity to Ca2 (see Chapter 25, Pharmacology o Cardiac Contractility). The increased chronotropy results rom a 1-mediated increase in the rate o phase 4 depolarization o sinoatrial node pacemaker cells. Both e ects contribute to increased cardiac output (recall that cardiac output heart rate stroke volume). Activation o 1-receptors also increases conduction velocity in the atrioventricular (AV) node because the 1-stimulated increase in Ca2 entry increases the rate o depolarization o AV node cells. 2-Adrenoceptors are expressed in smooth muscle (including bronchial smooth muscle), liver, skeletal muscle, and heart. In smooth muscle, receptor activation stimulates Gs, adenylyl cyclase, cAMP, and protein kinase A. Protein kinase A phosphorylates several contractile proteins, especially myosin light chain kinase. Phosphorylation o myosin light chain kinase reduces its a f nity or calciumcalmodulin, leading to relaxation o the contractile apparatus. 2-Adrenoceptor activation may also relax bronchial smooth muscle by Gs-independent activation o K channels. Increased K e ux leads to bronchial smooth muscle cell hyperpolarization and, there ore, opposes the depolarization necessary to elicit contraction. In hepatocytes, activation o the Gs signaling cascade initiates a series o intracellular phosphorylation events that result in glycogen phosphorylase activation and glycogen catabolism. The result o 2adrenoceptor stimulation o hepatocytes is, there ore, an increase in plasma glucose. In skeletal muscle, activation o these same signaling pathways stimulates glycogenolysis and promotes K uptake. Recent studies in cardiac myocytes suggest that 2-adrenoceptor-mediated activation o the G subunit o Gi leads to activation o phosphatidylinositide-3 kinase , which, in turn, activates the protein kinase B (also known as Akt) pathway that con ers anti-apoptotic activity. 3-Adrenoceptors are expressed in adipose tissue and in the gastrointestinal tract. Stimulation o 3-receptors leads to

an increase in lipolysis and thermogenesis in adipocytes and to a decrease in gastrointestinal tract motility. These physiologic actions have led to speculation that 3-agonists may be use ul in the treatment o obesity, noninsulin-dependent diabetes mellitus, and other potential indications, but such selective pharmacologic agents remain to be developed or clinical use.

Regulation of Receptor Response The ability o receptor agonists to initiate downstream signaling is related to the number o receptors activated, and changes in the density o receptors on the cell sur ace o ten alter the apparent e f cacy o an agonist. Thus, both short-term (desensitization) and long-term (down-regulation) changes in the number o unctional adrenoceptors are important in regulating tissue response (see Fig. 1-10). When an agonist activates an adrenoceptor, the dissociation o its associated heterotrimeric G protein subunits leads not only to downstream signaling, as discussed above, but also to a negative eedback mechanism that limits tissue responses. Activation o -adrenoceptors recruits GPCRspecif c protein kinases (GRKs), which phosphorylate serine and threonine residues in the carboxyl-terminal tail o the receptor. Protein kinase A and protein kinase C can also phosphorylate G protein-coupled receptors. The phosphorylated state o the receptor promotes the translocation to the membrane o a cytosolic protein called -arrestin, which binds to the intracellular domain o the receptor and sterically inhibits interaction between the receptor and the G protein. This e ectively silences receptor signaling. -Arrestins also recruit clathrin and the clathrin adaptor protein AP2 to the phosphorylated receptor, and this complex targets the adrenoceptors to clathrin-coated pits. These pits are pinched o rom the membrane with the help o the large GTPase dynamin, and the internalized receptors are then either rapidly recycled, targeted to endosomes and recycled more slowly, or degraded in lysosomes. Each o these processes is important in regulating tissue responsiveness on a short- or longterm basis. Over the last decade, evidence has suggested that -arrestins can turn on (rather than o ) novel signaling pathways by serving as sca old proteins or signaling complexes that promote G protein-independent pathways involving the activation o Erk1/2, Src, and small GTP-binding proteins. A newly discovered aspect o GPCR signaling is that some antagonists that block G protein signaling pathways may also unction as agonists in alternative signaling pathways such as -arrestin signaling. In this way, both the desensitization/ down-regulation and the signaling roles or -arrestins may be involved in physiologic and pathologic situations.

Physiologic and Pharmacologic Effects of Endogenous Catecholamines The endogenous catecholamines epinephrine and norepinephrine act as agonists at both - and -adrenoceptors. At supraphysiologic concentrations, dopamine can also act as an agonist at - and -receptors. The overall e ect o each catecholamine is complex and depends on the concentration o the agent and on tissue-specif c receptor expression.

Epinephrine

Epinephrine is an agonist at both - and -adrenoceptors. At low concentrations, epinephrine has predominantly 1 and 2

156 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

effects, while at higher concentrations, its 1 effects become more pronounced. Acting at 1-receptors, epinephrine increases cardiac contractile orce and cardiac output, with consequent increases in cardiac oxygen consumption and systolic blood pressure. Vasodilation mediated by 2-receptors causes a decrease in peripheral resistance and a decrease in diastolic blood pressure. Stimulation o 2-receptors also increases blood ow to skeletal muscle, relaxes bronchial smooth muscle, promotes glycogenolysis, and increases the concentrations o glucose and ree atty acids in the blood. Recent studies suggest that 1-receptors are responsible or vasodilation in large arteries such as the emoral and pulmonary arteries, while 2-receptors have a predominant role in vasodilation o the arterioles that contribute to peripheral vascular resistance. These 1 and 2 e ects are all components o the “f ght-or- ight” response. Epinephrine was used to treat acute asthmatic attacks shortly a ter its discovery more than 100 years ago; other drugs with higher selectivity or 2-receptors, and which are delivered directly to pulmonary 2-receptors by inhalation, are now more o ten used in the treatment o asthma, chronic obstructive pulmonary disease, and other pulmonary conditions. Epinephrine remains a drug o choice or the treatment o anaphylaxis. Locally injected epinephrine causes vasoconstriction and prolongs the action o local anesthetics; or example, it is o ten used in combination with a local anesthetic in dentistry. It is ine ective orally due to extensive f rst-pass metabolism. Epinephrine has a rapid onset and a brie duration o action when injected intravenously. Adverse consequences o rapid intravenous in usions include increased cardiac excitability that may lead to cardiac arrhythmias and excessive increases in blood pressure.

Norepinephrine

Norepinephrine is an agonist at 1- and 1-receptors but has relatively little effect at 2-receptors. Because o the lack o action at 2-receptors, systemic administration o norepinephrine increases not only systolic blood pressure ( 1 e ect) but also diastolic blood pressure and total peripheral resistance. Norepinephrine is used in the pharmacologic treatment o hypotension in patients with distributive shock, most requently due to sepsis.

Dopamine

Although dopamine is a prominent CNS neurotransmitter, systemic administration has ew CNS e ects because it does not readily cross the blood–brain barrier. Dopamine activates one or more subtypes o catecholamine receptor in peripheral tissues, and the predominant e ect is dependent on the local concentration o the compound. At low doses ( 2 g/kg per min), a continuous intravenous in usion o dopamine acts predominantly on D1 dopaminergic receptors in renal, mesenteric, and coronary vascular beds. D1 dopaminergic receptors activate adenylyl cyclase in vascular smooth muscle cells, leading to increased cAMP levels and vasodilation. At higher rates o in usion (2–10 g/kg per min), dopamine is a positive inotrope via its activation o 1-adrenergic receptors. At still higher rates o in usion ( 10 g/kg per min), dopamine acts on vascular 1-adrenergic receptors to cause vasoconstriction. Dopamine is used in the treatment o shock, particularly in states o shock caused by low cardiac output and accompanied by compromised renal unction leading to oliguria. However, e f cacy in protecting the kidneys has not been clearly demonstrated.

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic intervention is possible at each o the major steps in catecholamine synthesis, storage, reuptake, metabolism, and receptor activation. The ollowing discussion presents the various classes o agents in the order o their action on adrenergic pathways, rom neurotransmitter synthesis to receptor activation.

Inhibitors of Catecholamine Synthesis Inhibitors o catecholamine synthesis have limited clinical utility because such agents nonspecif cally inhibit the ormation o all catecholamines (see Fig. 11-1). -Methyltyrosine is a structural analogue o tyrosine that is transported into nerve terminals, where it inhibits tyrosine hydroxylase, the f rst enzyme in the catecholamine biosynthesis pathway. This agent is used occasionally in the treatment o hypertension associated with pheochromocytoma (a tumor o the enterochroma f n cells o the adrenal medulla that produces norepinephrine and epinephrine). Its clinical use is limited, however, because it causes signif cant orthostatic hypotension and sedation, and many other antihypertensive drugs with ewer adverse e ects are available or this indication.

Inhibitors of Catecholamine Storage Catecholamines originate rom two pools—de novo synthesis and recycled transmitter. An agent that inhibits catecholamine storage in vesicles generally has two sequential e ects. In the short term, the agent increases the net release o catecholamine rom the synaptic terminal, and thus mimics sympathetic stimulation (sympathomimetic ). Over a longer time period, however, the agent depletes the pool o available catecholamine and thus acts as a sympatholytic (inhibitor o sympathetic activity) (Fig. 11-4). Reserpine binds tightly to the vesicular antiporter VMAT at or very near the substrate-binding site on the cytoplasmic sur ace o the transporter. Although the time course o binding is relatively slow, the tight binding results in irreversible inhibition o the antiporter (see Figs. 11-1 and 11-2). VMAT inhibition causes the secretory vesicles to lose their ability to concentrate and store norepinephrine and dopamine. At low doses, reserpine causes neurotransmitter to leak into the cytoplasm, where the catecholamine is destroyed by MAO. At high doses, the rate o transmitter leak can be su f ciently high to overwhelm the MAO in the presynaptic neuron. Under these conditions, there is a high concentration o transmitter in the neuronal cytoplasm, and transmitter can exit rom the cytoplasm to the synaptic space through NET acting in reverse. The e ux o catecholamine has a transient sympathomimetic e ect. Because reserpine’s inhibition o VMAT is irreversible, new storage vesicles must be synthesized and transported to the nerve terminal to restore proper vesicular unction. The recovery phase may require days to weeks a ter an individual stops taking reserpine. Reserpine can also be used experimentally to assess whether drugs need to be concentrated in presynaptic terminals to exert their action. In the past, reserpine was used to treat hypertension. However, the irreversible nature o its action and its association with severe depression make it an unattractive agent now that more e f cacious and less toxic drugs are available or the treatment o hypertension. Nonetheless, there has

C h a p t e r 1 1 Adrenergic Pharmacology 157 A Acute e ffe ct of indire ct s ympa thomime tic Cytopla s m

S yna ptic cle ft NE

NE G G

NE NE

G

NET

VMAT

NE

NE

G G

NE NE NE

NE

NE

MAO Mitochondrion

NE

DOP GAL

B Chronic e ffe ct of indire ct s ympa thomime tic

G G

G NE

NET

VMAT

NE

G G

NE

MAO Mitochondrion

DOP GAL

FIGURE 11-4. Acute and chronic effects of indirect sympathomimetics. Indirect sympathomimetics have di erent e ects on sympathetic outf ow depending on whether they are administered acutely or chronically. A. Administered acutely, an indirect sympathomimetic such as guanethidine (G) displaces norepinephrine (NE) that is stored in the synaptic vesicles o adrenergic neurons. This results in a massive e f ux o norepinephrine through the NE transporter acting in reverse; the resultant f ooding o the synapse with norepinephrine causes marked sympathetic stimulation. B. Administered chronically, an indirect sympathomimetic such as guanethidine (G) is concentrated in synaptic vesicles and replaces norepinephrine. In addition, monoamine oxidase (MAO) degrades the small pool o norepinephrine that remains in the cytoplasm. Both o these e ects contribute to decreased sympathetic stimulation.

been some interest in the possibility that reserpine may be a use ul drug or the treatment o hypertension when used at doses lower than those associated with severe depression. Tyramine is a dietary amine that is ordinarily metabolized by MAO in the gastrointestinal tract and liver. In patients taking MAO inhibitors (MAOIs; see below), tyramine is absorbed in the gut, transported through the blood, and taken up by sympathetic neurons, where it is transported into synaptic vesicles by VMAT. Uptake o tyramine by the synaptic vesicles causes displacement o vesicular norepinephrine and nonvesicular release o norepinephrine rom the nerve terminal via reversal o NET. By this mechanism, an acute challenge with large amounts o dietary tyramine, or with modest dietary tyramine in patients taking MAOIs, can cause acute and massive release o norepinephrine rom nerve terminals. In turn, the massive norepinephrine release

causes a pressor response with markedly elevated systolic blood pressure. Fermented oods such as red wine and aged cheese possess high concentrations o tyramine; this is why, in the introductory case, Ms. S developed a hypertensive crisis shortly a ter her wine and cheese party. Although tyramine itsel is poorly retained in synaptic vesicles, its hydroxylated metabolite octopamine (the synthesis o which is catalyzed by vesicular dopamine -hydroxylase) can be stored at high concentrations in the vesicles. Under conditions o chronic MAOI treatment and modest dietary tyramine intake, norepinephrine may gradually be replaced by octopamine in storage vesicles. Because octopamine has little agonist activity at most mammalian adrenoceptors, postsynaptic responses to sympathetic stimulation may gradually be diminished, leading ultimately to postural hypotension. Migraine and cluster headaches have been associated with elevated levels o circulating neurotransmitters and neuromodulators, including tyramine and octopamine. Like tyramine, guanethidine is actively transported by NET into neurons, where it concentrates in transmitter vesicles and displaces norepinephrine, leading to gradual depletion o norepinephrine (Fig. 11-4). Like octopamine, guanethidine is not an agonist at postsynaptic adrenoceptors, so its vesicular release upon sympathetic stimulation does not elicit a postsynaptic response. In the past, guanethidine was used to treat uncontrolled hypertension. Guanethidine inhibits cardiac sympathetic nerves, leading to reduced cardiac output, and it blocks sympathetically mediated vasoconstriction, leading to reduced cardiac preload. Inhibition o these sympathetic responses by guanethidine can lead to symptomatic hypotension ollowing exercise or standing up (postural hypotension). Guanadrel also acts as a alse neurotransmitter. As with guanethidine, this agent can be used in the treatment o hypertension, but it is no longer a f rst-line agent. The adverse e ect prof le o guanadrel is similar to that o guanethidine. Amphetamine has several adrenergic actions: (1) it displaces endogenous catecholamines rom storage vesicles (similar to tyramine); (2) it is a weak inhibitor o MAO-A; (3) it competitively inhibits catecholamine reuptake mediated by NET and DAT; and (4) it is an agonist at the trace amine-associated receptor 1 (TAAR1), a Gs/Gq coupled receptor on presynaptic neurons. Stimulation o TAAR1 by amphetamine activates protein kinases A and C, causing DAT phosphorylation and thereby noncompetitively inhibiting dopamine reuptake. Although amphetamine binds to postsynaptic adrenergic receptors, the drug has little agonist action at - or -adrenoceptors. Amphetamine has marked behavioral e ects including increased alertness, decreased atigue, depressed appetite, and insomnia. Thus, it has been used to treat depression, attention-def cit hyperactivity disorder (ADHD), and narcolepsy (recurrent attacks o drowsiness and sleep during the daytime) and to suppress appetite. Its adverse e ects can be substantial, including atigue and depression ollowing the period o central stimulation. Ephedrine , pseudoephedrine , and phenylpropanolamine are structurally related agents that have some capacity to activate various adrenergic responses. Ephedrine has been used medically or the treatment o persistent hypotension. An herbal source o ephedrine (and various isomers) called ma huang was used to treat asthma in China or at least 2,000 years. Pseudoephedrine is used as an over-the-counter

158 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

decongestant and is ound in some cold remedies. Phenylpropanolamine was removed rom the over-the-counter market in the United States due to concerns about an association with cerebral hemorrhage. Methylphenidate , a structural analogue o amphetamine, is widely used in psychiatry to treat ADHD in children; its major e ect is thought to be related to enhanced attention. Amphetamine can cause psychological and physiologic dependence as well as tolerance. Amphetamine may cause paranoia and hallucinations. Methamphetamine (“crank” or “crystal meth”) is a major drug o abuse. See Chapter 15, Pharmacology o Serotonergic and Central Adrenergic Neurotransmission, or more detailed discussion o the pharmacology o amphetamine and related drugs.

Inhibitors of Catecholamine Reuptake Inhibitors o catecholamine reuptake can exert an acute and power ul sympathomimetic e ect by prolonging the time that released neurotransmitter remains in the synaptic cle t. Cocaine is a potent inhibitor o NET; unlike other uptake inhibitors (such as imipramine and uoxetine), cocaine essentially eliminates catecholamine transport (see Fig. 11-2). It is used occasionally as a local anesthetic because o its independent activity as an inhibitor o neuronal action potentials (see Chapter 12, Local Anesthetic Pharmacology); in addition, in this setting, cocaine promotes vasoconstriction due to its capacity to inhibit norepinephrine uptake. Cocaine is a controlled substance with high abuse potential. It is a major public health concern because o its role as an agent o abuse (see Chapter 19, Pharmacology o Drugs o Abuse). Tricyclic antidepressants (TCAs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) inhibit NET-mediated reuptake o norepinephrine into presynaptic terminals and thus promote accumulation o norepinephrine in the synaptic cle t. Because o their important role in the treatment o depression, TCAs, SNRIs, and other inhibitors o norepinephrine reuptake are discussed in more detail in Chapter 15.

Inhibitors of Catecholamine Metabolism Monoamine oxidase inhibitors (MAOIs) prevent secondary

deamination o catecholamines that are transported into presynaptic terminals or taken up into tissues such as the liver. In the absence o metabolism, more catecholamine accumulates in presynaptic vesicles or release during each action potential. Most MAOIs are oxidized by MAO to reactive intermediates, which then act as irreversible inhibitors o MAO. Nonselective agents in this class (i.e., agents that inhibit both MAO-A and MAO-B) include tranylcypromine , phenelzine , and iproniazid (the drug used in the introductory case; this drug was later withdrawn rom the market in the United States and some other countries). Selective inhibitors include clorgyline , which is selective or MAO-A, and selegiline and rasagiline , which are selective or MAO-B. Moclobemide is a reversible inhibitor o MAO-A. As with the tricyclic antidepressants, MAOIs are used to treat depression. Selegiline and rasagiline are also approved or the treatment o Parkinson’s disease; their mechanism o action may include both potentiation o dopamine in the remaining nigrostriatal neurons and decreased ormation o neurotoxic intermediates. As noted above, patients taking MAOIs should avoid eating certain ermented or aged oods containing large amounts o tyramine and other

monoamines, such as most cheeses and some f shes, poultry, bee , and wines, because MAOIs block oxidative deamination o these monoamines in the gastrointestinal tract and liver, allowing them to enter the circulation and precipitate a hypertensive crisis. Concomitant use o MAOIs and selective serotonin reuptake inhibitors (SSRIs) or SNRIs is also contraindicated, because this may precipitate the serotonin syndrome . This syndrome is characterized by agitation, restlessness, tremors, seizures, tachycardia, hypertension, and possibly coma and death. Serotonin syndrome may also occur with concomitant use o MAOIs and other drugs, such as meperidine, tramadol, and amphetamine. The reversible inhibitors o MAO-A may be less prone to adverse e ects and drug interactions. MAOIs and SSRIs are also discussed in Chapter 14, Pharmacology o Dopaminergic Neurotransmission, and Chapter 15.

Receptor Agonists Because adrenoceptors are important in mediating vascular tone, smooth muscle tone, and cardiac contractility, selective agonists and antagonists o these receptors are mainstays o therapy or hypertension, asthma, ischemic heart disease, heart ailure, and other conditions. In the ollowing discussion, the agents are organized according to receptor subtype specif city (see Table 11-1 or an overview o the relevant receptor subtypes).

-Adrenergic Agonists

The 1-selective adrenergic agonists increase peripheral vascular resistance and thereby maintain or elevate blood pressure. These drugs may also cause sinus bradycardia through activation o re ex vagal responses mediated by baroreceptors. Systemically administered 1-agonists, such as methoxamine , have limited clinical use but are sometimes employed to increase blood pressure in the treatment o shock. A number o topically administered 1-agonists, such as phenylephrine , oxymetazoline , and tetrahydrozoline , are used in the nonprescription remedies A rin® and Visine® (and others) to constrict vascular smooth muscle in the symptomatic relie o nasal congestion and ophthalmic hyperemia. Oxymetazoline is also a partial agonist at 2-receptors. Damage to the nasal mucosa and possible rebound hypersensitivity and return o symptoms o ten accompany extended use o these medications. Phenylephrine is also used intravenously in the treatment o distributive shock. Clonidine is an 2-receptor agonist that lowers blood pressure and decreases heart rate by acting in brainstem vasomotor centers to suppress sympathetic out ow to the periphery. Evidence supporting its capacity to decrease adverse cardiovascular outcomes in patients with hypertension is limited. Clonidine has limited utility in ameliorating symptoms o withdrawal rom ethanol and opioid drugs. Adverse e ects include bradycardia caused by decreased sympathetic activity and increased vagal activity, as well as dry mouth and sedation. Because sympathetic nervous system activation is an important mechanism in maintaining blood pressure on standing, postural hypotension may also complicate therapy with this drug. Other centrally acting 2-agonists include the seldom-used agents guanabenz and guanfacine . These agents have adverse e ect prof les similar to that o clonidine. Dexmedetomidine is an 2-receptor agonist whose capacity to cause sedation has been exploited as a benef cial e ect

C h a p t e r 1 1 Adrenergic Pharmacology 159

in surgical patients, because sedation is induced by this drug without additional respiratory depression. Suppression o sympathetic nervous system activity by dexmedetomidine helps to avoid swings in blood pressure in surgical patients, who are care ully monitored by anesthetists during surgical procedures. Dexmedetomidine may also possess analgesic properties. Note that the 2-mediated e ects o sedation and decreased sympathetic activity are adverse e ects o clonidine in the setting o outpatient treatment or hypertension but benef cial e ects o dexmedetomidine in the controlled setting o the surgical patient. -Methyldopa is a precursor (prodrug) to the 2-agonist -methylnorepinephrine. Dopamine -hydroxylase catalyzes the metabolism o methyldopa to methylnorepinephrine, and the -methylnorepinephrine is then released by the adrenergic nerve terminal, where it can act presynaptically as an 2-agonist. This action results in decreased sympathetic out ow rom the CNS and consequent lowering o blood pressure in hypertensive patients. Methyldopa is also a competitive inhibitor o DOPA decarboxylase, which converts DOPA to dopamine, and thereby reduces adrenergic neurotransmission in the peripheral nervous system. Because -methyldopa use can be associated with rare hepatotoxicity, autoimmune hemolytic anemia, and adverse CNS e ects, this drug is very rarely used in the treatment o hypertension in the United States, with one exception—there is considerable experience with methyldopa as an antihypertensive drug in pregnancy, and it is still used as a pre erred drug in that context.

-Adrenergic Agonists

Stimulation o 1-adrenergic receptors causes increases in the heart rate and the orce o cardiac muscle contraction, resulting in increased cardiac output, while stimulation o 2-adrenergic receptors causes relaxation o vascular, bronchial, and gastrointestinal smooth muscle. Isoproterenol is a nonselective -agonist. This drug lowers peripheral vascular resistance and diastolic blood pressure (a 2 e ect), while systolic blood pressure remains unchanged or slightly increased (a 1 e ect). Because isoproterenol is a positive inotrope (increases cardiac contractility) and chronotrope (increases heart rate), cardiac output is increased. Isoproterenol can be used to relieve bronchoconstriction in asthma ( 2 e ect). However, because isoproterenol is a nonselective activator o 1- and 2-adrenoceptors, its use or relie o bronchoconstriction is o ten accompanied by adverse cardiac e ects. Use o this drug in asthma has there ore been supplanted by newer 2-selective agonists (see below). Isoproterenol may occasionally be used to increase the heart rate in emergency situations o pro ound bradycardia, typically in anticipation o the placement o an electrical cardiac pacemaker. The overall e ect o dobutamine depends on the di erential e ects o the two stereoisomers contained in the racemic mixture (see Chapter 1 or a discussion o stereoisomers). The ( ) isomer acts as both an 1-agonist and a weak 1-agonist, whereas the ( ) isomer acts as both an 1-antagonist and a potent 1-agonist. The 1-agonist and antagonist properties e ectively cancel each other out when the racemic mixture is administered, and the observed clinical result is that o a selective 1-agonist. This agent has more prominent inotropic than chronotropic e ects, resulting in increased contractility and cardiac output. Dobutamine can

be used intravenously in the urgent treatment o severe heart ailure. It is also used as a diagnostic agent, in conjunction with imaging o the heart, in the investigation o ischemic heart disease. 2-Selective agonists are valuable in the treatment o asthma. These drugs represent pharmacologic improvements over epinephrine (an agonist at all adrenergic receptors) and isoproterenol (an agonist at 1- as well as 2-receptors) in that their e ects are more limited at nontarget tissues. It is particularly important that these selective drugs have limited capacity to stimulate 1-adrenoceptors in the heart and, there ore, limited capacity to produce adverse cardiac e ects. Specif city or the lung rather than the heart or other peripheral tissues has been urther enhanced by generally delivering these drugs via aerosols inhaled into the lungs. Administration o the drugs directly into the lungs lowers the amount o drug that reaches the systemic circulation, again limiting the activation o cardiac 1-receptors and skeletal muscle 2-receptors. The most important e ects o these agents are relaxation o bronchial smooth muscle and decrease in airway resistance. 2-Selective agonists are not completely specif c or airway 2-receptors, however, and adverse e ects can include skeletal muscle tremor (through 2-stimulation) and tachycardia (through 1-stimulation). Metaproterenol is the prototype 2-selective agonist. This drug is used to treat obstructive airway disease and acute bronchospasm. Terbutaline and albuterol are two other agents in this class that have similar e f cacy and duration o action. Salmeterol is a long-acting 2-agonist; its e ects last or about 12 hours. The clinical utility o 2-selective agonists is discussed more ully in Chapter 48, Integrative In ammation Pharmacology: Asthma.

Receptor Antagonists A broad spectrum o disease states respond to modulation o adrenoceptor activity, and antagonists at - and adrenoceptors are among the most widely used drugs in clinical practice.

-Adrenergic Antagonists

-Adrenergic antagonists block the binding o endogenous catecholamines to 1- and 2-adrenoceptors. These agents cause vasodilation, decreased blood pressure, and decreased peripheral resistance. The baroreceptor re ex usually attempts to compensate or the all in blood pressure, resulting in re ex increases in heart rate and cardiac output. An important laboratory tool since the 1950s, phenoxybenzamine is an alkylating agent that blocks both 1- and 2-receptors irreversibly. In addition, phenoxybenzamine inhibits catecholamine uptake into both adrenergic nerve terminals and extraneuronal tissues. Because o its many direct and indirect e ects on the sympathetic nervous system and target tissues, phenoxybenzamine, once used in the treatment o hypertension and benign prostatic hyperplasia (BPH), is now rarely used clinically. Some physicians use preoperative phenoxybenzamine to prepare patients with pheochromocytoma or surgery with the intent to decrease operative complications. Phenoxybenzamine has been ound to cause tumors in laboratory animals, although the implications o these f ndings or humans are unclear. Phentolamine is a reversible, nonselective -adrenoceptor antagonist. This drug can also be used in the preoperative

160 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

management o pheochromocytoma. Phentolamine was the pharmacologically ideal agent or use in the introductory case, because it blocked the -adrenergic–mediated vasoconstriction that caused Ms. S’s hypertension. However, most physicians have very little clinical experience with phentolamine, and other drugs are more requently used in the treatment o severe hypertension. Prazosin has a 1,000- old higher a f nity or 1-receptors than or 2-receptors. Its selective blockade o 1-receptors in arterioles and veins results in decreased peripheral vascular resistance and dilation o the venous (capacitance) vessels. The latter e ect decreases venous return to the heart; because o this reduction in cardiac preload, prazosin has little tendency to increase cardiac output and heart rate. Prazosin is an antihypertensive drug. Because patients may experience marked postural hypotension and syncope with the f rst dose, the drug is generally prescribed initially at a very low dose and is titrated to higher doses depending on the clinical response. Used in this manner, postural hypotension is uncommon, presumably due to the development o tolerance (by an unclear mechanism). Other agents in this class include terazosin and doxazosin; these agents have a longer hal -li e than prazosin, allowing less requent dosing. 1-Receptor antagonists are not o ten used clinically in the treatment o hypertension, because comparative studies have suggested that other antihypertensive medications, such as diuretics, may be more e ective. Because 1-adrenoceptors mediate contraction o genitourinary as well as vascular smooth muscle, some 1-antagonists have ound clinical application in the symptomatic treatment o benign prostatic hyperplasia (BPH). 1-Adrenoceptor antagonists may be more e f cacious than f nasteride (a 5 reductase inhibitor; see Chapter 30, Pharmacology o Reproduction) in the medical treatment o BPH. Also, their onset o action is relatively rapid, whereas that o 5 -reductase inhibitors is generally delayed by months or more. As noted above, there are three subtypes o the 1-receptor, namely 1A, 1B, and 1D. Evidence points to pre erential expression o the 1A-receptor in genitourinary smooth muscle. Tamsulosin is a relatively selective antagonist at 1A-receptors; however, the selectivity is modest: the drug binds with approximately six old higher a f nity to 1A- than to 1B-receptors. The increased selectivity o tamsulosin or 1A-receptors may decrease the incidence o orthostatic hypotension relative to that associated with prazosin and other nonsubtype-selective 1-adrenoceptor antagonists. However, this modest advantage has been demonstrated only at low doses o tamsulosin. Selective blockade o 2-autoreceptors by drugs such as yohimbine leads to increased release o norepinephrine, with subsequent stimulation o cardiac 1-receptors and peripheral vasculature 1-receptors. 2-Selective antagonists also cause increased insulin release through blockade o 2-receptors in the pancreatic islets, which suppress insulin secretion. Yohimbine has been used to treat erectile dys unction on the basis o very limited data suggesting possible clinical e f cacy.

-Adrenergic Antagonists

-Adrenergic antagonists block the positive chronotropic and inotropic actions o endogenous catecholamines at 1receptors, resulting in decreased heart rate and myocardial contractility. These drugs decrease blood pressure in hypertensive patients but generally do not lower blood pressure in normotensive individuals. Long-term use o -adrenoceptor blockers decreases peripheral vascular resistance, although

the mechanism o this e ect remains unclear. The decreases in peripheral vascular resistance and cardiac output both contribute to the antihypertensive e ect o these drugs. Nonselective -adrenoceptor antagonists also block 2-receptors in bronchial smooth muscle, which can cause li e-threatening bronchoconstriction in patients with asthma. In addition, nonselective -receptor blockade may mask symptoms o hypoglycemia in diabetic patients. For these reasons, selective inhibitors o 1-adrenoceptors have been developed. Pharmacologic antagonists at -adrenergic receptors can be divided into nonselective -antagonists, nonselective -antagonists with concomitant action as 1-antagonists, -adrenergic partial agonists, and 1-selective antagonists (Table 11-2). Selective blockers o 2-adrenergic receptors have not been developed clinically as there is no obvious indication or selective 2-receptor antagonism. Propranolol, nadolol, and timolol do not distinguish between 1- and 2-receptors in their binding a f nities. This is the origin o the term “nonselective -blockers.” At clinical doses, these drugs do not block -receptors. Nonselective blockers have been used or many years in the treatment o hypertension and angina. Although nonselective -blockers are relatively contraindicated in patients with asthma, these drugs are o ten well tolerated in patients with chronic obstructive pulmonary disease (COPD) and may be initiated cautiously in many such patients i they have a compelling indication (e.g., coronary artery disease). Nadolol is also e f cacious in the prevention o bleeding rom esophageal varices in patients with cirrhosis. It is pharmacologically attractive or this indication because it has a long hal -li e,

TABLE 11-2 Selectivity of Some -Adrenoceptor Antagonists DRUG

NOTES

Nonselective -Adrenergic Antagonists Propranolol

Short half-life

Nadolol

Long half-life

Timolol

Lipophilic, high CNS penetration

Nonselective - and

1-Antagonists

Labetalol

Also partial agonist at

Carvedilol

Intermediate half-life

2-receptors

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-Nonselective

Acebutolol

1-Selective

1-Selective

Adrenergic Antagonists

Esmolol

Short half-life (3–4 minutes)

Metoprolol

Intermediate half-life

Atenolol

Intermediate half-life

Celiprolol

Also agonist at

CNS, central nervous system.

2-receptors

C h a p t e r 1 1 Adrenergic Pharmacology 161

allowing once-daily administration, and, because the drug is excreted primarily by renal elimination without hepatic metabolism, no dosing adjustments are needed on account o hepatic insu f ciency. Penbutolol is an additional drug in this class. An ocular ormulation o timolol is used in the treatment o glaucoma; even when administered to the eye, systemic absorption o the drug may be su f ciently high to cause adverse e ects in susceptible patients. Levobunolol and carteolol are additional nonselective -blockers that are indicated or administration via eye drops in the treatment o glaucoma. Labetalol and carvedilol block 1-, 1-, and 2-receptors. Labetalol has two chiral centers; the clinically used drug is a combination o our stereoisomers that have di ering pharmacologic properties. Two o these isomers are inactive— (S,S) and (R,S). The (S,R) isomer is a power ul 1-blocker, and the (R,R) isomer is a nonselective blocker at 1-, 1-, and 2-receptors. Because the e ect and metabolism o these isomers may vary among individual patients, the relative proportion o 1- versus -blockade is variable. The 1-receptor blockade tends to lower peripheral resistance; -blockade also contributes to a decrease in blood pressure, as indicated above. An intravenous ormulation o labetalol is available or the lowering o blood pressure in patients with hypertensive emergencies. Drug-induced hepatitis is an unpredictable and idiosyncratic adverse e ect o labetalol. In addition to its action as an 1-, 1-, and 2-receptor blocker, carvedilol acts as a G protein-independent, arrestin biased ligand at both 1- and 2-adrenergic receptors. Moreover, while carvedilol is e f cacious in the outpatient management o hypertension, much interest in this drug has been due to its e f cacy in the management o heart ailure with decreased systolic unction. Carvedilol’s cardioprotective e ect may be related to its action as a -arrestin biased ligand at 1-adrenoceptors, leading to transactivation o the epidermal growth actor receptor. Pindolol is a partial agonist at 1- and 2-receptors. The drug blocks the action o endogenous norepinephrine at 1-receptors and is use ul in treating hypertension. As a partial agonist, pindolol also causes partial stimulation o 1-receptors, leading to overall smaller decreases in resting heart rate and blood pressure than those caused by pure antagonists. Acebutolol is a partial agonist at 1-adrenoceptors but has no e ect at 2-receptors. This agent is also used to treat hypertension and ischemic heart disease. While it has been suggested that partial agonists may be less likely to cause adverse e ects in patients with bradycardia, the clinical advantages o drugs in this category remain unclear. Esmolol, metoprolol, atenolol, and betaxolol are 1selective adrenergic antagonists. The elimination hal -li e is the main eature that distinguishes among these agents. Esmolol has an extremely short hal -li e (3–4 minutes); metoprolol and atenolol have intermediate hal -lives (4–9 hours). Because o its short hal -li e, esmolol may be sa er in unstable patients requiring -blockade. Esmolol is rapidly metabolized by esterases. Clinical trials have shown that some -blockers, including metoprolol, prolong li e expectancy in patients with mild to moderate heart ailure and in patients who have survived a f rst myocardial in arction (see Chapter 26, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure). Nebivolol is a novel 1-selective adrenergic antagonist that

has the ancillary property o promoting vasodilation via nitric oxide release rom endothelial cells. Many o the major adverse e ects o -adrenergic antagonists are a predictable extension o their pharmacologic e ects. Such e ects include worsening o bronchoconstriction in patients with asthma, decreased cardiac output in patients with decompensated heart ailure, and potentially impaired recovery rom hypoglycemia in diabetic patients receiving insulin. While 1-selective adrenergic antagonists may have a lower propensity to block 2-receptors in bronchial smooth muscle, the selectivity o these drugs is modest and may not be a clinically reliable sa eguard against adverse e ects. With chronic administration o -receptor antagonists, pharmacologic adaptations may occur that render cells hypersensitive to catecholamines when the drug is stopped suddenly.

CONCLUSION AND FUTURE DIRECTIONS Adrenergic pharmacology encompasses drugs that act at essentially every step o adrenergic neurotransmission, rom synthesis o catecholamines to stimulation o - and receptors. Other classes o drugs, such as L-channel Ca2 blockers, inter ere with e ector responses activated by these receptors. Novel drugs are being developed that selectively inhibit the downstream e ector pathways activated by adrenergic receptors. The drugs discussed in this chapter are mainstays o therapy or hypertension, angina, heart ailure, shock, asthma, pheochromocytoma, and other conditions. The benef cial pharmacologic actions o these drugs, as well as many o their important adverse e ects, can be anticipated rom knowledge o their molecular and cellular mechanisms o action and how these actions a ect the processes o adrenergic neurotransmission. While nine subtypes o adrenergic receptor have been identif ed, three in each o the major classes, the clinical relevance o these subtypes has not yet been ully determined and the pharmacologic implications o these discoveries have not been ully exploited. The development o novel, subtype-selective agonists and antagonists may lead to more e ective and less toxic therapies. Biased ligands represent an opportunity or the discovery o new drugs that may expand and diversi y the therapeutic options available to clinicians.

Acknowledgment We thank Freddie M. Williams, Timothy J. Turner, and Brian B. Ho man or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading DeWire SM, Ahn S, Le kowitz RJ, Shenoy SK. Beta-arrestins and cell signaling. Annu Rev Physiol 2007;69:483–510. (Review of novel mechanisms of signaling via seven transmembrane receptors.) Reiter E, Ahn S, Shukla AK, Le kowitz RJ. Molecular mechanism o arrestin-biased agonism at seven transmembrane receptors. Annu Rev Pharmacol Toxicol 2012;52:179–197. (Review of biased ligands that have the ability to alter the balance between G protein-dependent and -arrestindependent signal transduction.) Rosenbaum DM, Rasmussen SG, Kobilka BK. The structure and unction o G-protein-coupled receptors. Nature 2009;459:356–363. (Detailed review of the structure of adrenergic receptors.)

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166 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

Epine u rium Pe rin inee u rium m E do En done uriu um S chw wa nn n ce ll

12

Local Anesthetic Pharmacology Q u e n t in J . Ba c a , J o s h u a M . S ch u lm a n , a n d Ga ry R. S t rich a rt z 1

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INTROD DUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . 16 167– 7 16 1688 Disttribu Di buti tio ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1776 Ne e dl ddle llee iinje jje ct ti g LA cting Metabolism M etabolis and Excretionn . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 PHYSIOL LOGY OF NO OCI C CEPTION . . . . . . . . . . . . . . . . . . . . . . . . . . 167 1667 Administration of Local Anesthetics . . . . . . . . . . . . . . . . . . . . . 17 Ad 1766 Transm missionUnm ofm Pain Pa Unmy yeSensation llina ina na tee d fibe f be r bbundle uundle C fibe rs 169 )699 undl ((C Topi To pical Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 16 Firstt Pain and Sec Secon c oond ond P Pain Pa iinn . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Inf ltrati tionn Ane nesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Pain n Perce ercept ptiion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 ckad adee . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Mye lin a te lin t d fibe r (A fib fibe rs ) Peripheral Nerve Block Analgesia and Anesthesia . . . . . . My . . . .e lina Central Nerve Blockadee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 177 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 171 Intravenous Regional and Systemi micc An Anes esth thesia . . . . . . . . 17 1777 Molecular Determinants of Local Anesthetic Actio cttion . . . . . . . 17 1711 Majo j r Toxi jo oxi xici citi ci ties es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Aromatic Groupp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1711 I divi In dividu dual al Age g nts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1788 Amine Groupp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Ester-Linked Local Anest sthe heti tics cs . . . . . . . . . . . . . . . . . . . . . . . 178 Mechanism of Action of Local Anesthetics. . . . . . . . . . . . . . . 172 Amide-Linked Local Anestheticss . . . . . . . . . . . . . . . . . . . . . 17 1799 Anatomic Factors in Impulse Blockade . . . . . . . . . . . . . . . . 172 Voltage-Gated Sodium Channell . . . . . . . . . . . . . . . . . . . . . . 173 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 180 Other Receptors or Local Anestheticss . . . . . . . . . . . . . . . . 175 Su Sugg gges este tedd Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .180 Pharmacokinetics Ph ki i off L Locall Anesthetics A h i . . . . . . . . . . . . . . . . . . 176 Systemic Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

INTRODUCTION Local anesthetics (LAs) are a class o locally applied chemi-

cals, with similar molecular structures, that can disrupt nerve transmission, inhibit the perception o sensations (importantly, pain), blunt autonomic activity, and prevent movement. They are used or a variety o clinical applications and delivered via many di erent approaches, ranging rom topical application or burns and small cuts, to injections during dental care, to epidural and intrathecal (“spinal”) blocks during obstetric procedures and major surgery. Cocaine , the f rst local anesthetic, comes rom the leaves o the coca shrub (Erythroxylon coca). It was f rst isolated in 1860 by Albert Niemann, who noted its numbing powers. In 1886, Carl Koller introduced cocaine into clinical practice as a topical ophthalmic anesthetic. However, its addictive properties and toxicity prompted the search or substitutes. Procaine , the f rst o these substitutes to prove clinically use ul, was synthesized in 1905. Known as Novocain®, it is still used today, although less requently than some more recently developed LAs. Local anesthetics exert their effect by blocking voltagegated sodium channels, thus inhibiting the propagation of action potentials along neurons (see Chapter 8, Principles o Cellular Excitability and Electrochemical Transmission). By inhibiting action potential propagation, LAs prevent transmission o in ormation to and rom the central nervous

system (CNS). LA actions are not selective or pain f bers; they can also block conduction in other sensory f bers as well as motor and autonomic f bers and action potentials in skeletal and cardiac muscle. This nonselective blockade can serve other use ul unctions (see Chapter 24, Pharmacology o Cardiac Rhythm) or can be a source o toxicity.

PHYSIOLOGY OF NOCICEPTION Nociception occurs ollowing the activation o particular

primary sensory nerve f bers (nociceptors) by a noxious stimulus representing potential or actual tissue damage. Noxious stimuli include extreme temperatures, intense mechanical perturbations, harsh chemical environments, and certain molecules released by damaged cells (Fig. 12-1). Nociceptors are activated by noxious stimuli at ree nerve endings, which are located in the skin, deeper tissues such as joints and tendons, and viscera. When activated, nociceptors transmit impulses rom the periphery to the dorsal horn o the spinal cord, where the in ormation is subsequently processed through synaptic circuitry and transmitted to various parts o the brain. Thus, nociceptors are the f rst, or primary, cells in a chain o neurons ultimately responsible or pain perception. Because nociceptors, among other sensory f bers, transmit in ormation toward the brain, they are termed afferent neurons . 167

168 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

EM is a 24-year-old graduate student in o rganic che m is try. Wh ile h e is working in th e lab on e even in g, a bo ttle o con ce n tra te d hyd ro ch lo ric a cid a lls ro m a sh el above him an d s m ash es h is le t hand against the lab bench be ore it shatters and cuts his index f nger. Although he re exively jerks his hand away, s om e o the acid alls on th e f n g ertips o his h a n d . He im m e d ia te ly e e ls a lo ca lize d s h a rp p a in wh ere th e b ottle cut h is ha n d o llo wed b y a d i u s e stinging pain where the acid touched his skin. Although he begins to rinse his hand under running water, he still eels a slowly developing, burning, throbbing ache th ro u g h o u t h is h a n d . EM a n xio u s ly te le p h o n es 911 and is transported to the em ergency departm ent. On e xa m in th e e m e rg e n cy d e p a rtm e n t, h e is n o te d to h a ve a 1.5-cm la ce ra tio n o h is le t in d e x f n ge r a n d is o la te d a re a s o red , te n de r s kin w ith pa rtia lth ickn e s s b u rn o n th e f n g e rs e xp o s e d to a cid . He is in s e ve re p a in a n d h is la ce ra tio n w ill n e e d to b e s u tu re d . A te r co n tin u e d irriga tio n o th e a cid -e xp o s e d a re a s o h is h a n d , to pica l EMLA cre a m (e u te ctic m ixtu re o lo ca l a n e s th e tic—a co m b in a tio n o lid o ca in e a n d p rilo ca in e ) is a p p lie d to th e b u rn e d a re a s a n d a d ig ita l n e rve b lo ck o h is in d e x f n g e r is p e r o rm e d . A te r th e a re a is m a d e s te rile , 1 cc o 2% lid o ca in e w ith o u t e p in e p h rin e is in je cte d in to th e m e d ia l a n d

la te ra l a s p e cts o h is f n g e r n e a r th e w e b s p a ce to b lo ck th e d ig ita l n e rve s . Th e lid o ca in e in je ctio n is d ire cted to wa rd both the pa lm a nd the ba ck o h is ha nd in o rd er to b lo ck b o th th e d o rs al an d ve n tral co n trib utio n s to th e d ig ita l n e rve s th a t ru n d o w n th e la te ra l a n d m e d ia l a s p e cts o th e f n g e r. He n o tice s th a t th e s harp s tinging pain rom his cut aba tes f rs t, ollowe d b y the dull throbbing ach e in his f n ger. EM then los es s e n s a tio n to lig ht to uch o n his f n ge r a n d la te r n o te s th a t h e ca n n o t e e l th e n e e d le p ie rcin g h is s kin a s th e s utu re s a re a p p lie d . EM’s w o un d s h e a l ove r th e n e xt 2 w e e ks , a n d th e p a in , n o w p rim a rily a re s u lt o p o s ttra u m a tic in a m m a tio n , is w e ll co n tro lle d w ith o ra l ib u p ro e n . He is a b le to re tu rn to w o rk.

Questions 1 . Why did EM initially experience a stinging pain be ore the dull aching pain, and why did the stinging pain resolve more quickly than the dull pain a ter lidocaine administration? 2 . Why is epinephrine sometimes administered with lidocaine, and why was it not co-administered in this case? 3 . What is lidocaine’s mechanism o action? To which broader class o drugs does it belong?

Dors a l root ga nglion To bra in

1a The rma l Me cha nica l Che mica l

Fre e ne rve e ndings

2 Nocice ptor a ctiva tion

1b Ne a rby ce ll da ma ge

S pina l cord

Bra dykinin Endothe lin-1

Axon

P ros ta gla ndins S e rotonin, his ta mine

3 CGRP a nd s ubs ta nce P re le a s e d by a ctiva te d nocice ptors

4b Ma s t ce ll de gra nula tion 4a Blood ve s s e l dila tion

FIGURE 12-1. Nociceptor activation. Nociceptors transduce pain in ormation using a variety o mechanisms. Some receptors on the neuron transduce noxious stimuli (thermal, mechanical, or chemical) into electrical potentials. Other receptors are stimulated by substances that are released when nearby cells are injured (bradykinin, endothelin-1, ATP, prostaglandins). The release o K rom nearby damaged cells directly depolarizes nociceptor membranes. All o these stimuli cause nociceptor “sensitization,” which decreases the threshold or electrical activation. 1a. A noxious stimulus leads to nociceptor activation and action potential generation (2). 1b. Concurrent nearby cell damage causes nociceptor sensitization. 3. Activated nociceptors release substances, including substance P and calcitonin gene-related peptide (CGRP), that contribute to urther sensitization and that initiate inf ammatory responses to promote healing. For example: 4a. Blood vessel dilation promotes white blood cell recruitment to the area. 4b. Mast cell degranulation releases histamine and serotonin, thus increasing sensitization.

C h a p t e r 1 2 Local Anesthetic Pharmacology 169

Nociceptors are normally not activated by nonnoxious stimuli such as a breeze against one’s skin or a handshake (the nerves that are activated in these circumstances are termed tactile or low- or medium-threshold mechanoreceptors ). When a noxious stimulus is present at a ree nerve ending o a nociceptor, it activates specif c transducing receptors that generate inward currents and depolarize the primary neuron (Fig. 12-1; also see Chapter 8). I a noxious stimulus has an intensity above the nociceptor’s threshold (e.g., above a certain temperature), an action potential (AP) will be generated. Sensory in ormation is almost always encoded by trains o APs, and the requency o APs in a train increases as the stimulus intensity increases (e.g., at higher temperatures). I impulses rom a nociceptive a erent nerve are su f ciently requent, or i multiple nociceptors are activated, then the generating stimulus is perceived as “pain ul.” Although lower stimulus intensities may not be perceived, these “subliminal” sensory inputs are still transmitted to the CNS and can in uence uture sensory coding. Explicitly pain ul stimuli are able to induce long-lasting increases in responsiveness o the CNS and thus to e ect a “memory” o pain. Such central sensitization is one component o chronic pain (see Chapter 18, Pharmacology o Analgesia).

Transmission o Pain Sensation In their simplest orm, neurons are composed o dendrites, a cell body, and an axon. Axons transmit in ormation along the neuron to dendrites, which synapse with other neurons. Nerve axons are classif ed as A-f bers , B-f bers , or C-f bers , ranked in the order o decreasing diameter and having respectively decreasing conduction velocities. A-f bers and B-f bers are myelinated; while C-f bers are nonmyelinated, they are still encased in a peripheral glial Schwann cell that collects up to 10 axons in a Remak bundle (Table 12-1). Myelin is composed o the cell membranes o supportive cells in the nervous system (Schwann cells peripherally and oligodendrocytes in the CNS) that wrap tightly many times around neuronal axons to create an electrically insulating sheath that increases the velocity o impulse transmission. Regular, narrow spacings between the myelinated segments, called nodes of Ranvier, contain the voltage-gated

Na channels and are the locations where inward ionic current drives the APs o myelinated f bers. Distinct types o nociceptors are activated by specif c noxious stimuli and transmit in ormation along characteristic classes o axons. Nociceptor axons are generally either A -f bers or C-f bers . Thermal nociceptors are activated at temperatures above 45°C (noxious heat, C-f bers) or below 5°C (noxious cold, A -f bers). High-threshold mechanical nociceptors exclusively transmit in ormation indicating injurious orce on the skin (A - and some A -f bers). Polymodal nociceptors are activated by thermal, mechanical, and chemical stimuli (C-f bers).

First Pain and Second Pain

Myelinated A -f bers transmit impulses aster than nonmyelinated C-f bers do (Fig. 12-2). An A -f ber transmits impulses along its axon at a rate o 5–25 meters per second (m/s), while C-f bers transmit impulses at roughly 1 m/s. Impulse transmission is slower in C-f bers primarily because these f bers are nonmyelinated. The A -f bers transmit what is called f rst pain, which is perceived within seconds a ter an injury, is sharp in quality, and is highly localized on the body. The density o nociceptor A -f bers is high on the f ngertips, ace, and lips but relatively low on the back. A -f bers require a weaker stimulus or excitation than C-f bers do. The C-f bers transmit what is called second pain. Second pain is slower to develop, appearing many seconds a ter an insult, but lasts much longer than f rst pain; it o ten eels dull, throbbing, or burning, is poorly localized, and endures long a ter the stimulus ends. C-f ber nociceptors are o ten polymodal, which means that a single f ber can be activated by noxious thermal, mechanical, and chemical stimuli. In the case above, EM experienced an initial stinging f rst pain transmitted by myelinated A -f bers and a later burning and throbbing second pain transmitted by unmyelinated C-f bers. A erent activity conducted by C-nociceptors has a pronounced potential to create central sensitization, and it also activates regions o the brain, such as the amygdala and pre rontal cortex, that are involved in the a ective, subjective aspect o pain perception.

TABLE 12-1 Types o Peripheral Nerve Fibers FIBER TYPE

MYELINATED

DIAMETER ( m)

CONDUCTION VELOCITY (m/s)

FUNCTION

A ,A

Yes

6–22

10–85

Motor and proprioception (pressure, touch, position)

A

Yes

3–6

15–35

Muscle tone

A

Yes

1–4

5–25

First pain and temperature

B

Yes

3

3–15

Vasomotor, visceromotor, sudomotor, pilomotor

C (sympathetic)

No

0.3–1.3

0.7–1.3

Vasomotor, visceromotor, sudomotor, pilomotor

C (dorsal root)

No

0.4–1.2

0.1–2.0

Second pain and temperature

SENSITIVITY TO LIDOCAINE ,

Each peripheral nerve f ber type is responsible or transmitting one or more specif c modalities. For example, the nociceptors (A and C dorsal root f bers) are responsible or transmitting pain and temperature sensations. These f bers are not activated by pressure, light touch, or position changes. Myelin is an insulator that allows impulses to be conducted at aster speeds along axons. The nonmyelinated C-f bers have a slower conduction velocity than the myelinated f bers do. The di erent f ber types are a ected by local anesthetics with di ering degrees o sensitivity.

170 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

A Firs t a nd s e cond pa in (no block)

S e cond pa in

P

a

i

n

i

n

t

e

n

s

i

t

y

Firs t pa in

executive unction. The CNS uses e erent projections within the brain and spinal cord to modulate the incoming nociceptive signals and thus to modi y pain perception (see Chapter 18). For example, an athlete ocused on an important game might not eel the pain o an injury intensely until a ter the game is over. Her brain modulates the e ect o the input so that the same stimulus is perceived as less pain ul at certain times than at others.

P a inful s timulus

Time (s e c)

Analgesia and Anesthesia Analgesics are specif c inhibitors o pain pathways, whereas local anesthetics are nonspecif c inhibitors o peripheral

B Effe ct of Aδ fibe r block

n

s

i

t

y

sensory (including pain), motor, and autonomic nerve transmission. Analgesics have actions at specif c receptors on

P

a

i

n

i

n

t

e

3˚ ne uron proje cts to va rious re gions of the bra in

P a inful s timulus

Time (s e c)

P rima ry s oma tic s e ns ory corte x

2˚ ne uron s yna ps e s with 3˚ ne uron in tha la mus

P

a

i

n

i

n

t

e

n

s

i

t

y

C Effe ct of C fibe r block

P a inful s timulus

Time (s e c)

Ce re brum Ve ntra l pos te rior la te ra l nucle us of tha la mus

FIGURE 12-2. First and second pain. First pain, which is transmitted by A -f bers, is sharp and highly localizable. Second pain, which is transmitted by C-f bers, is slower in arriving, duller, and longer lasting (A). First pain can be prevented by selective blockade o A -f bers (B), and second pain can be prevented by selective blockade o C-f bers (C). Because A -f bers are more susceptible than C-f bers to blockade by local anesthetics, f rst pain o ten disappears at concentrations o anesthetic lower than those required to eliminate second pain.

Midbra in

P ons

Pain Perception Pain is the physical or emotional distress due to perceived actual or potential tissue injury. Pain perception is a complex process that involves activation o nociceptive neurons by a noxious stimulus and central regulation and interpretation o the nociceptive signals in the CNS. Impulses generated in the skin by nociceptor activation are conducted to the dorsal horn o the spinal cord. In the dorsal horn, the nociceptors orm synapses with interneurons and second-order neurons. The second-order neurons travel in the lateral areas o the spinal cord and project mainly to the thalamus, a gray matter structure just superior to the brainstem. The thalamus has cells that project to the somatosensory cortex o the parietal lobe and to other areas o the cortex (Fig. 12-3). Nociceptive signals as well as in ormation rom non-nociceptive a erent nerves are integrated centrally and interpreted as pain. The subjective sensation o pain is also altered by less tangible central actors including emotional state and higher

Me dulla 2˚ ne urons

Dors a l root ga nglion 1˚ ne uron (nocice ptor)

Ce rvica l s pina l cord 1˚ a nd 2˚ ne urons s yna ps e in dors a l horn of s pina l cord

FIGURE 12-3. Pain pathways. Primary (1°) nociceptors have cell bodies in the dorsal root ganglion and synapse with secondary (2°) a erent neurons in the dorsal horn o the spinal cord. Primary a erents use the neurotransmitter glutamate. The 2° a erents travel in the lateral areas o the spinal cord and eventually reach the thalamus, where they synapse with tertiary (3°) a erent neurons. The processing o pain is complex, and 3° a erents have many destinations including the somatosensory cortex (localization o pain) and the limbic system (emotional aspects o pain).

C h a p t e r 1 2 Local Anesthetic Pharmacology 171

primary nociceptors and in the CNS (see Chapter 18). For example, opioid analgesics activate opioid receptors, which signal cells to increase potassium conductance in postsynaptic neurons and to decrease calcium entry into presynaptic neurons. By these mechanisms, postsynaptic excitability and presynaptic transmitter release are reduced, and pain sensations are not transmitted as e ectively to the brain (or within it). Importantly, the transmission o other sensations and motor in ormation is not a ected. Local anesthetics act by a di erent mechanism. These agents inhibit conduction o action potentials in all a erent and e erent nerve f bers, usually in the peripheral nervous system. Thus, pain and other sensory modalities are not transmitted e ectively to the brain, and motor and autonomic impulses are not transmitted e ectively to muscles and peripheral organs.

hydrophobicity results in a decrease in permeability. This somewhat paradoxical behavior occurs because molecules that are extremely hydrophobic partition so strongly into the cell membrane that they remain there. The same strong hydrophobic orces that concentrate such molecules in the cell membrane cause them to dissociate very slowly rom that compartment. The LA binding site on the sodium channel contains both hydrophilic and hydrophobic residues that are located on transmembrane helices in three o the our domains that orm the channel’s pore. The f t is not very tight, however, resulting

A Es te r-linke d loca l a ne s the tic (proca ine ) Aroma tic group (R)

Es te r linka ge

PHARMACOLOGIC CLASSES AND AGENTS Local anesthetics prevent signal transmission by blocking the unction o sodium channels in excitable tissues. All LAs share this common mechanism o action and have similar chemical structures. The next section highlights the general principles o LA pharmacology and how the molecular properties o LAs a ect their unction. Specif c LA agents are discussed at the end o the chapter.

O

H+

H2 N H+

Ba s ic form

O NH +

O H2 N P rotona te d (a cidic) form B Amide -linke d loca l a ne s the tic (lidoca ine ) Aroma tic group (R)

Amide linka ge

H N

Te rtia ry a mine (R')

N

H+

O

Aromatic Group

To be e ective, a local anesthetic must partition into, di use across, and f nally dissociate rom the membrane into the cytoplasm; the compounds most likely to do so have moderate hydrophobicity. All local anesthetics contain an aromatic group that gives the molecule much o its hydrophobic character. Adding alkyl substituents on the aromatic ring, or on the amino nitrogen, also increases the hydrophobicity o these drugs. Biological membranes have a hydrophobic interior because o their lipid bilayer structure. The hydrophobicity o an LA molecule a ects the ease with which the drug passes through nerve cell membranes to reach its target site, which is the cytoplasmic side o the voltage-gated sodium channel (Fig. 12-5). Molecules with low hydrophobicity partition very poorly into the membrane because their solubility in the lipid bilayer is so low; such molecules are largely restricted to the polar aqueous environment. As the hydrophobicity o a series o drugs increases, the concentration in the membrane and the correlated permeability o the drugs through the cell membrane also increase. However, at a certain hydrophobicity, this relationship reverses, and a urther increase in

N

O

Molecular Determinants of Local Anesthetic Action All local anesthetics have three structural domains: an aromatic group, an amine group, and an ester or amide linkage connecting these two groups (Fig. 12-4). Local anesthetics are structurally classif ed as ester-linked LAs or amide-linked LAs . As discussed below, the structure o the aromatic group in uences the hydrophobicity o the drug, and the nature o the amine group in uences the charge o the drug. Both eatures def ne the rate o onset, potency, duration o action, and adverse e ects o an individual local anesthetic.

Te rtia ry a mine (R')

Ba s ic form

H+ H N

H+ N O

P rotona te d (a cidic) form

FIGURE 12-4. Prototypical local anesthetics. Procaine (A) and lidocaine (B) are prototypical ester-linked and amide-linked local anesthetics, respectively. Local anesthetics have an aromatic group on one end and an amine on the other end of the molecule; these two groups are connected by an ester (-RCOOR ) or amide (-RHNCOR ) linkage. In solution at high pH, the equilibrium between the basic (neutral) and acidic (charged) forms of a local anesthetic favors the basic form. At low pH, the equilibrium favors the acidic form. At intermediate (physiologic) pH, nearly equal concentrations of the basic and acidic forms are present. Generally, ester-linked local anesthetics are easily hydrolyzed to a carboxylic acid (RCOOH) and an alcohol (HOR ) in the presence of water and esterases. In comparison, amides are far more stable in solution. Consequently, amide-linked local anesthetics generally have a longer duration of action than do ester-linked anesthetics.

172 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

hydrophobicity are the clinically e ective molecules. In addition, excessively hydrophobic drugs have limited solubility in the aqueous solutions o their pharmaceutical ormulation, and even the molecules that do dissolve remain in the f rst membrane that is encountered, never reaching the target site in the axon membrane (despite their high a f nity or that site). Indeed, access o LAs rom the injection site to the axon is so poor, due to permeability barriers and removal by the local circulation, that LA concentrations 20- to 50- old higher than those e ective on an isolated nerve must be injected locally or a success ul block.

A P oorly hydrophobic loca l a ne s the tic LA

1

Linke r re gion

B Mode ra te ly hydrophobic loca l a ne s the tic Volta ge -ga te d Na + cha nne l LA 4

1

H+

Extra ce llula r

LA

Loca l a ne s the tic binding s ite

2 LA LA

Intra ce llula r 3

C Extre me ly hydrophobic loca l a ne s the tic LA

1

LA

2

FIGURE 12-5. Local anesthetic hydrophobicity, diffusion, and binding. Local anesthetics (LAs) act by binding to the cytoplasmic (intracellular) side o the voltage-gated Na channel. The hydrophobicity o a local anesthetic determines how e f ciently it di uses across lipid membranes and how tightly it binds to the Na channel and there ore governs its potency. A. Less hydrophobic LAs are unable to cross the lipid bilayer e f ciently: (1) hydrophilic neutral LA cannot enter the neuronal cell membrane because the LA is stable in the extracellular solution and has a high thermodynamic energy cost to entering the hydrophobic membrane. B. Moderately hydrophobic LAs are the most e ective agents: (1) neutral LA adsorbs to the extracellular side o the neuronal cell membrane; (2) LA di uses through the cell membrane to the cytoplasmic side; (3) LA di uses and binds to its binding site on the voltage-gated sodium channel; and (4) once bound, LA can switch between its neutral and protonated orms by binding and releasing protons. C. Extremely hydrophobic LAs become trapped in the lipid bilayer: (1) neutral LA adsorbs to the neuronal cell membrane (2), where it is unlikely to dissociate rom or di use out o the membrane.

in relatively low LA potency (IC50 o 10 3 to 10 5 M) and only weak stereoselectivity. In general, more hydrophobic drugs bind more tightly to the target site, increasing the potency o the drug. However, because o the practical need or the drug to di use across several membranes in order to reach the target site (see below), LAs with moderate

Amine Group

The amine group o a local anesthetic can exist in either the protonated (positively charged; acid) orm or the deprotonated (neutral; base) orm. The pKa is the pH at which the concentrations o a base and its conjugate acid are equal. LAs are weak bases; their pKa values range rom about 8 to 10. Thus, at the physiologic pH o 7.4, substantial amounts o both the protonated orm and the neutral orm coexist in solution. As the pKa o a drug increases, a larger raction o molecules exists in solution in the protonated orm at physiologic pH (see Chapter 1, Drug–Receptor Interactions). Protonation and deprotonation reactions are very rapid in solution (103 sec 1), but drugs in membranes or bound to proteins are protonated and deprotonated more slowly. The neutral orms o LAs di use across membranes much more easily than the positively charged orms do. However, the positively charged orms bind with much higher a f nity to the drugs’ target binding site. This site is located in the pore o the voltage-gated sodium channel and is accessible rom the intracellular, cytoplasmic entrance o the channel (Fig. 12-5B). Thus, moderately hydrophobic weak bases are e ective as local anesthetics because, at physiologic pH, a signif cant raction o the weak base molecules are in the neutral orm, which, because o its moderate hydrophobicity, can rapidly di use across membranes to enter nerve cells. Once the drug is inside the cell, it can then readily gain a proton, become positively charged, and bind to the sodium channel. The neutral species o LAs can also bind to the channel, but the dissociation o this species rom the channel is much aster and its a f nity is correspondingly lower. Some nonionizable LA drugs, such as benzocaine, are permanently neutral but are still able to block sodium channels. For these drugs, however, the block is weak and rapidly reversible.

Mechanism of Action of Local Anesthetics Anatomic Factors in Impulse Blockade

The peripheral nerve is composed o a collection o di erent types o nerve f bers (A-, B-, and C-f bers) surrounded by three protective membranes, or sheaths: the epineurium, perineurium, and endoneurium. Local anesthetic molecules must pass through these sheaths, which present the same permeation-limiting barriers as the nerve cell membranes, considered above, be ore they can reach the neuronal membranes to block conduction (Fig. 12-6). The sheaths are made up o connective tissue and cell membranes. LAs are injected outside the most external sheath, the epineurium, to avoid mechanical damage to the nerve, but the major barrier to LA penetration into the nerve is the perineurium, an epithelium-like tissue that bundles axons into separate ascicles. Recall that LAs a ect not only nociceptors but also

C h a p t e r 1 2 Local Anesthetic Pharmacology 173

Epine urium P e rine urium Endone urium S chwa nn ce ll

1

2

3

Ne e dle inje cting LA Unmye lina te d fibe r bundle (C fibe rs ) Mye lina te d fibe r (A fibe rs )

FIGURE 12-6. Peripheral nerve anatomy. 1. Local anesthetics (LAs) are injected or otherwise applied outside the peripheral nerve epineurium (the outermost sheath o connective tissue containing blood vessels, adipose tissue, f broblasts, and mast cells). 2. LA molecules must cross the epineurium to reach the perineurium, another epithelial membrane, which organizes nerve f bers into ascicles. The perineurium is the most di f cult layer or local anesthetics to penetrate because o the tight junctions between its cells. 3. LAs then pass through the endoneurium, which envelops the myelinated and unmyelinated f bers, Schwann cells, and capillaries. Only LAs that have passed through these three sheaths can reach the neuronal membranes where the voltage-gated sodium channels are located. Clinically, a high concentration o local anesthetic must be applied because only a raction o the molecules reach the target site.

other a erent and e erent, somatic, and autonomic nerve f bers. All o these f bers may be contained within a peripheral nerve, and conduction in all f bers can be blocked by local anesthetics. This is why, in the introductory case, EM experienced not only loss o pain sensation but also a more complete block o all sensation in his le t index f nger. In general, more proximal regions o the body (shoulder, thigh) are innervated by axons traveling relatively superf cially in a peripheral nerve, while more distal regions (hands, eet) are innervated by axons traveling closer to the core o the nerve. Because local anesthetics are applied to the outside o a peripheral nerve, external to the epineurium, the axons innervating more proximal areas are usually reached f rst by the local anesthetic that is di using radially into the nerve. Consequently, in the anatomic progression o unctional block, proximal areas are numbed be ore distal areas. For example, i a nerve block is applied in the brachial plexus, the shoulder and upper arm are numbed be ore the orearm, hand, and f ngers. During the onset o local anesthesia, di erent f ber types within a peripheral nerve are also blocked at di erent rates due to their intrinsic susceptibility to blockade. The general order in which unctional def cits occur is as ollows: f rst pain, second pain, temperature, touch, proprioception (pressure, position, or stretch), and f nally skeletal muscle tone and voluntary tension. This phenomenon is re erred to as differential functional blockade . In the introductory case, recall that EM’s f rst pain was blocked be ore his second pain,

and that block o both sensations preceded the loss o his other sensory modalities. Clinically, i a patient is still able to eel the sharp pain o a pinprick, then the degree o anesthesia is unlikely to be su f cient to block the transmission o long-lasting second pain. Because locomotor unction is resistant to e ects o relatively low concentrations o epidural local anesthetics, it is possible to block nociception with relatively little e ect on ambulation. The concentration o local anesthetic required to block sensory impulses without inducing a large motor blockade varies or di erent agents. With lidocaine, or example, it is impossible to block A -f bers without also blocking A -motor f bers (Table 12-1); in contrast, epidural bupivacaine can achieve analgesia at low concentrations without debilitating motor block. For this reason, dilute epidural bupivacaine is requently used during labor, as it relieves pain while still allowing the parturient to push with contractions.

Voltage-Gated Sodium Channel

Local anesthetics prevent impulse transmission by blocking sodium channels in neuronal membranes. The sodium channel exists in three main con ormational states: open, inactivated, and resting. In going rom the resting to the open state, the channel also moves through several transient “closed” con ormations. The resting neuronal membrane potential is 60 to 70 mV. At this potential, the channels are in equilibrium between the resting state (majority) and the inactivated state (minority). During an action potential, the resting channels move through the closed con ormations and f nally open brie y to allow sodium ions to enter the cell. This sodium in ux results in depolarization o the membrane. A ter a ew milliseconds, the open channel spontaneously undergoes a con ormational change to the inactivated state. This halts the in ux o sodium, and the membrane repolarizes. The inactivated state o the channel returns slowly to the resting state in the repolarized membrane. The time needed to make this transition largely determines the length o the re ractory period. During the absolute re ractory period, there are so ew Na channels in the resting state that, even i all the resting-state channels were simultaneously activated to the open state, the activation threshold would not be reached. Thus, no new action potentials can be generated during this period o time (Fig. 12-7A). Modulated Receptor Hypothesis

The di erent con ormational states o the sodium channel (resting, various closed, open, and inactivated) bind local anesthetics with di erent a f nities. This concept is known as the modulated receptor hypothesis (Fig. 12-7B and Table 12-2). Local anesthetics have a higher a f nity or the open and inactivated states o the sodium channel than or the resting state. Although the LA binds at a site in the channel’s pore, the molecular mechanism o channel inhibition involves not only the physical occlusion o the pore but also the restriction o the con ormational changes that underlie activation o the channel. The binding o drug to the closed states that occur during the sequential activation process seems to restrict the con ormational changes o the sodium channel, so that a drug-bound channel cannot undergo the ull range o motions necessary to open. For a drug-bound channel to reopen, the LA must dissociate rom the channel and thereby allow the channel to return to its resting state. This dissociation o drug (the rate

174 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

A

Intermediate closed conformation

Resting conformation

Open conformation

S 4 re gions

Extra ce llula r + + +

+ Volta ge

+ + +

Intra ce llula r

Volta ge

Inactivated conformation

Na + + + +

+ + +

+ + +

+ + +

Afte r 1 ms

+ + +

+ + +

Linke r re gion Volta ge (re fra ctory pe riod)

B

Intermediate closed conformation (high affinity for LA)

Resting conformation (low affinity for LA)

+ + +

+ + +

+ Volta ge Volta ge

+ + +

+ + +

Open conformation (high affinity for LA)

+ + +

LA

+ + + LA

Inactivated conformation (high affinity for LA)

+ + +

+ + + LA

S ta bilize d conforma tion

Volta ge (longe r re fra ctory pe riod)

FIGURE 12-7. Local anesthetic binding to different conformations (states) of the sodium channel. A. The sodium channel is composed o one polypeptide chain that has our repeating units. One region, known as the S4 region, has many positively charged amino acids (lysine and arginine). These residues give the channel its voltage dependence. At rest, the pore is closed. When the membrane is depolarized, the charged residues move in response to the change in the electric f eld. This results in several con ormational changes (intermediate closed states) that culminate in channel opening. A ter about 1 ms (the channel open time), the 3–4 amino acid “linker region” plugs the open channel, yielding the inactivated con ormation. The inactivated con ormation returns to the resting state only when the membrane is repolarized; this con ormational change involves the return o the S4 region to its original position and the expulsion o the linker region. The time required or the channel to return rom the inactivated state to the resting state is known as the refractory period ; during this period, the sodium channel is incapable o being activated. B. The binding o local anesthetic (LA) alters the properties o the intermediate orms assumed by the sodium channel. Sodium channels in any o the con ormations (resting, closed, open, or inactivated) can bind local anesthetic molecules, although the resting state has a low a f nity or LA, while the other three states have a high a f nity or LA. LA can dissociate rom the channel–LA complex in any con ormational state, or the channel can undergo con ormational changes while associated with the LA molecule. Ultimately, the channel–LA complex must dissociate and the sodium channel must return to the resting state in order to become activated. LA binding extends the re ractory period, including both the time required or dissociation o the LA molecule rom the sodium channel and the time required or the channel to return to the resting state.

o which varies among the di erent LAs) is slower than the normal recovery rom the inactivated to the resting channel con ormation in the absence o LA. Thus, by delaying the inactivated channel’s return to the resting state, LAs extend the re ractory period o the neuron by about 50- to 100- old. At high concentrations o LAs, the number o resting channels that are drug-bound (blocked) is su f cient to prevent impulse conduction altogether. Tonic and Phasic Inhibition

The di erential a f nity o local anesthetics or di erent states o the voltage-gated sodium channel has an important pharmacologic consequence: the degree o inhibition o sodium current by the LA depends on the requency o impulses in the nerve, such that the number o APs in a train is diminished under conditions where single impulses are una ected. When there is a long interval between action

potentials, any additional LA binding that occurred during the preceding impulse has time to ully reverse, and the level o inhibition o each impulse is the same; the inhibition is said to be tonic. When the interval between action potentials is short, drug dissociation rom additionally bound channels is incomplete, and the number o bound channels increases with each successive impulse; the inhibition is said to be phasic or use-dependent (Fig. 12-8). Tonic inhibition occurs when the time between action potentials is long compared to the time or dissociation o the LA rom the sodium channel. Assume, or example, that be ore an action potential arrives, an equilibrium has been established in which 5% o the sodium channels are bound by local anesthetic molecules. When an action potential arrives, the other 95% o the channels are available to open and subsequently to inactivate. During the brie impulse, some o these channels become bound by local anesthetic molecules.

C h a p t e r 1 2 Local Anesthetic Pharmacology 175

TABLE 12-2 Modulated Receptor Hypothesis CHANNEL STATE

AFFINITY FOR LOCAL ANESTHETIC

RELATIVE EFFECT ON CHANNEL

Resting

Low

Prevents channel opening (only at high LA concentrations)

Closed (several)

High

Prevents channel opening (major e ect)

Open

High

Blocks channel pore (minor e ect)

Inactivated

High

Extends re ractory period (major e ect)

The voltage-gated sodium channel can assume several di erent con ormations. Local anesthetics (LAs) have di erent a f nities or di erent con ormations o the channel; this di erential a f nity alters the kinetics o sodium channel activation (see Fig. 12-7).

However, in the relatively long time be ore the next impulse arrives at the LA-exposed region, the bound LA can dissociate rom the sodium channel, allowing those channels to return to the resting state. Thus, be ore the next action potential arrives, the 5% binding equilibrium is reestablished. The next action potential will there ore be blocked to the same extent as the previous one. Phasic inhibition occurs when there is not su f cient time between action potentials or this equilibrium to be reestablished. Rapidly arriving action potentials cause resting sodium channels to open and then inactivate, and some o these channels become bound by LAs. However, because there is not su f cient time between impulses or all the newly ormed LA–sodium channel complexes to dissociate, only some o the channels are able to return to the resting state. With each arriving action potential, more and more channels are blocked, until a new steady state o LA–sodium channel binding is reached. This is the phenomenon o phasic, or usedependent, inhibition. As more o the channels are bound by LA, ewer and ewer channels are available to open when the next action potential arrives. Consequently, action potential conduction is increasingly inhibited at higher frequencies of impulses. The clinical importance o this phenomenon is that tissue injury or trauma causes nociceptors in the area o injury to f re at high requency. In these situations, application o a local anesthetic tends to block local nociceptors in a phasic manner, inhibiting pain transmission to a greater extent than the transmission o other local sensory or motor impulses that are blocked only tonically.

Other Receptors for Local Anesthetics

In addition to blocking sodium channels, LAs have a wide range o other biochemical and physiologic e ects. Local anesthetics can interact with potassium channels, calcium channels, pacemaker channels, ligand-gated channels (such as the ionotropic glutamate receptors), transient receptor potential (TRP) channels, and several G protein-coupled receptors (including muscarinic cholinergic receptors, -adrenergic receptors, and receptors or substance P). Local anesthetics can also uncouple some G proteins rom their

A Tonic block (low fre que ncy s timula tion) De pola rize d

Voltage

Re s ting

0.5 Fraction of bound channels 0 LA in e quilibrium with s odium cha nne ls

Equilibrium re e s ta blis he d

Time B P ha s ic block (high fre que ncy s timula tion)

De pola rize d

Voltage

Re s ting

0.5 Fraction of bound channels 0 LA in e quilibrium with s odium cha nne ls

Ne w ba s e line e s ta blis he d

Time FIGURE 12-8. Tonic and phasic (use-dependent) inhibition. A. In tonic block, depolarizations occur with low requency, and there is su f cient time between depolarizations or equilibrium binding o local anesthetic (LA) molecules to the various states o the sodium channel to be reestablished. When a depolarization occurs, resting channels (which have low a f nity or LA) are converted into open channels and inactivated channels (both o which have high a f nity or LA). Thus, there is an increase in the number o LA-bound channels. Once the depolarization ends, there is su f cient time be ore the next depolarization or equilibrium between LA molecules and sodium channels to be reestablished, and virtually all o the channels return to the resting and unbound state. B. In phasic block, depolarizations occur with high requency, and there is not su f cient time between depolarizations or equilibrium to be reestablished. A ter each depolarization, a new baseline is established that has more LA-bound channels than the previous baseline, leading eventually to conduction ailure. Because high- requency stimulation o nociceptors occurs in areas o tissue damage, phasic (usedependent) block causes actively f ring nociceptors to be inhibited more e ectively than nerve f bers that are only occasionally f ring. The requency dependence o phasic block depends on the rate at which LA dissociates rom its binding site on the channel.

cell sur ace receptors and thus inhibit signal transduction. In most cases, these e ects are not signif cant because LAs have lower a f nity or these other receptors than or the sodium channel. But or some types o local anesthetics in some clinical situations, these alternate targets may have important therapeutic and toxic consequences. For example, in spinal anesthesia, a high concentration o local anesthetic is injected into the cerebrospinal uid, rom which the LA then di uses into the spinal cord. Neuropeptides (such as substance P) and small organic neurotransmitters (such as glutamate ) mediate the transmission o nociceptive impulses between the primary and secondary

176 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

a erent neurons in the dorsal horn o the spinal cord (see above). Evidence rom in vivo and in vitro studies indicates that receptors or substance P (NK-1) and bradykinin (B2) and the ligand-gated, ionotropic receptors or glutamate (AMPA and NMDA receptors; see Chapter 13, Pharmacology o GABAergic and Glutamatergic Neurotransmission) are all inhibited directly by local anesthetics. Combined with the anesthetic action that results rom the sodium channelblocking e ect o the LA, the overall result is a signif cantly increased pain threshold.

Pharmacokinetics of Local Anesthetics Systemic Absorption

Upon administration by injection or topical application, LAs di use to their sites o action. Local anesthetic molecules are also taken up by local tissues and removed rom the site o administration by the systemic circulation. The amount o local anesthetic that enters the systemic circulation and the potency o the LA together determine the systemic toxicity o the agent. Ideally, systemic absorption is minimized to avoid unnecessary toxicity. The vascularity o the delivery site, the drug concentration, the co-administration o a vasoconstrictor, and properties o the anesthetic solution (such as its viscosity) all in uence the rate and extent o systemic absorption o local anesthetics. Absorption is greater rom densely per used tissues. For example, intratracheal administration o vaporized local anesthetic leads to rapid and nearly complete systemic absorption because o local anesthetic contact with the highly per used lung epithelium.

Distribution

A ter absorption, local anesthetics are distributed throughout the body. Similar to the e ect o vascularity on the rate o absorption, the rate o distribution and the peak plasma concentration o anesthetic depend on the vascularity o the nearby tissue. This e ect is particularly important or injected local anesthetics, where distribution away rom the site o injection is a determinant o LA concentration at the intended site o action (and there ore duration o e ect) and o peak LA plasma concentration (and there ore risk o toxicity). For example, a larger total quantity o local anesthetic can be sa ely given in a peripheral nerve block than in an intercostal block because the high vascularity o the intercostal space results in more rapid distribution o the anesthetic, a higher peak plasma concentration o the anesthetic, and an increased risk o systemic toxicity. Common sites o local anesthetic administration, ranked in order o most vascular to least vascular, include intercostal, caudal epidural space, lumbar epidural space, brachial plexus, emoral nerve, and subcutaneous tissue. Vasoconstrictors (such as epinephrine) are o ten administered together with many short-acting or medium-acting local anesthetics. These adjunctive agents reduce blood ow to the area o injection by causing the smooth muscles o the vessels to contract and thereby slow the rate o removal o the LA. In doing so, vasoconstrictors both increase the concentration o anesthetic around the nerve and decrease the peak plasma concentration that is reached in the systemic circulation. The ormer e ect enhances the duration o action o the LA, and the latter e ect decreases the LA’s systemic toxicity. However, vasoconstriction can also lead to tissue hypoxia and damage i the oxygen supply to the area is reduced too severely. Thus, vasoconstrictors are not used

when LAs are administered in the extremities, because there is limited circulation to these areas. In the introductory case, EM was given lidocaine without epinephrine to avoid tissue hypoxia in his digit. In the circulation, LAs bind reversibly to two major plasma proteins: -1 acid glycoprotein (an acute-phase protein) and albumin. Local anesthetics also bind to the membranes o all cells in the blood. Binding to plasma proteins decreases as the pH decreases, suggesting that the neutral orm binds these proteins with higher a f nity. Tissue binding, largely due to membrane uptake and partitioning, occurs at the site o injection as well as other sites. The more hydrophobic the agent is, the greater the extent o tissue binding. The volume o distribution (Vd) indicates the extent to which a drug distributes to the tissues rom the systemic circulation. For the same amount o administered drug, a less hydrophobic LA (e.g., procaine) has a higher plasma concentration (i.e., less is stored in tissues) and there ore a smaller Vd. A more hydrophobic LA (e.g., bupivacaine) has a lower plasma concentration (i.e., more is stored in tissues) and there ore a larger Vd. Local anesthetics with a larger Vd are eliminated more slowly. (See Chapter 3, Pharmacokinetics, or a detailed discussion o the inverse relationship between Vd and the elimination hal -li e o a drug.)

Metabolism and Excretion

Ester-linked LAs are metabolized by tissue and plasma esterases (pseudocholinesterases). This process is ast (on the order o minutes), and the resulting products are excreted via the kidney. Amide-linked LAs are primarily metabolized in the liver by cytochrome P450 enzymes. The three major routes o hepatic metabolism are aromatic hydroxylation, N-dealkylation, and amide hydrolysis. Metabolites o amide-linked LAs are returned to the circulation and excreted by the kidney. Alterations in liver per usion can change the rate at which these agents are metabolized, as can induction or inhibition o the P450 enzymes. Metabolism is slowed in patients with cirrhosis or other liver diseases, and a standard dose o an amide-linked LA can lead to toxicity in such a patient. Some metabolism o amide-linked LAs can also occur extrahepatically, or example, in the lung and kidney.

Administration of Local Anesthetics The method o administration o local anesthetics can determine both the therapeutic e ect and the extent o systemic toxicity. The ollowing is an overview o the most common methods or administering local anesthetics.

Topical Anesthesia

Topical anesthetics provide short-term pain relie when applied to mucous membranes or skin. The drug must cross the epidermal barrier, with the stratum corneum (outermost layer o the epidermis) presenting the major obstacle, to reach the A -f bers and C-f bers in the epidermis. Once across the epidermis, local anesthetics are absorbed rapidly into the circulation, increasing the risk o systemic toxicity. A mixture o tetracaine, adrenaline (epinephrine), and cocaine, known as TAC, is a historically common anesthetic that was o ten used be ore suturing small cuts. Because o concern about cocaine toxicity and/or addiction rom this ormulation, alternatives such as EMLA are now used

C h a p t e r 1 2 Local Anesthetic Pharmacology 177

(see below). Recent ormulations o local anesthetics or transdermal administration include lidocaine patches that deliver the medication over 12–24 hours.

Inf ltration Anesthesia

Inf ltration anesthesia is used to numb an area o skin or a mucosal sur ace via injection. The local anesthetic is injected intradermally or subcutaneously, o ten at several neighboring sites near the area to be anesthetized. This technique produces numbness more rapidly than topical anesthesia, because the agent does not have to cross the epidermis. However, the injection can be pain ul due to the acidic pH o the local anesthetic solution that is needed to maintain the drug in an ionized, soluble, and chemically stable orm. Neutralization o the solution by addition o sodium bicarbonate can reduce the injection pain. The local anesthetics most commonly used or inf ltration anesthesia include lidocaine , procaine , and bupivacaine . Inf ltration o local anesthetics or dental procedures is discussed in Box 12-1.

Peripheral Nerve Blockade

Peripheral nerve blocks are increasingly used or pain control associated with trauma and surgery. Generally, local anesthetics are injected percutaneously using anatomic landmarks, ultrasound guidance, or uoroscopic techniques to sa ely deliver the anesthetic near a particular nerve. Anesthetics can be delivered as bolus injections (single shot or, occasionally, repeated nerve block) or as continuous in usions via catheters that are inserted near the target nerve. As in the case o the digital nerve block or EM, single shot injections may be used to provide short-term anesthesia or a procedure. Alternatively, emoral nerve blocks o ten use continuous in usions o local anesthetics to control pain rom a hip racture both prior to and or days a ter surgery.

This technique is particularly help ul in elderly patients who may be sensitive to adverse e ects o opioid medications, such as delirium and respiratory depression, that might otherwise be used to control pain. The choice o anesthetic typically depends on the desired duration o action. Peripheral nerve blocks are also used in the management o trauma. Intercostal nerve blocks can reduce pain a ter rib racture, which can improve lung mechanics and reduce rates o pulmonary complications such as pneumonia. Examples o other use ul peripheral blocks include transversus abdominis plane (TAP) block or the anterior abdominal wall; interscalene, cervical plexus, and brachial plexus blocks or shoulder and arm surgery; and emoral and popliteal blocks or the distal lower limb.

Central Nerve Blockade

This type o blockade, also called neuraxial blockade, involves delivery o drug near the spinal cord. Central nerve blockade includes both epidural and intrathecal (spinal) anesthesia. The early e ects o these procedures result primarily rom impulse blockade in spinal roots, but in later phases, anesthetic drug penetrates and may act within the spinal cord. Bupivacaine is particularly use ul as an epidural anesthetic during labor because, at low concentrations, it provides adequate pain relie without signif cant motor block. Reports o bupivacaine cardiotoxicity have led to decreased use o this agent in high concentrations ( 0.5% weight/ volume [w:v]), although the dilute solutions used in obstetrics are rarely toxic.

Intravenous Regional and Systemic Anesthesia

Local anesthetic can be injected intravenously into a distal extremity to provide regional anesthesia to that limb. Generally, venous blood is encouraged to drain rom the limb by

B O X 1 2 - 1 Lo ca l An e s th e tics in De n tis try Modern dentistry is predicated on the action o local anesthetics: without adequate pain control, patients could not com ortably undergo the majority o dental procedures. Not surprisingly, then, local anesthetics are the most commonly used drugs in dentistry. O ten, both an injected and a topical anesthetic agent are used in dental procedures; the injected agent blocks the sensation o pain during (and sometimes a ter) the procedure, while the topical agent allows or painless needle penetration when the injected agent is administered. Topical anesthetics are applied to mucous membranes and penetrate to a depth o 2 to 3 millimeters. Because topical anesthetics must di use across this distance, relatively high concentrations are used, and care must be taken to avoid local and systemic toxicity. Benzocaine and lidocaine—two commonly used topical anesthetics—are insoluble in water and poorly absorbed into the circulation, decreasing the likelihood o systemic toxicity. Injected anesthetics are administered either as local in ltrations or as eld or nerve blocks. In a local in ltration, the anesthetic solution is deposited at the site where the dental procedure will be per ormed. The solution bathes ree nerve endings at that site, blocking pain perception. In eld and nerve blocks, the

anesthetic solution is deposited more proximally along the nerve, away rom the site o incision. These techniques are used when larger regions o the mouth must be anesthetized. Numerous injected anesthetics are used in dental practice, and the choice o which agent to use or a given procedure ref ects actors such as the rate o onset, the duration o action, and the vasodilatory properties o the agent. Lidocaine is the most widely used injected anesthetic; it is notable or a rapid rate o onset, long duration o action, and extremely low incidence o allergic reaction. Mepivacaine is less vasodilating than most other local anesthetics, allowing it to be administered without a vasoconstrictor. This property makes mepivacaine ideally suited or pediatric dentistry, because it is “washed out” o the area o administration more rapidly than agents administered with vasoconstrictors. As a result, mepivacaine provides a relatively short period o so t-tissue anesthesia, minimizing the risk o inadvertent, sel -inf icted trauma rom biting or chewing on anesthetized tissue. Bupivacaine is a more potent and longer acting anesthetic than lidocaine or mepivacaine. It is used or lengthy dental procedures and or the management o postoperative pain. ■

178 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

elevating it above the level o the heart be ore a proximal tourniquet is applied and the anesthetic is injected into the vein. This allows high local concentrations o anesthetic to reach the nerves in the limb, while limiting the redistribution o the anesthetic and thus preventing systemic toxicity. This type o local anesthesia, also called Bier block, is occasionally used or arm and hand surgery. Systemic intravenous lidocaine is used perioperatively to reduce postoperative pain and is also administered or relie o chronic pain rom injury or disease (e.g., diabetic neuropathy). Relie o chronic pain o ten lasts or weeks ollowing a single, brie lidocaine in usion, even though the drug is cleared rom the circulation within a ew hours. The mechanism underlying this long duration o e ect remains uncertain.

Major Toxicities Local anesthetics can have both systemic and organ-specif c toxicities. These include hypersensitivity reactions as well as e ects on local tissues, the CNS, peripheral vasculature, and the heart. Toxicity associated with LAs range rom mild local tissue irritation to li e-threatening complications including CNS excitation and depression and cardiovascular collapse. Local anesthetics can cause local irritation. For example, skeletal muscle is sensitive to intramuscular LA injection. A ter intramuscular injection o LAs, plasma levels o creatine kinase are elevated, indicating damage to muscle cells. This e ect is usually reversible, and muscle regeneration is complete within a ew weeks o the injection. Peripheral nerve blocks and, particularly, spinal blocks with high concentrations o LAs (e.g., 5% lidocaine) requently cause local neurotoxicity. Some o this toxicity may result rom local in ammation o surrounding tissues, but there is also evidence or neuronal apoptosis, perhaps triggered by elevation o intracellular Ca 2. Local anesthetics can have serious e ects on the CNS. LAs are small amphipathic molecules that, as the uncharged species, can rapidly cross the blood–brain barrier. Initially, LAs produce signs o CNS excitement, including perioral numbness or a metallic taste at lower plasma concentrations, ollowed by shivering, twitching, and seizures at higher concentrations. Cellular studies show that some LAs release Ca 2 rom intracellular stores and thus stimulate the release o glutamate in the brain, resulting in the excitatory phase o CNS toxicity. CNS excitation is ollowed by depression. As the concentration o LA increases in the CNS, all neuronal pathways are blocked—excitatory as well as inhibitory— leading to CNS depression. Death can ultimately result rom respiratory ailure. CNS toxicity is most commonly associated with inadvertent intravascular injection o local anesthetic or with large doses o intrathecal anesthetic. Local anesthetics have complex e ects on the peripheral vasculature. Lidocaine, or example, initially causes vasoconstriction but can subsequently also cause vasodilation. Such biphasic actions may be attributable to separate e ects, respectively, on the vascular smooth muscle and on sympathetic nerves that innervate resistance arterioles. Bronchial smooth muscle is also a ected in a biphasic manner, with initial bronchoconstriction ollowed by bronchodilation. The early e ect may re ect LA-induced release o calcium ions into the cytoplasm rom intracellular stores, while the latter e ect may be caused by LA inhibition o plasma membrane sodium and calcium channels (see the ollowing discussion).

The cardiac e ects o LAs are complex due to their actions on multiple molecular targets, including Na , K , Ca2 , and pacemaker channels. An early e ect is to reduce the conduction velocity o the cardiac action potential through both conducting and nodal tissues. At very low concentrations, LAs can act as antiarrhythmic drugs because o their ability to prevent ventricular tachycardia and ventricular f brillation (this is an example o use-dependent block; see above). Lidocaine, or example, is used as both a local anesthetic and a class IB antiarrhythmic (see Chapter 24). Local anesthetics also cause a dose-dependent decrease in cardiac contractility (a negative inotropic e ect). The mechanism o this e ect is not entirely understood but may be caused by LA-mediated slow release o calcium rom the sarcoplasmic reticulum with a consequent reduction in the stores o calcium available to drive subsequent contractions. LAs can also directly block calcium channels in the plasma membrane. The combination o reduced intracellular calcium storage and decreased calcium entry may lead to decreased myocardial contractility. It has recently been shown that lipid emulsions injected into the circulation may help reverse CNS and cardiac toxicity rom LAs. This f nding has been demonstrated in animal models o local anesthetic toxicity and in many clinical case reports o success ul resuscitation a ter seizure or cardiac arrest related to local anesthetic overdose. While the mechanism o this e ect is unclear, it is thought that the lipid serves as a “sink” or the hydrophobic local anesthetic and allows the local anesthetic to redistribute away rom CNS and cardiac tissue and into the lipid. It has become common to have lipid emulsions immediately available or resuscitation in surgical centers where nerve blocks are per ormed. Hypersensitivity to local anesthetics is rare. This adverse e ect is usually mani ested as allergic dermatitis or asthma. LA-induced hypersensitivity occurs almost exclusively with ester-linked LAs. For example, a metabolite o procaine, para-aminobenzoic acid (PABA), is a known allergen (as well as the active agent in many sunscreens).

Individual Agents Having discussed the general properties o local anesthetics, this section presents examples o individual anesthetics in current clinical use, with an emphasis on the agents’ di erences in potency and elimination hal -li e.

Ester-Linked Local Anesthetics Procaine Procaine (Novocain®) is a short-acting, ester-linked LA

(Fig. 12-4A). Its low hydrophobicity allows or rapid removal o drug rom the site o administration via the circulation and results in little sequestration o drug in the local tissue surrounding the nerve. In the bloodstream, procaine is degraded rapidly by plasma pseudocholinesterases, and the metabolites are subsequently excreted in the urine. Procaine’s low hydrophobicity also causes it to dissociate rapidly rom its binding site on the sodium channel, accounting or the low potency o this agent. Procaine’s primary uses are in inf ltration anesthesia and in dental procedures. Occasionally, it is used in diagnostic nerve blocks. Procaine is rarely used or peripheral nerve block because o its low potency, slow onset, and short duration o action. The rapidly hydrolyzed, short-acting homologue o procaine, 2-chloroprocaine (Nesacaine®),

C h a p t e r 1 2 Local Anesthetic Pharmacology 179

is popular as an obstetric anesthetic that is sometimes administered epidurally to control pain during delivery. One o the metabolites o procaine is PABA, a compound required by some bacteria or purine and nucleic acid synthesis. The antibacterial sul onamides are structural analogues o PABA that competitively inhibit the synthesis o an essential metabolite in olate biosynthesis (see Chapter 33, Principles o Antimicrobial and Antineoplastic Pharmacology). Excess PABA can reduce the e ectiveness o sul onamides and there ore exacerbate bacterial in ections. As mentioned above, PABA is also an allergen. Tetracaine Tetracaine is a long-acting, highly potent, ester-linked LA. Its

long duration o action is caused by its high hydrophobicity— it has a butyl group attached to its aromatic group—which allows tetracaine to remain in the tissue surrounding a nerve or an extended period o time. Tetracaine’s hydrophobicity also promotes prolonged interaction with its binding site on the sodium channel, accounting or its higher potency than lidocaine and procaine. It is mainly used in spinal and topical anesthesia. Its e ective metabolism is slow, despite the potential or rapid hydrolysis by esterases, because it is released only gradually rom tissues into the bloodstream.

Cocaine Cocaine , the prototypical and only naturally occurring LA, is

ester-linked. It has an intermediate potency or nerve block (one-hal that o lidocaine) and a medium duration o action. Cocaine’s structure is slightly unusual or local anesthetics; its tertiary amine is part o a complex cyclic structure to which a secondary ester group is attached. Cocaine’s primary therapeutic uses are in ophthalmic anesthesia and as part o the topical anesthetic TAC (tetracaine, adrenaline, cocaine; see above). Like prilocaine (see below), cocaine has a marked vasoconstrictive action that results rom its inhibition o catecholamine uptake in synaptic terminals o both the peripheral and central nervous systems (see Chapter 11, Adrenergic Pharmacology). Inhibition o this uptake system is also the mechanism or cocaine’s proound cardiotoxic potential and or the psychotropic “high” associated with cocaine use. Cardiotoxicity and euphoria limit the value o cocaine as a local anesthetic.

Amide-Linked Local Anesthetics Lidocaine and Prilocaine Lidocaine , the most commonly used LA and the one used in

EM’s case, is an amide-linked drug o moderate hydrophobicity (Fig. 12-4B). It has a rapid onset o action and a medium duration o action (about 1–2 hours) and is moderately potent. Lidocaine has two methyl groups on its aromatic ring, bordering the amide bond, which enhance its hydrophobicity relative to procaine and slow its rate o hydrolysis. Lidocaine has a relatively low pKa, and a large raction o the drug is present in neutral orm at physiologic pH. This property allows rapid di usion o the drug through membranes and a rapid block. Lidocaine’s duration o action is based on two actors: its moderate hydrophobicity and its amide linkage. The amide linkage prevents degradation o the drug by esterases, and its hydrophobicity allows the drug to remain near the area o administration (i.e., in local tissue) or a long time. Its hydrophobicity also allows lidocaine to bind more tightly than procaine to the LA binding site

on the sodium channel, enhancing its potency. The vasoconstrictive e ects o co-administered epinephrine can extend lidocaine’s duration o action substantially. Lidocaine is used in inf ltration, peripheral nerve block, epidural, spinal, and topical anesthesia. The mechanism o antiarrhythmic action is its blocking o sodium channels in cardiac myocytes. Lidocaine’s slow metabolism in the circulation makes it a use ul Class IB antiarrhythmic (see Chapter 24). More potent amide-linked LAs, such as bupivacaine, bind too tightly to cardiac sodium channels to serve as use ul antiarrhythmics; such drugs cause either conduction blocks or tachyarrhythmias (see below). Lidocaine undergoes metabolism in the liver, where it is f rst N-dealkylated by P450 enzymes (see Chapter 4, Drug Metabolism). Subsequently, it undergoes hydrolysis and hydroxylation. Lidocaine’s metabolites have only weak anesthetic activity. The systemic toxic e ects o lidocaine are mani ested mainly in the CNS and heart. Adverse e ects can include drowsiness, tinnitus, twitching, and even seizures. CNS depression and cardiotoxicity occur at high plasma levels o the drug. Still, lidocaine has a higher therapeutic index than bupivacaine. Prilocaine is similar to lidocaine, except that it causes less vasodilation and, at high doses, can cause methemoglobinemia. Since it causes less vasodilation, it is o ten an anesthetic o choice when vasoconstrictive additives such as epinephrine must be avoided. Bupivacaine Bupivacaine is an amide-linked LA with a long duration o

action. It is highly hydrophobic (and there ore highly potent) as a result o a butyl group attached to the tertiary nitrogen. Dilute bupivacaine (0.125% w:v or lower) administered epidurally has more e ect on nociception than on locomotor activity. This property, combined with the drug’s long duration o action and high potency, has made bupivacaine use ul in spinal, epidural, and peripheral nerve blocks and in inf ltration anesthesia. Bupivacaine is metabolized in the liver, where it undergoes N-dealkylation by P450 enzymes. It has been used widely in low concentrations or labor and postoperative anesthesia because it provides 2–3 hours o pain relie with minimal motor blockade. However, because o its cardiotoxicity at higher concentrations, total acceptable doses must be care ully determined and not exceeded when used or these indications. (The drug blocks cardiac myocyte sodium channels during systole but is very slow to dissociate during diastole. Thus, it can trigger arrhythmias through the promotion o reentry pathways.) Bupivacaine contains a chiral center, and it exists as a racemic mixture o mirror-image R- and S-enantiomers. The R- and S-enantiomers have di erent a f nities or the sodium channel and, there ore, di erent cardiovascular e ects. The S-enantiomer has been separated and marketed as the sa er and less cardiotoxic levobupivacaine , as has its structurally homologous relative ropivacaine .

Articaine Articaine is a relatively new amide-linked LA that has several interesting structural eatures. First, along with prilocaine ,

articaine is unique among local anesthetics because o its secondary amine group. (Virtually all other LAs have a tertiary amine group.) Second, articaine is structurally unique because it contains an ester group bound to a thiophene ring;

180 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

the presence o the ester group means that articaine can be partially metabolized in the plasma by cholinesterases, as well as in the liver. Its rapid metabolism in the plasma may minimize its potential toxicity. Articaine is currently used in dentistry, where it is becoming an increasingly popular agent, and it may f nd additional uses as more studies o its clinical applications are per ormed. EMLA EMLA (eutectic mixture o local anesthetic) is a combination

o lidocaine and prilocaine that is delivered topically as a cream or patch. EMLA is use ul clinically because it has a higher concentration o local anesthetic per drop contacting the skin than standard topical preparations. It is e ective in a number o situations including venipuncture, arterial cannulation, lumbar puncture, and dental procedures, and is most commonly used in pediatric settings.

CONCLUSION AND FUTURE DIRECTIONS Local anesthetics are vital to the practice o medicine, surgery, and dentistry because they can block pain sensations regionally. Their clinical actions involve blocking pain neurons called nociceptors. Nociceptors are a erent neurons whose axons are classif ed as either A - or C-f bers. Local anesthetics block all types o nerve f bers in peripheral nerves, including those o nociceptors, by blocking voltagegated sodium channels in neuronal membranes. LAs act on sodium channels rom the cytoplasmic side o the membrane. In general, local anesthetics have an aromatic group that is connected to an ionizable amine via an ester or amide linkage. This structure is common to almost all local anesthetics and contributes to their unction. Both the hydrophobicity, attributable in large part to the aromatic ring and its substituents, and the ionizability (pKa) o the amine determine the potency o the LA and the kinetics o local anesthetic action. Molecules with pKa values o 8–10 (weak bases) are the most e ective as local anesthetics. The neutral orm can cross membranes to reach the LA binding site on the sodium channel, and the protonated orm is available to bind with high a f nity to that target site. The sodium channel exists in three main con ormational states: open, inactivated, and resting. There are also several transient “closed” states between the resting and open states. Local anesthetics bind with higher a f nity to the open and inactivated con ormations than to the resting con ormation o the sodium channel. This tight binding slows the return o the channel to the resting state a ter an action potential and

thereby extends the re ractory period, increasingly inhibiting the transmission o trains o action potentials occurring at high requency. Local anesthetics have actions beyond their inhibition o sodium channels. Some o these ancillary e ects show therapeutic promise and could potentially lead to other indications or LAs. For example, LAs have been reported to a ect wound healing, in ammation, thrombosis, hypoxia/ ischemia-induced brain injury, and bronchial hyperactivity. This is particularly important clinically since regional blocks using local anesthetics are becoming more common in surgery. LAs are also being investigated or use in chronic and neuropathic pain management, such as that seen in patients with diabetic neuropathy, postherpetic neuralgia, burns, cancer, and strokes. The development o ultralong-acting LAs (whose e ects could last or days) is continuing to be investigated: these studies involve altering LA structure at the molecular level, using a variety o drug delivery systems, and discovering new classes o neuronal impulse blockers. Lastly, a promising area o current discovery involves nociceptor-specif c LAs. Some o these experimental agents bind to particular sodium channel subtypes that are expressed pre erentially on A - or C-f bers. Others are charged anesthetics that typically cannot di use through neuronal cell membranes; co-administration o these anesthetics with agents that activate other ion channels ound pre erentially on nociceptors (such as TRPV1) allows the anesthetic molecules to cross the nociceptor membrane through these open channels in a modality-specif c ashion. Nociceptor-specif c LAs have the potential to block pain perception without a ecting motor, autonomic, or other neuronal signaling and may there ore be use ul in a variety o clinical settings.

Suggested Reading Berde CB, Strichartz GR. Local anesthetics. In: Miller RD, Cohen NH, Eriksson LI, Fleisher LA, Wiener-Kronish JP, Young WL, eds. Miller’s anesthesia. 8th ed. Philadelphia: Elsevier Churchill Livingstone; 2015. (A more complete mechanistic and, primarily, clinical summary.) Crystal CS, McArthur TJ, Harrison B. Anesthetic and procedural sedation techniques or wound management. Emerg Med Clin North Am 2007;25: 41–71. (A clinically oriented review that discusses how to administer LAs at various anatomic sites.) McLure HA, Rubin AP. Review o local anaesthetic agents. Minerva Anestesiol 2005;71:59–74. (A clear discussion o both general concepts and individual agents.) Mercado P, Weinberg GL. Local anesthetic systemic toxicity: prevention and treatment. Anesthesiol Clin 2011;29:233–242. (Review o LA toxicity and discussion o clinical use o lipid rescue therapy or LA toxicity.) Suzuki S, Gerner P, Colvin AC, Binshtok AM. C-f ber-selective peripheral nerve blockade. Open Pain J 2009;2:24–29. (Reviews research on agents that may have selectivity or C-f bers.)

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182 PRINCIPLES OF AUTONOMIC AND PERIPHERAL NERVOUS SYSTEM PHARMACOLOGY

IIC Principles of Central Nervous System Pharmacology

C GAB GA BAA re ce p tor

13 Pharmacology of GABAergic and Glutamatergic Neurotransmission B

GABA (+)) a nd d Be nzodia ze pine s (+) S tu a rt A. Fo rm a n , Hu a -J u n Fe n g , J a n e t Ch o u , J ia n re n M a o , a n d En g H. Lo comp co mpe e tit titiv ive a nta goniss ts (-) F luma ze nil (-))

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INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1884– INT 4–1885 Ch hloral lo Hydrate, e --H Hydroxybbut buty tyrri tty ric Ac A id i , a nd n Flunitraz azee pam m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1966 OVERVIEW OF GABAERGIC AND GL GLUT UTAM AMATERGIC NEUROTRANSMISS SIO ION N ..................... 184 PHYSIOLO AT LOGY OF GL G UTAMAT A ERGIC Ba rbb itur ura tte s (+)) F Furo Fu uro o s e mi os mide de (-)) UROTR N MISSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1966 PHYS PH YSIO IOL LOGY OF GABAERGIC NEUROTRANSMISSION N . . . . . . 185 NEUROTRANS Glutam amat atee Me M tabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1966 GABA Metabolism and Transportt . . . . . . . . . . . . . . . . . . . . . . . 186 G ut Gl utam amat atee Recee pt ptors an andd Trran ansp sportee rss . . . . . . . . . . . . . . . . . . 1966 GABA GA BA Rec ecep epto tors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 1866 Io ono notr trop oppic Glu luta lu tama mate tee Recc epptto tors ors . . . . . . . . . . . . . . . . . . . . . . 1966 Ionotropic GABA Recept ptor ors: s: GABA ABAA a nd d GABAC . . . . . . . 18 1866 Metabotropic Glutamate ate Receptors Recept . . . . . . . . . . . . . . . . . . . 1977 Meta Me tabo botropiic GABA Receptors: GABAB . . . . . . . . . . . . . . . 188 Glutamate Transporterss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1988 PHARMACOLOGIC CLASSES AND AGENTS AFFECTIN NG AR OGY OF GABAERGIC NEUROTRANSMISSION. . . . . . . . . . . . . . . . . . . . . . 189 PATHOPHYSIOLOGY AND PHARMACOLOGY GLUTAMATERGIC GLUT UTAM AMAT ATER ERGI GIC C NE NEUROTRANSMISSION NEUR UROT OTRA RANS NSMI SMISS M SSIO ION N . . .Ne . . . uros . . . . . .te . . roid . . . ds 198 Inhibitors of GABA Metabolism and Transport . . . . . . . . . . . . 189 GL s8 ((+) Neurodegenerative Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . 198 GABAA Receptor Agonists and Antagonists . . . . . . . . . . . . . . 190 Stroke and Trauma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 GABAA Receptor Modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Benzodiazepiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Hyperalgesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Barbituratess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Opioid Tolerancee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 Etomidate, Propofol, and Alphaxalone . . . . . . . . . . . . . . . . . 195 GABAB Receptor Agonists and Antagonists . . . . . . . . . . . . . . 195 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 200 Nonprescription Uses of Drugs That Alter GABA Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .201 Physiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Ethanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

INTRODUCTION Inhibitory and excitatory neurotransmitters regulate almost every behavioral process, including consciousness, sleep, learning, memory, and all sensations. Inhibitory and excitatory neurotransmitters are also implicated in pathologic processes such as epilepsy and the neurotoxicity associated with stroke. The interactions among ion channels, the receptors that regulate these channels, and amino acid neurotransmitters in the central nervous system (CNS) constitute the molecular basis or these processes. This chapter discusses the physiology, pathophysiology, and pharmacology o -aminobutyric acid (GABA) and glutamate neurotransmission. Together, these molecules are the two most important amino acid neurotransmitters in the CNS.

184

OVERVIEW OF GABAERGIC AND GLUTAMATERGIC NEUROTRANSMISSION The CNS has high concentrations o certain amino acids that bind to postsynaptic receptors and thereby act as inhibitory or excitatory neurotransmitters. O the two main classes o neuroactive amino acids, -aminobutyric acid (GABA) is the major inhibitory amino acid, and glutamate is the primary excitatory amino acid. Amino acid neurotransmitters elicit inhibitory or excitatory responses by altering the conductance o one or more ionselective channels. Inhibitory neurotransmitters induce a net outward current, generally hyperpolarizing the membrane. For example, inhibitory neurotransmitters may open K channels or Cl channels to induce K e f ux or Cl inf ux, respectively.

C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission

PHYSIOLOGY OF GABAERGIC NEUROTRANSMISSION GABA unctions as the primary inhibitory neurotransmitter in the mature mammalian CNS. The cell membranes o most vertebrate CNS neurons and astrocytes express GABA receptors, which decrease neuronal excitability through several mechanisms. Because o their widespread distribution, GABA receptors inf uence many neural circuits and unctions. Drugs that modulate GABA receptors a ect arousal and attention, memory ormation, anxiety, sleep, and muscle tone. Modulation o GABA signaling is also an important mechanism or treatment o ocal or widespread neuronal hyperactivity in epilepsy.

Questions 1 . What are the signs o barbiturate poisoning, and how are these signs explained by the drugs’ mechanism o action? 2 . How do barbiturates act to control epileptic seizures and to induce sleep? 3 . How does the patient’s age a ect the extent o CNS depression caused by barbiturates? 4 . What is the interaction o barbiturates and ethanol that results in pro ound CNS and respiratory depression?

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Either type o ion movement—the loss o intracellular cations or the gain o intracellular anions—results in membrane hyperpolarization and decreased membrane resistance (Fig. 13-1), respectively moving the membrane potential urther below its threshold value and reducing the ability o inward currents to depolarize the membrane. Excitatory amino acid neurotransmitters induce a net inward current, generally depolarizing the membrane. For example, excitatory neurotransmitters may open cationspeci c channels, such as a sodium channel, and thereby cause a net inf ux o sodium ions that depolarizes the membrane. An excitatory (depolarizing) response could also result i a neurotransmitter closes potassium “leak channels” to reduce the outward f ow o potassium ions and thereby depolarize the membrane (see Chapter 8, Principles o Cellular Excitability and Electrochemical Transmission). Pharmacologic agents that modulate GABAergic neurotransmission, including benzodiazepines and barbiturates , are drug classes o major clinical importance. In comparison, pharmacologic agents targeting glutamatergic neurotransmission remain largely experimental. The balance o the discussion is, there ore, addressed at GABAergic physiology and pharmacology; the pathophysiology and pharmacology o glutamatergic neurotransmission is discussed at the end o the chapter.

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S B, a 70-ye a r-o ld m a n , is h a vin g tro u b le s le e p in g . He re ca lls th a t h is s is te r h a s b e e n p re s crib e d p h e n o b a rb ita l, a b a rb itu ra te , to co n tro l h e r e p ile p tic s e izu re s , a n d th a t b a rb itu ra te s a re s o m e tim e s a ls o p re s crib e d a s s le e p in g p ills . He d e cid e s to ta ke “ ju s t a e w ” w ith s o m e a lco h o l to h e lp h im s le e p . S h o rtly a te rwa rd , S B is ru s h e d to th e e m e rg e n cy d e p a rtm e n t a te r h is s is te r f n d s h im m in im a lly re s p o n s ive . On e xa m in a tio n , h e is d i f cu lt to a ro u s e a n d dys a rth ric, w ith a n u n s te a d y ga it a n d im p a ire d a tte n tio n a n d m e m o ry. His re s p ira to ry ra te is a p p roxim a te ly s ix s h a llo w b re a th s p e r m in u te . Th e p a tie n t is s u b s e q u e n tly in tu b a te d to p ro te ct h im ro m a s p ira tin g ga s tric co n te n ts . Activa te d ch a rco a l is a d m in is te re d th ro u g h a n a s o ga s tric tu b e to lim it u rth e r a b s o rp tio n o p h e n o b a rb ita l. He a ls o re ce ive s

185

FIGURE 13-1. Effects of inhibitory and excitatory neurotransmitters on ion conductances. A. Inhibitory neurotransmitters hyperpolarize membranes by inducing a net outward current, by promoting either an inf ux o anions (e.g., opening a Cl channel) or an e f ux o cations (e.g., opening a K channel). Opening o chloride or potassium channels also decreases the membrane resistance and thereby lowers the Vm response to excitatory currents, a process called shunting. The decreased membrane resistance results in decreased responsiveness (i.e., a smaller change in Vm per change in current) because Vm im rm, where Vm is the membrane potential, im is the excitatory current, and rm is the membrane resistance. B. Excitatory neurotransmitters depolarize membranes by inducing a net inward current, either by enhancing inward current (e.g., opening a Na or Ca 2 channel) or by reducing outward current (e.g., closing a K channel). Potassium channel closure, independent o changes in the resting membrane potential, also increases the resting membrane resistance and renders the cell more responsive to excitatory postsynaptic currents.

186 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

GABA Metabolism and Transport The synthesis o GABA is mediated by glutamic acid decarboxylase (GAD), which catalyzes the decarboxylation o glutamate to GABA in GABAergic nerve terminals (Fig. 13-2A). Thus, the amount o GABA in brain tissue correlates with the amount o unctional GAD. GAD requires pyridoxal phosphate (vitamin B6) as a co actor. GABA is packaged into presynaptic vesicles by a vesicular transporter (VGAT). (The same transporter, VGAT, is also expressed in nerve terminals that release glycine, another inhibitory neurotransmitter.) In response to an action potential and the presynaptic elevation o intracellular Ca2 , GABA is released into the synaptic cle t by usion o GABA-containing vesicles with the presynaptic membrane. Termination o GABA action at the synapse depends on the removal o GABA rom the extracellular space. Neurons and glia take up GABA via specif c GABA transporters (GATs) in the cell membrane. Four GATs have been identif ed, GAT-1 through GAT-4, each with a characteristic distribution in the CNS. Within cells, the widely distributed mitochondrial enzyme GABA transaminase (GABA-T) catalyzes the conversion

o GABA to succinic semialdehyde (SSA), which is oxidized to succinic acid by SSA dehydrogenase. Succinic acid enters the Krebs cycle to become -ketoglutarate, and GABA-T regenerates glutamate rom -ketoglutarate (Fig. 13-2A).

GABA Receptors GABA mediates its neurophysiologic e ects by binding to GABA receptors. There are two types o GABA receptors. Ionotropic GABA receptors (GABAA and GABAC) are multisubunit membrane proteins that bind GABA and open an intrinsic chloride ion channel. Metabotropic GABA receptors (GABAB) are heterodimeric G protein-coupled receptors that activate neuronal potassium channels through second messengers.

Ionotropic GABA Receptors: GABAA and GABAC

The most abundant GABA receptors in the CNS are ionotropic GABAA receptors, which are members o the super amily o ast neurotransmitter-gated ion channels. This super amily includes peripheral and neuronal nicotinic acetylcholine receptors (nAChRs), serotonin type 3A/B (5-HT3A/B) receptors,

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FIGURE 13-2. Glutamate and GABA synthesis and metabolism. A. Glutamate synthesis and metabolism are intertwined with GABA synthesis and metabolism. In one pathway for glutamate synthesis, -ketoglutarate produced by the Krebs cycle serves as a substrate for the enzyme GABA transaminase (GABA-T), which reductively transaminates intraneuronal -ketoglutarate to glutamate. The same enzyme also converts GABA to succinic semialdehyde. Alternatively, glutamate is converted to GABA by the enzyme glutamic acid decarboxylase (GAD), changing the major excitatory neurotransmitter to the major inhibitory transmitter. GABA-T is irreversibly inhibited by vigabatrin; by blocking the conversion of GABA to succinic semialdehyde, this drug increases the amount of GABA available for release at inhibitory synapses. GABA-T, GABA transaminase; SSADH, succinic semialdehyde dehydrogenase; GAD, glutamic acid decarboxylase. B. Glutamate transporters in neuronal [Gt(n)] and glial [Gt(g)] cell membranes sequester glutamate (Glu) from the synaptic cleft into their respective cells. In the glial cell, the enzyme glutamine synthetase transforms glutamate into glutamine (Gln). Glutamine is then transferred to the neuron, which converts it back to glutamate via mitochondria-associated glutaminase.

C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission

P icrotoxin (-)

FIGURE 13-3. Schematic representation of the GABAA receptor. A. The pentameric structure o the GABAA receptor. Each o the f ve subunits is typically one o three predominant subtypes: , , or . Activation requires the simultaneous binding o two GABA molecules to the receptor, one to each o the two binding sites at the inter ace o the and subunits. Each subunit o the GABAA receptor has our membrane-spanning regions and a cysteine loop in the extracellular N-terminal domain (depicted as a blue segment and a dashed line). B. Drug binding sites on the GABAA receptor. For most o the exact locations schematically indicated in this diagram, the current evidence is largely indirect. ( ) indicates agonist or allosteric modulator action at the GABAA receptor; ( ) indicates competitive or noncompetitive antagonist action.

and glycine receptors. Like other members o this super amily, GABAA receptors are pentameric transmembrane glycoproteins that are assembled to orm a central ion pore surrounded by ve subunits, each o which has our membrane-spanning domains (Fig. 13-3A). Sixteen di erent GABAA receptor subunits are currently known ( 1–6, 1–3, 1–3, , , , and ). The number o pentameric ion channels that could be ormed by potential combinations o 16 subunits is very large, but only about 20 di erent subunit combinations have been identi ed in native GABAA receptors. Importantly, receptors containing di erent subunit combinations display distinct distributions at the cellular and tissue levels, and evidence is accumulating that di erent GABAA receptor subtypes play distinct roles in speci c neural circuits. Most synaptic GABAA receptors consist o two subunits, two subunits, and one subunit. “Extrasynaptic” GABAA receptors have also been identi ed on dendrites, axons, and neuronal cell bodies. These o ten contain 5 subunits together with a subunit or a subunit.

t

Ne uros te roids (+)

n

Ba rbitura te s (+)

e

Furos e mide (-)

P e nicillin (-)

r

Fluma ze nil (-)

GABA (+) a nd compe titive a nta gonis ts (-)

r

Be nzodia ze pine s (+)

u

B

c

GABAA re ce ptor

-

C

l

N

The ve subunits o GABAA receptors surround a central chloride-selective ion pore that opens in the presence o GABA. GABA and other agonists bind to two sites, which are located in extracellular portions o the receptor-channel complex at the inter ace between the and subunits. GABAA receptors also contain a number o modulatory sites where other endogenous ligands and/or drugs bind (Fig. 13-3B). In many cases, the presence o these sites and the impact o ligand binding depend on the receptor subunit composition. GABAA receptor-channel activation ollows the binding o two molecules o GABA, one to each o the receptor’s agonist sites (Fig. 13-3). Fast inhibitory postsynaptic currents (IPSCs) are responses activated by very brie (high- requency) bursts o GABA release at synapses. Uptake by GAT removes GABA rom the synapse in less than 1 ms; IPSCs deactivate over about 12–20 ms, a rate that is determined by both closure o the GABAA receptor ion channel and dissociation o GABA rom the receptor. Prolonged occupation o the agonist sites by GABA also leads to GABAA receptor desensitization, a transition to an inactive agonist-bound state (Fig. 13-4). During burst (or “phasic”) ring, the presynaptic nerve membrane releases “quanta” ( 1 mM) o GABA by exocytosis o synaptic vesicles, resulting in transient, largeamplitude inhibitory postsynaptic potentials (IPSPs). The di usion o GABA away rom synaptic cle ts also results in low concentrations o GABA (up to a ew M) in cerebrospinal f uid and interstitial spaces. Thus, GABA also activates extrasynaptic GABAA receptors and thereby induces a baseline “tonic” inhibitory current in many neurons. Because the internal chloride concentration [Cl ]in o mature neurons is lower than the extracellular Cl concentration [Cl ]out, activation o chloride-selective channels (increasing conductance) shi ts the neuronal transmembrane voltage toward the Cl equilibrium potential (ECl 70 mV). This Cl f ux hyperpolarizes or stabilizes the postsynaptic cell near its normal resting membrane potential (Vm 65 mV), reducing the likelihood that excitatory stimuli will initiate action potentials. Open Cl channels attenuate the change in membrane potential caused by excitatory synaptic currents, an e ect called shunting. This is the molecular explanation or the inhibitory e ects o GABA signaling via GABAA receptors.

C

1 s ubunit

A

187

0

3

M GABA

30

M GABA

300 M GABA

Time (s ha de d ba r = 1 s e c) FIGURE 13-4. Effects of GABA on GABAA-mediated chloride conductance. Increasing concentrations o GABA induce both larger Cl currents and more rapid receptor desensitization. The latter phenomenon can be observed as the rapid decline rom the peak current during continuous exposure to 300 M GABA (right panel ). In each panel, the shaded bar indicates the 1-second period during which GABA was applied. Although individual presynaptic endings release GABA or much shorter times, the cumulative GABA released rom many presynaptic neurons, stimulated by trains o invading action potentials, can persist or seconds.

188 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

In mature neurons, the chloride gradients are maintained by a potassium-chloride (K -Cl ) co-transporter (KCC2). In immature neurons o etal and neonatal brain, the Cl gradient may be reversed due to a di erence in the Cl transporting pumps (sodium-potassium-chloride [Na -K -Cl ] co-transporter, NKCC1). In neurons expressing NKCC1, GABAA receptors may mediate the outward f ow o Cl ions, constituting an inward current and thus depolarizing the neuron. Thus, drugs that activate or potentiate GABAA receptors may have an excitatory action during brain development rather than the inhibitory e ect they have in mature neurons. The molecular role o GABAA receptors in neurons is consistent with their known physiologic roles in CNS disease and with their pharmacology. Drugs that inhibit GABAA receptors produce seizures in animals, and mutations in GABAA receptor subunits that impair activation at the molecular level are associated with inherited human epilepsy syndromes. Conversely, endogenous or exogenous substances that enhance the activation o GABAA receptors reduce neuronal excitability and may impair numerous CNS unctions. Recent evidence indicates that GABAA receptors are also expressed in peripheral tissues such as airway epithelium. Activation o these receptors may enhance smooth muscle relaxation (bronchodilation) and could represent a uture therapy or asthma. Certain endogenous substances such as taurine and steroids (also known as neurosteroids) allosterically modulate GABAA receptor activity. The steroid hormones deoxycorticosterone and progesterone are metabolized in the brain to produce pregnenolone, dehydroepiandrosterone (DHEA), 5 -dihydrodeoxycorticosterone (DHDOC), 5 -tetrahydrodeoxycorticosterone (THDOC), and allopregnanolone. Neurosteroids do not act through nuclear receptors like most steroid hormones; instead, they alter GABAA receptor unction by binding to allosteric sites on the receptor protein, causing increased GABAA receptor activation. DHDOC and THDOC are thought to modulate brain activity during stress. Menstrual variations in allopregnanolone, a metabolite o progesterone, contribute to perimenstrual (catamenial) epilepsy. Sul ation o pregnenolone and DHEA results in neurosteroids that inhibit GABAA receptors. Another endogenous substance that enhances GABAA receptor activity is oleamide , a atty acid amide ound in the cerebrospinal f uid o sleep-deprived animals.

GABAB re ce ptor

Injection o oleamide into normal animals induces sleep, in part through potentiation o GABAA receptors. Another group o ionotropic GABA receptors, GABAC, are ormed by three subunits that are not ound in GABAA receptors ( 1–3). GABAC receptors are also pentameric ligand-gated chloride channels, but their distribution in the CNS is restricted primarily to the retina. GABAC receptors display distinct pharmacologic properties that di er rom those o most GABAA receptors. No drugs currently in use target GABAC receptors.

Metabotropic GABA Receptors: GABAB

GABAB receptors are G protein-coupled receptors that are

expressed at lower levels than GABAA receptors and are ound principally in the spinal cord (Fig. 13-5). They unction as a receptor complex composed o principal GABAB1 and GABAB2 subunits and auxiliary potassium channel tetramerization domain (KCTD) subunits. The principal subunits determine the cell sur ace expression and distribution o the receptors to axonal and dendritic sites. The auxiliary subunits inf uence the agonist potency and the kinetics o the receptor response. The GABAB receptor interacts with heterotrimeric G proteins, leading to the dissociation o their subunit, which directly activates K channels and inhibits the opening o voltage-gated Ca2 channels (Fig. 13-5). GABAB receptor activation also leads to inhibition o adenylyl cyclase and concomitant reduction in cAMP, but this seems to have only minor e ects on cellular excitability. At GABAergic synapses, GABAB receptors are expressed both presynaptically and postsynaptically. Presynaptic “autoreceptors” modulate neurotransmitter release by reducing Ca2 inf ux, while postsynaptic GABAB receptors produce slow IPSPs through activation o G protein-activated “inward recti er” K channels (GIRKs). The slower rates o activation and deactivation o GABAB currents in comparison to GABAA currents are due to the relatively slow second messenger signal transduction mechanisms. Because K has an equilibrium potential near 90 mV, activation o K channels by GABAB-coupled G proteins inhibits neuronal ring. Thus, like increased Cl conductance, increased K conductance drives the neuronal transmembrane voltage toward “resting” potentials, reduces the requency o action potential initiation, and shunts excitatory currents.

Effe ctor prote in (P LC or AC)

GABA

Ca 2+

β

γ

β +

K Clos e s

Ca 2+ cha nne l

Ope ns

K+ cha nne l

α γ

GTP GTP -GDP e xcha nge

α β

γ

β

γ

GDP

α GTP

GTP -GDP e xcha nge

FIGURE 13-5. Downstream signaling of the GABAB receptor. GABAB receptor activation alters cytoplasmic G proteins that then dissociate into and

subunits, the latter o which bind directly to K or Ca 2 channels (leftward arrow). The released subunits are linked to second messenger systems such as adenylyl cyclase (AC) or phospholipase C (PLC) (rightward arrow). The increased K e f ux leads to slow, long-lasting inhibitory postsynaptic potentials. The reduced Ca 2 inf ux may account or the ability o GABAB autoreceptors to inhibit presynaptic neurotransmitter release. The GABAB receptor unctions as an obligate heterodimer o GABAB1 and GABAB2 subunits, each o which is a seven-transmembrane-spanning G protein-coupled receptor (not shown).

C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission

PHARMACOLOGIC CLASSES AND AGENTS AFFECTING GABAERGIC NEUROTRANSMISSION Pharmacologic agents acting on GABAergic neurotransmission a ect GABA metabolism, transport, or receptor activity. The majority o pharmacologic agents a ecting GABAergic neurotransmission act on the ionotropic GABAA receptor. Several drug classes can regulate GABAA receptors by interacting with the GABA binding sites or with allosteric sites (Fig. 13-3). Therapeutic agents that activate GABAA receptors are used or sedation, anxiolysis, hypnosis (general anesthesia), neuroprotection ollowing stroke or head trauma, and control o epilepsy. Other agents that modulate GABAergic transmission are used only or experimental purposes (Table 13-1).

Inhibitors of GABA Metabolism and Transport Tiagabine is a competitive inhibitor o the GABA transporters

in neurons and glia, where it may act selectively on GAT-1. Epilepsy is the major clinical indication or tiagabine. By inhibiting GABA reuptake, tiagabine increases both synaptic and extrasynaptic GABA concentrations. The result is

nonspeci c agonism o both ionotropic and metabotropic GABA receptors, with the major e ects at GABAA receptors. Tiagabine is an oral medication that is rapidly absorbed with 90% bioavailability and is highly protein bound. Metabolism is hepatic, primarily by CYP3A4. Tiagabine does not induce cytochrome P450 enzymes, but its metabolism is inf uenced by concomitant use o either inducers or inhibitors o CYP3A4. Adverse e ects o tiagabine are those o high GABA activity, including con usion, sedation, amnesia, and ataxia. Tiagabine potentiates the action o GABAA receptor modulators such as ethanol, benzodiazepines, and barbiturates. -Vinyl GABA (vigabatrin) is a “suicide inhibitor” o GABA transaminase (GABA-T, see Fig. 13-2). Administration o this drug blocks the conversion o GABA to succinic semialdehyde, resulting in high intracellular GABA concentrations and increased synaptic GABA release. Like the e ect o tiagabine, enhancement o GABA receptor unction by -vinyl GABA is not selective because GABA concentrations are increased wherever GABA is released, including the retina. Vigabatrin is used in the treatment o epilepsy, and it is being investigated or treatment o drug addiction, panic disorder, and obsessive-compulsive disorder. Adverse e ects

TABLE 13-1 Partial List of Agents That Modulate GABAergic Transmission DRUG CLASS

PRESUMED MECHANISM

EFFECTS

Allylglycine

Inhibits glutamic acid decarboxylase

Convulsant

Isoniazid

Inhibits pyridoxal kinase (antivitamin B6 e ect)

Convulsant at high doses

Inhibits GABA and glycine release

Convulsant

Tiagabine

Inhibits GAT-1

Anticonvulsant

Vigabatrin

Inhibits GABA transaminase

Anticonvulsant

Muscimol

GABAA receptor agonist

Anticonvulsant, mimics psychosis

Gaboxadol

GABAA receptor agonist

Anticonvulsant

Bicuculline

Competitive antagonist

Convulsant

Gabazine

Competitive antagonist

Convulsant

Picrotoxin

Noncompetitive antagonist, pore blocker, occludes the chloride channel

Convulsant

Benzodiazepines

Potentiate GABA binding

Anticonvulsant, anxiolytic

Barbiturates

Increase GABA e f cacy, weak agonist

Anticonvulsant, anesthetic

GABAB receptor agonist

Muscle relaxant

GABA Synthesis

GABA Release Tetanus toxin

GABA Metabolism and Transport

GABAA Receptor Agonists

GABAA Receptor Antagonists

GABAA Receptor Modulators

GABAB Receptor Agonists Baclo en

189

190 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

o -vinyl GABA include drowsiness, con usion, and headache. The drug has been reported to cause bilateral visual f eld de ects associated with irreversible di use atrophy o the peripheral retinal nerve f ber layer. This appears to result rom accumulation o the drug in retinal nerves.

Benzodiazepines act as positive allosteric modulators by enhancing GABAA receptor channel gating in the presence o GABA (Fig. 13-6). Benzodiazepines increase the frequency of channel opening in the presence o low GABA concentrations, and, at GABA concentrations similar to those in synapses,

GABAA Receptor Agonists and Antagonists A

m

)

100

Benzodiazepines are high-a f nity, highly selective drugs that

bind at a single site on GABAA receptors containing 1, 2, 3, or 5 subunits and a subunit. In molecular studies, benzodiazepine potency correlates with hydrophobicity. However, benzodiazepines are highly bound to plasma proteins such as albumin, and hydrophobicity enhances protein binding and thereby reduces the drugs’ ree concentration and transport across the blood–brain barrier. There ore, highly protein-bound benzodiazepines may appear less potent in vivo even though they display higher potency in molecular studies. Furthermore, in clinical states associated with low albumin, such as acute hemodilution or liver dys unction, the clinical potency o benzodiazepines may be dramatically increased.

u m xi a m % ( t n e r r

Low GABA + mida zola m

Low GABA a lone

C

l

-

c

u

40 20

100

10 -8

10 -7

10 -6

10 -5

10 -4

10 -3

80

xi

m

u

m

)

GABA + high-dos e pe ntoba rbita l

60

t

(

%

m

a

GABA + ma xima l mida zola m

40

u

r

r

e

Benzodiazepines

60

B

GABA a lone

c l

Benzodiazepines and barbiturates are modulators o GABAA receptors that act at allosteric binding sites to enhance GABAergic neurotransmission (Fig. 13-3B). Benzodiazepines have sedative, hypnotic, muscle relaxant, amnestic, and anxiolytic e ects. At high doses, benzodiazepines can cause hypnosis and stupor. However, when used alone, these drugs rarely cause atal CNS depression. Barbiturates constitute a large group o drugs that were f rst introduced in the midtwentieth century and continue to be used, albeit with diminishing requency, or control o epilepsy, as general anesthetic induction agents, and or control o intracranial hypertension.

80

Mida zola m or pe ntoba rbita l (Mola r)

C

GABAA Receptor Modulators

Low GABA + pe ntoba rbita l

0 10 -9

n

Agonists such as muscimol and gaboxadol activate the GABAA receptor by binding directly to the GABA binding site. Muscimol, f rst derived rom hallucinogenic Amanita muscaria mushrooms, is a ull agonist at many GABAA receptor subtypes and is used primarily as a research tool. Purif ed muscimol (as well as other GABAA receptor agonists) does not induce hallucinations, which are probably caused by other actors rom Amanita muscaria. Gaboxadol at high concentrations is a partial agonist at synaptic GABAA receptors; at low concentrations, gaboxadol selectively activates extrasynaptic receptors containing 4, 3, and subunits. Gaboxadol was initially approved or treatment o epilepsy and anxiety, but therapeutic doses were associated with ataxia and sedation. Lower gaboxadol doses, which activate extrasynaptic receptors, induce slow-wave sleep in laboratory animals. Human trials o gaboxadol or treatment o insomnia were halted in 2007 due to concerns about adverse e ects such as hallucinations, disorientation, sleepwalking, and sleep-driving. Bicuculline and gabazine are competitive antagonists that bind at the GABA sites on GABAA receptors. Picrotoxin is a noncompetitive inhibitor o GABAA receptors that blocks the ion pore. All o these GABAA antagonists induce seizures and are used exclusively or research; they also illustrate the importance o the tonic activity o GABAA receptors in maintaining a state o relatively normal excitability in the CNS.

20 0 10 -7

10 -6

10 -5

10 -4

10 -3

GABA (Mola r) FIGURE 13-6. Effects of benzodiazepines and barbiturates on GABAA receptor activity. A. Both benzodiazepines and barbiturates enhance GABAA receptor activation (measured experimentally by Cl current), but with di erent potencies and e f cacies. Midazolam (a benzodiazepine) maximally enhances by about three old the current evoked by 10 M GABA (low GABA). In contrast, the anesthetic barbiturate pentobarbital increases the current evoked by 10 M GABA to a much greater extent (near that o a maximal GABA response), but its maximal e ect requires concentrations greater than 100 M. Thus, benzodiazepines such as midazolam are highpotency, low-e f cacy modulators o GABAA receptor activity, while barbiturates such as pentobarbital are low-potency, high-e f cacy modulators. B. Another way to compare the e f cacy o benzodiazepines and barbiturates is to measure the degree to which they enhance the sensitivity o GABAA receptors to GABA. Maximally e ective concentrations o midazolam shi t the GABA concentration–response curve modestly to the le t, reducing the EC50 (increasing the potency) o GABA by about two old. In contrast, highdose pentobarbital causes a much greater shi t to the le t, reducing the EC50 o GABA by approximately 20- old. Pentobarbital at high concentrations also directly activates GABAA receptors, even in the absence o GABA (note the nonzero Cl current at 10 7 M GABA). In contrast, the benzodiazepines do not have direct agonist activity.

C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission

receptor deactivation is slowed. Both actions result in a net increase in Cl inf ux. In addition, GABAA receptors in the open state have a higher a f nity or GABA than in the closed state, so the ability o benzodiazepines to avor channel openness results, secondarily, in an apparently higher agonist a f nity. Benzodiazepines do not activate native GABAA receptors in the absence o GABA, but they do activate certain mutant receptors and enhance maximal activation by partial agonists, indicating that they are weak positive allosteric agonists (Fig. 13-7). This mechanism is consistent with the known location o the benzodiazepine binding site at the inter ace between the external domains o the and subunits. This site is a structural homologue o the two GABA agonist sites at the inter aces between the and subunits. In GABA concentration–response studies, benzodiazepines shi t the response curve to the le t, increasing the apparent potency o GABA by up to three old (Fig. 13-6B). This is a smaller allosteric e ect than that caused by other modulators, such as barbiturates or other general anesthetics (see etomidate, below). The limited e f cacy o benzodiazepines is accompanied by a reduced potential or atal overdose. However, the margin o sa ety decreases when benzodiazepines are co-administered with alcohol or other sedative/hypnotics. Clinical Applications

Benzodiazepines are used as sleep enhancers, anxiolytics, sedatives, antiepileptics, and muscle relaxants, and or treatment o ethanol withdrawal symptoms (Table 13-2). Benzodiazepines achieve an anxiolytic e ect by inhibiting synapses in the limbic system, a CNS region that controls emotional behavior and is characterized by a high density o GABAA receptors. Benzodiazepines such as diazepam and alprazolam are used to mitigate chronic, severe anxiety and the anxiety associated with some orms o depression and schizophrenia. Because o the potential or the development o tolerance, dependence, and addiction, benzodiazepine use should be intermittent. In acute-care settings, such as in preparation or invasive procedures, midazolam is requently used as a rapid-onset and short-acting anxiolytic/sedative/amnestic. Benzodiazepines are o ten adequate as sedatives or brie ,

m xi n

t

(

%

m

a

80 60

Ma xima l GABA re s pons e

P 4S + mida zola m P 4S a lone

40

e r r

B

20

Curre nt from s ponta ne ous ly a ctive muta nt GABAA cha nne ls

Mida zola m a lone a ctiva te s curre nt

-

C

l

c

u

uncom ortable procedures associated with minimal sharp pain, such as endoscopy. When combined with opioids, however, a synergistic potentiation o both sedation and respiratory depression can occur. Given prior to general anesthesia, benzodiazepines reduce the requirement or hypnotic agents. Many benzodiazepines, including estazolam, f urazepam, quazepam, temazepam, and triazolam, and other benzodiazepine site agonists, including the so-called z-drugs (zolpidem, zaleplon, zopiclone , and eszopiclone ), are prescribed or treatment o insomnia. Benzodiazepines both acilitate sleep onset and increase the overall duration o sleep. They also alter the distribution o the various sleep stages: they increase the length o stage 2 non-rapid eye movement (NREM) sleep (the light sleep that normally comprises approximately hal o sleeping time) and decrease the length o REM sleep (the period characterized by requent dreams) and slow-wave sleep (the deepest level o sleep). A ter extended use, these e ects may diminish because o tolerance. In a healthy individual, hypnotic doses o benzodiazepines induce respiratory changes comparable to those present during natural sleep and do not cause signif cant cardiovascular changes. Patients with either pulmonary or cardiovascular disease may experience signif cant respiratory or cardiovascular depression because o medullary depression rom otherwise therapeutic doses o these drugs. Patients who have su ered brain damage rom stroke or head trauma may also become pro oundly sedated with these drugs. The sedative benzodiazepines di er in their rates o onset, durations o e ect, and tendencies to cause rebound insomnia when withdrawn. For example, f urazepam is a long-acting benzodiazepine that acilitates sleep onset and maintenance and increases sleep duration. Although it does not cause signif cant rebound insomnia, its long elimination hal -li e (about 74 hours) and the accumulation o active metabolites may cause daytime sedation. Triazolam is a ast-onset benzodiazepine that also decreases the time needed to all asleep. Intermittent rather than chronic administration o this drug is recommended to lessen the rebound insomnia associated with its discontinuation. The so-called z-drugs such as zolpidem are unique among sedatives used or insomnia in selectively interacting with GABAA receptors containing

100

u

m

)

A

191

P icrotoxin blocks curre nt

0

Time (s e c) FIGURE 13-7. Evidence that benzodiazepines enhance the GABAA receptor channel opening probability. A. When GABAA receptors are activated using saturating concentrations o the partial agonist P4S, midazolam increases the peak current. This indicates that the P4S e cacy (the maximal channel opening probability) is increased by the addition o midazolam. B. GABAA receptors containing a certain point mutation are spontaneously active, which can be demonstrated by the loss o current caused by picrotoxin (a noncompetitive GABAA receptor antagonist). When these mutant receptors are exposed to midazolam, the amount o current increases, indicating that midazolam directly inf uences the opening o GABAA receptors. This e ect is not observed in wild-type channels, which exhibit only rare spontaneous openings.

192 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 13-2 Clinical Uses and Relative Duration o Action o Several Benzodiazepines BENZODIAZEPINE

CLINICAL USES

DURATION OF ACTION

Clorazepate

Anxiety disorders, seizures

Short-acting (3–8 hours)

Midazolam

Preanesthetic, IV general anesthetic

Short-acting (3–8 hours)

Alprazolam

Anxiety disorders, phobias

Intermediate-acting (11–20 hours)

Lorazepam

Anxiety disorders, status epilepticus, IV general anesthetic

Intermediate-acting (11–20 hours)

Chlordiazepoxide

Anxiety disorders, alcohol withdrawal

Long-acting (1–3 days)

Clobazam

Anxiety disorders, seizures

Long-acting (1–3 days)

Clonazepam

Seizures

Long-acting (1–3 days)

Diazepam

Anxiety disorders, status epilepticus, muscle relaxant, IV general anesthetic, alcohol withdrawal

Long-acting (1–3 days)

Triazolam

Insomnia

Short-acting (3–8 hours)

Estazolam

Insomnia

Intermediate-acting (11–20 hours)

Temazepam

Insomnia

Intermediate-acting (11–20 hours)

Flurazepam

Insomnia

Long-acting (1–3 days)

Quazepam

Insomnia

Long-acting (1–3 days)

1 subunits. This selectivity is associated with reduced muscle relaxant and anxiolytic actions, but tolerance and amnesia remain as potential adverse e ects. Benzodiazepines also have antiepileptic e ects. Clonazepam is requently used or this indication, because the anticonvulsant e ects o clonazepam are not accompanied by signi cant psychomotor impairment. Drugs used in the treatment o epilepsy are discussed urther in Chapter 16, Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System. Clobazam is a unique benzodiazepine whose structure is di erent rom classic benzodiazepines. It is a long-acting benzodiazepine used or selective anxiolysis and seizure control. Compared with other benzodiazepines, this agent is less sedating and has ewer negative e ects on cognition. Benzodiazepines reduce skeletal muscle spasticity by enhancing the activity o inhibitory interneurons in the spinal cord. Diazepam is used to alleviate muscle spasms caused

by physical trauma as well as muscle spasticity associated with neuromuscular degenerative disorders such as multiple sclerosis. The high doses required or these e ects also requently cause sedation. Pharmacokinetics and Metabolism

Benzodiazepines can be administered via oral, transmucosal, intravenous, and intramuscular routes. The lipophilic nature o benzodiazepines explains their rapid and complete absorption. Although these drugs and their active metabolites are bound to plasma proteins, they do not compete with other protein-bound drugs. Benzodiazepines are metabolized by hepatic microsomal cytochrome P450 enzymes, speci cally CYP3A4, and subsequently excreted in the urine as glucuronides or oxidized metabolites. Prolonged benzodiazepine administration does not signi cantly induce hepatic drug-metabolizing enzyme activity. However, other drugs that inhibit CYP3A4 activity (e.g., ketoconazole and macrolide antibiotics) may enhance the e ects o benzodiazepines, while drugs that induce CYP3A4 (e.g., ri ampicin, omeprazole, ni edipine) may reduce their e ectiveness. Patients with impaired hepatic unction, including the elderly and the very young, may experience prolonged e ects rom benzodiazepine administration. Some benzodiazepine metabolites (e.g., desmethyldiazepam) remain pharmacologically active and are cleared more slowly than the parent drug. Adverse E ects

The adverse e ects o benzodiazepines are primarily related to their therapeutic e ects in undesirable settings: amnesia, oversedation, and ataxia. In patients with insomnia, rare but sometimes dangerous adverse e ects o benzodiazepines and z-drugs include sleepwalking, sleep-driving, and sleepeating. The relative sa ety o benzodiazepines derives rom their limited e cacy in modulating GABAA receptors. High doses o benzodiazepines rarely cause death unless administered with other drugs, such as ethanol, CNS depressants, opioid analgesics, or tricyclic antidepressants. The enhanced CNS depression seen with concomitant ethanol and benzodiazepine use is due to both synergistic e ects on GABAA receptors and ethanol-mediated inhibition o CYP3A4. The latter e ect occurs when ethanol is consumed rapidly, decreasing benzodiazepine clearance. Benzodiazepine overdose can be reversed by a benzodiazepine antagonist such as f umazenil. Although f umazenil has minimal clinical e ects on its own, it antagonizes the e ects o benzodiazepine agonists by competing or occupancy o high-a nity benzodiazepine sites on GABAA receptors (Fig. 13-3B). In patients with benzodiazepine dependence, f umazenil can cause a severe withdrawal syndrome. This drug does not block the e ects o barbiturates or ethanol. Tolerance and Dependence

Chronic benzodiazepine use induces tolerance, which is mani ested as a decrease in the e cacy o both benzodiazepines and barbiturates. Animal models suggest that tolerance to benzodiazepines results rom decreased expression o benzodiazepine (GABAA) receptors at synapses. Another proposed mechanism or tolerance involves uncoupling o the benzodiazepine binding site rom the GABA site. Sudden cessation a ter chronic benzodiazepine administration can result in a withdrawal syndrome characterized by con usion, anxiety, agitation, and insomnia.

C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission

Barbiturates

The CNS sites a ected by barbiturates are widespread, including the spinal cord, brainstem (cuneate nucleus, substantia nigra, reticular activating system), and brain (cortex, thalamus, cerebellum). Barbiturates reduce neuronal excitability primarily by increasing GABA-mediated inhibition via GABAA receptors. Barbiturate-enhanced GABAergic transmission in the brainstem suppresses the reticular activating system (discussed in Chapter 9, Principles o Nervous System Physiology and Pharmacology), causing sedation, amnesia, and loss o consciousness. Heightened GABAergic transmission at motor neurons in the spinal cord relaxes muscles and suppresses ref exes. Selectivity or GABAA receptor subtypes containing speci c subunit combinations has not been demonstrated or barbiturates. The stoichiometry o barbiturate binding sites on GABAA receptors is variable. The anesthetic barbiturates thiopental, pentobarbital, and methohexital act as both agonists at GABAA receptors and as enhancers o receptor responses to GABA. Anticonvulsant barbiturates such as phenobarbital produce ar less direct agonism on native GABAA receptors. The direct GABAA receptor activation is not mediated by GABA binding sites but depends on barbiturate-speci c sites in the subunits o the receptor. At clinically relevant concentrations o barbiturates, the degree o membrane hyperpolarization due to direct activation o GABAA receptors is ar less than that rom enhancement o GABA agonism. The major action o the barbiturates is to enhance the e cacy o GABA by increasing the time that the Cl channel stays open, permitting a much greater inf ux o Cl ions or each activated channel (Fig. 13-6A). This leads to a greater degree o hyperpolarization and to decreased excitability o the target cell. The GABA-enhancing action o barbiturates is greater than that o the benzodiazepines (Fig. 13-6B). The direct-activating and GABA-enhancing actions o barbiturates may be associated with di erent binding sites or, as shown or etomidate (see below), may ref ect actions at a single class o sites. In keeping with their relative e cacy or GABA potentiation, overdoses o the low-e cacy benzodiazepines are deeply sedating but rarely dangerous, whereas barbiturate overdose may produce pro ound hypnosis, coma, respiratory depression, and death i supportive therapy is not provided. Barbiturates a ect not only GABAA receptors but also receptors involved in excitatory neurotransmission. Barbiturates decrease activation o glutamate-sensitive AMPA receptors (see Fig. 13-8B), thereby reducing both membrane depolarization and neuronal excitability. At anesthetic concentrations, pentobarbital also decreases the activity o voltage-dependent Na channels, inhibiting high- requency neuronal ring. Clinical Applications

Be ore the discovery o benzodiazepines, the sedative/hypnotic e ects o barbiturates were commonly used to treat insomnia or anxiety. Benzodiazepines have largely replaced barbiturates in most clinical applications because benzodiazepines are sa er, cause less tolerance, have ewer withdrawal symptoms, and induce less pro ound e ects on drug-metabolizing enzymes. Barbiturates are still used or induction o general anesthesia, as antiepileptic agents, and or neuroprotection (Table 13-3). The lipid-soluble barbiturates, such as thiopental, methohexital, and pentobarbital, are used to induce general anesthesia. These drugs enter the brain rapidly a ter intravenous

N

A

193

1 s ubunit

Te tra me ric s tructure

C B Gluta ma te /AMPA/Ka ina te Alcohols , vola tile a ne s the tics ?

Gluta ma te /NMDA Glycine

Ba rbitura te

Na +

+

K

AMPA/Ka ina te re ce ptor

Zn 2+

P he ncyclidine Ca 2+

Mg 2+

+

K

NMDA re ce ptor

FIGURE 13-8. Schematic representation of the ionotropic glutamate receptors. A. All three ionotropic glutamate receptors are tetrameric complexes composed o the same (termed homomeric) or di erent (termed heteromeric) subunits. The structure on the right shows one ionotropic glutamate receptor subunit, which spans the membrane three times and has a partially spanning hairpin turn that, when juxtaposed with homologous turns rom the other three subunits, orms the lining o the ion channel’s pore. B. Major binding sites on the AMPA/kainate and NMDA classes o ionotropic glutamate receptors are shown. Although there is indirect evidence or the location o many o the drug binding sites that are schematically indicated in this diagram, the def nitive localization o these sites remains to be determined.

administration and then redistribute to less highly per used tissues. This redistribution away rom the CNS results in a short duration o action a ter a single bolus administration. The anesthetic barbiturates are also discussed in Chapter 17, General Anesthetic Pharmacology. Barbiturates such as phenobarbital serve as e ective antiepileptics. As discussed in Chapter 16, seizures are characterized by rapidly depolarizing CNS neurons that repeatedly re action potentials. Barbiturates reduce epileptic activity both by enhancing GABA-mediated synaptic inhibition and by inhibiting AMPA receptor-mediated excitatory transmission. Phenobarbital is used to treat ocal and tonic–clonic seizures at concentrations that produce minimal sedation. The pro ound suppression o neuronal activity by highdose barbiturates can produce electroencephalographic silence, known as barbiturate coma. This state is associated with signi cantly reduced brain oxygen consumption and reduced cerebral blood f ow. These e ects can protect the brain rom ischemic damage in pathologic conditions associated with reduced oxygen delivery (e.g., hypoxia, pro ound anemia, shock, brain edema) or increased oxygen demand (e.g., status epilepticus). To produce barbiturate coma, bolus

194 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 13-3 Clinical Uses and Relative Duration of Action of Several Barbiturates DURATION OF ACTION

BARBITURATE

CLINICAL USES

Methohexital

Anesthesia induction and short-term maintenance

Ultrashort-acting (5–15 minutes)

Thiopental

Anesthesia induction and short-term maintenance, emergency seizure treatment

Ultrashort-acting (5–15 minutes)

Amobarbital

Insomnia, preoperative sedation, emergency seizure treatment

Short-acting (3–8 hours)

Pentobarbital

Insomnia, preoperative sedation, emergency seizure treatment

Short-acting (3–8 hours)

Secobarbital

Insomnia, preoperative sedation, emergency seizure treatment

Short-acting (3–8 hours)

Phenobarbital

Treatment o seizures, status epilepticus

Long-acting (days)

hormones, bile salts, cholesterol, and vitamins D and K, although the concomitant administration o barbiturates with these agents slows their biotrans ormation because o substrate competition or the metabolizing enzymes. Elderly patients (who o ten have impaired liver unction) and patients with severe liver disease have reduced barbiturate clearance; even normal doses o sedative/hypnotics may have signif cantly greater CNS e ects in these patients, as experienced by SB in the introductory case. Because acidic compounds such as phenobarbital are excreted aster in alkaline urine, administration o intravenous sodium bicarbonate increases clearance. Adverse Effects

The duration o action o a barbiturate is determined by the rapidity with which it is redistributed rom the brain to other, less vascular compartments, particularly to muscle and at.

administration is ollowed by in usion (or multiple additional boluses) to maintain the CNS concentration o drug at therapeutic levels. Pharmacokinetics and Metabolism

Barbiturates, like benzodiazepines, can be administered orally or intravenously. Oral administration may be associated with signif cant f rst-pass metabolism and reduced bioavailability. Methohexital can also be absorbed transmucosally. The ability o a barbiturate to cross the blood–brain barrier and enter the CNS is largely determined by its lipid solubility. Consequently, termination o the drug’s acute CNS e ects depends primarily on its redistribution rom the brain, f rst to such highly per used areas as the splanchnic circulation, then to skeletal muscle, and, f nally, to poorly per used adipose tissue. As a result, bolus administration o a barbiturate that redistributes rapidly causes only a short-lived e ect on the CNS. Chronic administration o the lipophilic barbiturates may have a prolonged e ect because o the high uptake capacity o adipose tissue, which leads to a high volume o distribution and a long elimination hal -li e. Barbiturates undergo extensive hepatic metabolism beore renal excretion. The cytochrome P450 enzymes that metabolize barbiturates are CYP3A4, CYP3A5, and CYP3A7. Chronic barbiturate use greatly up-regulates the expression o these enzymes, thereby accelerating the metabolism o barbiturates (contributing to tolerance) and other substrates or these enzymes. Barbiturate use can thus enhance the metabolism o other sedative/hypnotics, as well as benzodiazepines, phenytoin, digoxin, oral contraceptives, steroid

The multiplicity o sites at which barbiturates act, coupled with their low selectivity and high e f cacy or enhancing GABAA receptor activation, contributes to the relatively low therapeutic index o these drugs. Unlike benzodiazepines, high doses o barbiturates can cause atal CNS and respiratory depression. The anesthetic barbiturates such as pentobarbital are more likely to induce pro ound CNS depression than the antiepileptic barbiturates such as phenobarbital (Table 13-4). In addition, as exemplif ed by the case o SB, the concomitant administration o barbiturates and other CNS depressants, o ten ethanol, results in CNS depression more severe than that caused by barbiturates alone. Tolerance and Dependence

Repeated and extended misuse o barbiturates induces tolerance and physiologic dependence. Prolonged barbiturate use increases the activity o cytochrome P450 enzymes and accelerates barbiturate metabolism, thereby contributing to the development o tolerance to barbiturates and cross-tolerance to benzodiazepines, other sedative/hypnotics, and ethanol. TABLE 13-4 Comparison of Pentobarbital and Phenobarbital PENTOBARBITAL

PHENOBARBITAL

Routes o administration

Oral, IM, IV, rectal

Oral, IM, IV

Duration o action

Short-acting (1–4 hours)

Long-acting (days)

Suppression o spontaneous neuronal activity

Yes

Minimal

Activity at GABAA receptor

Major: Increases e f cacy o GABA by increasing open time o Cl channel Minor: Direct GABAA receptor activation

Increases e f cacy o GABA by increasing open time o Cl channel

Activity at glutamate receptor

Noncompetitive antagonist at AMPA receptor (2–3 times more potent than phenobarbital)

Noncompetitive antagonist at AMPA receptor

Therapeutic uses

Preoperative sedation Emergency treatment or seizures

Antiepileptic

C h a p t e r 1 3 Pharmacology of GABAergic and Glutamatergic Neurotransmission

Development o physiologic dependence results in a drug withdrawal syndrome characterized by tremors, anxiety, insomnia, and CNS excitability. I le t untreated, these withdrawal e ects may progress to seizures and cardiac arrest.

Etomidate, Propofol, and Alphaxalone

Etomidate , propofol, and alphaxalone are drugs used or in-

duction o general anesthesia. Etomidate and propo ol are also discussed in Chapter 17. Like barbiturates, these intravenous anesthetics act primarily on GABAA receptors. Etomidate is particularly use ul during induction o anesthesia in hemodynamically unstable patients. Propo ol is the most widely used anesthetic induction agent in the United States. It is used both or single-bolus induction o anesthesia and or maintenance via continuous intravenous in usion. Alphaxalone is a neurosteroid that is rarely used clinically.

Mechanisms of Action

Like barbiturates, etomidate, propo ol, and alphaxalone enhance activation o GABAA receptors by GABA and, at high concentrations, can act as agonists. For etomidate, both o these actions display similar stereoselectivity. Quantitative analysis indicates that both actions are caused by etomidate binding at a single set o two identical allosteric sites per receptor. Similar mechanisms are hypothesized to account or the actions o propo ol and alphaxalone. Etomidate and propo ol act selectively at GABAA receptors that contain 2 and 3 subunits. Based on knock-in animal experiments, in which 3 subunits are expressed as transgenes, 3-containing receptors are the most important or the hypnosis and muscle relaxation associated with general anesthesia. Alphaxalone shows little selectivity among synaptic GABAA receptors but is more potent at extrasynaptic receptors that contain subunits. Pharmacokinetics and Metabolism

Both etomidate and propo ol induce anesthesia rapidly a ter bolus intravenous injection. Like barbiturates, these hydrophobic drugs cross the blood–brain barrier rapidly. The CNS e ect o a bolus dose lasts or only several minutes, because redistribution to muscle and other tissues rapidly reduces the CNS drug concentrations. Propo ol has an extremely large volume o distribution, and prolonged continuous in usions may be used without signif cant changes in the apparent clearance o the drug. Metabolism o etomidate and propo ol is primarily hepatic. Adverse Effects

Etomidate inhibits the synthesis o cortisol and aldosterone. Suppression o cortisol production is thought to contribute to mortality among critically ill patients who receive prolonged etomidate in usions. Etomidate is generally used only or single-dose induction o anesthesia, not or anesthesia maintenance. It is also used rarely at subhypnotic doses or treatment o metastatic cortisol-producing tumors. The major toxicity o propo ol as a general anesthetic is depression o cardiac output and vascular tone. Hypotension is observed in patients who are hypovolemic or, as with many elderly patients, dependent on vascular tone to maintain blood pressure. Propo ol is ormulated in a lipid emulsion, and hyperlipidemia has been reported in patients receiving prolonged in usions or sedation. There is growing evidence in etal and newborn animal models that positive GABAA receptor modulators lead to

195

neurotoxicity and later neurodevelopmental problems. The mechanism suggested or this toxicity is the depolarizing e ect o GABAA receptors in some etal and neonatal neurons (see earlier discussion), resulting in excitotoxicity in the presence o GABAA receptor enhancers. These data have raised concerns about potential damage to the brains o human etuses and neonates who are exposed to general anesthetics. Long-term clinical studies are underway to assess the unctional impact o these e ects.

GABAB Receptor Agonists and Antagonists Baclofen is the only compound currently in clinical use

that targets GABAB receptors. It was f rst synthesized as a GABA analogue and screened or antispastic action be ore GABAB receptors were discovered. Subsequently, it was ound that baclo en is a selective GABAB receptor agonist. It is used primarily or treatment o spasticity associated with motor neuron diseases (e.g., multiple sclerosis) or spinal cord injury. Oral baclo en is e ective or mild spasticity. Severe spasticity may be treated with intrathecal baclo en therapy using doses that are ar lower than those required systemically. By activating metabotropic GABA receptors in the spinal cord, baclo en stimulates downstream second messengers to act on Ca2 and K channels. Although baclo en is prescribed primarily or treatment o spasticity, clinical observations suggest that it also modulates pain and cognition, and it is being investigated as a therapy or drug addiction. Baclo en is absorbed slowly a ter oral administration; peak plasma concentrations are reached a ter 90 minutes. It has a modest volume o distribution and does not readily cross the blood–brain barrier. Baclo en is primarily cleared rom the circulation in unmodif ed orm in the urine; about 15% o the drug is metabolized by the liver and then excreted in bile. The elimination hal -time is about 5 hours in patients with normal renal unction, and dosing is typically three times daily. A ter intrathecal injection and in usion, spasmolytic e ects are observed at 1 hour and peak e ects are observed at 4 hours. Adverse e ects o baclo en include sedation, somnolence, and ataxia. These are worsened when baclo en is taken with other sedative drugs. Reductions in renal unction may precipitate toxicity as drug levels rise. Baclo en overdose can produce blurry vision, hypotension, cardiac and respiratory depression, and coma. Tolerance apparently does not develop to oral baclo en. In contrast, dosing requirements a ter initiation o intrathecal baclo en o ten increase over the f rst 1 to 2 years. Withdrawal rom baclo en therapy, especially rom intrathecal in usion, can precipitate acute hyperspasticity, rhabdomyolysis, pruritus, delirium, and ever. Withdrawal has also led to multiorgan ailure, coagulation abnormalities, shock, and death. I withdrawal symptoms persist, e ective treatments reportedly include benzodiazepines, propo ol, intrathecal opioid administration, and restarting baclo en.

Nonprescription Uses of Drugs That Alter GABA Physiology Ethanol

Ethanol acts as an anxiolytic and sedative by causing CNS depression, but it is not without signif cant potential toxicity. Ethanol appears to exert its e ects by acting on multiple

196 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

targets, including GABAA and glutamate receptors. Ethanol increases GABAA-mediated Cl inf ux and inhibits the excitatory e ects o glutamate at NMDA receptors. Ethanol interacts synergistically with other sedatives, hypnotics, antidepressants, anxiolytics, anticonvulsants, and opioids. Ethanol tolerance and dependence are associated with changes in GABAA receptor unction. In animal models, chronic ethanol administration blunts the ethanol-mediated potentiation o GABA-induced Cl inf ux in the cerebral cortex and cerebellum. Acute tolerance to ethanol occurs without a change in the number o GABAA receptors, but chronic ethanol exposure alters GABAA receptor subunit expression in the cortex and cerebellum. Changes in the subunit composition o GABAA receptors may be responsible or the changes in receptor unction associated with chronic ethanol use. Other mechanisms proposed or the development o tolerance to ethanol include post-translational modi cations o GABAA receptors and changes in second messenger systems, or example, alterations in the expression patterns o di erent iso orms o protein kinase C (PKC). The upregulation o NMDA receptor expression that occurs with prolonged ethanol use may account or the hyperexcitability associated with ethanol withdrawal. Benzodiazepines, such as diazepam and chlordiazepoxide , reduce the tremors, agitation, and other e ects o acute alcohol withdrawal. Use o these medications in a patient experiencing withdrawal rom chronic alcohol abuse can also prevent the development o withdrawal seizures (delirium tremens).

Chloral Hydrate, -Hydroxybutyric Acid, and Flunitrazepam

Chloral hydrate is an older sedative–hypnotic rarely used today

to alleviate insomnia. It has occasionally been employed to incapacitate individuals against their will, or example, to acilitate the commission o a crime. Gamma ( )-hydroxybutyric acid (GHB) is a GABA isomer that has clinical utility as a sedative and treatment or narcolepsy but nds much wider illicit use as a recreational drug and “date rape” drug. There is recent evidence that GHB acts in part by activating GABAB receptors, but it is also an endogenous molecule that may act as a neurotransmitter at other receptors that have not yet been identi ed. Like barbiturates, high doses o GHB can produce deep sedation and coma, and its e ects are exacerbated by ethanol. Flunitrazepam (Rohypnol®) is a ast-acting benzodiazepine that can cause amnesia and thereby prevent an individual’s recall o events that occurred under the drug’s inf uence. This drug has also been reported to acilitate “date rape.”

PHYSIOLOGY OF GLUTAMATERGIC NEUROTRANSMISSION Glutamatergic synapses exist throughout the CNS. The binding o glutamate to its receptors initiates excitatory neuronal responses associated with motor neuron activation, acute sensory responses including the development o elevated pain sensation (hyperalgesia), synaptic changes involved in certain types o memory ormation, and cerebral neurotoxicity rom brain ischemia as well as unctional de cits rom spinal cord injury. Although the clinical applications o glutamate pharmacology are currently limited, it is anticipated that glutamate pharmacology will become an increasingly important area o neuropharmacology.

Glutamate Metabolism Glutamate synthesis occurs via two distinct pathways. In one pathway, -ketoglutarate ormed in the Krebs cycle is transaminated to glutamate in CNS nerve terminals, a step that is directly linked to GABA conversion (Fig. 13-2A). Alternatively, glutamine produced and secreted by glial cells is transported into nerve terminals and converted to glutamate by glutaminase (Fig. 13-2B). Glutamate is released via calcium-dependent exocytosis o transmitter-containing vesicles. Glutamate is removed rom the synaptic cle t by glutamate reuptake transporters located on presynaptic nerve terminals and on the plasma membrane o glial cells. These transporters are Na -dependent and have a high a nity or glutamate. In glial cells, the enzyme glutamine synthetase converts glutamate to glutamine, which is recycled into adjacent nerve terminals or conversion back to glutamate. Glutamine generated in glial cells can also enter the Krebs cycle and undergo oxidation; the resulting -ketoglutarate enters neurons to replenish the -ketoglutarate consumed during glutamate synthesis (Fig. 13-2B).

Glutamate Receptors and Transporters As with GABA receptors, glutamate receptors are divided into ionotropic and metabotropic subgroups.

Ionotropic Glutamate Receptors

Ionotropic glutamate receptors mediate ast excitatory synaptic responses. These receptors are multisubunit, cation-selective channels that, on activation, permit the f ow o Na , K , and, in some channels, Ca2 ions across plasma membranes. Ionotropic glutamate receptors are thought to be tetramers composed o di erent subunits, with each subunit containing helical domains that span the membrane three times, in addition to a short sequence that orms the channel’s pore when the entire tetramer is assembled (Fig. 13-8A). There are three main subtypes o glutamate-gated ion channels, classi ed according to their activation by the selective agonists AMPA, kainate , and NMDA. The diversity o ionotropic receptors arises rom di erences in amino acid sequence because o alternative mRNA splicing and posttranscriptional mRNA editing and rom the use o di erent combinations o subunits to orm the receptors (Table 13-5). AMPA ( -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are located throughout the CNS, particularly in the hippocampus and cerebral cortex. Four AMPA receptor subunits (GluR1–GluR4) have been identi ed (Table 13-5). AMPA receptor activation results primarily in Na inf ux (as well as some K e f ux), allowing these receptors to regulate ast, excitatory postsynaptic depolarization at glutamatergic synapses (Fig. 13-8B). Although most AMPA receptors in the CNS have low Ca2 permeability, the absence o certain subunits (such as GluR2) in the receptor complex increases the Ca2 permeability o the channel. Calcium entry through AMPA receptors may play a role in long-term changes in neuronal phenotype and in neuronal damage associated with stroke. Kainate receptors are expressed throughout the CNS, particularly in the hippocampus and cerebellum. Five kainate receptor subunits have been identi ed (Table 13-5). Like AMPA receptors, kainate receptors allow Na inf ux and K e f ux through channels that possess rapid activation and deactivation kinetics. The combination o subunits in the

C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission

197

TABLE 13-5 Classif cation o Ionotropic Glutamate Receptor Subtypes IONOTROPIC GLUTAMATE RECEPTOR SUBTYPE

SUBUNITS

AGONISTS

ACTIONS

AMPA

GluR1 GluR2 GluR3 GluR4

Glutamate or AMPA

Increase Na and Ca 2 inf ux, increase K e f ux; note that receptors with GluR2 have ion channels with low Ca 2 permeability

Kainate

GluR5 GluR6 GluR7 KA1 KA2

Glutamate or kainate

Increase Na inf ux, increase K e f ux

NMDA

NR1 NR2A NR2B NR2C NR2D

Glutamate or NMDA and glycine and membrane depolarization

Increase Ca 2 inf ux, increase K e f ux

kainate receptor complex determines whether the channel is also permeable to Ca2 . Experiments using receptor-selective agents have allowed the assignment o speci c unctions to kainate receptors in di erent regions o the CNS. NMDA (N-methyl-D-aspartate) receptors are expressed primarily in the hippocampus, cerebral cortex, and spinal cord. These receptors consist o multisubunit oligomeric transmembrane complexes. NMDA receptor activation, which requires simultaneous binding o glutamate and glycine, opens a channel that allows K e f ux as well as Na and Ca2 inf ux (Fig. 13-8B). In NMDA receptors that are occupied by glutamate and glycine, Mg2 ions block the channel pore in the resting membrane (Fig. 13-8B). Depolarization o the membrane concurrent with agonist binding is required to relieve this voltage-dependent Mg2 block. Either trains o postsynaptic action potentials or activation o AMPA/ kainate receptors in adjacent regions o the membrane can cause postsynaptic membrane depolarization that unblocks the Mg2 -bound NMDA receptor. There ore, NMDA receptors di er rom the other ionotropic glutamate receptors in two important respects—they require the binding o multiple Me ta botropic gluta ma te re ce ptor

Ne urotra ns mitte r N binding re gions Effe ctor prote in (P LC or AC)

ligands or channel activation, and their gating depends on more intense presynaptic activity than that required to open AMPA or kainate receptors.

Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluR) consist o a seven-

transmembrane-spanning domain protein that is coupled via G proteins to various e ector mechanisms (Fig. 13-9). At least eight subtypes o metabotropic glutamate receptors exist; each belongs to one o three groups (groups I, II, and III) according to its sequence homology, signal transduction mechanism, and pharmacology (Table 13-6). Group I receptors cause neuronal excitation either through phospholipase C (PLC) activation and IP3-mediated release o intracellular Ca2 or through activation o adenylyl cyclase and cAMP generation. (The di erence arises rom the coupling o di erent G proteins to the receptors.) Groups II and III receptors inhibit adenylyl cyclase and decrease cAMP production (Table 13-6). These second messenger pathways subsequently regulate ion f uxes o other channels. For example, metabotropic glutamate receptor activation in the

Gluta ma te Ion cha nne l

Glu 1

Ca 2+

4 3 C G prote in binding re gions

α GTP

β

α β

γ

2 GTP -GDP e xcha nge

GDP

α

γ

2

GTP

GTP -GDP e xcha nge

β

γ

β +

K

Clos e s

K+ cha nne l

γ Ope ns

Ca 2+ cha nne l

FIGURE 13-9. Schematic representation and downstream signaling o metabotropic glutamate receptors. Le t panel: Metabotropic glutamate receptors are seven-transmembrane-spanning proteins with an extracellular ligand binding site and an intracellular G protein binding site. Right panel: 1-2. Ligand binding to the metabotropic glutamate receptor results in GTP association with the subunit o the G protein. The GTP-associated subunit then dissociates rom the dimer. 3. G and G can then activate e ector proteins such as adenylyl cyclase (AC) and phospholipase C (PLC). 4. G subunits can also open or close ion channels directly.

198 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 13-6 Metabotropic Glutamate Receptor (mGluR) Subtypes and Their Actions GROUP

SUBTYPE

ACTIONS

I

mGluR1 mGluR5

Activates adenylyl cyclase → increases cAMP (mGluR1 only) Increases PLC activity → PIP2 hydrolysis → increases IP3 and DAG → increases Ca 2 levels, stimulates PKC Inhibits K channels

II

mGluR2 mGluR3

Inhibits adenylyl cyclase → decreases cAMP Inhibits voltage-sensitive Ca 2 channels Activates K channels

III

mGluR4 mGluR6 mGluR7 mGluR8

Inhibits adenylyl cyclase → decreases cAMP Inhibits voltage-sensitive Ca 2 channels

Group I mGluRs activate adenylyl cyclase and phospholipase C (PLC), while group II and group III mGluRs inhibit adenylyl cyclase. The downstream effects of mGluRs on ion channels are complex and varied. Some of the main actions on ion channels are listed. Note that the actions of group I receptors are generally excitatory, while those of groups II and III receptors are generally inhibitory.

transporter expression and unction on the pathogenesis o neurologic disorders including cerebral ischemia, epilepsy, spinal cord injury, amyotrophic lateral sclerosis, AIDS neuropathy, and Alzheimer’s disease.

PATHOPHYSIOLOGY AND PHARMACOLOGY OF GLUTAMATERGIC NEUROTRANSMISSION Under physiologic conditions, termination o glutamate receptor activation occurs via transmitter reuptake by presynaptic and glial transporters, transmitter di usion out o the synaptic cle t, or receptor desensitization. As described below, increased release or decreased reuptake o glutamate in pathologic states can lead to a positive eedback cycle involving increased intracellular Ca2 levels, cellular damage, and urther glutamate release. Together, these processes can lead to excitotoxicity, def ned as neuronal death caused by excessive cellular excitation. Excitotoxicity has been implicated as a pathophysiologic mechanism in many diseases, including neurodegenerative syndromes, stroke and trauma, hyperalgesia, and epilepsy. Although the clinical applications o interrupting excitotoxicity remain limited, it is hoped that better understanding o glutamate-induced excitotoxicity will lead to the development o new approaches or treatment o these diseases.

Neurodegenerative Diseases hippocampus, neocortex, and cerebellum increases neuronal f ring rates by inhibiting a hyperpolarizing K current. Presynaptic mGluRs, such as group II and III receptors in the hippocampus, can unction as inhibitory autoreceptors that inhibit presynaptic Ca2 channels and thereby limit presynaptic release o glutamate. (There are also presynaptic ionotropic cholinergic receptors in the CNS that act to modulate the release o glutamate.)

Glutamate Transporters

Maintenance o a physiologic range o extracellular glutamate concentration is necessary to prevent glutamate overexcitation and neurotoxicity. Regulation o extracellular glutamate is primarily carried out by an e f cient, high-capacity glutamate transporter system, because clearance o extracellular glutamate via glutamate metabolism and di usion is negligible. To date, at least f ve cell membrane glutamate transporter proteins have been cloned. Glutamate transporters are denoted by the common name excitatory amino acid transporter (e.g., EAAT1). Among the cell membrane glutamate transporters, EAAT1 (GLAST), EAAT2 (GLT1), and EAAT3 (EAAC1) are particularly relevant to the regulation o glutamate uptake in broad CNS regions. EAAC1 is generally considered a neuronal transporter, whereas GLAST and GLT1 are primarily astroglial transporters, although both GLAST and GLT1 have also been localized to neuronal cells during neurologic development. Because the homeostasis o extracellular glutamate concentration is critically regulated by neuronal and glial transporters, reduced glutamate transporter expression and/or unction would be expected to increase extracellular glutamate concentration, with excessive subsequent activation o glutamate receptors and excitotoxicity. To date, numerous studies have shown a detrimental e ect o reduced glutamate

Elevated levels o dysregulated glutamate (i.e., excitotoxicity) have been implicated in Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). In ALS, motor neurons degenerate in the ventral horn o the spinal cord, brainstem, and motor cortex, resulting in weakness and atrophy o skeletal muscles. The pathogenesis o this disease and the reasons or the selective pattern o neurodegeneration remain uncertain; mechanisms currently proposed or cell death in ALS include excitotoxicity and oxidative stress. The CNS areas a ected in ALS express diverse populations o AMPA and NMDA receptors as well as glutamate reuptake transporters. Patients with ALS have impaired glutamate transporters in the spinal cord and motor cortex. These abnormal glutamate transporters permit the accumulation o high glutamate concentrations in the synaptic cle t, possibly leading to motor neuron death via excitotoxicity. NMDA receptors can mediate neuronal survival or death, depending on whether the receptor is synaptic or extrasynaptic. Activation o synaptic NMDA receptors promotes cell survival by up-regulating CaM kinase and activating certain mitogen-activated protein kinase (MAPK) signaling pathways. The subsequent expression o growth actors (e.g., BDNF) modulates post-translational targets and causes long-term phenotypic changes through transcriptional modif cation in target neurons. In contrast, overstimulation o extrasynaptic NMDA receptors inactivates the CREB (cAMP response element binding protein) pathway and promotes neuronal apoptosis. Riluzole is a voltage-gated sodium channel blocker that prolongs survival and decreases disease progression in ALS. Although the exact mechanism o action is uncertain, it appears that riluzole acts in part by reducing Na conductance and thereby decreasing glutamate release. The drug may also directly antagonize NMDA receptors.

C h a p t e r 1 3 Pharmacology of GABAergic and Glutamatergic Neurotransmission

Excitotoxicity rom excessive glutamate release has also been implicated in progression o dementia in Alzheimer’s disease. Memantine is a noncompetitive NMDA receptor antagonist (channel blocker) used in the treatment o this disease. In clinical studies, memantine slows the rate o clinical deterioration in patients with moderate to severe Alzheimer’s disease. In Parkinson’s disease, reduced dopaminergic transmission to the striatum results in the overactivation o glutamatergic synapses in the CNS. Excessive glutamatergic neurotransmission contributes to the clinical signs o Parkinson’s disease, as discussed in Chapter 14, Pharmacology o Dopaminergic Neurotransmission. Amantadine is a noncompetitive blocker o NMDA receptor channels, similar in action to memantine. Although amantadine is not an e ective treatment as a single agent, the combination o amantadine and levodopa reduces the severity o dyskinesia in Parkinson’s disease by 60%. It is not clear, however, whether the e ect o amantadine derives solely rom NMDA receptor blockade.

Stroke and Trauma In ischemic stroke, interruption o blood f ow to the brain leads to de cits in oxygen supply and glucose metabolism that trigger excitotoxicity (Fig. 13-10). In hemorrhagic stroke, high concentrations o glutamate are ound in blood leaking into the brain. In traumatic brain injury, the direct Is che mia O2

Dis rupte d ion gra die nts

Impa ire d Na +-couple d gluta ma te tra ns porte rs

Incre a s e d s yna ptic gluta ma te

NMDA-R a ctiva tion

rupture o brain cells can release high intracellular stores o glutamate and K into the restricted extracellular space. Dysregulation o excitatory transmitters such as glutamate leads to widespread membrane depolarization, elevation o intracellular Na and Ca2 concentrations, and triggering o glutamate release rom adjacent neurons. Increasing glutamate levels activate Ca2 -permeable NMDA and AMPA receptor-coupled channels. Ultimately, the resultant accumulation o intracellular Ca2 activates many Ca2 -dependent degradation enzymes (e.g., DNAses, proteases, phosphatases, and phospholipases) that lead to neuronal cell death. Although the highly Ca2 -permeable NMDA receptor was originally viewed as the major contributor to neuronal cell death caused by Ca2 overload, AMPA receptors have also been implicated. Clinical trials o NMDA and AMPA receptor antagonists in patients with stroke have not been success ul to date and, in some cases, have led to schizophrenia-like e ects, memory impairment, and neurotoxic reactions. Future pharmacologic research will be directed at the development and use o drugs with ewer adverse e ects, such as the noncompetitive NMDA receptor antagonist memantine or drugs targeted to speci c subunits o the NMDA or AMPA receptor complex. Glutamate released during ischemic or traumatic brain damage can also activate metabotropic glutamate receptors. In animal models o stroke, pharmacologic antagonism o the mGluR1 receptor subtype acilitates recovery and survival o hippocampal neurons and prevents memory and motor loss caused by trauma. These ndings suggest that the mGluR1 subunit may represent a potential target or pharmacologic intervention (Figs. 13-10 and 13-11).

Epilepsy

ATP

Me mbra ne de pola riza tion

199

AMPA-R a ctiva tion

mGluR a ctiva tion

Seizures can result rom overstimulation o glutamatergic pathways, beginning with overactivation o AMPA receptors and progressing to overactivation o NMDA receptors. In animal models, inhibition o AMPA receptor activation prevents seizure onset, whereas NMDA receptor antagonists decrease seizure intensity and duration. Lamotrigine , a drug used in the treatment o re ractory ocal seizures (see Chapter 16), stabilizes the inactivated state o the voltage-gated Na channel and thereby reduces membrane excitability, the number o action potentials in a burst, glutamate release, and glutamate receptor activation. Felbamate is another antiepileptic that has a variety o actions, including the inhibition o NMDA receptors. Because o associated aplastic anemia and hepatotoxicity, its use is restricted to patients with reractory seizures.

intra ce llula r Ca 2+

Hyperalgesia Activa tion of DNa s e s , prote a s e s , phos pha ta s e s , phos pholipa s e s

Intra ce llula r a nd me mbra ne da ma ge

Da ma ge by fre e ra dica ls

Mitochondria l da ma ge

Re le a s e of pro-a poptotic fa ctors

FIGURE 13-10. Role of glutamate receptors in excitotoxicity. A multiplicity of damaging cellular processes occur as a consequence of the decreased ATP levels that result from impaired oxidative metabolism or from the oxidative damage caused by activated neutrophils that invade an ischemic region; only glutamate-mediated processes are depicted here.

Hyperalgesia is the increased perception o pain, o ten in response to stimuli that, under normal conditions, cause little or no pain. Hyperalgesia can occur in the presence o peripheral nerve injury, inf ammation, surgery, and diseases such as diabetes. Although hyperalgesia is reversed in most cases when the underlying pathophysiology has resolved, it may persist even in the absence o an identi ed organic source, leading to chronic pain that is physically crippling and psychologically debilitating. Evidence is accumulating that glutamatergic transmission contributes to the development and/or maintenance o hyperalgesia. NMDA receptors enhance synaptic transmission

200 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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Postsynaptic neuron Re le a s e of re trogra de me s s e nge rs le a ding to incre a s e d pre s yna ptic tra ns mitte r re le a s e

FIGURE 13-11. Interactions among metabotropic, AMPA, and NMDA classes of glutamate receptors. Action potentials depolarize the plasma membrane of presynaptic neurons, leading to opening of voltage-gated Ca 2 channels and ultimately to glutamate release into the synaptic cleft. Studies have proposed a “tonic” physiologic role for activation of the metabotropic glutamate receptor (mGluR) during low-frequency stimulation of postsynaptic neurons by glutamate. In contrast, high-frequency presynaptic stimulation “phasically” activates AMPA receptors (1) and thereby induces the prolonged membrane depolarization required to relieve the Mg2 blockade of NMDA receptors (2). Calcium entering the cell via activated, Mg2 -free NMDA receptors (3) is then able to activate downstream kinases independently of the mGluR. Kinases associated with the postsynaptic densities, which act to scaffold the ionotropic receptors to the membrane, phosphorylate AMPA receptor subunits and thereby cause a change in that receptor’s composition (not shown). AMPA-R, AMPA receptor; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate; mGluR, metabotropic glutamate receptor; NMDA-R, NMDA receptor; PIP2, phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C.

between nociceptive a erent bers and neurons in the dorsal horn o the spinal cord. As discussed in Chapter 18, Pharmacology o Analgesia, experimental hyperalgesia o ten involves a phenomenon called central sensitization, in which repeated nociceptive stimuli in the periphery lead to progressively increasing excitatory postsynaptic responses in postsynaptic pain neurons in the super cial dorsal horn. One mechanism by which this synaptic potentiation occurs involves postsynaptic NMDA receptors that, when stimulated chronically, increase the strength o excitatory connections between presynaptic and postsynaptic neurons in spinal pain circuits. In turn, the Ca2 inf ux through activated NMDA receptors acts on special localized kinases to e ect a phosphorylation-induced switch o the subunits o the AMPA receptor, allowing more Ca2 to enter through AMPA receptors. Increased intracellular Ca2 also activates Ca2 -sensitive transcription actors, such as CREB, and induces changes in protein synthesis via ribosomes located right at the synaptic terminals. Experimental NMDA receptor antagonists can both prevent and reverse central sensitization in patients. Many o these antagonists, however, also inhibit a wide range o ast excitatory synaptic pathways in the CNS. For this reason, current NMDA receptor drug development ocuses on intraspinal or extradural administration o NMDA receptor antagonists to limit the e ects o the drug to the dorsal horn o the spinal cord. The high density o kainate receptors in sensory neurons may also modulate transmitter release, providing another uture pharmacologic target or the relie o chronic pain. Regulation o glutamate transporters has also been implicated in the central mechanisms o nociceptive processing. Preclinical studies have demonstrated that the expression o spinal glutamate transporters is altered ollowing peripheral nerve injury and contributes to neuropathic pain behavior in rats. This altered transporter expression is mediated, at least in part, through a tyrosine kinase receptor (TrkB) and intracellular mitogen-activated protein kinases. Moreover, peripheral nerve injury signi cantly reduces spinal glutamate uptake activity, supporting the hypothesis that spinal glutamate transporters, via regulation o regional glutamate homeostasis, are involved in the mechanisms o nerve injury-induced neuropathic pain behaviors.

Opioid Tolerance

Recent studies have demonstrated that chronic morphine administration regulates the expression o spinal glutamate transporters, which contributes to the mechanisms o morphine tolerance and associated neuronal apoptosis and hyperalgesia. Because neuropathic pain and opioid tolerance share a common glutamatergic mechanism, regulation o glutamate transporter expression and unction could be an important approach to preventing and reversing glutamate overexcitation and neurotoxicity in neuropathic pain and opioid tolerance.

CONCLUSION AND FUTURE DIRECTIONS GABA and glutamate represent the major inhibitory and excitatory neurotransmitters in the CNS, respectively. Most drugs that act on GABAergic neurotransmission enhance GABAergic activity and thereby depress CNS unctions.

C h a p t e r 1 3 Pharmacology of GABAergic and Glutamatergic Neurotransmission

Modulation o GABAergic transmission can occur either presynaptically or postsynaptically. Drugs acting at presynaptic sites primarily target GABA synthesis, degradation, and reuptake. Drugs acting postsynaptically a ect GABA receptors directly, either by occupying the GABA binding site or by an allosteric mechanism. Each o the three main GABA receptor types has a distinct pharmacology. The GABAA receptor is targeted by the largest number o drugs, including GABA binding-site agonists, benzodiazepines, barbiturates, general anesthetics, and neuroactive steroids. GABAB receptors are currently targeted by only a ew therapeutic agents, which are used to treat spasticity. GABAB receptors have recently been ound to inf uence pain, cognition, and addictive behavior, and interest is growing in drugs that modulate these receptors. GABAC receptors have not yet been developed as a target o pharmacologic agents. To improve sa ety and reduce adverse e ects, including ataxia, tolerance, and physical dependence, development o new anxiolytics and sedatives has aimed or low-e cacy compounds (e.g., benzodiazepines) as well as compounds with selective activity at GABAA receptor subtypes. Animal models with selectively mutated GABAA receptor subunits have revealed that sedation/hypnosis is produced by enhancing the activity o receptors containing 1 subunits. In contrast, anxiolysis is produced by modulation o 2- or 3-containing receptors, and amnesia is associated with 5-containing receptors. There is also evidence or distinct pharmacology and physiology o synaptic GABAA receptors containing di erent subunits. Because o the potential role o excitatory neurotransmission in a number o pathologic processes, such as neurodegenerative diseases, stroke, trauma, hyperalgesia, and epilepsy, glutamate receptors have become important targets or drug development. The diversity o glutamate receptors and receptor subunits constitutes a potential advantage or the development o glutamate receptor antagonists that are selective or a particular receptor subtype. In the uture, highly selective antagonists or glutamate receptor subtypes could potentially protect the CNS in stroke, prevent hyperalgesia a ter tissue trauma, and treat epileptic seizures. Although neurotransmitter receptors comprise the traditional targets or drug development, recent experimental studies suggest that targeting sca olding proteins may also be a promising area or treatment o stroke and other diseases. Postsynaptic cytoskeletal proteins such as postsynaptic density protein-95 (PSD-95) comprise an important part o the dendrite sca olding structure, and PSD-95 mediates the intracellular signaling that occurs a ter glutamate receptor activation. In the context o excitotoxicity, PSD-95 can ampli y the initial NMDA signal into deleterious cascades o nitric oxide generation. Blockade o PSD-95 reduces ischemic brain injury a ter experimental stroke in rats.

201

A clinical trial testing this approach as a therapy or ischemic stroke is now ongoing. Emerging opportunities also exist or regulation o glutamate transporter expression and activity. This approach may minimize the pathologic impact o glutamate overload while retaining the physiologic role o glutamate. Current research is exploring the cellular and molecular mechanisms o transporter expression and unction in relation to the pathogenesis o neuropathic pain, opioid-related problems, and other neurologic disorders. In addition, studies on the role o glutamate transporter regulation in opioid tolerance and dependence may provide new insights into the cellular mechanisms o substance abuse.

Acknowledgment We thank Gary R. Strichartz or his valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Aarts M, Liu Y, Liu L, et al. Treatment o ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 2002; 298:846–850. (Scaffolding proteins as therapeutic targets for glutamate excitotoxicity and neuropathic pain.) Besancon E, Guo S, Lok J, Tymianski M, Lo EH. Beyond NMDA and AMPA glutamate receptors: emerging mechanisms or ionic imbalance and cell death in stroke. Trends Pharmacol Sci 2008;29:268–275. (This review expands on traditional concepts of excitotoxicity to include newly discovered mechanisms of cell death.) Foster AC, Kemp JA. Glutamate- and GABA-based CNS therapeutics. Curr Opin Pharmacol 2006;6:7–17. (General overview of pharmacologic strategies in GABAergic and glutamatergic neurotransmission.) Herd MD, Belelli D, Lambert JJ. Neurosteroid modulation o synaptic and extrasynaptic GABAA receptors. Pharmacol Ther 2007;116:20–34. (Reviews physiology of neurosteroids and their interactions with GABAA receptors.) Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 2003;4:399–415. (Advances in pathophysiology of excitotoxicity in stroke.) Mizuta K, Xu D, Pan Y, et al. GABAA receptors are expressed and acilitate relaxation in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2008;294:L1206–L1216. (Points to a role for GABAA receptors in airway tone.) Olsen RW, Sieghart W. GABAA receptors: subtypes provide diversity o unction and pharmacology. Neuropharmacology 2008;56:141–148. (Reviews different GABAA receptor subtypes and their physiologic and pharmacologic roles.) Rudolph U, Knof ach F. Beyond classical benzodiazepines: novel therapeutic potential o GABAA receptor subtypes. Nat Rev Drug Discov 2011;10:685–697. (Reviews evidence that different GABAA receptor subtypes mediate different benzodiazepine effects.) Werner FM, Coveñas R. Classical neurotransmitters and neuropeptides involved in generalized epilepsy: a ocus on antiepileptic drugs. Curr Med Chem 2011;18:4933–4948. (Discusses the linked roles of GABA and glutamate and their receptors in epilepsy.)

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C h a p t e r 1 3 Pharmacology o GABAergic and Glutamatergic Neurotransmission 205

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David G. S t a n d a e rt a n d Vic t oInd r irereW. Indire e ct S u n g pa thw hway hw ay

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INTR INTR TRO ODU UCTION & CAS SE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206–207 BIOC BI OCH HEMISTRY M AND CEL E L BIOLOGY OF DOPA DO P MINERGIC I NEU EURO EU R TRAN TRANSM SMIS ISSION . . . . . . . . . . . . . . . . . . 206 Doopa pami mine m ne Stora torage ge,, Re Release, Reuptake, and Inactivation acc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 D pa Do pami mine ne Rec ecep epto tors rss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 To s pina i l Central Pathw hways . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 hw motor ne Dopamine urons DOPA DO PAM MINE AND CONTROL OF MOVEMENT: PARKINSON’S DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Physiology Ph i l off Nigrostriatal Ni i l Pathways P h . . . . . . . . . . . . . . . . . . . . 211 2 Pathophysiology.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 Pathophysiology Pharmacologic Classes and Agents . . . . . . . . . . . . . . . . . . . . . 213 Dopamine Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Dopamine Receptor Agonistss . . . . . . . . . . . . . . . . . . . . . . . . 214

INTRODUCTION Dopamine (DA) is a catecholamine neurotransmitter that is the therapeutic target or a number o important central nervous system (CNS) disorders, including Parkinson’s disease and schizophrenia. DA is also a precursor or the other catecholamine neurotransmitters norepinephrine and epinephrine. The machinery o catecholamine neurotransmission has a number o components that are shared among members o the class, including biosynthetic and metabolic enzymes. There are also components that are specialized or the individual members o the class, including reuptake pumps and presynaptic and postsynaptic receptors. This chapter presents the principles that underlie current therapies or diseases that directly or indirectly involve changes in dopaminergic neurotransmission. The chapter begins with a discussion o the biochemistry and cell biology o dopaminergic neurotransmission and the localization o the major DA systems in the brain. Following this background, the chapter explores the physiology, pathophysiology, and pharmacology o Parkinson’s disease , which results rom the specif c loss o neurons in one o these DA systems, and schizophrenia , which is currently treated, in part, with drugs that inhibit dopaminergic neurotransmission. 206

Dopa paa mi mine n rgg ic input from S Ncc

Parkins o n's d is e as e Dire ct pa thw hway inhibite d a nd indire c t pa thway a ctiv tivaa te te d, d, both lee a d ing to re duce d move me ntt Glut Gl utaa ma m a te te rgi rgicc in n put put from cortexx

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Dire ct pa thway ay Activity y re duce d d,, ACh be caInhibitors a us e of losofs Dopamine of Metab abol oliism is is m . . . . . . . . . . . . . . . . . . . 215 D1 s timula timu ti imula la l tion ti tio ion

Nondopaminergic Pharmacoolo loggy in in D2 D Move ve me nt inhibite dDiseasee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Parkinson’s 2155 Treatment of Pattie ient nts with Par arki kins nson on’ss ct .pa p. .thway DIndire is isee ase . . . . y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Dopa Do D pa mi min in e rgiic iinputt Activity Acti Ac tivi vity ty y i incre nc c re e a s e d d, DOPAMINE AND DISORDERS OF THOUGHT: from S Nc be e ca c a us e of re r e l e a s e o f SCHIZOPHRENIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 D2 2 iinhib inhibitio nhib nh hibit ibititio iion n Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Mov Mo Move ve me nt inhibite d and Agents . . . . . . . . . . . . . . . . . . . . . 217 Pharmacologic Classes Typical Antipsychotic Agentss . . . . . . . . . . . . . . . . . . . . . . . . 217 Atypical Antipsychotic Agentss . . . . . . . . . . . . . . . . . . . . . . . 220 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 220 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .221

BIOCHEMISTRY AND CELL BIOLOGY OF DOPAMINERGIC NEUROTRANSMISSION Dopamine belongs to the catecholamine

amily o neurotransmitters. In addition to dopamine, this amily includes norepinephrine (NE) and epinephrine (EPI). As the name suggests, the basic structure o the catecholamines consists o a catechol (3,4-dihydroxybenzene) moiety connected to an amine group by an ethyl bridge (Fig. 14-1A). Recall rom Chapter 9, Principles o Nervous System Physiology and Pharmacology, that catecholaminergic pathways in the brain have “single source-divergent” organization, in that they arise rom small clusters o catecholamine neurons that give rise to widely divergent projections. CNS catecholamines modulate the unction o point-to-point neurotransmission and a ect complex processes such as mood, attentiveness, and emotion. The neutral amino acid tyrosine is the precursor or all catecholamines (Fig. 14-1B). The majority o tyrosine is obtained rom the diet; a small proportion may also be synthesized in the liver rom phenylalanine . The f rst step in the synthesis o DA is the conversion o tyrosine to L-DOPA (L-3,4-dihydroxyphenylalanine , or levodopa ) by oxidation o the 3 position on the benzene ring. This reaction is catalyzed by the enzyme tyrosine hydroxylase (TH), a erro

C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission

Ma rk S is a 55-ye a r-o ld m a n w h o g o e s to s e e h is p h ys icia n b e ca u s e h e n o tice s a tre m o r in h is rig h t h a n d th a t h a s d e ve lo p e d g ra d u a lly o ve r a n u m b e r o m o n th s . He f n d s h e ca n ke e p th e h a n d q u ie t i h e co n ce n tra te s o n it, b u t th e s h a kin g q u ickly re a p p e a rs i h e is d is tra cte d . His h a n d w ritin g h a s b e co m e s m a ll a n d d i f cu lt to re a d , a n d h e h a s tro u b le u s in g a co m p u te r m o u s e . His w i e co m p la in s th a t h e n e ve r s m ile s a n ym o re a n d th a t h is a ce is b e co m in g e xp re s s io n le s s . S h e a ls o s a ys th a t h e wa lks m o re s lo w ly a n d h e h a s tro u b le ke e p in g u p w ith h e r. As Mr. S e n te rs th e e xa m in a tio n ro o m , h is d o cto r n o tice s th a t h e is wa lkin g h u n ch e d ove r a n d h a s a s h o rt, s h u in g ga it. Th e d o cto r f n d s o n p h ys ica l e xa m in a tio n th a t Mr. S h a s in cre a s e d to n e a n d co g w h e e l rig id ity in h is u p p e r e xtre m itie s ,

(iron containing)-enzyme that consists o our identical subunits o approximately 60 kDa each. In addition to Fe2 , TH also requires the co actor tetrahydrobiopterin, which is oxidized to dihydrobiopterin in the course o the reaction. Importantly, oxidation o tyrosine to L-DOPA is the ratelimiting step in the production not only o DA but o all catecholamine neurotransmitters. The next and f nal step in the synthesis o DA is the conversion o L-DOPA to DA by the enzyme aromatic L-amino acid decarboxylase (AADC). AADC cleaves the carboxyl group rom the -carbon o the ethylamine side chain, liberating carbon dioxide. AADC requires the co actor pyridoxal phosphate. Although AADC is sometimes re erred to as “DOPA decarboxylase,” it is promiscuous in its ability to cleave carboxyl groups rom the -carbons o all aromatic amino acids and is involved in the synthesis o noncatechol transmitters, such as serotonin. AADC is abundant in the brain. It is expressed by dopaminergic neurons, but it is also present in nondopaminergic cells and glia. Furthermore, AADC is expressed throughout the body in almost all cell types. In dopaminergic neurons, the end product o the catecholamine synthetic pathway is dopamine. In cells that secrete the catecholamine NE, DA is converted to NE by the enzyme dopamine -hydroxylase . In other cells, NE may subsequently be converted to epinephrine by phenylethanolamine N-methyltransferase . Dopaminergic neurons lack both o these enzymes, but it is important to keep in mind the entire pathway o catecholamine biosynthesis because pharmacologic manipulation o DA biosynthesis can also alter the production o NE and EPI. For a more complete discussion o the last two steps in NE and EPI synthesis, see Chapter 11, Adrenergic Pharmacology.

Dopamine Storage, Release, Reuptake, and Inactivation DA is synthesized rom tyrosine in the cytoplasm o the neuron and then transported into secretory vesicles or storage and release (Fig. 14-2). Two separate molecular pumps are required or the transport o DA into synaptic vesicles.

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p a rticu la rly o n th e rig h t s id e , a n d th a t h e is s ig n if ca n tly s lo w e r th a n n o rm a l a t p e r o rm in g ra p id a lte rn a tin g m o ve m e n ts . Th e p h ys icia n d e te rm in e s th a t Mr. S ’s s ym p to m s a n d s ig n s m o s t like ly re p re s e n t th e e a rly s ta g e s o Pa rkin s o n’s d is e a s e , a n d s h e p re s crib e s a tria l o le vo d o p a .

Questions 1 . How does the selective loss of dopaminergic neurons result in symptoms such as those Mr. S is experiencing? 2 . What will be the effect of levodopa on the course of Mr. S’s disease? 3 . How will Mr. S’s response to levodopa change over time? 4 . Is levodopa the best choice for Mr. S at this stage of his disease?

A proton ATPase concentrates protons in the vesicle, creating an electrochemical gradient characterized by a low intravesicular pH (i.e., a high proton concentration) and an electropositive vesicle interior. This gradient is exploited by a proton antiporter, the vesicular monoamine transporter (VMAT), which allows protons to move down the gradient (out o the vesicle) while simultaneously transporting DA into the vesicle against its concentration gradient. Upon nerve cell stimulation, the DA storage vesicles use with the plasma membrane in a Ca2 -dependent manner, releasing DA into the synaptic cle t. In the cle t, DA can bind to both postsynaptic DA receptors and presynaptic DA autoreceptors (see below). Several mechanisms exist to remove synaptic DA and terminate the signaling produced by the neurotransmitter. Most o the DA released into the synaptic cle t is transported back into the presynaptic cell by a 12-transmembrane domain protein, the dopamine transporter (DAT). DAT belongs to the amily o catecholamine reuptake pumps. DA reuptake involves transport o the neurotransmitter against its concentration gradient and there ore requires an energy source. For this reason, the DAT couples dopamine reuptake to the cotransport o Na down its concentration gradient into the cell. In act, both Na and Cl are co-transported with DA into the cell. Because the Na gradient is maintained by the Na /K -ATPase pump, DA reuptake depends indirectly on the presence o a unctioning Na /K pump. DA taken up into the presynaptic cell can either be recycled into vesicles or urther use in neurotransmission (by VMAT) or degraded by the action o the enzymes monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT) (Fig. 14-3). MAO is a key enzyme that unctions to terminate the action o catecholamines in both the brain and the periphery. MAOs exist in two iso orms: MAO-A, which is expressed in the brain as well as the periphery, and MAO-B, which is concentrated in the CNS. Both iso orms o MAO can degrade dopamine as well as a wide range o monoamine compounds. Under normal conditions, MAO-B is responsible or catabolizing most CNS dopamine. The di erent roles played by the iso orms o MAO are therapeutically important. Selective inhibition o MAO-B is used to augment the unction o

208 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

CNS dopamine and generally is well tolerated. Inhibition of MAO-A, on the other hand, retards the breakdown of all central and peripheral catecholamines; as noted in Chapter 11, MAO-A inhibition may lead to life-threatening toxicity when combined with catecholamine releasers such as the indirect-acting sympathomimetic tyramine found in certain wines and cheeses. Synaptic DA that is not taken up into the presynaptic cell can either diffuse out of the synaptic cleft or be degraded by the action of COMT. COMT is expressed in the brain, liver,

A

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R

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O

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OH NH2

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FIGURE 14-1. Catecholamine synthesis. A. Catecholamines consist of a catechol nucleus with an ethylamine side chain (R group). The R group is ethylamine in dopamine, hydroxyethylamine in norepinephrine, and N-methyl hydroxyethylamine in epinephrine. B. Dopamine is synthesized from the amino acid tyrosine in a series of step-wise reactions. In cells that contain dopamine -hydroxylase, dopamine can be further converted to norepinephrine; in cells that also contain phenylethanolamine N-methyltransferase, norepinephrine can be converted to epinephrine.

FIGURE 14-2. Dopaminergic neurotransmission. Dopamine (DA) is synthesized in the cytoplasm and transported into secretory vesicles by the action of a nonselective monoamine-proton antiporter (VMAT) that is powered by the electrochemical gradient created by a proton ATPase. Upon nerve cell stimulation, DA is released into the synaptic cleft, where the neurotransmitter can stimulate postsynaptic dopamine receptors and presynaptic dopamine autoreceptors. DA is transported out of the synaptic cleft by the selective, Na -coupled dopamine transporter (DAT). Cytoplasmic DA is retransported into secretory vesicles by VMAT or degraded by the enzyme monoamine oxidase (MAO).

C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission Ne urotra ns mitte r

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Catechol-Omethyltransferase (COMT)

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Homova nillic a cid (HVA) Ma jor me ta bolite (e xcre te d in urine )

FIGURE 14-3. Catecholamine metabolism. Dopamine is metabolized to homovanillic acid (HVA) in a series of reactions. Dopamine is oxidized to dihydroxyphenylacetic acid (DOPAC) by sequential action of the enzymes monoamine oxidase (MAO) and aldehyde dehydrogenase (AD). CatecholO-methyltransferase (COMT) then oxidizes DOPAC to HVA. Alternatively, dopamine is methylated to 3-methoxytyramine by COMT and then oxidized to HVA by MAO and AD. HVA, the most stable dopamine metabolite, is excreted in the urine.

kidney, and heart; it inactivates catecholamines by adding a methyl group to the hydroxyl group at the 3 position o the benzene ring. In the CNS, COMT is expressed primarily by neurons. The sequential action o COMT and MAO degrades DA to the stable metabolite homovanillic acid (HVA), which is excreted in the urine (Fig. 14-3).

Dopamine Receptors Dopamine receptors are members o the G protein-coupled amily o receptor proteins. The properties o dopamine receptors were originally classif ed by their e ect on the ormation o cyclic AMP (cAMP): activation o D1 class receptors leads to increased cAMP, while activation o D2 class receptors inhibits cAMP generation (Fig. 14-4). Subsequent studies led to cloning o the receptor proteins, revealing f ve distinct receptors, each encoded by a separate gene. All known DA receptors have the typical structure o G protein-coupled receptors, with seven-transmembrane domains. The D1 class contains two dopamine receptors (D1 and D5), while the D2 class contains three receptors (D2, D3, and D4). There are two alternative orms o the D2 protein, D2S (i.e., short) and D2L (i.e., long), which represent alternate splice variants o the same gene; their di erence lies in the third cytoplasmic loop, which a ects G protein interaction but not dopamine binding.

209

The f ve di erent dopamine receptor proteins have distinct distributions in the brain (Fig. 14-5). Both D1 and D2 receptors are expressed at high levels in the striatum (caudate and putamen), where they play a role in motor control by the basal ganglia , as well as in the nucleus accumbens (see Chapter 19, Pharmacology o Drugs o Abuse) and olfactory tubercle . D2 receptors are also expressed at high levels on anterior pituitary gland lactotrophs , where they regulate prolactin secretion (see Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland). D2 receptors are thought to play a role in schizophrenia because many antipsychotic medications have high a f nity or these receptors (see below), although the localization o the D2 receptors involved remains to be elucidated. D3 and D4 receptors are structurally and unctionally related to D2 receptors and may also be involved in the pathogenesis o schizophrenia. High levels o D3 receptors are expressed in the limbic system, including the nucleus accumbens and ol actory tubercle, while D4 receptors have been localized to the frontal cortex, diencephalon, and brainstem. D5 receptors are distributed sparsely and expressed at low levels, mainly in the hippocampus , olfactory tubercle , and hypothalamus . Regulation o cAMP ormation is the def ning characteristic o the dopamine receptor classes, but dopamine receptors can also a ect other aspects o cellular unction depending on their localization and linkage to second messenger systems. Most dopamine receptors are expressed on the sur ace o postsynaptic neurons at dopaminergic synapses. The density o these receptors is tightly controlled through regulated insertion and removal o dopamine receptor proteins rom the postsynaptic membrane. DA receptors are also expressed presynaptically on the terminals o dopaminergic neurons. Presynaptic dopamine receptors, most o which are o the D2 class, serve as autoreceptors . These autoreceptors sense dopamine over ow rom the synapse and reduce dopaminergic tone, both by decreasing DA synthesis in the presynaptic neuron and by reducing the rate o neuronal f ring and dopamine release. Inhibition o DA synthesis occurs through cAMP-dependent downregulation o TH activity, while the inhibitory e ect on DA release and neuronal f ring is due, in part, to a separate mechanism involving the modulation o K and Ca2 channels. Increased K channel opening results in a larger current that hyperpolarizes the neuron, so that a larger depolarization is needed to reach the f ring threshold. Decreased Ca2 channel opening results in decreased levels o intracellular Ca2 . Because Ca2 is required or synaptic vesicle tra f cking to and usion with the presynaptic membrane, decreases in intracellular Ca2 levels result in decreased dopamine release.

Central Dopamine Pathways Most central dopaminergic neurons originate in discrete areas o the brain, as shown in Figure 14-6 (see also Fig. 9-8), and have divergent projections. Three major pathways can be distinguished. The largest DA tract in the brain is the nigrostriatal system, which contains about 80% o the brain’s DA. This tract projects rostrally rom cell bodies in the pars compacta o the substantia nigra to terminals that richly innervate the caudate and putamen, two nuclei that are collectively called the striatum. The striatum is named or the striped appearance o the white f ber tracts that run through it; the substantia nigra is named or the dark

210 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

D1 Receptor Family

D2 Receptor Family N

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FIGURE 14-4. Dopamine receptor families. The f ve dopamine receptor subtypes (D1–D5) can be classif ed into two major amilies o receptors. The D1 receptor amily has a long C-terminal tail and a short cytoplasmic loop between transmembrane helices 5 and 6, whereas the D2 receptor amily has a short C-terminal tail and a long cytoplasmic loop between helices 5 and 6. Stimulation o the D1 amily is excitatory, increasing cAMP and intracellular Ca 2 levels and activating protein kinase C (PKC). Stimulation o the D2 amily is inhibitory, decreasing cAMP and intracellular Ca 2 levels and hyperpolarizing the cell. The f ve receptor subtypes exhibit distinctive patterns o distribution in the central nervous system; the major areas o distribution are listed or each subtype. Within the D2 receptor subtype, there are D2S and D2L iso orms (not shown). IP3, inositol trisphosphate; PIP2, phosphatidylinositol-4,5-bisphosphate.

pigmentation that results from the decomposition of DA to melanin. Dopaminergic neurons of the nigrostriatal system are involved in the stimulation of purposeful movement. Their degeneration results in the abnormalities of movement that are characteristic of Parkinson’s disease.

D1

D5

Medial to the substantia nigra is an area of dopaminergic cell bodies in the midbrain called the ventral tegmental area (VTA). The VTA has widely divergent projections that innervate many forebrain areas, most notably the cerebral cortex, the nucleus accumbens, and other limbic structures.

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FIGURE 14-5. Location of dopamine receptors in the brain. The location o the f ve dopamine receptor subtypes in the human brain, as determined by localization o receptor mRNAs in corresponding regions o the rat brain, is shown in orange in coronal section. Both D1 and D2 receptors are localized in the caudate and putamen (the striatum), nucleus accumbens, amygdala, ol actory tubercle, and hippocampus. In addition, D1 receptors are present in the cerebral cortex, whereas D2 receptors are present in the substantia nigra, ventral tegmental area, and hypothalamus. AM, amygdala; C, caudate; Cx, cerebral cortex; H, hypothalamus; HIPP, hippocampus; nAc, nucleus accumbens; OT, ol actory tubercle; P, putamen; SN, substantia nigra; VTA, ventral tegmental area.

C h a p t e r 1 4 Pharmacology o Dopaminergic Neurotransmission

211

o dopaminergic systems is not always specif c to one system, many o the adverse e ects o drugs that act on these systems can be predicted based on their e ects on the other dopaminergic systems.

DOPAMINE AND CONTROL OF MOVEMENT: PARKINSON’S DISEASE Physiology of Nigrostriatal Pathways Hypotha la mus Ve ntra l te gme nta l a re a

Are a pos tre ma S ubs ta ntia nigra

FIGURE 14-6. Central dopamine pathways. Dopaminergic neurons originate in a number o specif c nuclei in the brain. Neurons that originate in the hypothalamus and project to the median eminence (blue arrow) are tonically active and inhibit prolactin secretion. Neurons that project rom the substantia nigra to the striatum (dashed arrows) regulate movement. Dopaminergic neurons that project rom the ventral tegmental area to the limbic system and pre rontal cortex (solid black arrows) are thought to have roles in the regulation o mood and behavior. The area postrema contains a high density o dopamine receptors, and stimulation o these receptors activates the vomiting centers o the brain.

These systems play an important and complex (as yet poorly understood) role in motivation, goal-directed thinking, regulation o a ect, and positive rein orcement (reward). Derangement o these pathways may be involved in the development o schizophrenia ; as discussed below, the blocking o dopaminergic neurotransmission can lead to a remission in psychotic symptoms. (See Chapter 19 or a more complete discussion o the reward pathway.) DA-containing cell bodies in the arcuate and paraventricular nuclei o the hypothalamus project axons to the median eminence o the hypothalamus. This system is known as the tubero-infundibular pathway. Dopamine is released by these neurons into the portal circulation connecting the median eminence with the anterior pituitary gland and tonically inhibits the release o prolactin by pituitary lactotrophs. A ourth anatomic structure, the area postrema located in the oor o the ourth ventricle, is also a target o dopaminergic therapies. The area postrema contains only a modest number o intrinsic dopamine neurons but a high density o dopamine receptors (mostly o the D2 class). The area postrema is one o the circumventricular organs that unction as blood chemoreceptors. Unlike the rest o the brain, the blood vessels in the circumventricular organs are enestrated, allowing communication between the blood and CNS (i.e., the circumventricular organs are “outside” the blood– brain barrier [BBB]). Stimulation o DA receptors in the area postrema activates the vomiting centers o the brain and is one o the causes o emesis . Drugs that block dopamine D2 receptors are used to treat nausea and vomiting. A derangement in any o these dopaminergic systems can result in disease. Parkinson’s disease, which arises rom dysregulated dopamine neurotransmission, and schizophrenia, which may also result rom abnormal dopamine neurotransmission, are two such examples. These two diseases, and the pharmacologic interventions used to treat them, are highlighted below. Because the pharmacologic manipulation

The basal ganglia have a crucial role in the regulation o purpose ul movement and are a site o the pathology in Parkinson’s disease. The basal ganglia do not connect directly to spinal motor neurons and thus do not directly control the individual movements o muscles. They appear to unction instead by assisting in learning coordinated patterns o movement and by acilitating the execution o learned motor patterns. Dopamine has a central role in the operation o this system, including signaling when desired movements are executed success ully and driving the learning process. Anatomically, the basal ganglia orm a reentrant loop by receiving input rom the cerebral cortex, processing this in ormation in the context o dopaminergic input rom the substantia nigra, and sending in ormation back to the cortex by way o the thalamus. The internal circuitry o the basal ganglia consists o several components. The striatum (caudate and putamen) is the primary input nucleus o the system, while the globus pallidus pars interna and substantia nigra pars reticulata are the output nuclei. These are interconnected through two internuclei, the subthalamic nucleus and the globus pallidus pars externa. Much o the in ormation processing per ormed by the basal ganglia occurs in the striatum. The cortical inputs to this structure are excitatory and use glutamate as a transmitter. The striatum is also the target o the dopaminergic nigrostriatal pathway. The neurons in the striatum are o several types. The majority o neurons are “medium spiny” neurons. These cells are studded with spines that receive input rom corticostriatal axons. These medium spiny neurons release the inhibitory transmitter GABA and send their projections to two downstream targets, orming the direct pathway and the indirect pathway (Fig. 14-7). The striatum also contains several small but important populations o interneurons, including neurons that release acetylcholine. These interneurons participate in the intercommunication between the direct and indirect pathways. The balance o activity between the direct and indirect pathways regulates movement. The direct pathway, ormed by striatal neurons expressing primarily dopamine D1 receptors, projects directly to the output o the basal ganglia, the internal segment o the globus pallidus . The latter neurons tonically inhibit the thalamus, which in turn, sends excitatory projections to the cortex that initiate movement. In this manner, activation o the direct pathway disinhibits the thalamus; that is, the activation of the direct pathway stimulates movement. The indirect pathway, ormed by striatal neurons expressing predominantly D2 receptors, projects to the external segment o the globus pallidus, which in turn, inhibits neurons in the subthalamic nucleus . The neurons in the subthalamic nucleus are excitatory glutamatergic neurons that project to the internal segment o the globus pallidus. As a result o this multistep pathway, activation o the indirect pathway disinhibits neurons o the subthalamic nucleus, which, in turn, stimulate neurons in the internal segment o

212 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Normal Ba la nce d a ctivity of dire ct a nd indire ct pa thwa ys Motor corte x

Gluta ma te rgic input from corte x

P uta me n D1

Dire ct pa thwa y (e na ble s move me nt)

ACh

D2

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FIGURE 14-7. Effect of Parkinson’s disease on dopaminergic pathways that regulate movement. Two principal pathways in the basal ganglia regulate movement: the direct pathway, which enables movement, and the indirect pathway, which inhibits movement. Dopamine stimulates the direct pathway and inhibits the indirect pathway, yielding a net bias that allows purpose ul movement. Excitatory pathways are shown in blue, and inhibitory pathways are shown in black. The direct pathway signals rom putamen to GPi to thalamus to cortex, while the indirect pathway signals rom putamen to GPe to STN to GPi to thalamus to cortex. GPi, internal segment o the globus pallidus; GPe, external segment o the globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus. Inset: Both direct and indirect pathway neurons in the putamen receive inputs rom the nigrostriatal dopaminergic system (dotted blue arrow) and rom cortical glutamatergic systems (solid blue arrow), process these inputs in the context o local cholinergic inf uences (ACh), and transmit a GABAergic output (not shown). Degeneration o dopaminergic neurons in the substantia nigra results in understimulation o the direct (movement-enabling) pathway and underinhibition o the indirect (movement-inhibiting) pathway. The net result is a paucity o movement. Dotted gray arrow indicates decreased activity caused by understimulation, and thick black arrow indicates increased activity caused by underinhibition.

the globus pallidus to inhibit the thalamus; that is, the activation of the indirect pathway inhibits movement. The di erential expression o D1 and D2 receptors within the two pathways leads to di ering e ects o dopaminergic stimulation. Increased levels o dopamine in the striatum tend to activate the D1-expressing neurons o the direct pathway while inhibiting the D2-expressing neurons o the indirect pathway. Notice that both o these e ects promote movement. The opposite e ect occurs in Parkinson’s disease, a state o dopamine def ciency: the direct pathway shows reduced activity, while the indirect pathway is overactive, leading to reduced movement. This model o basal ganglia unction is greatly simplif ed, o course, but it has been use ul in developing a deeper understanding o how the basal ganglia work. An important prediction o the model is that, in Parkinson’s disease, the indirect pathway (and, in particular, the subthalamic nucleus) should be overactive. This prediction has been proven

directly by in vivo electrical recordings in patients with Parkinson’s disease. Furthermore, surgical therapies that target the subthalamic nucleus, such as deep brain stimulation in this location, are now o ten used to treat Parkinson’s disease when pharmacologic treatments are inadequate.

Pathophysiology In Parkinson’s disease, there is a selective loss o dopaminergic neurons in the substantia nigra pars compacta (Fig. 14-7). The extent o loss is pro ound, with at least 70% o the neurons destroyed at the time symptoms f rst appear; o ten, 95% o the neurons are missing at autopsy. The destruction o these neurons results in the core motor eatures o the disease: bradykinesia, or slowness o movement; rigidity, a resistance to passive movement o the limbs; impaired postural balance, which predisposes to alling; and a characteristic tremor when the limbs are at rest.

C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission

The mechanisms underlying the destruction o DA neurons in the substantia nigra in Parkinson’s disease are not ully understood. Both environmental actors and genetic in uences have been implicated. In 1983, the unexpected development o Parkinson’s disease in abusers o the synthetic opioid meperidine (see Chapter 18, Pharmacology o Analgesia) yielded the f rst agent known to produce Parkinson’s disease directly and the strongest evidence that environmental actors can cause Parkinson’s disease. These individuals, who tended to be young and otherwise healthy, suddenly developed severe, levodopa-responsive parkinsonian symptoms. The cases were all linked to a single contaminated batch o meperidine that had been synthesized in a makeshi t lab. The contaminant was ound to be 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which orms as an impurity in the synthesis o meperidine when its manu acture is carried out or too long and at too high a temperature. Studies in nonhuman primates have shown that MPTP is oxidized in the brain to MPP (1-methyl-4-phenyl-pyridinium), which is selectively toxic to neurons in the substantia nigra. Despite extensive searches, it does not appear that there is any signif cant amount o MPTP present in the everyday environment, and MPTP itsel is not the cause o most cases o Parkinson’s disease. There may, however, be other environmental actors that have a more subtle e ect on development o the disease, such as exposure to certain pesticides. Recent research has established that genetic actors contribute to Parkinson’s disease. The best-studied examples are amilies with mutations in or overexpression o the protein -synuclein, which lead to autosomal dominant orms o Parkinson’s disease. While the unction o this protein is not clear, it appears to be involved in the ormation o neurotransmitter vesicles and the release o dopamine in the brain. At least our other genes have been identif ed as causing Parkinson’s disease in one or more amilies. These genetic discoveries have provided important clues into the biology o Parkinson’s disease and have allowed the development o transgenic mouse and ruit y models that serve as a plat orm or developing new treatments. Although these genetic discoveries have provided insight into the biology o Parkinson’s disease, it is important to note that all o the di erent genetic causes identif ed so ar account or less than 10% o cases, and most cases are still o unknown cause. The etiology o Parkinson’s disease in most patients is likely multi actorial, with contributions rom both genetic and environmental actors.

Pharmacologic Classes and Agents Parkinson’s disease is a progressive neurodegenerative disorder. Loss o dopaminergic neurons likely begins a decade or more be ore the symptoms become apparent, and this loss continues relentlessly. Currently available treatments are mostly symptomatic, meaning that they treat the symptoms but do not alter the underlying degenerative process. Symptomatic treatments are very use ul and can restore unction and quality o li e or many years, but ultimately, the progression o the disease leads to increasing di f culty in managing the symptoms. In addition, some eatures o Parkinson’s disease do not respond well to current medications, particularly the nonmotor symptoms (such as cognitive impairment and dementia) that characterize the late stages o the disease and that result rom an extension o the disease process rom the dopaminergic system to other areas o the brain. The goal o

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much current research is the development o neuroprotective and neurorestorative therapies, which might delay or eliminate the need or symptomatic treatment and avoid the late complications o the disorder. Most o the pharmacologic interventions currently used in Parkinson’s disease are aimed at restoring DA levels in the brain. In general, medications used in the management o Parkinson’s disease can be divided into DA precursors, DA receptor agonists, and inhibitors o DA degradation. There is a smaller but still use ul role or the existing nondopaminergic therapies, such as anticholinergic agents that modi y the unction o striatal interneurons.

Dopamine Precursors

Levodopa was f rst used to treat Parkinson’s disease over

40 years ago and is still the most e ective treatment or the disease. DA itsel is not suitable because it cannot cross the BBB. However, DA’s immediate precursor, L-DOPA (levodopa), is readily transported across the BBB by the neutral amino acid transporter (see Chapter 9); once in the CNS, L-DOPA is converted to dopamine by the enzyme AADC. Thus, L-DOPA must compete with other neutral amino acids or transport across the BBB, and its availability in the CNS may be compromised by recent high-protein meals (see the introductory case in Chapter 9). Orally administered levodopa is readily converted into dopamine by AADC in the gastrointestinal tract. This metabolic process both diminishes the amount o levodopa that can reach the blood–brain barrier or transport into the CNS and increases the peripheral adverse e ects that result rom the generation o dopamine in the peripheral circulation (predominantly nausea, due to binding o this dopamine to receptors in the area postrema). When levodopa is administered alone, only 1–3% o the administered dose reaches the CNS unchanged. In order to boost the levels o levodopa available to the brain and reduce the adverse e ects o peripheral levodopa metabolism, levodopa is almost always administered in combination with carbidopa , an inhibitor o AADC (Fig. 14-8). Carbidopa effectively prevents the conversion of levodopa to DA in the periphery. Importantly, because carbidopa is not able to cross the BBB, it does not inter ere with the conversion o levodopa to DA in the CNS. Carbidopa increases the raction o orally administered levodopa available in the CNS rom 1–3% (without carbidopa) to 10% (with carbidopa), allowing a signif cant reduction in the dose o levodopa and reducing the incidence o peripheral adverse e ects. Many patients with Parkinson’s disease show remarkable symptomatic improvement when prescribed the combination o levodopa and carbidopa, especially during the early phase o the disease. In act, an improvement in symptoms ollowing the initiation o levodopa therapy is considered diagnostic o Parkinson’s disease. Over time, however, the e ectiveness o levodopa declines. Continued use results in both tolerance and sensitization to the medication, maniested as a drastic narrowing o the therapeutic window. As patients continue on levodopa therapy, they require higher doses to produce a clinically signif cant improvement in symptoms. They develop uctuations in motor unction that include periods o reezing and increased rigidity, known as “o ” periods, alternating with periods o normal or even dyskinetic (excess involuntary) movement, known as “on” periods. These “on” periods generally occur shortly a ter the

214 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY Periphery

Brain

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FIGURE 14-8. Effects of carbidopa, COMT inhibitors, and MAO-B inhibitors on the peripheral and central metabolism of levodopa. Orally administered levodopa (L-DOPA) is metabolized in the peripheral tissues and in the gastrointestinal (GI) tract by aromatic L-amino acid decarboxylase (AADC), catecholO-methyltrans erase (COMT), and monoamine oxidase A (MAO-A; not shown). This metabolism substantially reduces the e ective dose o levodopa available to the brain and substantially increases the adverse peripheral e ects o the drug. Carbidopa is an AADC inhibitor that cannot cross the blood–brain barrier. When levodopa is administered in combination with carbidopa, a greater raction o the levodopa is available to the brain. There ore, a smaller dose o levodopa is required or clinical e f cacy, and the drug has less severe adverse e ects in the periphery. By inhibiting COMT in the periphery, entacapone and tolcapone similarly increase the raction o peripheral levodopa available to the brain. L-DOPA is transported across the blood–brain barrier by the L-neutral amino acid transporter (LNAA) and metabolized to dopamine (DA) by AADC. Within the brain, DA is metabolized by COMT and MAO-B. Tolcapone (COMT inhibitor) and selegiline and rasagiline (selective MAO-B inhibitors) augment the e ectiveness o levodopa therapy by inhibiting the metabolism o DA in the brain. 3-O-MD, 3-O-methylDOPA; DOPAC, dihydroxyphenylacetic acid; 3MT, 3-methoxytyramine.

administration o levodopa/carbidopa, when a large bolus o dopamine is delivered to the striatum. The dyskinetic adverse e ect o “on” periods can be overcome initially by taking smaller doses o medication, although this increases the likelihood o “o ” periods. “O ” periods tend to occur as plasma levels o levodopa decline and can be compensated or by increasing either the dose or requency o levodopa or by using agents that slow the degradation o dopamine (described below). As the disease progresses, these symptoms become increasingly di f cult to manage. The most pro ound adverse e ect o levodopa is its propensity to cause dyskinesias , or uncontrollable rhythmic movements o the head, trunk, and limbs. These appear in at least hal o all patients within 5 years o starting the drug, and they generally worsen as the disease progresses. Similar to the “on/o ” phenomenon, dyskinesias are usually linked to levodopa dosing, mostly occurring at times o maximal levodopa plasma concentrations. Accordingly, dyskinesias can also be managed initially by using smaller doses o levodopa more requently. Un ortunately, as the disease progresses, continued therapy leads to worsening o both the dyskinesias and the “on/o ” phenomenon, to the point where one or the other is almost always present. Although levodopa-induced dyskinesias and uctuations in motor unction are complex and poorly understood, at least two actors are thought to contribute to these adverse e ects. First, the continued destruction o dopaminergic neurons as Parkinson’s disease progresses results in the striatum’s increasing inability to store dopamine e ectively and reduces the ability o dopamine terminals to bu er the synaptic concentrations o dopamine. Second, chronic therapy with levodopa appears to cause adaptations in postsynaptic neurons in the striatum. Dopamine concentrations in striatal synapses

are normally tightly regulated. The large uctuations in dopamine concentration produced by intermittent oral levodopa administration induce changes in the cell sur ace expression o dopamine receptors and in postreceptor signaling events. These postsynaptic adaptations alter the cell’s sensitivity to synaptic dopamine levels, urther accentuating responses associated with high (“on” period, dyskinesia) and low (“o ” period, akinesia) transmitter concentrations. The predictable decline in e f cacy and increase in adverse e ects that result rom prolonged levodopa therapy have led to discussions about the appropriate time to begin treatment o Parkinson’s disease with levodopa and the relative merits o delaying the use o this drug in the early stages o the disease. Recent studies have suggested that there may be advantages to initial treatment with therapies other than levodopa, particularly the dopamine receptor agonists (see below), but these alternatives can lead to more severe adverse e ects than levodopa, at least in some patients. In addition, most patients who are initially treated with other therapies generally require levodopa treatment at some point. Levodopa remains the most e ective therapy or Parkinson’s disease and should be initiated as soon as other therapies are unable to control parkinsonian symptoms e ectively. Further delays in levodopa therapy are associated with reduced rates o symptom control and increased mortality.

Dopamine Receptor Agonists

Another strategy or enhancing dopaminergic neurotransmission is to target the postsynaptic DA receptor directly through the use o DA receptor agonists. The earliest therapies in this class were ergot derivatives such as bromocriptine (D2 agonist) and pergolide (D1 and D2), but these have been ound to induce adverse e ects, including f brosis o cardiac

C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission

valves, and have largely been abandoned in avor o nonergot agonists such as pramipexole , ropinirole , and rotigotine (all D3 D2). As a class, DA receptor agonists have several advantages. Because they are nonpeptide molecules, they do not compete with levodopa or other neutral amino acids or transport across the BBB. Furthermore, because they do not require enzymatic conversion by AADC, they remain e ective late in the course o Parkinson’s disease. All o the dopamine receptor agonists in current use have hal -lives longer than that o levodopa, which allows or less requent dosing and a more uni orm response to the medications. The major limitation to the use o the dopamine receptor agonists is their tendency to induce unwanted adverse e ects, which may include nausea, peripheral edema, and hypotension. All o the dopamine agonists may also produce a variety o adverse cognitive e ects, including excessive sedation, vivid dreams, and hallucinations, particularly in elderly patients. Dopamine receptor agonists may also trigger symptoms o the dopamine dysregulation syndrome, in which patients exhibit impaired impulse control. Common mani estations include pathological gambling, overspending, compulsive eating, and hypersexuality. These behaviors may be socially destructive and require discontinuation o the medications. Recent studies have examined the use o pramipexole and ropinirole as initial monotherapy or Parkinson’s disease. It was thought that, because the dopamine agonists have longer hal -lives than levodopa, they might be less likely to induce “o ” periods. These studies show that use o the dopamine receptor agonists as initial treatment or Parkinson’s disease does delay the onset o “o ” periods and dyskinesias, but there is also an increased rate o adverse e ects compared to initial treatment with levodopa. At present, many practitioners use dopamine agonists as the initial treatment or Parkinson’s disease, especially in younger individuals.

Inhibitors of Dopamine Metabolism

A third strategy that has been employed to treat Parkinson’s disease involves the inhibition o DA breakdown. Inhibitors o both MAO-B (the iso orm o MAO that predominates in the striatum) and COMT have been used as adjuvants to levodopa in clinical practice (Fig. 14-8). Selegiline is an MAO inhibitor that, in low concentrations, is selective or MAO-B. It does not inter ere with the peripheral metabolism o monoamines by MAO-A, and it avoids the toxic e ects o dietary tyramine and other sympathomimetic amines that are associated with nonselective MAO blockade (see Chapter 15, Pharmacology o Serotonergic and Central Adrenergic Neurotransmission). A drawback o selegiline is that this drug orms a potentially toxic metabolite, amphetamine, which can cause sleeplessness and con usion, especially in the elderly. Rasagiline , a newer MAO-B inhibitor that does not orm toxic metabolites, is also approved in the United States. Both rasagiline and selegiline improve motor unction in Parkinson’s disease when used alone, and both can augment the e ectiveness o levodopa therapy. There has also been interest in the question o whether MAO inhibitors can limit the ormation o reactive ree radicals associated with dopamine catabolism and thereby alter the rate o disease progression, but clinical trials o both drugs searching or a “neuroprotective” e ect have been inconclusive so ar.

215

Tolcapone and entacapone inhibit COMT and thereby in-

hibit the degradation o levodopa as well as DA. Tolcapone is a highly lipid-soluble agent that can cross the BBB, while entacapone distributes only to the periphery. Both drugs decrease the peripheral metabolism o levodopa and thereby make more levodopa available to the CNS. Tolcapone has the additional property o crossing the blood–brain barrier e ectively and inhibiting central as well as peripheral COMT. In clinical trials, both tolcapone and entacapone have been shown to reduce the “o ” periods that are associated with decreasing plasma levodopa levels. Although the central e ect o tolcapone is an advantage (Fig. 14-8), there have been several reports o atal hepatic toxicity associated with tolcapone, and it must be used with great care. In practice, thereore, entacapone is the most widely used COMT inhibitor.

Nondopaminergic Pharmacology in Parkinson’s Disease

Amantadine, trihexyphenidyl, and benztropine are all drugs that do not clearly a ect dopaminergic pathways but are nonetheless e ective in the treatment o Parkinson’s disease. Amantadine was developed and is marketed primarily as an antiviral that reduces the length and severity o in uenza A inections (see Chapter 38, Pharmacology o Viral In ections). In patients with Parkinson’s disease, however, amantadine is used to treat levodopa-induced dyskinesias that develop late in the course o the disease. The mechanism by which amantadine reduces dyskinesia is thought to involve blockade o excitatory NMDA receptors. Trihexyphenidyl and benztropine are muscarinic receptor antagonists that reduce cholinergic tone in the CNS. They reduce tremor more than bradykinesia and are there ore more e ective in treating patients or whom tremor is the major clinical mani estation o Parkinson’s disease. These anticholinergic drugs are thought to act by modi ying the actions o striatal cholinergic interneurons, which regulate the interactions o direct and indirect pathway neurons. They also cause a range o anticholinergic adverse e ects, which may include dry mouth, urinary retention, and most importantly, impairment o memory and cognition.

Treatment of Patients with Parkinson’s Disease The treatment o patients with Parkinson’s disease is an individualized process that must take into account not only the extent o symptoms but also the patient’s age, occupation, activities, and perceived disabilities. There is at present no laboratory test that can specif cally conf rm the diagnosis; instead, diagnosis is based on history and physical examination, along with laboratory studies to exclude other possible diagnoses. In patients with early disease, it may be appropriate to recommend a nonpharmacologic approach to treatment that emphasizes exercise and li estyle modif cation. Almost all patients eventually require treatment with medication. In patients with mild symptoms, MAO-B inhibitors, amantadine, or anticholinergic medications may be considered. When symptoms are more advanced, a dopaminergic therapy is indicated. Levodopa is the most e ective therapy, but many younger patients are treated f rst with a dopamine agonist in the hope o delaying the onset o motor uctuations. Advanced disease with uctuations requires polypharmacy, o ten including levodopa, dopamine agonists, entacapone, MAO-B inhibitors, and amantadine.

216 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

It is important to be vigilant or the development o cognitive symptoms and adverse e ects, which may require modif cation o the therapeutic approach.

DOPAMINE AND DISORDERS OF THOUGHT: SCHIZOPHRENIA Pathophysiology Schizophrenia is a thought disorder characterized by one or more episodes o psychosis (impairment in reality testing). Patients may mani est disorders o perception, thinking, speech, emotion, and/or physical activity. Schizophrenic symptoms are divided into two broad categories. Positive symptoms involve the development o abnormal unctions; these symptoms include delusions (distorted or alse belie s and misinterpretation o perceptions), hallucinations (abnormal perceptions, especially auditory), disorganized speech, and catatonic behavior. Negative symptoms involve the reduction or loss o normal unctions; these symptoms include a ective f attening (decrease in the range or intensity o emotional expression), alogia (decrease in the uency o speech), and avolition (decrease in the initiation o goal-directed behavior). The American Psychiatric Association criteria or schizophrenia are listed in Box 14-1.

Schizophrenia typically begins to a ect individuals in their late teens and early 20s. The disorder a ects males and emales equally. Approximately 4.75 million individuals su er rom schizophrenia in the United States, and 100,000 to 150,000 new cases are diagnosed annually. A genetic component o the disease has been demonstrated, but concordance among identical twins is only 50%. Schizophrenia, there ore, appears to have a multi actorial etiology, with both genetic and environmental components. The model that is most commonly cited to explain the pathogenesis o schizophrenia is the dopamine hypothesis , which states that the illness is caused by increased and dysregulated levels o DA neurotransmission in the brain. This hypothesis arises rom the empiric observation that treatment with DA receptor antagonists, specif cally D2 antagonists, relieves a number o the symptoms o schizophrenia in many, but not all, patients with the disease. The DA hypothesis is supported by several additional clinical observations. First, some patients taking drugs that increase DA levels or that activate dopamine receptors in the CNS, including amphetamines , cocaine , and apomorphine , develop a schizophrenia-like state that subsides when the dose o the drug is lowered. Second, hallucinations are a known adverse e ect o levodopa therapy or Parkinson’s disease. Finally, researchers have been able to correlate decreased

B O X 1 4 - 1 Crite ria fo r Sch izo p h re n ia , fro m th e Dia g n o s tic a n d S ta tis tica l Ma n u a l o f Me n ta l Dis o rd e rs , Fifth Ed itio n (DS M-5) A. Characteristic symptoms: Two (or more) o the ollowing, each present or a signif cant portion o time during a 1-month period (or less i success ully treated). At least one o these should include 1–3: 1. 2. 3. 4. 5.

Delusions Hallucinations Disorganized speech Grossly disorganized or catatonic behavior Negative symptoms (i.e., diminished emotional expression or avolition)

B. Social/occupational dys unction: For a signif cant portion o the time since the onset o the disturbance, one or more major areas o unctioning, such as work, interpersonal relations, or sel -care, are markedly below the level achieved be ore the onset (or when the onset is in childhood or adolescence, ailure to achieve expected level o interpersonal, academic, or occupational achievement). C. Duration o 6 months: Continuous signs o the disturbance persist or at least 6 months. This 6-month period must include at least 1 month o symptoms (or less i success ully treated) that meet Criterion A (i.e., active-phase symptoms) and may include periods o prodromal or residual symptoms. During these prodromal or residual periods, the signs o the disturbance may be mani ested by only negative symptoms or two or more symptoms listed in Criterion A present in an attenuated orm (e.g., odd belie s, unusual perceptual experiences).

D. Schizoa ective and mood disorder exclusion: Schizoa ective disorder and mood disorder with psychotic eatures have been ruled out because either (1) no major depressive, manic, or mixed episodes have occurred concurrently with the activephase symptoms, or (2) i mood episodes have occurred during active-phase symptoms, their total duration has been brie relative to the duration o the active and residual periods. E. Substance/general medical condition exclusion: The disturbance is not attributable to the direct physiologic e ects o a substance (e.g., a drug o abuse, a medication) or a general medical condition. F. Relationship to a global developmental delay or autism spectrum disorder: I there is a history o autism spectrum disorder or other communication disorder o childhood onset, the additional diagnosis o schizophrenia is made only i prominent delusions or hallucinations are also present or at least 1 month (or less i success ully treated). Classif cation o longitudinal course (the minimum observation period is 1 year; see DSM-5 or ull description): 1. 2. 3. 4. 5. 6. 7. 8.

First episode, currently in acute episode First episode, currently in partial remission First episode, currently in ull remission Multiple episodes, currently in acute episode Multiple episodes, currently in partial remission Multiple episodes, currently in ull remission Continuous Unspecif ed ■

Reprinted with permission rom the American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Arlington, VA: American Psychiatric Association; 2013.

C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission

DA metabolite levels, and by extension decreased DA levels, with clinical improvement in some schizophrenic symptoms. The dysregulation o dopaminergic neurotransmission in schizophrenia is thought to occur at specif c anatomic locations in the brain. The mesolimbic system is a dopaminergic tract that originates in the ventral tegmental area and projects to the nucleus accumbens in the ventral striatum, parts o the amygdala and hippocampus, and other components o the limbic system. This system is involved in the development o emotions and memory, and some hypothesize that mesolimbic hyperactivity is responsible or the positive symptoms o schizophrenia. This hypothesis is supported by positron emission tomography (PET) scans o the brains o patients displaying the earliest signs o schizophrenia; these PET images show changes in blood ow to the mesolimbic system that re ect changes in the level o unctioning o this system. Dopaminergic neurons o the mesocortical system originate in the ventral tegmental area and project to regions o the cerebral cortex, particularly the pre rontal cortex. Because the pre rontal cortex is responsible or attention, planning, and motivated behavior, the hypothesis has been advanced that the mesocortical system plays a role in the negative symptoms o schizophrenia. All o the evidence implicating DA in the pathogenesis o schizophrenia is circumstantial, however, and much o it is con icting. Changes in DA levels, particularly in the mesolimbic and mesocortical systems, could simply reect downstream consequences o a pathologic process in a hereto ore undiscovered pathway. One hypothesis involving such an upstream process suggests that an imbalance in glutamatergic neurotransmission plays an important role in schizophrenia. This model is supported by the observation that phencyclidine (PCP) (see Chapter 19), an antagonist at NMDA receptors, causes symptoms similar to those o schizophrenia. In act, the syndrome seen in patients taking PCP chronically—consisting o psychotic symptoms, visual and auditory hallucinations, disorganized thought, blunted a ect, withdrawal, psychomotor retardation, and an amotivational state—has components o both the positive and negative symptoms o schizophrenia. Dopaminergic neurons and excitatory glutamatergic neurons o ten orm reciprocal synaptic connections, which could account or the e f cacy o DA receptor antagonists in schizophrenia. Even i this hypothesis is correct, at present, there are no use ul therapies or schizophrenia that act at glutamate receptors. Glutamate is the primary excitatory transmitter in the brain, and urther research will be required to identi y drugs that are su f ciently selective or use in schizophrenia and that have an acceptable adverse e ect prof le.

Pharmacologic Classes and Agents Although the biological basis o schizophrenia remains controversial, a number o drugs are e ective in treating the illness. When success ul, these medications can lead to a remission o psychosis and allow the patient to integrate into society. Patients only rarely return completely to their premorbid state, however. Drugs used in the management o psychosis are o ten called neuroleptics or antipsychotics . Although these terms are requently used interchangeably, they have slight yet important di erences in connotation. The term neuroleptic emphasizes the drugs’ neurological actions that are commonly mani ested as adverse e ects o treatment. These adverse e ects, o ten called extrapyramidal e ects ,

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result rom DA receptor blockade in the basal ganglia and include the parkinsonian symptoms o slowness, sti ness, and tremor. The term antipsychotic denotes the ability o these drugs to abrogate psychosis and alleviate disordered thinking in schizophrenic patients. The antipsychotics may be urther divided into typical antipsychotics , older drugs with prominent actions at the D2 receptor, and atypical antipsychotics , a newer generation o drugs with less prominent D2 antagonism and consequently ewer extrapyramidal e ects.

Typical Antipsychotic Agents

The history o the typical antipsychotic drugs dates back to the approval o chlorpromazine in 1954. Approval was based on observations o the drug’s e ectiveness in schizophrenia, but there was little understanding o its mechanism o action. In the 1960s, as the role o DA in the brain became better understood, the ability o the typical antipsychotic drugs to block dopaminergic neurotransmission in the CNS was f rst elucidated. A f nity binding studies per ormed in the 1980s demonstrated that both therapeutic e f cacy and extrapyramidal adverse e ects o the typical antipsychotics correlate directly with the a f nity o these drugs or D2 receptors. As shown in Figure 14-9, drugs with higher a f nity or D2 receptors, as represented by lower dissociation constants, tend to require smaller doses to control psychotic symptoms and alleviate schizophrenia. Mechanism o Action

Although the typical antipsychotics block D2 receptors in all o the CNS dopaminergic pathways, their mechanism o action as antipsychotics appears to involve antagonism o mesolimbic, and possibly mesocortical, D2 receptors. As described above, one hypothesis holds that the positive symptoms o schizophrenia correlate with hyperactivity o the mesolimbic system, and antagonism o mesolimbic dopamine receptors could alleviate these symptoms. The typical antipsychotics are relatively less e ective at controlling the negative symptoms o schizophrenia. This relative lack o e f cacy at treating the negative symptoms could relate to the hypothesis that the negative symptoms correlate with hypoactivity o mesocortical neurons, because the antagonist action o the antipsychotics would not be expected to correct dopaminergic hypoactivity. Many o the adverse e ects o the typical antipsychotics are likely mediated by binding o these drugs to D2 receptors in the basal ganglia (nigrostriatal pathway) and pituitary gland (see below). The typical antipsychotics all into several structural classes, o which the most prominent are the phenothiazines and the butyrophenones (Fig. 14-10). Chlorpromazine is the prototypical phenothiazine, and haloperidol is the most widely used butyrophenone. Despite di erences in structure and D2 receptor a f nity, all typical antipsychotics have similar clinical e f cacy at their standard doses. In general, aliphatic phenothiazines (such as chlorpromazine) are less potent antagonists at D2 receptors than are butyrophenones, thioxanthenes (phenothiazines in which a nitrogen in the phenothiazine nucleus is substituted by a carbon), or phenothiazines unctionalized with a piperazine derivative (such as f uphenazine ). For all o these drugs, the clinical dose can be adjusted to account or the in vitro D2 receptor binding a f nity, so that e f cacy is una ected by potency at clinically useul doses. However, the potency o the typical antipsychotics is critical in determining the drugs’ adverse e ects prof les.

218 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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Antips ychotic drug dos e (mg/da y) FIGURE 14-9. Antipsychotic potency of dopamine receptor antagonists. Over at least three orders o magnitude, the clinically e ective dose o the typical antipsychotics is proportional to the dissociation constant o the drugs at D2 receptors. (Note that a higher dissociation constant represents a lower binding a f nity.) Atypical antipsychotics such as clozapine and remoxipride (blue diamonds) are exceptions to this rule; these agents have clinical e ects at a dose lower than that predicted by their dissociation constants. Data points represent the mean dissociation constant (averaged over multiple studies) at the most common clinically e ective dose. The dotted line represents the best f t to the data or all o the typical antipsychotics (blue circles).

Adverse Effects

The adverse e ects o typical antipsychotic drugs can be divided into two broad categories: those caused by antagonist action at dopamine D2 receptors outside the mesolimbic and mesocortical systems (on-target e ects) and those caused by nonspecif c antagonist action at other receptor types (o -target e ects). Given the broad distribution o dopamine receptor expression, it is not surprising that dopamine receptor antagonists have a wide range o on-target adverse e ects. As noted above, the most prominent o these e ects are o ten re erred to as extrapyramidal effects . Because endogenous stimulation o dopamine D2 receptors inhibits the indirect pathway within the basal ganglia, antagonism o D2 receptors by typical antipsychotic drugs can disinhibit the indirect pathway and thereby induce parkinsonian symptoms. Such symptoms can sometimes be treated with the nondopaminergic therapies or Parkinson’s disease, such as amantadine and anticholinergic drugs. Dopaminergic drugs are o ten ine ective because o the high a f nity o the antagonists or the D2 receptor and because, when used in this setting, dopaminergic drugs could cause a relapse o schizophrenic symptoms. The most severe adverse e ect o the typical antipsychotics is the so-called neuroleptic malignant syndrome (NMS), a rare but li e-threatening syndrome characterized by catatonia, stupor, ever, and autonomic instability; myoglobinemia and death occur in about 10% o these cases. NMS is most commonly associated with the typical antipsychotic

drugs that have a high a f nity or D2 receptors, such as haloperidol. It can also be seen in patients with Parkinson’s disease who abruptly discontinue dopaminergic medications, emphasizing the importance o dopamine in the causation o NMS. The symptoms are thought to arise at least in part rom the actions o the antipsychotics on the dopaminergic systems in the hypothalamus, which are essential or the body’s ability to control temperature. Treatment with antipsychotics and other dopamine antagonists can also cause abnormal movements, a condition known as tardive dyskinesia . This condition is observed most requently a ter prolonged treatment with drugs that have a high a f nity or the D2 receptor, such as haloperidol. It is occasionally seen in patients a ter only short-term treatment and has been reported to occur a ter a single dose o a D2 receptor antagonist. The syndrome is characterized by repetitive, involuntary, stereotyped movements o the acial musculature, arms, and trunk. The exact mechanism is unknown, but it is believed to involve adaptive hypersensitivity o D2 receptors in the striatum, which, in turn, results in excessive dopaminergic activity. Antiparkinsonian drugs can exacerbate tardive dyskinesia, and discontinuation o antiparkinsonian drugs can ameliorate the symptoms. Administration o high doses o high-potency typical antipsychotics can temporarily suppress the disorder, presumably by overcoming the adaptive response in striatal neurons, but may in the long run lead to worsening o symptoms. In many cases, cessation o all typical antipsychotic medications will

C h a p t e r 1 4 Pharmacology o Dopaminergic Neurotransmission

219

prolactin secretion. Antagonism o D2 receptors increases

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FIGURE 14-10. Chemical structures o the typical antipsychotics. The structure o the phenothiazines is based on a common skeleton, with two variable unctional groups. Chlorpromazine, the f rst approved antipsychotic, has substituted aminopropyl (R1) and chloride (R2) side groups. Piperazine (in blue box)-substituted phenothiazines, such as uphenazine, are signif cantly more potent than aliphatic-substituted phenothiazines, such as chlorpromazine. The ourth structure represents the skeleton o a thioxanthene, which substitutes a carbon (in blue box) or the phenothiazine nitrogen. As illustrated by the structure o haloperidol, butyrophenones (in blue box) are structurally distinct rom phenothiazines and thioxanthenes.

lead to slow reversal o the striatal adaptations, with eventual improvement in the symptoms o tardive dyskinesia. Some patients, however, are le t with a permanent and irreversible movement disorder. Some adverse e ects o typical antipsychotics are thought to be caused by antagonist action at dopamine receptors in the pituitary gland, where dopamine tonically inhibits

prolactin secretion, leading to amenorrhea, galactorrhea, and alse-positive pregnancy tests in women and to gynecomastia and decreased libido in men. Other adverse e ects o the typical antipsychotics result rom nonspecif c antagonism o muscarinic and -adrenergic receptors. Antagonism o peripheral muscarinic pathways causes anticholinergic e ects, including dry mouth, constipation, di f culty urinating, and loss o accommodation (see Chapter 10, Cholinergic Pharmacology). -Adrenergic antagonism can cause orthostatic hypotension and, in men, ailure to ejaculate. Sedation can also occur because o inhibition o central -adrenergic pathways in the reticular activating system. When sedation inter eres with normal unctioning during chronic antipsychotic use, it is considered an adverse e ect. In the acutely psychotic patient, however, sedation may be part o the drug’s intended spectrum o action. The adverse e ect prof les o the typical antipsychotics depend on their potency. High-potency drugs (whose clinical doses are only a ew milligrams) tend to have ewer sedative e ects and cause less postural hypotension than drugs with lower potency (i.e., drugs that require high doses to achieve a therapeutic e ect). On the other hand, lower potency typical antipsychotics tend to cause ewer extrapyramidal adverse e ects. These observations can be rationalized by the act that high-potency drugs have high a f nity or D2 receptors and are there ore more selective in their action. Thus, these drugs are more likely to cause on-target adverse e ects mediated by D2 receptors (i.e., extrapyramidal e ects) and less likely to cause o -target adverse e ects mediated by muscarinic and -adrenergic receptors (i.e., anticholinergic e ects, sedation, and postural hypotension). Conversely, low-potency typical antipsychotics do not bind D2 receptors as tightly and cause ewer extrapyramidal e ects, while their lower selectivity results in more prominent anticholinergic and antiadrenergic e ects.

Pharmacokinetics, Metabolism, and Drug Interactions

As with many drugs active in the CNS, the typical antipsychotics are highly lipophilic. In part because o this lipophilicity, typical antipsychotics tend to be metabolized in the liver and to exhibit both high binding to plasma proteins and high f rst-pass metabolism. The drugs are generally ormulated as oral or intramuscular dosage orms. The latter are use ul in treating acutely psychotic patients who may be a danger to themselves or others, while the oral ormulations are generally used or chronic therapy. Elimination hal -lives o the typical antipsychotics are erratic because their kinetics o elimination typically ollow a multiphasic pattern and are not strictly f rst-order. In general, however, the hal -lives o most typical antipsychotics are on the order o 1 day, and it is common practice to ollow a once-daily dosing regimen. Two drugs, haloperidol and f uphenazine , are available as decanoate esters. These highly lipophilic drugs are injected intramuscularly, where they are slowly hydrolyzed and released. The decanoate ester dosage orms provide a long-acting ormulation that can be administered every 3 to 4 weeks. These ormulations are particularly use ul or treating poorly adherent patients. Because typical antipsychotics are antagonists at dopamine receptors, it is logical that these drugs should interact prominently with antiparkinsonian drugs that act either by

220 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

increasing synaptic dopamine concentrations (levodopa) or through direct stimulation o dopamine receptors (dopamine agonists). Antipsychotics inhibit the action o both o these drug classes, and the administration o typical antipsychotics to patients with Parkinson’s disease o ten leads to a marked worsening o parkinsonian symptoms. In addition, typical antipsychotics potentiate the sedative e ects o benzodiazepines and centrally active antihistamines. Because the latter are pharmacodynamic e ects that result rom the nonspecif c binding o typical antipsychotics to cholinergic and adrenergic receptors, the low-potency typical antipsychotics tend to mani est more pronounced sedative e ects than their high-potency counterparts.

Atypical Antipsychotic Agents

The so-called atypical antipsychotics have e f cacy and adverse e ect prof les that di er rom those o the typical antipsychotics. The nine principal atypical antipsychotics available in the United States are risperidone , clozapine , olanzapine , quetiapine , ziprasidone , aripiprazole , iloperidone , lurasidone , and asenapine . All o these drugs are more e ective than the typical antipsychotics at treating the negative symptoms o schizophrenia. Long-term treatment trials comparing typical antipsychotics to atypical antipsychotics show similar treatment e f cacy between the classes and similar rates o discontinuation due to adverse events, although the types o adverse events di er. Atypical antipsychotics cause signif cantly milder extrapyramidal symptoms than typical antipsychotics but have a much higher incidence o other adverse e ects such as metabolic dys unction, weight gain, and sedation. The atypical antipsychotics have a relatively low a f nity or D2 receptors; unlike the typical antipsychotics, their a f nity or D2 receptors does not correlate with their clinically e ective dose (Fig. 14-9). Three main hypotheses have emerged to explain this discrepancy. The 5-HT2 hypothesis states that antagonist action at the serotonin 5-HT2 receptor (see Chapter 15), or antagonist action at both 5-HT2 and D2 receptors, is critical or the antipsychotic e ect o the atypical antipsychotics. This hypothesis is based on the f nding that the US Food and Drug Administration (FDA)approved atypical antipsychotics are all high-a f nity 5-HT2 receptor antagonists. It is not clear, however, how 5-HT2 antagonism contributes to the antipsychotic e ect. The second model, the D4 hypothesis, is based on the f nding that many o the atypical antipsychotics are also dopamine D4 receptor antagonists. This model suggests that selective D4 antagonism, or a combination o D2 and D4 antagonism, is critical to the mechanism o action o the atypical antipsychotics. Quetiapine does not act as a D4 receptor antagonist, however, so the D4 hypothesis cannot account or the mechanism o action o all atypical antipsychotics. The f nal hypothesis states that the atypical antipsychotics exhibit a milder extrapyramidal adverse e ect prof le because o their relatively rapid dissociation rom the D2 receptor. As described in Chapter 2, Pharmacodynamics, the binding a f nity (Kd) o a drug is equal to the ratio o its rate o dissociation rom the receptor (ko ) to its rate o association to the receptor (kon): D

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Because o their rapid o -rates, atypical antipsychotics bind D2 receptors more transiently than typical antipsychotics do. This could allow the atypical antipsychotics to inhibit the low-level, tonic dopamine release that may occur in the mesolimbic system. However, the drugs would be displaced by a surge o dopamine, as would occur in the striatum during the initiation o movement. Thus, extrapyramidal adverse e ects would be minimized. The atypical antipsychotics comprise a structurally diverse set o drugs. Their receptor-binding prof les also di er, as summarized in the Drug Summary Table. As noted above, these agents all show combined antagonist properties at dopamine D2 and serotonin 5-HT2 receptors, and most o the drugs are also dopamine D4 receptor antagonists. Clozapine has a distinct pharmacology; it binds D1–D5 receptors and 5-HT2 receptors and it blocks 1-adrenergic, H1, and muscarinic receptors as well. Clozapine has been used therapeutically in patients who have ailed other antipsychotic drugs, whether or lack o e f cacy or intolerable adverse e ects. Clozapine has not been used as a f rst-line agent because o a small but signif cant risk o agranulocytosis (approximately 0.8% per year) and seizures. The administration o clozapine requires requent monitoring o white blood cell counts and close ollow-up. Although the atypical antipsychotics are primarily approved or use in schizophrenia and other primary psychotic disorders, they have also been used in the management o psychosis associated with Parkinson’s disease and dementia. In Parkinson’s disease, quetiapine has proved particularly use ul because it does not seem to worsen the motor eatures o the disease. The atypical agents can also be used in managing patients with dementia, although epidemiological studies have shown that this use is associated with an increased risk o stroke and cerebral vascular disease; thereore, the risks and benef ts o the therapies in this setting must be weighed care ully. A subset o the atypical antipsychotics (aripiprazole, lurasidone, and asenapine) have potent activity in blocking 5-HT7 serotonergic receptors and 2A and 2C adrenergic receptors. These drugs have clinically signif cant e ects on symptoms o mood and anxiety and are used more o ten to treat depression and bipolar disorder than to treat schizophrenia.

CONCLUSION AND FUTURE DIRECTIONS Treatments or both Parkinson’s disease and schizophrenia modulate dopaminergic neurotransmission in the CNS. In Parkinson’s disease, the degeneration o dopaminergic neurons that project to the striatum is responsible or motor symptoms, including resting tremor, rigidity, and bradykinesia. In this disease, the direct pathway—which enables movement—is understimulated, whereas the indirect pathway—which inhibits movement—is disinhibited. Pharmacologic treatment o Parkinson’s disease depends on agents that increase dopamine release or activate dopamine receptors in the caudate and putamen and thereby help restore the balance between the direct and indirect pathways. Schizophrenia is treated by inhibiting dopamine receptors at various sites in the limbic system. The pathophysiology o schizophrenia is not ully understood, and

C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission

this lack o knowledge about etiology limits rational drug development. The clinical e ectiveness o the various antipsychotic agents has provided use ul clues, however. In particular, the pharmacology o the typical antipsychotic agents has ormed the basis o the dopamine model o schizophrenia, which posits that dysregulated levels o dopamine in the brain play a role in the pathophysiology o the disease. The e ectiveness o the atypical antipsychotic agents, which a ect the unction o several di erent receptor types, has highlighted the act that the dopamine hypothesis is a simplif cation. The atypical agents represent an attractive new modality or treating schizophrenia because they have ewer extrapyramidal e ects and are more e ective or some disease symptoms than the typical antipsychotics. Future developments in the treatment o Parkinson’s disease and schizophrenia are ocused on creating more selective agents within the current drug classes and on better elucidating the underlying pathophysiology o the disorders. New dopamine receptor agonists with higher selectivity, particularly those that bind D1 receptors, may one day provide more e ective treatment or Parkinson’s disease with less severe adverse e ects. The development o newer antipsychotics with increased receptor selectivity may similarly expand the therapeutic options or treating schizophrenia. Because Parkinson’s disease involves the death o dopaminergic neurons, much e ort is currently directed at neuroprotective drugs that may slow the progression o the disease. Further research into a potential role or a glutamate def cit in the pathophysiology o schizophrenia may yield new therapeutics or this disorder. For example, the development o selective glutamate receptor agonists may one day complement or even replace the use o dopamine receptor antagonists. Another important advance in the treatment o schizophrenia will likely result rom the elucidation o models or the mechanism o the atypical antipsychotics, which will allow rational development o more e ective drugs.

221

Acknowledgment We thank Joshua M. Galanter or his valuable contributions to this chapter in the First and Second Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Albin RL, Young AB, Penney JB. The unctional anatomy o basal ganglia disorders. Trends Neurosci 1989;12:366–375. (A classic article that describes the concept o “direct” and “indirect” pathways.) Connolly BS, Lang AE. Pharmacological treatment o Parkinson disease: a review. JAMA 2014;16:1670–1683. (A comprehensive, evidence-based evaluation o current therapies.) George M, Amrutheshwar R, Rajkumar RP, Kattimani S, Dkhar SA. Newer antipsychotics and upcoming molecules or schizophrenia. Eur J Clin Pharmacol 2013;69:1497–1509. (A review o recently approved antipsychotic agents.) Goldman JG, Weintraub D. Advances in the treatment o cognitive impairment in Parkinson’s disease. Mov Disord 2015;30:1471–1489. (A review o current treatments and uture therapeutic opportunities and challenges.) Howes OD, Kambeitz J, Kim E, et al. The nature o dopamine dys unction in schizophrenia and what this means or treatment. Arch Gen Psychiatry 2012;69:776–786. (A review o the evidence or dopamine dys unction in schizophrenia, emphasizing the presence o presynaptic de ects.) Kalia LV, Kalia SK, Lang AE. Disease-modi ying strategies or Parkinson’s disease. Mov Disord 2015;30:1442–1450. (A review o current clinical trials o disease-modi ying therapies or Parkinson’s disease.) Naber D, Lambert M. The CATIE and CUtLASS studies in schizophrenia: implications or clinicians. CNS Drugs 2009;23:649–659. (Discusses the major trials comparing typical and atypical antipsychotics on the basis o e f cacy, adverse e ect prof les, and cost.) Suchowersky O, Reich S, Perlmutter J, et al. Practice parameter: diagnosis and prognosis o new onset Parkinson disease (an evidence-based review). Report o the Quality Standards Subcommittee o the American Academy o Neurology. Neurology 2006;66:968–975. (This “parameter,” as well as several others published in the same issue, represents the product o a care ul review o the evidence or the e ectiveness o various treatments or Parkinson’s disease.) Thenganatt MA, Jankovic J. Parkinson disease subtypes. JAMA Neurol 2014;4:499–504. (A comprehensive literature review o the spectrum o eatures seen in Parkinson disease.) Trinh J, Farrer M. Advances in the genetics o Parkinson disease. Nat Rev Neurol 2013;8:445–454. (A review o the rapidly evolving genetics o Parkinson’s disease.)

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C h a p t e r 1 4 Pharmacology of Dopaminergic Neurotransmission 223

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226 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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IINTRODUCTION N & CA AS S5H ET. . . . . . . . . . . . . . . . . . . . . . . . . . . . 227–228 –2 5HT BIOCHEMISTRY AND D PHYSI PHYSIOLO OLOGY O LOGY OF SEROTON SEROTONERGIC ROT RO OT O T GIC IC C 5H 5HT HT T AND CENTRAL ADRENER ERG ERG RGIC NEUROTRANSMISSION ANSMIS NSM . . . . . . . 228 2 MAOI MA AOI O Serotooni ninn Sy Synt nthe hesi e ssis esi is and and Re R Regu eeggu gula lati tion on . . . . . . . . . . . . . . . . . . . . . 228 2 Serotonin ceepto e pt ptorrs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 3 5HT T1BB re ce ptor Rece 5HT PATHOPHYSIOL OGY OF F AFFE A AFF AFFECTIVE AFFECTIV AFFECTI E DISORDERS D DE . . . . . . . . . . . . 231 3 (a u tore ore ce ptor) LOGY 5-hydroxyindole 5 hydro ydroxyind d xyin i d l Clinical Charaacteristics C cteristics ccss of Affe Affecttive Affec t Disorders . . . . . . . . . . 231 23 a ce ta lde hhyde d The M Th T Monoamine i Th Theory off Depression D . . . . . . . . . . . . . . . . . . 233 2 Limitations of the Monoamine Theoryy . . . . . . . . . . . . . . . . . 233 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 233 Inhibitors of Serotonin Storage . . . . . . . . . . . . . . . . . . . . . . . . . 234 Inhibitors of Serotonin Degradation . . . . . . . . . . . . . . . . . . . . . 234

INTRODUCTION This chapter introduces the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT), which is a target or many drugs used to treat psychiatric disorders related to depression and anxiety. Some o these medications also a ect norepinephrine (NE) neurotransmission, and both neurotransmitter pathways are believed to be central to the modulation o mood. The various mechanisms by which drugs can alter serotonin and norepinephrine signaling are discussed. Although many such drugs unction as antidepressants or anti-anxiety medications, interventions in this pharmacologic group are also e ective treatments or migraine headache, irritable bowel syndrome, and other conditions. Lithium and some other drugs used to treat bipolar disorder are also discussed. The major mood disorders are def ned by the presence o depressive and/or manic or hypomanic episodes. Patients with recurrent depressive episodes and no history o mania or hypomania are said to have major depressive disorder (MDD); patients who have experienced at least one manic or hypomanic episode, with or without an additional history o depressive episodes, are said to have bipolar disorder (BD). The li etime prevalence o MDD is approximately 17%, whereas that o BD is 1% to 2%. MDD can occur as

Reuptake Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2355 Tric Tr icyc y liic An yc Anti tide depr e pr preessa e s s nts (T ( CAs)) . . . . . . . . . . . . . . . . . . . . . 236 Sele Se lect ctiv ive Serotonin Reuptake Inhibitors (SSR RIs)) . . . . . . . . 23 2366 Serotonin-Norepine phri hrine ne Reu eupt ptak akee In I hibitors (SNRIs). . . 237 Norepinephrine-Selective Reuptake Inhibitors (NRIs)) . . . 23 2377 Atypical Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Serotonin Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 Serotonin Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . 238 Mood Stabilizers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 Lithium m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 240

Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 an isolated illness or can be comorbid with other diseases such as stroke, dementia, diabetes, cancer, and coronary artery disease. Although twin studies suggest that up to 1/3 o the risk or MDD is heritable, environmental stress such as early traumatic experiences are also associated with risk. Aging and cerebral atherosclerosis are also associated with late-onset depression in the elderly. In addition to genetic and environmental triggers, many classes o drugs can precipitate or exacerbate depressive episodes (e.g., inter eron, glucocorticoids, and chemotherapeutic agents). BD has a particularly strong heritable risk, even though environmental actors are o ten triggers or the mood episodes themselves. Although mania is a characteristic o BD, patients spend signif cant periods o their lives depressed, and depressive symptoms are strongly associated with the elevated risk or suicide among mood disorders. (O note, in the majority o suicides, a physician [not necessarily a psychiatrist] will have seen the patient less than 1 month be ore the suicide.) Both MDD and BD are major causes o morbidity worldwide, resulting in lost productivity and substantial use o medical resources. The World Health Organization (WHO) projects that a major depression will be the leading cause o disease burden by the year 2030, ahead o ischemic heart disease, road tra f c incidents, and cerebrovascular disease. 227

228 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Mary R is a 27-year-old o f ce worker who pre se nts to her prim a ry care phys ician, Dr. Le e , w ith a n 8-lb . w e ig h t lo s s ove r th e p re vio u s 2 m o n th s . Ms . R te a r u lly explains that she is plagued by near-coneelings o sadness and by a sense o helplessstant eeling ness and inadequacy at work. She eels so terrible that she has not had a good night o sleep in m ore than a m onth. She no longer enjoys living and has recently becom e scared wh en n ew tho ughts o s uicide e nte r her m ind. Ms. R tells Dr. Lee that she had elt like this once be ore, but it had pass ed a ter several m onths. Dr. Lee asks her about her sleep patterns, appetite levels, ability to concentrate, energy level, m ood, interest level, and eelings o guilt. He asks her specif c questions about thoughts o suicide, particularly whether she has orm ed a specif c plan and whether she has ever attem pted suicide. Dr. Lee explains to Ms. R that s he has m a jor depre ss ive dis orde r, likely ca us e d by specif c abnorm alities in the unction o her brain circuitry, and he prescribes the antidepressant uoxetine. Tw o w e e ks la te r, Ms . R ca lls to in d ica te th a t th e m e d icin e is n o t w o rkin g . Dr. Le e e n co u ra g e s h e r to co n tin u e ta kin g th e m e d icin e , a n d a te r 2 m o re wee ks , Ms . R b eg in s to e el b etter. S h e no lo n g er eels s a d a n d d e m o ra lize d ; th e e e lin g s o h e lp le s s n e s s

BIOCHEMISTRY AND PHYSIOLOGY OF SEROTONERGIC AND CENTRAL ADRENERGIC NEUROTRANSMISSION Serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) have critical roles in modulating mood, the sleep–wake cycle, motivation and reward, cognitive processing, pain perception, neuroendocrine unction, and other physiologic processes. Serotonergic projections to the spinal cord modulate pain perception, visceral regulation, and motor control, while projections to the orebrain are important in modulating mood, cognition, and neuroendocrine unction. The noradrenergic system modulates vigilance, stress responses, neuroendocrine unction, pain control, and sympathetic nervous system activity. The wide variety o behavioral and psychological processes regulated by these two neurotransmitters explains the similarly wide variety o disorders that can be treated by medications that alter the levels or postsynaptic signaling o 5-HT and/or NE. 5-HT and NE are primarily released rom nonsynaptic neuronal varicosities. Unlike synapses, which orm tight contacts with specif c target neurons, varicosities release large amounts o neurotransmitter rom vesicles into the extracellular space, establishing concentration gradients o neurotransmitter in the projection areas o the varicosities. 5-HT-containing cells within the raphe nuclei and NE-containing cells within the locus ceruleus project broadly throughout the cerebral cortex, while dopamine has a more ocused pattern o projections. Each o these systems has prominent presynaptic autoreceptors that control local transmitter concentrations. This autoregulation results in coordinated f ring, which causes spontaneous

a n d in a d e q u a cy th a t p re vio u s ly p la g u e d h e r h a ve d im in is h e d . In a ct, w h e n s h e re tu rn s to s e e Dr. Le e 6 w e e ks la te r, s h e re p o rts e e lin g m u ch b e tte r. S h e n o lo n g e r n e e d s m u ch s le e p a n d is a lwa ys u ll o e n e rg y. S h e is n o w co nvin ce d th a t s h e is th e m o s t in te llig e n t p e rs o n in h e r co m p a ny. S h e p ro u d ly te lls Dr. Le e th a t s h e h a s re ce n tly p u rch a s e d a n e w s p o rts ca r a n d g o n e o n a la rg e s h o p p in g s p re e . A te r ta kin g a m o re d e ta ile d h is to ry, Dr. Le e te lls Ms . R th a t s h e m a y b e h a vin g a m a n ic e p is o d e a n d , in co n s u lta tio n w ith a p s ych ia tris t, p re s crib e s lith iu m a n d g ra d u a lly ta p e rs th e u oxe tin e . Ms . R is h e s ita n t to ta ke th e n e w m e d ica tio n , a rg u in g th a t s h e e e ls f n e a n d th a t s he is con cern ed abo ut the ad ve rs e e ects o lithium .

Questions 1 . How is a depressive episode di erent rom occasionally “ eeling blue”? 2 . What caused Ms. R’s mania? Why is it necessary to treat bipolar disorder i the patient “ eels good”? 3 . Why is there a delay in the onset o f uoxetine’s therapeutic e ect? 4 . What speci c concerns might Ms. R have about the adverse e ects o lithium?

and synchronous waves o activity that can be measured as f ring requencies; or example, the cells within the raphe nuclei usually f re at rates between 0.3 and 7 spikes per second. Because the requency o basal (tonic) f ring does not change rapidly and the quanta o neurotransmitter released with each discharge are airly well conserved, the neurotransmitter concentration in the vicinity o the varicosities is maintained within a narrow range. The mean concentration establishes the baseline tone o activity in the target neurons that receive 5-HT and NE projections. In addition, specif c stimuli can elicit rapid bursts o f ring that are superimposed on the baseline tonic activity. Di usely projecting systems can thus provide two types o in ormation: a rapid and discrete neuronal f ring akin to more traditional neurotransmission and a slower tonic f ring that presumably allows or integration o in ormation over a longer period o time.

Serotonin Synthesis and Regulation Serotonin is synthesized rom the amino acid tryptophan by the enzyme tryptophan hydroxylase (TPH), which converts tryptophan to 5-hydroxytryptophan. Aromatic L-amino acid decarboxylase then converts 5-hydroxytryptophan to serotonin (Fig. 15-1A). These enzymes are present throughout the cytoplasm o serotonergic neurons, both in the cell body and in cell processes. Serotonin is concentrated and stored within vesicles located in axons, cell bodies, and dendrites. The biochemistry o norepinephrine synthesis and regulation is discussed in Chapter 11, Adrenergic Pharmacology. For review, the synthesis o norepinephrine is summarized in Figure 15-1B.

C h a p t e r 1 5 Pharmacology o Serotonergic and Central Adrenergic Neurotransmission

A

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OH 5-Hydroxytryptophan

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Aromatic L-amino acid decarboxylase

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229

The serotonin metabolic cycle (Fig. 15-2) involves synthesis, uptake into synaptic vesicles, exocytosis, reuptake into the cytoplasm, and then either uptake into vesicles or degradation. The metabolic cycle o norepinephrine is summarized in Figure 15-3. Importantly, regulation o the levels o 5-HT and NE neurotransmission can occur at any o these steps. For all monoamines, the f rst synthetic step is rate-limiting. Thus, 5-HT synthesis is rate-limited by tryptophan hydroxylase (TPH), and DA and NE synthesis is rate-limited by tyrosine hydroxylase (TH). Both enzymes are tightly regulated by inhibitory eedback via autoreceptor-mediated signaling. 5-HT presynaptic autoreceptors respond to locally increased 5-HT concentrations by Gi protein signaling, which decreases TPH activity and serotonergic neuron f ring. Although other explanations exist, this autoregulatory loop could be one explanation or the observed time course o clinical action o antidepressants, which is discussed below (see “The Monoamine Theory o Depression”). 5-HT is transported into vesicles by the vesicular monoamine transporter (VMAT). The transporter is a nonspecif c monoamine transporter that is important or the vesicular packaging o dopamine (DA) and epinephrine (EPI) as well

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FIGURE 15-1. Synthesis of serotonin and norepinephrine. A. 5-Hydroxytryptamine (serotonin) is synthesized rom the amino acid tryptophan in two steps: the hydroxylation o tryptophan to orm 5-hydroxytryptophan by tryptophan hydroxylase and the subsequent decarboxylation o this intermediate to produce 5-hydroxytryptamine (5-HT) by aromatic L-amino acid decarboxylase. Tryptophan hydroxylase is the rate-limiting enzyme in this pathway. B. Norepinephrine is synthesized rom the amino acid tyrosine in a three-step process similar to the synthetic pathway or serotonin. Tyrosine is f rst oxidized to L-DOPA by the enzyme tyrosine hydroxylase and then decarboxylated to dopamine. A ter dopamine is transported into the synaptic vesicle, it is hydroxylated by the enzyme dopamine -hydroxylase to orm norepinephrine. The same enzyme decarboxylates 5-hydroxytryptophan and L-DOPA; it is known generically as aromatic L-amino acid decarboxylase. Tyrosine hydroxylase is the rate-limiting enzyme in this pathway.

5HT 5HT

FIGURE 15-2. Presynaptic regulation of serotonin neurotransmission. Serotonin (5-HT) is synthesized rom tryptophan in a two-reaction pathway: the rate-limiting enzyme is tryptophan hydroxylase. Both newly synthesized 5-HT and recycled 5-HT are transported rom the cytoplasm into synaptic vesicles by the vesicular monoamine transporter (VMAT). Neurotransmission is initiated by an action potential in the presynaptic neuron, which eventually causes synaptic vesicles to use with the plasma membrane in a Ca 2 -dependent manner. 5-HT is removed rom the synaptic cle t by a selective 5-HT transporter (SERT) as well as by nonselective reuptake transporters (not shown). 5-HT can stimulate 5-HT1B autoreceptors on the presynaptic membrane to provide eedback inhibition. Cytoplasmic 5-HT is either sequestered in synaptic vesicles by VMAT or degraded by mitochondrial monoamine oxidase (MAO).

230 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

monoamines (such as tyramine) in the liver and gut. The two iso orms, MAO-A and MAO-B, di er according to substrate specif city: MAO-A oxidizes 5-HT, NE, and DA, and MAO-B pre erentially oxidizes DA. Monoamine oxidases inactivate monoamines by oxidative deamination, using a covalently attached avin adenine dinucleotide (FAD) coactor as an electron acceptor. Catechol-O-methyltransferase (COMT) in the extracellular space is another important degradation enzyme or monoamines.

Aroma tic L-a mino a cid tra ns porte r Tyros ine

Na + Tyros ine

L-DOPA Action pote ntia l

Dopa mine DA

Serotonin Receptors

Adrenergic neuron

ATP

H+

ADP

NE tra ns porte r Na + NE

DA

Ca 2+

DA NE

H+ VMAT

α 2 (a utore ce ptor)

a dre ne rgic re ce ptor

NE

MAO NE

DOP GAL

FIGURE 15-3. Presynaptic regulation of norepinephrine neurotransmission. Norepinephrine in the synaptic vesicle is derived from two sources. First, dopamine synthesized from tyrosine is transported into the vesicle by the vesicular monoamine transporter (VMAT). Inside the vesicle, dopamine is converted to norepinephrine by dopamine -hydroxylase. Second, recycled NE is transported from the cytoplasm into the vesicle, also by VMAT (not shown). Neurotransmission is initiated by an action potential in the presynaptic neuron, which eventually causes synaptic vesicles to fuse with the plasma membrane in a Ca 2 -dependent manner. NE is removed from the synaptic cleft by a selective norepinephrine transporter (NET) as well as by nonselective reuptake transporters (not shown). NE can stimulate 2adrenergic autoreceptors to provide feedback inhibition. Cytoplasmic NE that is not sequestered in synaptic vesicles by VMAT is instead degraded to 3,4-dihydroxyphenylglycoaldehyde (DOPGAL) by monoamine oxidase (MAO) on the outer mitochondrial membrane.

as 5-HT. Reserpine , an indole alkaloid historically used to treat hypertension and certain psychiatric symptoms, binds irreversibly to VMAT and thereby inhibits the packaging o DA, NE, EPI, and 5-HT into vesicles. Selective serotonin reuptake transporters recycle 5-HT rom the extracellular space back into the presynaptic neuron. Selective monoamine reuptake transporters are 12-transmembranespanning proteins that couple neurotransmitter transport to the transmembrane sodium gradient. Unlike VMAT, which is a nonspecif c monoamine transporter, the individual monoamine reuptake transporters show selectivity, high a f nity, and low capacity or each individual monoamine. The selective monoamine transporters, which include the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT), are also capable o transporting the other monoamines, although less e f ciently. Once 5-HT is returned to the neuronal cytoplasm, the neurotransmitter is transported into vesicles via VMAT or degraded by the monoamine oxidase (MAO) system. MAOs are mitochondrial enzymes that regulate the levels o monoamines in neural tissues and inactivate circulating and dietary

Fi teen 5-HT receptors have been characterized, and all but one are G protein-coupled (Table 15-1). In general, the 5-HT1 class o receptors inhibits cellular activity via the Gi pathway (thus decreasing adenylyl cyclase activity and opening K channels), the 5-HT2 class increases signaling through the Gq pathway to cause phosphatidylinositol turnover, and the 5-HT4, 5-HT6, and 5-HT7 classes signal through the Gs pathway to stimulate adenylyl cyclase. The only known ligand-gated ion channel is the 5-HT3 receptor. 5-HT1A receptors are expressed both on serotonergic cell bodies in the raphe nuclei (autoreceptors) and on postsynaptic neurons in the hippocampus and act to hyperpolarize neurons via the Gi pathway (as described above). Presynaptic 5-HT1B receptors are expressed on serotonergic nerve terminals, where they autoinhibit 5-HT neurotransmission. 5-HT2A and 5-HT2C signaling is excitatory and lowers the threshold or neuronal f ring. The various serotonin receptors are expressed di erentially throughout the brain and are di erentially innervated by raphe projections. For example, a subset o 5-HT projections to the cortex stimulates postsynaptic 5-HT2A receptors, while other projections to the limbic system stimulate postsynaptic 5-HT1A receptors. There is considerable overlap o receptor subtype expression, however, and the physiologic signif cance o this overlap is unclear. The signaling mechanisms o norepinephrine (adrenergic) receptor subtypes are discussed in Chapter 11 and reviewed in Table 15-1.

TABLE 15-1 Signaling Mechanisms of Serotonin and Norepinephrine Receptor Subtypes 5-HT RECEPTOR SUBTYPE SIGNALING MECHANISMS 5-HT1A,B*,D,E,F

↓ cAMP, ↑ K channel opening

5-HT2A,B,C

↑ IP3, DAG

5-HT3

Ligand-gated ion channel

5-HT4,6,7

↑ cAMP

NE RECEPTOR SUBTYPE 1

↑ IP3, DAG

2*

↓ cAMP

1,2

↑ cAMP

Abbreviations: cAMP, cyclic AMP; DAG, diacylglycerol; IP3, inositol 1,4,5trisphosphate *5-HT1B serotonin receptors and 2-adrenergic receptors are presynaptic autoreceptors important for feedback inhibition.

C h a p t e r 1 5 Pharmacology o Serotonergic and Central Adrenergic Neurotransmission

PATHOPHYSIOLOGY OF AFFECTIVE DISORDERS Major depressive disorder (MDD) and bipolar disorder (BD) are characterized by mood dysregulation. MDD is typif ed by single or recurrent depressive episodes, whereas BD is def ned by the presence o mania or hypomania as well as periods o depression. The monoamine hypothesis proposes that decreased serotonin and/or norepinephrine levels cause mood disorders, based largely on the molecular mechanism o action o known antidepressants as well as animal models suggested to correspond to depression or mania. More current research suggests that these disorders re ect complex disturbances in neural circuit activity rather than a simple chemical imbalance. However, because the underlying etiologies o these disorders are still not well understood at a physiologic or molecular level, diagnostic criteria rely solely on clinical evaluation. To date, despite intriguing f ndings rom neuroimaging and transcriptomic studies, no reliable biomarkers or these disorders have been identif ed. The American Psychiatric Association diagnostic criteria or MDD and BD are summarized in Boxes 15-1 and 15-2.

Clinical Characteristics of Affective Disorders Major depressive disorder (MDD) is characterized by single

or recurrent episodes o depressed mood, social isolation (including apathy, decreased ability to experience pleasure, and eelings o worthlessness), and characteristic somatic

231

symptoms (decreased energy, changes in appetite and sleep, muscle pain, and slowing o movement with speech latency). Episodes are sometimes precipitated by major li e events or stresses, although they may also occur spontaneously. A single depressive episode must last 2 weeks or longer and must inter ere signif cantly with the patient’s daily unctions, such as work and personal relationships. An episode is not considered to be MDD i it is due to a general medical condition such as hypothyroidism or Cushing’s disease. In all depressed patients, it is crucial to determine whether there is any suicidality and whether there is evidence o psychosis. Although psychosis is more typical o BD, severely depressed patients may become psychotic, and either suicidality or psychosis is an indication or prompt psychiatric evaluation in a secure setting. Psychotic depression is among the most severe and disabling orms o MDD. SSRIs and antipsychotics are considered f rst-line agents or this subtype o depression, but patients may require electroconvulsant therapy i the symptoms are re ractory to f rst-line agents. A manic episode is associated with irritable, elevated, or euphoric mood, as well as increased overall activity. Associated symptoms o ten include an in ated sense o sel -worth (termed grandiosity) and distractibility. Rather than speech latency and so t speech, as seen in depression, there is increased, rapid, and loud speech that is o ten di f cult to interrupt. Rather than the sense o atigue and need or sleep seen in depression, there is o ten decreased need or sleep. At the extreme, patients may not sleep at all, and rather than eeling tired, they eel energized. Manic episodes are also characterized by disorganized, racing thoughts, o ten to point where

B O X 1 5 - 1 Crite ria fo r Ma jo r De p re s s ive Dis o rd e r (MDD), a b b re via te d fro m th e Dia g n o s tic a n d S ta tis tica l Ma n u a l o f Me n ta l Dis o rd e rs , Fifth Ed itio n (DS M-5) A. Five (or more) o the ollowing symptoms have been present during the same 2-week period and represent a change rom previous unctioning; at least one o the symptoms is either (1) depressed mood or (2) loss o interest or pleasure. 1. Depressed mood most o the day, nearly every day, as indicated by either subjective report (e.g., eels sad, empty, hopeless) or observation made by others (e.g., appears tear ul). 2. Markedly diminished interest or pleasure in all, or almost all, activities most o the day, nearly every day (as indicated by either subjective account or observation). 3. Signif cant weight loss when not dieting or weight gain (e.g., a change o more than 5% o body weight in a month), or a decrease or increase in appetite nearly every day. 4. Insomnia or hypersomnia nearly every day. 5. Psychomotor agitation or retardation nearly every day (observable by others, not merely subjective eelings o restlessness or being slowed down). 6. Fatigue or loss o energy nearly every day.

7. Feelings o worthlessness or excessive or inappropriate guilt (which may be delusional) nearly every day (not merely sel -reproach or guilt about being sick). 8. Diminished ability to think or concentrate, or indecisiveness, nearly every day (either by subjective account or as observed by others). 9. Recurrent thoughts o death (not just ear o dying), recurrent suicidal ideation without a specif c plan, or a suicide attempt or a specif c plan or committing suicide. B. The symptoms cause clinically signif cant distress or impairment in social, occupational, or other important area o unctioning. C. The episode is not attributable to the physiological e ects o a substance or to another medical condition. D. The occurrence o the major depressive episode is not better explained by schizoa ective disorder, schizophrenia, schizophreni orm disorder, delusional disorder, or other specif ed and unspecif ed schizophrenia spectrum and other psychotic disorders. E. There has never been a manic episode or a hypomanic episode. ■

Reprinted with permission rom the American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Arlington, VA: American Psychiatric Association; 2013.

232 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

B O X 1 5 - 2 Crite ria fo r Bip o la r Dis o rd e r (BD), a b b re via te d fro m th e Dia g n o s tic a n d S ta tis tica l Ma n u a l o f Me n ta l Dis o rd e rs , Fifth Ed itio n (DS M-5) BIPOLAR I DISORDER

BIPOLAR II DISORDER

For a diagnosis o bipolar I disorder, it is necessary to meet the ollowing criteria or a manic episode. The manic episode may have been preceded by and may be ollowed by hypomania or major depressive episodes.

For a diagnosis o bipolar II disorder, it is necessary to meet the ollowing criteria or a current or past hypomanic episode andd the ollowing criteria or a current or past depressive episode.

Manic Episode:

A. A distinct period o abnormally and persistently elevated, expansive, or irritable mood and abnormality and persistently increased goal-directed activity or energy, lasting at least 4 consecutive days and present most o the day, nearly every day. B. During the period o mood disturbance and increased energy or activity, three (or more) o the ollowing symptoms ( our i the mood is only irritable) represent a noticeable change rom usual behavior and have been present to a signi cant degree:

A. A distinct period o abnormally and persistently elevated, expansive, or irritable mood and abnormality and persistently increased goal-directed activity or energy, lasting at least 1 week and present most o the day, nearly every day (or any duration o hospitalization is necessary). B. During the period o mood disturbance and increased energy or activity, three (or more) o the ollowing symptoms ( our i the mood is only irritable) are present to a signi cant degree and represent a noticeable change rom usual behavior: 1. Inf ated sel -esteem or grandiosity. 2. Decreased need or sleep (e.g., eels rested a ter only 3 hours o sleep). 3. More talkative than usual or pressure to keep talking. 4. Flight o ideas or subjective experience that thoughts are racing. 5. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. 6. Increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation (i.e., purposeless non-goal-directed activity). 7. Excessive involvement in activities that have a high potential or pain ul consequences (e.g., engaging in unrestrained buying sprees, sexual indiscretion, or oolish business investments). C. The mood disturbance is su ciently severe to cause marked impairment in social or occupational unctioning or to necessitate hospitalization to prevent harm to sel or others, or there are psychotic eatures. D. The episode is not attributable to the psychological e ects o a substance (e.g., a drug o abuse, a medication, other treatment) or to another medical condition.

Hypomanic Episode:

1. Inf ated sel -esteem or grandiosity. 2. Decreased need or sleep (e.g., eels rested a ter only 3 hours o sleep). 3. More talkative than usual or pressure to keep talking. 4. Flight o ideas or subjective experience that thoughts are racing. 5. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. 6. Increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation. 7. Excessive involvement in activities that have a high potential or pain ul consequences (e.g., engaging in unrestrained buying sprees, sexual indiscretion, or oolish business investments). C. The episode is associated with an unequivocal change in unctioning that is uncharacteristic o the individual when not symptomatic. D. The disturbance in mood and the change in unctioning are observable by others. E. The episode is not severe enough to cause marked impairment in social or occupational unctioning or to necessitate hospitalization. I there are psychotic eatures, the episode is, by de nition, manic. F. The episode is not attributable to the psychological e ects o a substance (e.g., a drug o abuse, a medication, other treatment). ■

Reprinted with permission rom the American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Arlington, VA: American Psychiatric Association; 2013.

patients cannot stay on topic or more than a ew seconds. While not core eatures o mania, these episodes may be associated with psychosis (delusions or hallucinations). Mania is associated with high risk or adverse outcomes (e.g., tra f c accident, arrest, or psychiatric hospitalization), particularly in the absence o treatment. When some symptoms o a manic episode and a depressive episode are present simultaneously, the depressive or manic episode is said to have “mixed eatures.”

I a patient has manic symptoms or at least 4 days without such an adverse outcome, and without causing signif cant distress to the patient, it is then by def nition a hypomanic episode (literally, a “little mania”). In the introductory case, there is insu f cient detail to determine whether Ms. R has experienced signif cant adverse consequences yet. I Dr. Lee had not intervened, her symptoms might be expected to worsen and her risk or such consequences would increase.

C h a p t e r 1 5 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission

Although BD is characterized by manic symptoms (either mania or hypomania), the disorder is also notable or depression, which may be prolonged and debilitating. The depressive episodes may occur be ore any mania is experienced, and these patients are o ten mistakenly diagnosed with MDD. Patients with BD sometimes experience rapid “switches” into mania when taking antidepressants (as in the case of Ms. R), or more frequent mood episodes referred to as rapid cycling. The drug classes used to treat BD are discussed at the end o the pharmacology section and in the past were re erred to as mood stabilizers . More recently, such drugs may be described in terms o their relative antidepressant or antimanic properties, or their ability to prevent such episodes. In many patients with BD, combinations o medications are required to achieve adequate control o mood symptoms and recurrences.

The Monoamine Theory of Depression The biological basis or depression began to be understood in the 1940s and 1950s, when keen observers noticed that imipramine , iproniazid, and reserpine had unexpected e ects on mood. In the late 1940s, the tricyclic drug imipramine was developed or use in the treatment o psychotic patients, but it was subsequently noted to have strong antidepressant e ects. Imipramine pre erentially blocks the 5-HT transporter (SERT), and its active metabolite desipramine pre erentially blocks the NE transporter (NET). By these mechanisms, imipramine allows 5-HT and NE to persist in the extracellular space at higher concentrations and or longer durations, yielding increased activation o 5-HT and NE receptors. In 1951, the antituberculosis drug iproniazid was shown to have antidepressant e ects. Iproniazid inhibits monoamine oxidase (MAO) and thereby prevents the degradation o 5-HT, NE, and DA. The resulting increase in cytosolic neurotransmitter leads to increased neurotransmitter uptake into vesicles and, consequently, to greater release o neurotransmitter a ter exocytosis. In the 1950s, the antihypertensive agent reserpine was noted to induce depression in 10–15% o patients. Researchers then ound that reserpine could induce depressive symptoms in animal models as well as in humans. Reserpine depletes 5-HT, NE, and DA in presynaptic neurons by inhibiting the transport o these neurotransmitters into synaptic vesicles. The drug binds irreversibly to VMAT and ultimately destroys the vesicles. The 5-HT, NE, and DA that accumulate in the cytoplasm are degraded by mitochondrial MAO. The resulting decrease in monoamine neurotransmission is thought to be responsible or inducing a depressed mood. The f ndings described above strongly suggested that the central monoaminergic serotonin and norepinephrine systems are involved in the pathogenesis o depression. The monoamine theory of depression holds that depression results rom pathologically decreased serotonin and/or norepinephrine neurotransmission. Based on this hypothesis, it ollows that increasing serotonin and/or norepinephrine neurotransmission could ameliorate or reverse depression. As a biological disease related to long-term pathologic alterations in monoamine activity, MDD should thus be treatable by medications.

233

Limitations of the Monoamine Theory

Although nearly all o the antidepressants are pharmacologically active at their molecular and cellular sites o action almost immediately, their ull antidepressant e ects are generally not seen until the drugs have been administered or 6 or more weeks o continuous treatment. Similarly, although reserpine rapidly depletes neurotransmitter in monoaminergic systems, it takes several weeks o continuous treatment with reserpine to induce depression. The unexplained delay in the onset o ull e ect o these drugs remains a central conundrum and strong challenge to the monoamine theory. In some patients, drugs that selectively increase 5-HT neurotransmission decrease depressive symptoms, while drugs that selectively increase NE neurotransmission have little or no e ect. In other patients, drugs a ecting the NE system are more benef cial than those a ecting the 5-HT system. Overall, each individual drug is e ective in about 70% o patients with depression, and drugs that have markedly di erent e f cacies in blocking the reuptake o NE and/ or 5-HT may have similar clinical e ectiveness when tested in large populations. These clinical observations are not easily explained by the monoamine theory. The time lag in the clinical e ectiveness o antidepressants may re ect autoregulatory mechanisms in presynaptic monoaminergic neurons and/or in postsynaptic neural circuitry. Acute treatment with antidepressants actually produces a decrease in neuronal f ring in the locus ceruleus and/or raphe nucleus (depending on the drug), due to acute eedback inhibition via 5-HT1A and 2 autoreceptors on 5-HT- and NE-containing neurons, respectively. This causes a concomitant, acute decrease in the synthesis and release o 5-HT and NE. In contrast, chronic use o antidepressants causes the inhibitory autoreceptors themselves to be down-regulated, leading to enhancement o neurotransmission. The change in autoreceptor sensitivity takes several weeks to occur, consistent with the time-course o the therapeutic response in patients. This could explain the lag in ull therapeutic response; only a ter chronic antidepressant therapy does the gradual desensitization o autoreceptors allow increased neurotransmission (Fig. 15-4). Although speculative, this hypothesis regarding changes in monoamine receptor sensitivity o ers an explanation or the delay in onset o the therapeutic action o uoxetine experienced by Ms. R. Recent research has also suggested that chronic, but not acute, antidepressant administration increases neurogenesis (i.e., the birth o new neurons) in the hippocampus and that some clinical e ects o antidepressants may be mediated by neurogenesis. Other research has implicated e ects on neurotrophic actors, such as brain-derived neurotrophic actor (BDNF). The role o neurogenesis and neurotrophic actors in mood disorders is currently an area o intense investigation.

PHARMACOLOGIC CLASSES AND AGENTS Serotonergic and central adrenergic neurotransmission are modulated by a broad range o agents that target storage, degradation, and reuptake o the neurotransmitters. Other agents target the neurotransmitter receptors. Because serotonin is involved in multiple physiologic processes, both centrally

234 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY Neurotransmitter Synthesis

Neurotransmitter Release

Postsynaptic Effect

(NE and/or 5HT)

A Be fore tre a tme nt

Pos ts yna ptic re ceptor

Low leve l of s ignaling

P res ynaptic a utore ce ptor

FIGURE 15-4. Postulated mechanism o the delay in onset o the therapeutic e ect o antidepressant medications. A. Before treatment, neurotransmitters are released at pathologically low levels and exert steady-state levels of autoinhibitory feedback. The net effect is an abnormally low baseline level of postsynaptic receptor activity (signaling). B. Short-term use of antidepressant medication results in increased release of neurotransmitter and/or increased duration of neurotransmitter action in the synaptic cleft. Both effects cause increased stimulation of inhibitory autoreceptors, with increased inhibition of neurotransmitter synthesis and increased inhibition of exocytosis. The net effect is to dampen the initial effect of the medication, and postsynaptic receptor activity remains at pretreatment levels. C. Chronic use of antidepressant medication results in desensitization of the presynaptic autoreceptors. Thus, the inhibition of neurotransmitter synthesis and exocytosis is reduced. The net effect is enhanced postsynaptic receptor activity, leading to a therapeutic response. NE, norepinephrine; 5-HT, serotonin; TCA, tricyclic antidepressant; SSRI, selective serotonin reuptake inhibitor; SNRI, serotonin-norepinephrine reuptake inhibitor.

Inhibitors o Serotonin Storage NE a nd/or 5HT tra ns porte r

B Acute tre a tme nt

Low leve l of s ignaling

TCA, SSRI, or SNRI

C Long-te rm tre a tme nt

The ra pe utic leve l of s igna ling

TCA, SSRI, or SNRI

and peripherally, pharmacologic agents that alter serotonergic signaling have diverse actions on the brain (mood, sleep, migraines), on the gastrointestinal (GI) system, and on core temperature and hemodynamics (serotonin syndrome). Many o these biological e ects are discussed as the pharmacologic agents are introduced, although the emphasis is on agents that regulate mood.

Amphetamine and related drugs inter ere with the ability o synaptic vesicles to store monoamines such as serotonin (see Chapter 11). Thus, amphetamine, methamphetamine, and methylphenidate displace 5-HT, DA, and NE rom their storage vesicles. For atypical depression and or depression in the elderly, stimulants such as amphetamine , methylphenidate , and modaf nil have proved to be use ul as second-line agents, in part because o their combined e ects on serotonin, norepinephrine, and dopamine. Amphetamine, methylphenidate, dextroamphetamine , and lisdexam etamine are also widely used in the treatment o attention-def cit hyperactivity disorder (ADHD). Although it may seem counterintuitive that a hyperactivity disorder such as ADHD could be treated by drugs that increase catecholamine levels, this f nding makes sense in light o the di ering roles o central versus peripheral NE. In the pre rontal cortex, increased NE promotes attention and higher cognitive processes, while peripheral increases in NE increase heart rate and blood pressure and can induce tremors. These drugs have substantial potential or substance abuse; because the inactive prodrug lisdexam etamine is converted relatively slowly to the active compound dextroamphetamine by ratelimiting hepatic metabolism, it may have less abuse potential than other amphetamine derivatives. Fen uramine and dex en uramine are halogenated amphetamine derivatives that are modestly selective or 5-HT storage vesicles. These drugs were used brie y in the United States or appetite suppression, but severe cardiac toxicity led to their withdrawal. Another amphetamine derivative, methylenedioxymethamphetamine (MDMA), is both a selective serotonin storage inhibitor and a 5-HT receptor ligand. It is not approved or use in medical practice but is a signif cant drug clinically due to its illicit use (as “Ecstasy”).

Inhibitors o Serotonin Degradation The major pathway or serotonin degradation is mediated by MAO; accordingly, MAO inhibitors (MAOIs) have signif cant e ects on serotonergic neurotransmission. The MAOIs are classif ed according to their specif city or the MAO-A and MAO-B isoenzymes and according to the reversibility or irreversibility o their binding. The older MAOIs are nonselective, and most older MAOIs, such as iproniazid, phenelzine , and isocarboxazid, are irreversible inhibitors. Newer

C h a p t e r 1 5 Pharmacology o Serotonergic and Central Adrenergic Neurotransmission

MAOIs, such as moclobemide , bef oxatone , and bro aromine , are selective or MAO-A and bind reversibly. Selegiline , a selective MAO-B inhibitor at low doses (see Chapter 14, Pharmacology o Dopaminergic Neurotransmission), also inhibits MAO-A at higher doses. MAOIs block the deamination o monoamines by binding to and inhibiting the FAD co actor o MAO (Fig. 15-5). By inhibiting the degradation o monoamines, MAOIs increase the 5-HT and NE available in the cytoplasm o presynaptic neurons. The increase in cytoplasmic levels o these monoamines leads not only to increased uptake and storage o 5-HT and NE in synaptic vesicles but also to some constitutive leakage o the monoamines into the extracellular space. As noted in Chapter 11, the most toxic adverse e ect o MAOI use is systemic tyramine toxicity. Because GI and hepatic MAO metabolizes tyramine, consumption o oods that contain tyramine, such as processed meats, aged hard cheeses, and red wine, can lead to excess levels o circulating tyramine. Tyramine is an indirect sympathomimetic that can stimulate the release o large amounts o stored catecholamines by reversing the reuptake transporters. This uncontrolled catecholamine release can induce a hypertensive crisis characterized by headache, tachycardia, nausea, cardiac arrhythmia, and stroke. The older MAOIs are no longer considered f rst-line therapy or depression because o the potential or systemic tyramine toxicity; they should be prescribed only to patients able to commit to a tyramineree diet. The newer MAOIs (i.e., the reversible inhibitors o MAO-A [RIMAs] that bind reversibly to MAO) are displaced by high concentrations o tyramine, resulting in signif cantly more tyramine metabolism and hence less tyramine toxicity. Selegiline has been approved as a transdermal patch, thus bypassing the GI system. Transdermal selegiline can maximally inhibit brain MAO-A (and MAO-B) at doses that reduce gastrointestinal MAO-A activity by only 30–40%, thus reducing the risk o a tyramine-induced hypertensive crisis and allowing patients greater dietary reedom. MAOIs, like other antidepressants, can precipitate manic or hypomanic episodes in some bipolar patients. All antidepressant drugs, including MAOIs, are hydrophobic and cross the blood–brain barrier. They are well absorbed orally and are metabolized to active metabolites by the liver. These metabolites are subsequently inactivated by acetylation, also in the liver. Excretion is primarily via renal clearance. The older, irreversibly binding MAOIs are cleared rom the circulation as complexes with MAO and are e ectively inactivated only when new enzyme is synthesized. Because o the extensive e ects o MAOIs on cytochrome P450 enzymes in the liver, they can cause numerous drug– drug interactions. All members o a patient’s medical team must prescribe other drugs with caution when a patient is taking an MAOI.

Reuptake Inhibitors Serotonergic tone is maintained at steady state by the balance between transmitter release and reuptake. Thus, inhibitors o the serotonin reuptake transporter decrease the reuptake rate, resulting in a net increase in the concentration o 5-HT in the extracellular space. These drugs alleviate the symptoms o a variety o common psychiatric conditions, including depression, anxiety, and obsessive-compulsive

235

A Dopa mine

Reserpine DA

Serotonin-norepinephrine reuptake inhibitors (SNRIs)

H+

Tricyclic antidepressants (TCAs)

VMAT

Na + NE

DA NE NE

MAOI NE

DOP GAL

Reserpine

B Serotonin-norepinephrine reuptake inhibitors (SNRIs)

S e rotonin 5HT

Tricyclic antidepressants (TCAs)

H+

Selective serotonin reuptake inhibitors (SSRIs)

VMAT

Na + 5HT

5HT 5H 5HT

5HT

MAOI 5HT1B re ce ptor (a utore ce ptor)

5HT

5HT

5-hydroxyindole a ce ta lde hyde 5HT 5HT

FIGURE 15-5. Sites and mechanisms o action o antidepressant drugs. The sites o action o antidepressant drugs and o reserpine (which can induce depression) are indicated in noradrenergic neurons (A) and serotonergic neurons (B). Monoamine oxidase inhibitors (MAOIs) inhibit the mitochondrial enzyme monoamine oxidase (MAO); the resulting increase in cytosolic monoamines leads to increased vesicular uptake o neurotransmitter and to increased release o neurotransmitter during exocytosis. Tricyclic antidepressants (TCAs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) inhibit both the norepinephrine transporter (NET) and the serotonin transporter (SERT), thereby increasing the levels o both NE and 5-HT in the synaptic cle t. Selective serotonin reuptake inhibitors (SSRIs) specif cally inhibit the SERT-mediated reuptake o 5-HT. TCAs, SNRIs, and SSRIs increase the duration o neurotransmitter action in the synaptic cle t, leading to increased downstream signaling. Reserpine, which can induce depression in humans and in animal models, blocks the VMAT-mediated uptake o monoamines into synaptic vesicles, which ultimately destroys the vesicles.

236 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

disorder. Four classes o reuptake inhibitors are in use: the nonselective tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and the newer norepinephrineselective reuptake inhibitors (NRIs). Each class is discussed below, ollowed by a discussion o atypical antidepressants that do not all clearly into one o these our categories.

Tricyclic Antidepressants (TCAs)

The TCAs derive their name rom their common chemical backbone, consisting o three rings that include two aromatic rings attached to a cycloheptane ring. The prototype TCA is imipramine , and other members o this class include amitriptyline , desipramine , nortriptyline , and clomipramine (which is a f rst-line agent or obsessive-compulsive disorder). TCAs with secondary amines pre erentially a ect the NE system, whereas those with tertiary amines primarily a ect the 5-HT system. Tetracyclic antidepressants, which include maprotiline , have also been developed but are not widely used. Tetracyclic antidepressants tend to be more selective or the NE system. TCAs inhibit the reuptake o 5-HT and NE rom the extracellular space by blocking 5-HT and NE reuptake transporters, respectively. These agents do not a ect DA reuptake (Fig. 15-5). The molecular mechanism o transporter inhibition remains to be elucidated. Because the increased time spent by neurotransmitter in the extracellular space leads to increased receptor activation, the reuptake inhibitors cause enhancement o postsynaptic responses. Despite widely varying a f nities or 5-HT and NE reuptake transporters, the TCAs are markedly similar in their clinical e f cacy. The TCAs are also use ul or treating pain syndromes and are o ten used or this indication at lower doses than those needed to produce antidepressant e ects. They are particularly use ul in the treatment o migraine headaches, other somatic pain disorders, and chronic atigue syndrome. The adverse e ect prof le o TCAs results rom their ability to bind a number o channels and receptors in addition to their therapeutic targets. The most dangerous adverse e ects o TCAs involve the cardiovascular system. TCAs a ect sodium channels in a quinidine-like manner. The quinidinelike adverse e ects o TCAs (and TCA overdose in particular) include potentially lethal conduction delays, such as rst-degree atrioventricular and bundle branch blocks. There ore, TCAs should always be prescribed with caution in patients at risk o attempting suicide, and an electrocardiogram (ECG) should be examined to rule out conduction system disease prior to starting TCAs. TCAs can also act as antagonists at muscarinic (cholinergic), histamine, adrenergic, and dopamine receptors. The anticholinergic e ects are most prominent and include symptoms typical o muscarinic acetylcholine receptor blockade: nausea, vomiting, anorexia, dry mouth, blurred vision, con usion, constipation, tachycardia, and urinary retention. The antihistaminergic e ects include sedation, weight gain, and con usion (in the elderly). The antiadrenergic e ects include orthostatic hypotension, re ex tachycardia, drowsiness, and dizziness. Orthostatic hypotension is an especially signif cant risk or elderly patients, and TCA use must be monitored care ully in such patients. Finally, TCAs may also precipitate mania in patients with BD.

Selective Serotonin Reuptake Inhibitors (SSRIs)

In 1987, the treatment o depression was revolutionized with the introduction o selective serotonin reuptake inhibitors (SSRIs). The f rst SSRI to be approved by the US Food and Drug Administration (FDA) was f uoxetine ; this drug is still one o the most widely prescribed SSRIs. Other SSRIs include citalopram, its more active S-enantiomer escitalopram, f uvoxamine , paroxetine , and sertraline . Although the e ectiveness o the SSRIs is similar to that o the TCAs or the treatment o depression, their greater sa ety in overdose and lower adverse e ect prof le has made them f rst-line agents or the treatment o depression, as well as or anxiety disorders. In particular, SSRI overdoses produce relatively benign e ects compared to the potential lethality o TCA overdoses. SSRIs are also used in the treatment o panic disorder, generalized anxiety, obsessive-compulsive disorder, and posttraumatic stress disorder (PTSD). Because o their propensity to diminish or delay orgasm, they have also been used or the treatment o premature ejaculation. The SSRIs are similar to the TCAs in their mechanism o action, except that the SSRIs are signif cantly more selective or 5-HT transporters (Fig. 15-5B). Inhibition o serotonin reuptake increases serotonin levels in the extracellular space, thereby increasing 5-HT receptor activation and enhancing postsynaptic responses. At low doses, SSRIs are believed to bind primarily to 5-HT transporters, whereas at higher doses, they can lose selectivity and also bind to NE transporters. Despite widely varying chemical structures, the SSRIs have clinical e f cacies similar to the TCAs and to one another. Thus, the choice o drug o ten depends on issues such as cost and tolerability o adverse e ects. In addition, because o the variability o individual patient responses to individual antidepressants, a patient may need to try more than one SSRI to f nd the most e ective drug: despite their common mechanism, one SSRI may be e ective a ter another one ails. Because the SSRIs are more selective or serotonin reuptake than the TCAs at clinically e ective doses, they have ar ewer adverse e ects. SSRIs lack signif cant cardiotoxicity (although one, citalopram, has rarely been associated with QTc prolongation at higher doses) and they do not bind as avidly to muscarinic (cholinergic), histamine, adrenergic, or dopamine receptors. As a consequence, SSRIs are generally better tolerated than TCAs. The enhanced selectivity o the SSRIs also means that these agents have a higher therapeutic index than the TCAs. The SSRIs are not entirely without adverse e ects, however. All SSRIs can cause some degree o sexual dys unction, diminishing libido and/or delaying orgasm. Another common adverse e ect is GI distress; sertraline is more o ten associated with diarrhea, while paroxetine is associated with constipation. A more serious adverse e ect o the SSRIs is serotonin syndrome , a rare but dangerous elevation o 5-HT levels that can occur when both an SSRI and an MAOI are administered concurrently. The clinical mani estations o serotonin syndrome include hyperthermia, muscle rigidity, myoclonus, and rapid f uctuations in mental status and vital signs. SSRIs can also cause bleeding complications in a very small percentage o patients and have occasionally been associated with hyponatremia. Abrupt withdrawal rom SSRIs can cause SSRI-discontinuation syndrome, which is characterized by anxiety, dysphoria, gastrointestinal u-like symptoms, insomnia, depersonalization, and rank suicidality.

C h a p t e r 1 5 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission

Finally, as with TCAs and MAOIs, SSRIs can sometimes cause a “switch” from depression to mania or hypomania in patients with BD. The uoxetine that Ms. R was prescribed or MDD was likely responsible or her subsequent manic episode. The mechanism o the SSRI-induced switch rom depression to mania or hypomania is unknown.

Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs)

Although SSRIs are use ul f rst-line agents or the treatment o depression, there is a signif cant patient population that does not respond, or responds only partially, to SSRIs. Also, although TCAs are o ten use ul in cases in which somatic pain is a signif cant concern, the broad receptor prof le o TCAs makes them di f cult to prescribe in medically complicated or ragile patients. A newer class o drugs, the serotonin-norepinephrine reuptake inhibitors (SNRIs), is proving to be use ul in such patients. The SNRIs presently consist o venlafaxine , its active metabolite desvenlafaxine , duloxetine , and milnacipran. Venla axine and desvenla axine block the 5-HT reuptake transporter and the NE reuptake transporter in a concentration-dependent manner; at low doses, they behave as SSRIs, but at higher doses, they also increase extracellular NE levels. Duloxetine also inhibits NE and 5-HT reuptake specif cally and has been approved or the treatment o depression as well as neuropathic pain and other pain syndromes. Milnacipran is a selective NE and 5-HT reuptake inhibitor that is approved or the treatment o f bromyalgia based on clinical trials in which it improved symptoms o pain and dysphoria.

Norepinephrine-Selective Reuptake Inhibitors (NRIs) Atomoxetine is an NE-selective reuptake inhibitor that is used

in the treatment o ADHD. It is thought to improve ADHD symptoms by blocking NE reuptake and thereby increasing NE levels in the pre rontal cortex. (Note that methylphenidate and amphetamines are also thought to improve ADHD symptoms by increasing NE levels in the pre rontal cortex, acting via increased NE release.) Atomoxetine has several advantages over the amphetamines, including a lower abuse/addiction potential and a longer plasma hal -li e that allows or once-daily dosing. Atomoxetine increases peripheral as well as central NE levels and thus increases heart rate and blood pressure.

Atypical Antidepressants Several drugs that interact with multiple targets and are indicated or the treatment o depression are sometimes re erred to as “atypical antidepressants.” These agents include bupropion, mirtazapine, and trazodone; tianeptine and agomelatine are approved in Europe but not the U.S. Most recently, intravenous ketamine has been shown to have antidepressant e ects. They are categorized together here only because they do not f t conveniently into other categories. These agents are newer than the TCAs and act by several di erent mechanisms, and some have unknown or incompletely characterized mechanisms o action. Bupropion, an aminoketone, is particularly use ul or the treatment o atypical depression. This drug appears to act mechanistically like the amphetamines, with weak occupancy o the dopamine transporter in the human brain. Its ull mechanism o action is not well understood, however, in part because o its extensive metabolism to active metabolites

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that have e ects on nicotinic acetylcholine (nACh) receptors. Bupropion is one o the antidepressants with the ewest sexual adverse e ects. The principal contraindication to the use o bupropion is a predisposition to seizures, since it lowers the seizure threshold. Thus, bupropion is generally contraindicated in patients with seizure disorders, electrolyte abnormalities, or eating disorders (since these can cause electrolyte imbalances). Mirtazapine , a tetracyclic molecule, blocks postsynaptic 5-HT2A and 5-HT2C receptors and the presynaptic 2-adrenergic autoreceptor and presumably decreases neurotransmission at 5-HT2 synapses while increasing NE neurotransmission. Mirtazapine is an e ective anxiolytic and hypnotic, as well as an orexigenic (appetite stimulant), making it a particularly use ul antidepressant or the elderly population (who o ten present with insomnia and weight loss) and or other patients with weight loss and depression. Trazodone , a phenylpiperazine, blocks postsynaptic 5-HT2A and 5-HT2C receptors while also inhibiting the serotonin transporter. It is discussed in more detail below. Tianeptine is a tricyclic antidepressant with anxiolytic e ects. Despite its structure, this drug acts as a selective enhancer (rather than inhibitor) o serotonin reuptake. The mechanism o tianeptine’s action as an antidepressant is poorly understood: possible mechanisms include e ects on neuroplasticity through increasing BDNF, e ects on glutamate receptors, and modulation o and opioid receptors. Tianeptine is currently approved or human use in Europe. Agomelatine , a structural analogue o melatonin, is an agonist at the melatonin receptors 1 and 2 and an antagonist at the 5-HT2C receptor. It has no e ect on the reuptake o monoamines and does not a ect extracellular serotonin levels, although its antagonism o 5-HT2C receptors increases dopamine and norepinephrine release. Agomelatine is well tolerated and generally lacks sexual adverse e ects. A distinguishing eature o agomelatine is its ability to modulate circadian rhythms and related physiologic e ects through its actions at melatonin receptors. Ketamine is an antagonist at glutamatergic N-methylD-aspartate (NMDA) receptors. This agent was originally developed as an anesthetic (see Chapter 17, General Anesthetic Pharmacology). It also has antidepressant e ects with onset within hours but o set by 72 hours; such e ects have been demonstrated even in patients who have ailed to respond to standard antidepressants. The e ectiveness o ketamine in treating depression challenges the monoamine theory o depression, as ketamine appears to lack a direct e ect on the serotonergic system and, mechanistically, its actions are likely to involve regulation o mTOR-mediated translational control o synaptic proteins and regulation o GSK3 , one o the putative targets o the mood stabilizer lithium. While concerns exist regarding the abuse potential o this drug and its need or repeated IV administration, better understanding o the molecular pathways a ected by ketamine may point the way to a new class o antidepressants. Overall, the atypical antidepressants have relatively ew adverse e ects and demonstrate similar clinical e f cacies despite their widely heterogeneous mechanisms o action and molecular targets. Indeed, such heterogeneity poses challenges or traditional monoaminergic models o antidepressant e ect.

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Serotonin Receptor Agonists Ergots are naturally occurring serotonin receptor agonists. Several dozen structurally similar ergots are elaborated by the rye rust ungus Claviceps purpurea. Many naturally occurring ergot alkaloids produce intense vasoconstriction by acting as agonists at serotonin receptors in vascular smooth muscle. This action was responsible or ergotism—described during the Middle Ages as “St. Anthony’s Fire”—in which consumers o ungus-in ected grains experienced severe peripheral vasoconstriction leading to necrosis and gangrene. In modern times, several ergot alkaloids have been employed clinically. The semisynthetic ergot lysergic acid diethylamide (LSD) produces hallucinations and sensory dys unction at doses as small as 50 g in humans. 5-HT receptor subtype-selective agonists have become therapeutics o increasing interest in the past decade. These agents are used primarily to treat anxiety and migraine headaches. Buspirone is a nonbenzodiazepine anxiolytic that does not bind to GABA receptors but rather acts as a 5-HT1A-selective partial agonist. It is nonsedating with moderate anxiolytic properties. Although it is o ten not as clinically e ective as other anxiety treatments such as a benzodiazepine, buspirone is nonetheless a use ul option in some patients because it is nonaddictive, does not have abuse potential, and is nonsedating. Vilazodone , a piperazine class antidepressant approved by the FDA in 2011, is an example o a newer generation o antidepressants designed to unction as combined partial agonists at the 5-HT1A receptor, which mediates negative eedback circuitry, and reuptake inhibitors at the 5-HT transporter (SERT). The dual activity at 5-HT1A and SERT is expected to enhance endogenous adaptation mechanisms leading to increased serotonergic neurotransmission. Following a similar rationale, but more mixed in its actions, vortioxetine , a bisarylsul anyl amine class antidepressant approved by the FDA in 2013, is a 5-HT1A agonist; a 5-HT1B partial agonist; an antagonist at 5-HT3, 5-HT7, and 5-HT1D receptors; and a SERT inhibitor. Whether these two drugs represent agents with truly novel mechanisms or are simply additional SSRIs with similar or di erent adverse-e ect prof les requires urther evaluation. Migraine headaches are believed to be precipitated by cerebral vasodilation with subsequent activation o small pain f bers. A class o selective serotonin agonists (5-HT1 agonists) has been ound to be particularly e ective in the treatment o migraine headaches, presumably because o their potent vasoconstrictive e ects. Sumatriptan is the prototype 5-HT1D agonist in this group, known collectively as the triptans , which also includes rizatriptan, almotriptan, frovatriptan, eletriptan, and zolmitriptan. The triptans, as well as the less selective ergot alkaloid ergotamine , act on 5-HT1 receptors in the vasculature to alter intracranial blood ow. These agents are most use ul or acute migraine attacks when taken at the onset o an episode rather than as prophylaxis. They must be taken early in a migraine (ideally, at the time o the aura) to e ectively block the activation o pain receptors. The triptans are thought to activate both 5-HT1D and 5-HT1B receptors. In the CNS, both receptor subtypes are present on presynaptic endings o a variety o neurons in the vasculature. Relatively ew 5-HT2 agonists are used clinically. Trazodone is a prodrug used in the treatment o depression and insomnia that is converted into meta-chlorophenylpiperazine (mCPP), a selective 5-HT2A/2C agonist. Trazodone is used

principally as an anxiolytic and hypnotic (sleep-inducing) agent because the higher doses required or its antidepressant e ect are usually oversedating. The ergot derivative methysergide is a partial agonist at 5-HT2 receptors that also has adrenergic and muscarinic e ects; this agent is no longer available in the United States. Serotonin and serotonin receptors are abundant in the GI tract. Serotonin is a critical regulator o GI motility, mediated in large part by 5-HT4 receptors. Cisapride , a 5-HT4 agonist that also enhances acetylcholine release rom the myenteric plexus, induces gastric motility. However, cisapride has been withdrawn in the United States due to sa ety concerns; it can cause QT prolongation and cardiac arrhythmias as a consequence o hERG K channel blockade.

Serotonin Receptor Antagonists Serotonin receptor antagonists are increasingly important therapeutics. Like many receptor ligands, these drugs show varying degrees o receptor subtype selectivity and o ten cross-react with adrenergic, histamine, and muscarinic receptors. This property can limit their clinical utility because o intolerable adverse e ects. Ketanserin is a 5-HT2A/2C antagonist with substantial 1-adrenergic antagonist activity. It reduces blood pressure to a similar degree as -blockers and has been used topically to reduce intraocular pressure in glaucoma. This drug is available in Europe. Ondansetron is a 5-HT3 antagonist. This drug is o interest because, o all the currently identif ed monoamine receptors, only 5-HT3 is an ionotropic receptor belonging to the nicotinic acetylcholine super amily o pentameric receptors. 5-HT3 receptors are expressed in the enteric nervous system, the nerve endings o the vagus, and the CNS, particularly the chemoreceptor trigger zone. Ondansetron is a potent antiemetic and is widely used as an adjunct to cancer chemotherapy and to treat re ractory nausea. As predicted by its mechanism o action, it has little e ect on nausea caused by vertigo. Irritable bowel syndrome (IBS) is believed to be a disorder o GI motility, particularly in the colon. Patients experience episodes o diarrhea, constipation, or both, with signif cant GI cramping. The 5-HT4 antagonists tegaserod and prucalopride enhance GI motility and are e ective in treating the constipation associated with IBS; tegaserod was withdrawn rom the market in 2007 because o concerns about an increased risk o myocardial in arction and stroke. Alosetron is a 5-HT3 antagonist that decreases serotonergic tone in intestinal cells, thus reducing motility. It is particularly use ul or diarrhea associated with IBS, although it carries a “black box” warning because it may cause severe ischemic colitis.

Mood Stabilizers The discovery by the Australian psychiatrist John F.J. Cade o the therapeutic e ect o lithium on “psychotic excitement” (corresponding to the modern concept o mania) in 1949 is o ten considered to be the ounding event o modern psychopharmacology. However, historians now note that over hal a century earlier, the Danish neurologist Carl Lange published studies on the treatment o “periodical depressions” with lithium, suggesting that he may more accurately be considered the ounding ather o lithium therapy or treatment o mood disorders.

C h a p t e r 1 5 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission

Notwithstanding this debate, Cade’s publication in 1949 led to the discovery (or rediscovery) o lithium’s remarkable psychopharmacological e ects, which continue to be investigated today. Pursuing an idea about the basis or psychiatric susceptibility that he had ormulated as a prisoner o war during World War II, Cade sought to understand the basis or the elevated toxicity o urine samples rom patients with mental disorders that he had observed when such samples were injected intraperitoneally into guinea pigs. In ormed by the work o the English physician Sir Alred B. Garrod, who had ormally introduced lithium salts into the materia medica or the treatment o “gouty mania” in 1859 (apparently, independently o Lange’s work), Cade ortuitously selected lithium urate, the most water-soluble orm o uric acid, to determine whether the elevated urine toxicity was due to increased levels o uric acid. Having ruled out di erences in urea levels, even though the administration o urea alone exhibited the same convulsant toxicity as whole urine, Cade observed less urea toxicity than expected a ter administering lithium urate. Subsequent investigation led him to determine that lithium had a protective e ect on urea toxicity and that, administered alone, lithium was capable o causing a reversible lethargic and sedating response in guinea pigs. Next, in a medical leap o aith, based on the rationale that the sedating e ects could be benef cial or treating psychosis, Cade went on to test lithium treatment in 10 patients with mania, 6 with dementia praecox (schizophrenia), and 3 with melancholia (depression). An eventual double-blind, placebocontrolled, discontinuation trial o lithium in BD patients by the Danish psychiatrists Poul C. Baastrup and Mogens Schou conf rmed Cade’s earlier f ndings; as they concluded in their published f ndings in 1970, “lithium is the f rst drug demonstrated as a clear-cut prophylactic agent against one o the major psychoses.” These ounding discoveries in psychopharmacology sparked intense research on the biochemical e ects o lithium and the mechanisms by which this drug exerts its antimanic e ects. Although research on lithium has provided some insights, the mechanisms responsible or its psychiatric e ects remain poorly understood. In the 1970s, some researchers considered the possibility that mania could be related to epilepsy, because both disorders exhibit episodic patterns involving neural overactivity, grossly speaking. Subsequent research did not bear out this relationship, but certain antiepileptic drugs such as carbamazepine and valproic acid were ound to have some e f cacy in the treatment o BD. Carbamazepine and valproic acid (see Chapter 16, Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System) are used to treat mania and to prevent uture mood episodes, while lamotrigine is used to prevent subsequent depressive episodes. Traditionally, the term mood stabilizer has been used to re er to lithium, valproic acid, and carbamazepine; this term is now used less o ten in the setting o a broadening pharmacopeia or bipolar disorder. In particular, antipsychotics have also been shown to be e ective or the treatment o manic episodes. More recently, some second-generation antipsychotics have demonstrated e f cacy in treating or preventing bipolar depressive episodes as well. (Some are also used in the treatment o MDD.)

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Lithium

Lithium, commonly administered as lithium carbonate, is a

monovalent cation that is similar in electrochemical properties to sodium and potassium. At therapeutic concentrations o 0.4 to 1.0 mEq/L, lithium enters cells via Na channels. Because lithium can mimic other monovalent cations, as well as the divalent cation magnesium due to its water hydration shell, it has the potential to disrupt a number o proteins and transporters that require specif c cation co actors. Lithium exerts numerous e ects at the intracellular level. Its e ect on the regeneration o inositol or second messenger signaling is particularly well studied, although this e ect is not necessarily central to its therapeutic actions. In the inositol lipid pathway, G protein-coupled receptors (such as 5-HT2 receptors) activate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to the signaling molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 signaling is terminated by conversion to inositol 4,5-bisphosphate (IP2), either directly or via an IP4 intermediate. Lithium inhibits both the inositol phosphatase that dephosphorylates IP2 to inositol phosphate (IP1) and the inositol phosphatase that dephosphorylates IP1 to ree inositol. Because ree inositol is essential or the regeneration o PIP2, lithium e ectively blocks the phosphatidylinositol signaling cascade in the brain; this is the basis or the “inositol depletion hypothesis.” Although inositol circulates reely in blood, it cannot cross the blood–brain barrier. The two mechanisms o inositol synthesis in CNS neurons—regeneration rom IP3 and de novo synthesis rom glucose-6-phosphate—are both inhibited by lithium. By blocking the regeneration o PIP2, lithium decreases central adrenergic, muscarinic, and serotonergic neurotransmission. Disruption o the phosphatidylinositol signaling cascade was previously thought to be the primary mechanism o lithium’s mood-stabilizing action. However, recent studies suggest that other actions o lithium may also be relevant. These actions include increasing 5-HT neurotransmission by enhancing neurotransmitter synthesis and release; decreasing NE and DA neurotransmission by inhibiting neurotransmitter synthesis, storage, release, and reuptake; inhibiting adenylyl cyclase by decoupling G proteins rom neurotransmitter receptors; and altering electrochemical gradients across cell membranes by substituting or Na and/or blocking K channels. Possible neurotrophic e ects o lithium are also under investigation. Recent studies indicate that lithium blocks glycogen synthase kinase 3 (GSK3) activity. GSK3 is a key enzyme involved in regulating the WNT signaling pathway, which controls adult neurogenesis and is a regulator o multiple neuroplasticity mechanisms. Growing pharmacologic and genetic evidence rom preclinical models, as well as analysis o patient-derived samples, support the likelihood that GSK3 inhibition is involved in lithium’s antimanic and antidepressant actions. Immediately upon the introduction o lithium into clinical use or treating mood disorders, it was recognized that the drug has a narrow therapeutic window (desired range o 0.4 to 1.0 mEq/L, although the optimal level remains a subject o dispute). This recognition led patients such as Ms. R to be concerned about lithium’s potential adverse e ects. Acute lithium intoxication—a clinical syndrome characterized by nausea, vomiting, diarrhea, renal ailure, neuromuscular dys unction, ataxia, tremor, con usion, delirium, and seizures—is a medical emergency that may require

240 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

dialysis or treatment. Hyponatremia or the administration o nonsteroidal anti-in ammatory drugs (NSAIDs) can lead to increased lithium reabsorption in the proximal tubule and can thereby elevate plasma lithium concentrations to toxic levels. Long-term lithium treatment has also been associated with increased risk or renal insu f ciency. Lithium’s inhibition o K entry into myocytes can lead to abnormalities in membrane repolarization, resulting in abnormal T waves on ECG. In addition, the transmembrane electrical potential is shi ted because inhibition o K entry into cells leads to extracellular hyperkalemia and intracellular hypokalemia. This shi t in transmembrane potential exposes patients to a greater risk o sudden cardiac arrest rom small changes in potassium balance. Antidiuretic hormone and thyroid-stimulating hormone both activate adenylyl cyclase, which is inhibited by lithium. By this mechanism, lithium treatment can also lead to nephrogenic diabetes insipidus , hypothyroidism, and goiter. Separate rom the tremor associated with acute lithium intoxication, long-term lithium treatment can cause a tremor consisting o involuntary rhythmic oscillation (8–12 Hz), most o ten o the hands and upper limbs at rest, that is dosedependent and nonprogressive. The pathophysiologic basis or this tremor remains to be elucidated; there is some evidence that it is both peripherally and centrally mediated. The central mechanisms may involve motor neurons in the cortex and serotonergic neurons in the brainstem. Given the wide range o adverse e ects that may accompany lithium treatment and the euphoria that may be associated with manic or hypomanic episodes, many patients are hesitant to begin treatment. Care ul serum monitoring and lithium dose titration can help to avoid some, i not all, o the adverse e ects discussed above, although this requires peripheral blood sampling on a regular basis. Despite its drawbacks, lithium remains one o the most e ective agents or treating BD. Lithium and a limited number o other mood-stabilizing drugs (see Drug Summary Table) help to prevent depressive episodes as well as mania, and lithium remains the only drug demonstrated to reduce suicide risk in patients with bipolar disorder. Ongoing preclinical research aims to identi y lithium “mimetics” with greater e f cacy, higher therapeutic index, and reduced adverse e ects through clarif cation o lithium’s therapeutically relevant mechanism o action and identif cation o adjunctive therapeutic agents that can enhance lithium’s mood-stabilizing activity.

CONCLUSION AND FUTURE DIRECTIONS This chapter discusses central monoamine neurotransmission—primarily serotonin pathways but also norepinephrine and, to a lesser extent, dopamine pathways. Serotonin is a critical mediator o mood and anxiety and is also involved in the pathophysiology o migraine headache and IBS. The ocus o the chapter is on the antidepressant class o medications. The monoamine theory o depression has been an intellectual ramework or conceptualizing the pathophysiology and treatment o MDD, although this theory is clearly an oversimplif cation. Therapy with drugs that increase synaptic concentrations o 5-HT and NE is still e ective in many cases o MDD and orms the basis o treatment

or this disorder. The delay between initiation o treatment and maximal clinical improvement may occur because o slow changes in presynaptic autoreceptor sensitivity and/or slow changes in postsynaptic neural circuitry. However, the necessity o directly modulating monoamine levels is being increasingly challenged by a subset o emerging drugs with antidepressant activity (e.g., tianeptine and agomelatine), and the monoamine theory is also challenged by agents with a more rapid albeit short-lived onset o therapeutic e ect (e.g., ketamine). TCAs, SSRIs, MAOIs, and other antidepressants have similar clinical e f cacies when tested on groups o patients, although individual patients may respond to one drug and not to another. TCAs nonselectively inhibit 5-HT and NE reuptake transporters (in addition to other receptors); SSRIs selectively block 5-HT reuptake transporters; SNRIs selectively block 5-HT and NE reuptake transporters; and MAOIs inhibit the degradation o both 5-HT and NE. The choice o antidepressant medication or an individual patient depends on the two goals o f nding an e ective agent or that patient and minimizing adverse e ects. SSRIs have become the most commonly prescribed antidepressants because o their avorable therapeutic index and are the f rst-line pharmacologic choice or treatment o MDD, anxiety, obsessivecompulsive disorder, and posttraumatic stress disorder. The mechanisms underlying e ective therapies in BD are even less well understood. Emerging research supports a role or common genetic variation in determining the risk or developing BD, although only a modest number o risk variants have been identif ed to date. Agents used to treat BD include lithium, some antiepileptics, and antipsychotics. Recent advances in drug development or the treatment o MDD have ocused on a deeper understanding o the mechanism o action o current drugs, the physiology o their molecular targets, and e orts to identi y drugs with a more rapid onset o e ect. Pharmacogenomic approaches have ailed to convincingly implicate genetic variants that a ect the likelihood o treatment response, though such e orts continue with the goal o better matching o drugs to patients by identi ying patients who are particularly likely or particularly unlikely to respond to or tolerate a specif c drug. Drug targets beyond the monoamine systems are also showing promise, including agents targeting melatonergic and glutamatergic neurotransmission. Preclinical work in rodent models is beginning to elucidate novel targets with antidepressant-like activity, which urther supports the notion o adaptive changes in neuroplasticity as key mediators o antidepressant e ects. Some o this work involves pharmacologic agents targeting epigenetic mechanisms and glucocorticoid signaling. More generally, emerging understanding o the complex but ultimately tractable genetic liability, which may cross traditional diagnostic boundaries, is likely to point the way to entirely novel treatment targets.

Acknowledgment We thank Mireya Nadal-Vicens, Jay H. Chyung, Timothy J. Turner, Miles Berger, and Bryan Roth or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

C h a p t e r 1 5 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission

Suggested Reading Beaulieu JM, Caron MG. Looking at lithium: molecular moods and complex behaviour. Mol Interv 2008;8:230–241. (Review of the likely mechanism[s] of action of lithium.) Berger M, Gray J, Roth BL. The expanded biology o serotonin. Annu Rev Med 2009;60:355–366. (Broad review of the role of serotonin in modulating physiologic processes.) Dayan P, Huys QJ. Serotonin in a ective control. Annu Rev Neurosci 2009;32:95–126. (Review of serotonergic neurotransmission from the viewpoints of evolution and computational and systems neuroscience.) Insel T, Cuthbert B, Garvey M, et al. Research domain criteria (RDoC): toward a new classif cation ramework or research on mental disorders. Am J Psychiatry 2010;167:748–751. (Overview of a new framework for advancing research on MDD, BD, and other mental disorders that aims to revolutionize diagnosis and treatment; see also http://www.ted.com/talks /thomas_insel_toward_a_new_understanding_of_mental_illness.) Krishnan V, Nestler EJ. The molecular neurobiology o depression. Nature 2008;455:894–902. (Current understanding of mood disorders and targets for new antidepressant drugs.)

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Nestler EJ. Epigenetic mechanisms o depression. JAMA Psychiatry 2014;71:454–456. (Reviews possible epigenetic etiologies of depression.) Richelson E. Pharmacology o antidepressants. Mayo Clin Proc 2001; 76:511–527. (Broad and thorough overview of the molecular mechanisms and cellular targets of first-generation antidepressant medications.) Schioldann J. History of the introduction of lithium into medicine and psychiatry: birth of modern psychopharmacology 1949. Adelaide: Adelaide Academic Press; 2009. (Review of founding experiments on the psychopharmacology of lithium.) Schloesser RJ, Martinowich K, Manji HK. Mood-stabilizing drugs: mechanisms o action. Trends Neurosci 2012;35:36–46. (Reviews primary targets of mood stabilizers and downstream molecular and cellular mechanisms of action.) Vialou V, Feng J, Robison AJ, Nestler EJ. Epigenetic mechanisms o depression and antidepressant action. Annu Rev Pharmacol Toxicol 2013;53: 59–87. (Describes the emerging role for neuroepigenetic mechanisms and chromatin-mediated neuroplasticity in the pathophysiology and treatment of MDD.)

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242 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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C h a p t e r 1 5 Pharmacology o Serotonergic and Central Adrenergic Neurotransmission 243

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248 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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INTRODUCTION N & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249–2 249–25 –250 Thha a la la mus m usD Tha PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 (s e izure ure focus focus ) PATHOPHYSIOLOGY O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Patthophysiologgy of Foc ocal al Sei eizures . . . . . . . . . . . . . . . . . . . . . . 251 Pathophysiollogy of Secondary Seconda Generalized Seizuress . . . . . . 252 Pathophysiology of Primary Generalized Seizures 253 2 PHAR PH ARMA MACO COLO LOGI GIC C CL CLAS ASSE SES S AN AND D AGEN AGENTS TS . . . . . . . . . . . . . . . . 25 2533 Drugs That Enhance Na Channel-Mediated Inhibition . . . . 254 Phenytoin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Carbamazepine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Oxcarbazepinee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Lamotrigine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Lacosamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Drugs That Enhance K Channel-Mediated Inhibition . . . . . 258 Ezogabine (Retigabine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

INTRODUCTION With over 10 billion neurons and an estimated 1014 synaptic connections, the human brain boasts unparalleled electrical complexity. Unlike myocardial tissue, where electrical signals spread synchronously through a syncytium of cells, proper functioning of the brain requires distinct isolation of electrical signals and thus demands a far higher level of regulation. Control of this complex function begins at the level of the ion channel and is further maintained through the effects of these ion channels on the activity of highly organized neuronal networks. Abnormal function of ion channels and neural networks can result in rapid, synchronous, and uncontrolled spread of electrical activity, which is the basis of a seizure . A seizure can present with a variety of symptoms and result from a variety of causes. A single seizure should be distinguished from epilepsy, which refers to the condition in which an individual has a tendency toward recurrent seizures (i.e., a patient who has had a single seizure does not

T-typ typee Caa 2+ bi Calcium . . . . . . . . . . . . . . . . . . . . 25 2588 Drugs That Inhibit iuum u Channel Etho Et E hosu suuxi ximi mide dee . . . . . . . . . . .cha . . .nne . . . l. . . . . . . . . . . . . . . . . . . . . . 258 (blocke (bl (b l k d) d) Va llp lpr pr proic rroi oiicc Acid idd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2588 Gabape bapen n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 bapentin Pregabalin lin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2588 Drugs That hat En Enhance ce GA GABA-Mediated GABA-M -M Mediated Inhibition . . . . . . . . . . 259 3 Benzodiazep epi pines nes (Diazepa (Diazepam, paam, Lorazepam, Loraz pam, Mid Midazolam, Mi Clonazepam, Clonazepam pam pa m Clo Clobazam) Clobaz baza ) . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Barbiturates (Phenobarbital). . . . . . . . . . . . . . . . . . . . . . . . . 259 Vigabatrinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Drugs That Inhibit Glutamate Receptors . . . . . . . . . . . . . . . . . 259 Felbamatee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 Ruf namidee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 Perampanel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 Drugs with Mechanisms Under Investigation. . . . . . . . . . . . . 260 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 260 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .260

necessarily have epilepsy). Seizure symptoms vary according to the location of seizure activity and may include prominent motor symptoms and loss of consciousness (as seen in tonic– clonic seizures), paroxysmal alterations in nonmotor functions (e.g., sensation, olfaction, vision), or changes in higher order functions (e.g., emotion, memory, language, insight). This chapter explores the molecular mechanisms by which the brain maintains precise control over the spread of electrical activity and how various abnormalities can undermine these physiologic mechanisms and lead to seizures. The various classes of antiepileptic drugs are then discussed, with an emphasis on molecular mechanisms for restoring inhibitory function in the brain and suppressing seizure activity.

PHYSIOLOGY The normal human brain, in the absence of any lesions or genetic abnormalities, is capable of undergoing a seizure. Acute changes in the availability of excitatory neurotransmitters 249

250 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

J o n a rrive s in th e e m e rg e n cy d e p a rtm e n t w ith h is b ro th e r Ro b a t 9:12 PM. Be ca u s e h is b ro th e r is s till to o le th a rg ic to s p e a k, J o n re la ys m o s t o th e s to ry to th e a tte n d in g p hys icia n . Th e tw o h a d b e e n wa tch in i g te le vis io n w h e n J o n n o tice d th a t h is 40-ye a r-o ld b ro th e r s e e m e d to b e d a yd re a m in g . Ne ve r m is s in g a n o p p o rtu n ity to te a s e , J o n b e ga n ch id in g h is b ro th e r o r “ s p a cin g o u t.” Bu t in s te a d o th e b o is te ro u s la u g h th a t h e wa s s o u s e d to , J o n o b s e rve d o n ly a co n u s e d , a lm o s t e a r u l s ta re . J o n re ca lls th a t h is b ro th e r’s rig h t h a n d s u d d e n ly b e ga n to b e n d in to a n a w kwa rd p o s itio n a n d th e n to s h a ke . Th e s h a kin g g re w w o rs e , p ro g re s s in g g ra d u a lly ro m th e h a n d to th e a rm a n d th e n to th e e n tire rig h t s id e o th e b o d y. J o n th e n n o tice d th a t Ro b ’s b o d y s ti e n e d , a lm o s t a s i h e w e re a tte m p tin g to co n tra ct e ve ry m u s cle in h is b o d y. Th is s u s ta in e d co n tra ctio n la s te d o r a b o u t 15 s e co n d s a n d wa s o llo w e d b y s h a kin g m o ve m e n ts o a ll o u r lim b s th a t la s te d a n o th er 30 s e co n d s o r s o . Th e re q u e n cy o th e s h a kin g s lo w e d a te r s e ve ra l m in u te s , a n d Ro b th e n b e ca m e lim p , b e ga n b re a th in g ve ry h e a vily, a n d re m a in e d u n re s p o n s ive . Ro b re ga in e d co n s cio u s n e s s o n th e wa y to th e e m e rg e n cy d e p a rtm e n t.

(e.g., caused by ingestion o the toxin domoate , which is a structural analogue o glutamate) or changes in the e ect o inhibitory neurotransmitters (e.g., caused by injection o penicillin, a GABAA antagonist) can result in massive seizure activity in the otherwise healthy human brain. These examples demonstrate that the complex circuits within the brain exist in a balance between excitatory and inhibitory actors and that changes in either category o these control mechanisms can lead to major dys unction. In the CNS, two important elements normally involved in the f ne-tuning o neuronal signaling also unction to prevent the repetitious and synchronous f ring characteristic o a seizure. At the cellular level, a “re ractory period” induced by Na channel inactivation and K channel-mediated hyperpolarization prevents abnormal repetitive f ring in neuronal cells. As discussed in Chapter 8, Principles o Cellular Excitability and Electrochemical Transmission, action potentials are propagated by voltage-sensitive ion channels. A ter initiation in the axon hillock, the action potential is propagated by alternating currents o depolarizing Na in ux and hyperpolarizing K e ux. Throughout the course o an action potential (Fig. 16-1), the Na channels exist in three distinct states: (1) a closed state be ore activation, (2) an open state during depolarization, and (3) an inactivated state shortly a ter the peak o depolarization. Because Na channels adopt the inactivated state in response to depolarization, action potentials are intrinsically sel -limiting—Na channels will not recover rom the inactivated state until the membrane is su f ciently repolarized. K channel opening repolarizes the cell, but the high K e ux transiently hyperpolarizes the membrane beyond its resting potential, urther increasing the time be ore a new action potential can be generated. Thus, under physiologic

At th e h o s p ita l, a m a g n e tic re s o n a n ce im a g in g (MRI) s ca n s h o w s a s m a ll n e o p la s m in Ro b ’s le t tem po ra l lo b e. Becau s e the n eo p las m app ears to be benign, Rob, ollowing the advice o his physician, decid e s n o t to u n d e rg o s u rg e ry. Th e p o te n tia l b e n e f ts an d ris ks o variou s antiepilep tic drug s are discus sed , in clu d in g p h e nyto in , ca rb a m a ze p in e , va lp ro ic a cid , a n d la m o trig in e , a n d it is d e cid e d to s ta rt Ro b o n a regim en o carbam azepine to prevent urther seizures.

Questions 1 . By what mechanisms can a ocal neoplasm result in a seizure? 2 . Is there any clinical signif cance to the ear ul, blank stare? 3 . What is the signif cance o the order o spread o the seizure rom the hand to the arm and then to the leg? 4 . The generalized seizure that ollowed the right-sided shaking included a tonic phase (sti ening) ollowed by a clonic phase (shaking). What occurs at the molecular level to cause these symptoms? 5 . Why was carbamazepine chosen as the antiepileptic treatment or Rob’s seizures?

conditions, the biochemical properties o Na and K channels impose a limit on the requency o f ring and help to prevent the repetitive f ring characteristic o many seizure types. Beyond the single-cell level, neural networks ensure the specif city o neuronal signaling by restricting the e ects o a given action potential to a def ned area. Even a strong train o action potentials, i contained within about 1,000 neurons, will not generate seizure activity. This is quite a remarkable eat, given the close proximity o neurons in the CNS and the act that a single neuron in the neocortex may have more than 1,000 postsynaptic connections. As seen in the simplif ed neural network in Figure 16-2, the f ring neuron activates immediately neighboring neurons in addition to interneurons that transmit inhibitory (GABA) signals to surrounding neurons. This contrast o local amplif cation and surrounding cell inhibition results in what is re erred to as surround inhibition. Surround inhibition is essential to the normal unction o the nervous system, because this phenomenon not only amplif es local signals but also provides insulation and protection against synchronicity in surrounding areas. Many seizure disorders appear to result rom disruption o this intricate balance.

PATHOPHYSIOLOGY Because the pathophysiologic mechanisms underlying seizure disorders are only beginning to be determined, seizures are in part classif ed according to their clinical mani estations. There has been a tendency to consider seizures as a dichotomous process involving the whole brain or part o the brain and re erred to as generalized or focal accordingly. This is probably an oversimplif cation. In act, a seizure can involve a neuronal network that is restricted to one

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hemisphere or that gradually or rapidly involves both hemispheres, and seizures involving both hemispheres may be asymmetric and may not involve the whole cortex. Thus, the term generalized should not be taken to mean “the whole brain” but rather to indicate that the involved neuronal network is bilaterally distributed. When seizures involve part o the brain, they are said to be focal. Depending on the location and size o the seizure ocus, there may be alteration in awareness, in which case the seizure is re erred to as focal dyscognitive or “ ocal with alteration in awareness.” (Note that this roughly corresponds to the previously termed complex partial seizure, a label that has been abandoned). In addition to helping the clinician def ne the underlying neuroanatomy, these associated symptoms have implications or the extent o disability and or def ning appropriate treatments, including surgery (Table 16-1). Whether a seizure involves one hemisphere or two, all seizures share the common characteristic o abnormal synchronous discharges. For this to occur, protective mechanisms must be compromised at the cellular and network levels. The direct causes o these changes can be primary (e.g., genetic abnormalities such as ion channel de ects), secondary (e.g., changes in the neuronal environment induced by toxins, autoantibodies, or acquired lesions such as strokes or neoplasms), or a combination o the two (e.g., ebrile seizures in children).

FIGURE 16-2. Surround inhibition prevents synchronization of adjacent neurons. In this simplif ed neuronal circuit, Neuron A sends excitatory projections (light yellow) to proximal neurons such as B. In addition to activating nearby neurons, Cell A also activates GABAergic interneurons (C) that send inhibitory projections (dark yellow) to surrounding neurons (D). This type o circuit creates an “inhibitory surround” (light brown), so that action potentials generated by Neuron A, even i rapid and robust, are unable to activate surrounding circuits.

Pathophysiology of Focal Seizures The ocal seizure (Fig. 16-3A) occurs in three specif c steps: (1) initiation at the cellular level by an increase in electrical activity, (2) synchronization o surrounding neurons, and (3) spread to adjacent regions o the brain. Seizures are initiated by a sudden depolarization within a group o neurons. This sudden change, called a paroxysmal depolarizing shift (PDS), lasts up to 200 ms and results in the generation o an abnormally rapid train o action potentials. Changes in the extracellular milieu, attributable, or instance, to a space-occupying lesion (such as in the introductory case), can have major e ects on neuronal burst activity. For example, the space-occupying lesion could cause an increase in extracellular K , which would blunt the e ects o K -mediated a ter-hyperpolarization by decreasing the magnitude o the K gradient between the outside and inside o the cell. Similarly, an increase in excitatory neurotransmitters or modulation o excitatory receptors by other exogenous molecules could increase burst activity. Increased burst activity could also result rom properties intrinsic to the cell, such as abnormal channel conductance or altered membrane characteristics. Because o surround inhibition, local discharges are o ten contained within a so-called focus and do not induce symptomatic pathology. These local discharges can be seen

252 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 16-1 Classif cation o Epileptic Seizures TYPE OF SEIZURE

SYMPTOMS/KEY FEATURES

Focal Seizures Focal seizure without altered awareness

Symptoms vary depending on location o abnormal activity in the brain: involuntary, repetitive movement (motor cortex), paresthesias (sensory cortex), f ashing lights (visual cortex), etc. Consciousness is preserved Spread to ipsilateral regions within cortex (e.g., “J acksonian march”)

Focal seizure with altered awareness

Symptoms typically result rom abnormal activity in the temporal lobe (amygdala, hippocampus) or rontal lobe Altered consciousness (cessation o activity, loss o contact with reality) O ten associated with involuntary “automatisms” ranging rom simple repetitive movements (lip smacking, hand wringing) to highly skilled activity (driving, playing musical instrument) Impaired memory o ictal phase Classically preceded by an aura

Focal seizure with secondary generalization

Initially mani ests with symptoms o ocal seizure with or without altered awareness Evolves into a tonic–clonic seizure with sustained contraction (tonic) ollowed by rhythmic movements (clonic) o all limbs Loss of consciousness Preceded by aura

Primary Generalized Seizures Absence seizure (petit mal)

Sudden, brie interruption o consciousness Blank stare Occasional motor symptoms such as lip smacking, rapid blinking Not preceded by an aura

Myoclonic seizure

Brie (1 second or less) muscle contraction; symptoms may occur in individual muscle or generalize to all muscle groups o the body (the latter can result in alling) Associated with systemic disease states such as uremia, hepatic ailure, hereditary degenerative conditions, Creutz eldt–J akob disease

Tonic–clonic (grand mal) seizure

Symptoms as described above, but onset is abrupt and not preceded by symptoms o ocal seizure

on an electroencephalogram (EEG) as sharp interictal spikes . Identif cation o these spikes can be use ul in locating the seizure ocus in a patient who is not actively undergoing a seizure. There are several pathways, however, whereby the seizure ocus can override surround inhibition. Repetitive f ring o neurons increases extracellular K . As described above, this weakens K -mediated hyperpolarization, allowing the seizure activity to spread. Rapidly f ring neurons also open depolarization-sensitive NMDA channels (see Chapter 13, Pharmacology o GABAergic and Glutamatergic Neurotransmission) and accumulate Ca2 in their synaptic terminals, both o which increase the likelihood o signal propagation and local synchronization. In many cases, it appears that the most important compromise o surround inhibition occurs at the level o GABAergic transmission. Decreases in GABA-mediated inhibition—because of exogenous factors, degeneration of GABAergic neurons, or changes at the receptor level—are major factors that aid in the synchronization of a seizure focus. I the synchronizing ocus is su f ciently strong, the abnormal, synchronized f ring rom a small neural network will begin to spread to neighboring regions o the cortex. During this spread to neighboring areas, the patient may experience an aura , a conscious “warning” o the spread o the seizure. In the introductory case, Rob’s aura mani ested as a blank, ear ul stare. Although the aura is usually stereotypical or a given patient, a wide variety o auras exist. These include a sense o ear and con usion, disturbances o memory

(e.g., déjà vu) or language, altered sensations, or an ol actory hallucination. As the seizure continues to spread, it can lead to additional clinical mani estations; the specif c mani estation depends on the brain regions that become involved. In the introductory case, the clinical symptoms initially started with shaking o the hand and progressed to the arm and then to the leg. This is a J acksonian march (named a ter the English neurologist Hughlings Jackson, who f rst described the symptoms), where the clinical symptoms result rom spread o synchronous activity across the motor homunculus.

Pathophysiology o Secondary Generalized Seizures Focal seizures may become generalized by spreading along di use connections to involve both cerebral hemispheres. This is known as a secondary (or secondarily) generalized seizure (Fig. 16-3B). Typically, seizures spread to distant sites by ollowing normal circuits, and this spread can occur through several pathways. U f bers connect various regions o the cortex; the corpus callosum allows or spread between hemispheres; and thalamocortical projections provide a pathway or di use synchronized spread throughout the brain. Once seizure activity spreads to involve both hemispheres, a patient usually loses consciousness. Among the secondarily generalized seizures, the tonic– clonic subtype is the most common. In the introductory clinical case, Rob underwent a period where he appeared to

C h a p t e r 1 6 Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System 253

This oscillatory pattern (when involving the motor cortex) results in clonic or shaking movements o the body. With time, the GABA-mediated inhibition prevails, and the patient becomes accid and remains unconscious during the postictal period until normal brain unction returns.

A Foca l s e izure S e izure focus

Pathophysiology of Primary Generalized Seizures

B S e conda ry ge ne ra lize d s e izure S e izure focus

Tha la mus

C P rima ry ge ne ra lize d s e izure

Tha la mus (s e izure focus )

FIGURE 16-3. Pathways of seizure propagation. A. In a ocal seizure, paroxysmal activity begins in a seizure ocus (purple) and spreads to adjacent areas via di use neuronal connections. When activity is conf ned to one region o the cortex that serves a basic unction, such as motor movement or sensation, and there is no change in the patient’s mental status, the seizure is re erred to as a focal seizure without altered awareness. Seizures that involve brain regions serving more complex unctions, such as language, memory, and emotions, are re erred to as focal seizures with altered awareness. B. In a secondary generalized seizure, paroxysmal activity begins in a ocus but then spreads to subcortical areas. Di use connections rom the thalamus then synchronize the spread o activity to both hemispheres. C. Primary generalized seizures, such as the absence seizure, result rom abnormal synchronization between thalamic and cortical cells (see Fig. 16-5B) or rom neuronal networks that rapidly involve the bilateral hemispheres.

be contracting every muscle in his body, ollowed by an episode o uncontrolled shaking o all our limbs. These clinical symptoms can be understood at the level o abnormal channel activity (Fig. 16-4). The initial phase o the tonic–clonic seizure is associated with a sudden loss o GABA input, which leads to a long train o f ring lasting or several seconds. This sustained, rapid f ring mani ests clinically as contraction o both agonist and antagonist muscles and is re erred to as the tonic phase. Eventually, as GABA-mediated inhibition begins to be restored, AMPA-mediated and NMDA-mediated excitation starts to oscillate with the inhibitory component.

Primary generalized seizures di er rom ocal seizures in both pathophysiology and etiology (Fig. 16-3C). In contrast to the ocal seizure, where synchronicity begins with sudden trains o action potentials within an aggregate o neurons and subsequently spreads to adjacent regions, the primary generalized seizure emanates rom central brain regions and then spreads rapidly to both hemispheres. These seizures do not necessarily begin with an aura (which is an important method o clinically distinguishing primary generalized seizures rom ocal seizures that secondarily generalize). Currently, the best understood o the primary generalized seizures is the absence seizure (also known as the petit mal seizure ). Absence seizures are characterized by sudden interruptions in consciousness that are o ten accompanied by a blank stare and occasional motor symptoms, such as rapid blinking and lip smacking. Absence seizures are thought to result rom abnormal synchronization o thalamocortical and cortical cells. The underlying pathophysiology o absence seizures is based on the observation that patients experiencing absence seizures have EEG readings somewhat similar to the patterns generated during slow-wave (stage 3) sleep. Relay neurons connecting the thalamus to the cortex exist in two di erent states depending on the level o wake ulness (Fig. 16-5A). During the awake state, these neurons unction in transmission mode , whereby incoming sensory signals are aith ully transmitted to the cortex. During sleep, however, the transient, bursting activity o a unique, dendritic T-type calcium channel alters incoming signals so that output signals to the cortex have an oscillatory f ring rate, which, on an EEG, has a characteristic “spike and wave” readout. In this slow-wave sleep state, sensory in ormation is not transmitted to the cortex. For reasons not yet understood, absence seizures are associated with activation o the T-type calcium channel during the awake state (Fig. 16-5B). This channel is active only when the cell is hyperpolarized, and several mechanisms can mediate activation o the channel during the awake state. These mechanisms include an increase in intracellular K , an increase in GABAergic input rom the reticular nucleus, or a loss o excitatory input. A variety o studies have shown that the activity o the T-type calcium channel in the relay neurons is essential to the 3-per-second spike-and-wave activity observed in absence seizures. Because o its important pathophysiologic role, the T-type calcium channel is a primary target in the pharmacologic treatment o absence seizures.

PHARMACOLOGIC CLASSES AND AGENTS The current approach to treating a patient with epilepsy depends in part on the type o seizure(s) experienced by the patient. An appropriate antiepileptic drug regimen will take into account whether a patient is having ocal seizures, with or without secondary generalization, or primary

V

)

254 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Clonic phase

Postictal phase

0 -20 -40 -60 -80

Action pote ntia ls

1 sec

M

e

m

b

r

a

n

e

vo

l

t

a

g

e

(

m

Tonic phase

vi

t

y

S odium (AMPA-R)

e

l

a

c

t

i

Chloride (GABAA-R)

C

h

a

n

n

Ca lcium (NMDA-R) Ca lcium (g Ca )

FIGURE 16-4. Abnormal channel activity in the tonic–clonic seizure. The tonic phase of the tonic–clonic seizure is initiated by a sudden loss of GABAmediated surround inhibition. Loss of inhibition results in a rapid train of action potentials, which manifests clinically as tonic contraction of the muscles. As GABAergic innervation is restored, it begins to oscillate rhythmically with the excitatory component. The oscillation of excitatory and inhibitory components manifests clinically as clonic movements. The postictal phase is characterized by enhanced GABA-mediated inhibition.

generalized seizures. In addition, or patients with ocal seizures, there is an attempt to determine whether the seizures are caused by an identif able ocal lesion that can be removed surgically or ablated by other means. Mechanistically, the e f cacy o antiepileptic drugs (AEDs) centers on manipulation o ion channel activity. As discussed above, physiologic protection against repetitive f ring occurs via inhibition at two levels: the cellular level (e.g., Na channel inactivation) and the network level (e.g., GABA-mediated inhibition). Accordingly, currently available AEDs all into f ve main categories: (1) drugs that enhance Na channel-mediated inhibition, (2) drugs that enhance K channel-mediated inhibition, (3) drugs that inhibit calcium channels, (4) drugs that enhance GABA-mediated inhibition, and (5) drugs that inhibit glutamate receptors. Although AEDs all into several di erent mechanistic classes, it is important to keep in mind that the therapeutic e f cacy o many o the AEDs is only partially explained by the known mechanisms described below, primarily because the AEDs act pleiotropically. Valproic acid, or example, stabilizes Na channels, but the drug also has an e ect on T-type calcium channels and may have e ects on GABA metabolism as well. Thus, although in vitro studies may suggest that a drug is best suited or the treatment o one particular type o seizure, other seizure types may also respond to the drug. (One benef t o this pleiotropy is that many o the drugs are interchangeable, to the extent that minimization o adverse e ects is o ten the main clinical criterion underlying the choice o AED.) The classif cation below is shown only or simplicity and is based on the primary target o the drug. A list o the drugs discussed here and their multiple mechanisms o action is provided in Table 16-2.

Drugs That Enhance Na Channel-Mediated Inhibition Each neuron in the brain is equipped with the machinery to prevent rapid, repetitive f ring. As discussed above, depolarization o the neuronal membrane results in sodium channel inactivation. This inactivation o the Na channel provides a

key checkpoint in the prevention o repetitive f ring within a potential seizure ocus. The AEDs phenytoin, carbamazepine , oxcarbazepine , lamotrigine , lacosamide , and valproic acid act directly on the Na channel (Fig. 16-6A) to increase channel inactivation, thereby enhancing inhibition at the single-cell level. In general, antiepileptic drugs that act exclusively on Na channels show strong specif city or the treatment o ocal and secondary generalized seizures. This is consistent with their molecular prof le. The Na channel blockers act in a use-dependent manner, much like the action o lidocaine on peripheral nerves (see Chapter 12, Local Anesthetic Pharmacology). Thus, neurons that f re rapidly are particularly susceptible to inhibition by this class o drug. Conversely, many Na channel blockers (particularly those that act only at the Na channel, such as phenytoin) have little e ect on absence seizures. Presumably, the thalamocortical cells activated during an absence seizure have a slow f ring rate, such that Na channel blockers do not have a use-dependent e ect on the Na channels in these cells.

Phenytoin

Phenytoin acts directly on the Na channel to slow the rate

o channel recovery rom the inactivated state to the closed state. As described above, the Na channel exists in three con ormations—closed, open, and inactivated—and the probability o a channel existing in each state depends on the membrane potential (Fig. 16-1; see also Fig. 12-7). By slowing the rate o recovery rom the inactivated state to the closed state, phenytoin increases the threshold or action potentials and prevents repetitive f ring. This has the e ect o stabilizing the seizure ocus by preventing the paroxysmal depolarizing shi t (PDS) that initiates the ocal seizure. In addition, phenytoin prevents the rapid spread o seizure activity to other neurons, accounting or its e f cacy in secondarily generalized seizures. Importantly, phenytoin targets Na channels in a usedependent manner (see Fig. 12-8). Thus, only channels that are opened and closed at high requency (i.e., those involved

C h a p t e r 1 6 Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System 255

A

1. Awa ke

2. S low-wa ve s le e p

3. Typica l a bs e nce s e izure (EEG)

EEG

S ingle s pike s

Burs ts V m 0 5

5

0

m

V

Tha la mic firing 100 ms

100 ms

T-type Ca 2+ cha nne l a ctivity

Volta ge -ga te d Na + cha nne l a ctivity

FIGURE 16-5. Mechanism of absence seizure. A. EEG recordings of paCe re bra l corte x

B

2

1

3

Tha la mus

in the PDS) are likely to be inhibited. This use-dependency lessens the e ects o phenytoin on spontaneous neuronal activity and avoids many o the adverse e ects observed with GABAA potentiators (which are not use-dependent). Because o its use-dependent blockade, as well as its ability to prevent sudden rapid f ring, phenytoin is a major drug o choice or ocal seizures and tonic–clonic seizures. It is not used in absence seizures. The complex pharmacokinetics and drug interactions o phenytoin play a decisive role in the choice between phenytoin and similarly acting drugs such as carbamazepine. Phenytoin is metabolized by the liver and, at typical doses, has a plasma hal -li e o about 24 hours. Phenytoin is also highly (95%) protein bound to albumin. Phenytoin metabolism shows properties o saturation kinetics, whereby small increases in doses above a certain level can cause large and o ten unpredictable increases in plasma drug concentration (see Chapter 3, Pharmacokinetics). These increases in plasma phenytoin concentration increase the risk o adverse

tients experiencing absence seizures are similar to “sleep spindle” patterns generated during slow-wave sleep. The 3-per-second oscillatory pattern is generated by the burst activity of a dendritic T-type calcium channel in the thalamus. 1. During the awake state, relay neurons of the thalamus are in “transmission mode,” in which incoming signals are faithfully transmitted to the cortex as single spikes. These signals to the cortex register on the EEG as small, desynchronized, low-voltage waves. 2. During slow-wave sleep, signals relayed through the thalamus are altered because of the bursting activity of a dendritic T-type calcium channel. During this stage, called burst mode, sensory information is not transmitted to the cortex. 3. Absence seizures result from abnormal activation of the T-type calcium channel during the awake state, resulting in a similar spike-and-wave EEG pattern. B. The absence seizure is generated by a self-sustaining cycle of activity between the thalamus and the cortex. Synchronicity is initiated by hyperpolarization of the thalamic relay neurons (white). This occurs normally during slowwave sleep and is caused by GABAergic input from the reticular thalamic nucleus (purple). The factors that cause hyperpolarization in relay neurons during an absence seizure are poorly understood. 1. Hyperpolarization of relay neurons induces burst activity of the T-type calcium channel, resulting in synchronous depolarization in the cortex via excitatory connections. This large depolarization in the cortex registers as a spike-and-wave pattern on the EEG. 2. Excitatory input from the cortex (light yellow ) activates the reticular thalamic neurons (dark yellow ). 3. The activated GABAergic reticular neurons hyperpolarize the thalamic relay neurons and reinitiate the cycle.

e ects, including ataxia, nystagmus, incoordination, con usion, gingival hyperplasia, megaloblastic anemia, hirsutism, acial coarsening, and a systemic skin rash. Phenytoin inactivation by the hepatic microsomal P450 enzyme system is susceptible to alteration by several drugs. Drugs that inhibit the P450 system, such as chloramphenicol, cimetidine, disulf ram, and isoniazid, increase phenytoin plasma concentration. Carbamazepine, an antiepileptic drug that induces the hepatic P450 system, increases the metabolism o phenytoin, thereby lowering phenytoin plasma concentration when these drugs are used concurrently. Similarly, phenytoin, because o its ability to induce the hepatic P450 system, increases the metabolism o drugs that are inactivated by this system. Some o these drugs include oral contraceptives, quinidine, doxycycline, cyclosporine, methadone, and levodopa.

Carbamazepine

Although structurally unrelated to phenytoin, carbamazepine appears to exert its antiseizure activity in a manner similar

256 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 16-2 Currently Known Targets of Antiepileptic Drugs

DRUG

SODIUM CHANNELS

POTASSIUM CHANNELS

T-TYPE CALCIUM CHANNELS

HIGH-VOLTAGEACTIVATED CALCIUM CHANNELS

GABA SYSTEM

GLUTAMATE RECEPTORS

Main effects on ion channels Phenytoin Carbamazepine Oxcarbazepine Lamotrigine Lacosamide Ezogabine Zonisamide Ethosuximide

Main effects on GABA mechanisms Benzodiazepines Vigabatrin Tiagabine

Main effects on glutamate mechanisms Perampanel

Mixed actions Valproic acid Gabapentin Pregabalin Levetiracetam Topiramate Felbamate Ruf namide Phenobarbital

to phenytoin. That is, carbamazepine is a Na channel blocker that slows the rate o recovery o Na channels rom the inactivated state to the closed state. This has the e ect o suppressing a seizure ocus (by preventing the PDS) as well as preventing rapid spread o activity rom the seizure ocus. A metabolite o carbamazepine, 10,11-epoxycarbamazepine, also acts to slow Na channel recovery and may be responsible or some o the therapeutic e ects o the drug. Carbamazepine is o ten the drug o choice or ocal seizures because o its dual action in the suppression o seizure oci and the prevention o spread o activity. Carbamazepine was chosen or the treatment o Rob’s seizures because his tumor was a specif c ocus or his seizure onset, and carbamazepine is likely the most e ective drug or preventing spread o activity rom that ocus. The hal -li e o

carbamazepine is initially between 10 and 20 hours and is reduced with chronic treatment (because o P450 induction), requiring patients to take several doses daily or convert to an extended-release ormulation, which remains twice daily. Metabolism o carbamazepine is linear (i.e., it exhibits f rstorder kinetics); this property makes carbamazepine a more attractive choice than phenytoin or patients with potential drug interactions. Individuals o Asian ancestry should be screened or HLA-B*1502 be ore starting carbamazepine, because the presence o HLA-B*1502 predicts a higher risk or the development o Stevens-Johnson syndrome.

Oxcarbazepine

Oxcarbazepine is structurally related to carbamazepine, also

acts via the Na channel, and has a similar clinical prof le.

C h a p t e r 1 6 Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System 257 A S e izure focus

Drug tre a tme nt

Action pote ntia ls (tra ns mis s ion inhibite d)

1 3

2

+

+

+

NMDA-R (clos e d)

+

Felbamate

+

Barbiturate or benzodiazepine Cl-

+

Los s of inhibitory s urround

GABAA cha nne l (ope n)

Gabapentin HVA Ca 2+ cha nne l (clos e d)

Phenytoin, carbamazepine, or lamotrigine

Volta ge -ga te d Na + cha nne l (ina ctiva te d)

Cl-

B

Benzodiazepine (clonazepam)

GABAA cha nne l (ope n)

Ethosuximide or valproic acid

1

T-type Ca 2+ cha nne l (blocke d)

Tha la mus (s e izure focus )

2

3

FIGURE 16-6. Mechanisms of pharmacotherapy for seizures. A. The ocal seizure (1) results rom rapid, uncontrolled neuronal f ring and a loss o surround inhibition (2). Antiepileptic drugs act at our molecular targets to enhance inhibition and prevent spread o synchronous activity (3). Barbiturates and benzodiazepines prevent seizure spread by acting on the GABAA receptor to potentiate GABA-mediated inhibition. Na channel inhibitors such as phenytoin, carbamazepine, and lamotrigine prevent rapid neuronal f ring by selectively prolonging Na channel inactivation in rapidly f ring neurons (see Figs. 12-7 and 12-8). Felbamate suppresses seizure activity by inhibiting the NMDA receptor and thereby decreasing glutamate-mediated excitation. Gabapentin decreases release o excitatory neurotransmitter by inhibiting the high-voltage-activated (HVA) calcium channel. B. The absence seizure (1) is caused by a sel -sustaining cycle o activity generated between thalamic and cortical cells (2). Antiepileptic drugs prevent this synchronous thalamocortical cycle (3) by acting at two molecular targets. Clonazepam, a benzodiazepine, potentiates GABAA channels in the reticular thalamic nucleus, thus decreasing the activation o the inhibitory reticular neurons and decreasing the hyperpolarization o the thalamic relay neurons. T-type calcium channel inhibitors such as ethosuximide and valproic acid prevent the burst activity o thalamic relay neurons that is required or synchronous activation o cortical cells.

It is rapidly converted to an active metabolite, eslicarbazepine , which was recently approved as a separate drug. Oxcarbazepine distinguishes itsel rom carbamazepine by being a somewhat less potent P450 enzyme inducer, although clinically the same considerations apply. It has a somewhat lower risk or rash and a higher risk o clinically signif cant hyponatremia.

Lamotrigine

As with the other Na channel agents, lamotrigine acts to stabilize the neuronal membrane by slowing Na channel recovery rom the inactivated state. It is hypothesized that lamotrigine may act by other undetermined mechanisms as well, because it appears to have broader clinical e f cacy.

For example, in addition to treating ocal and tonic–clonic seizures, lamotrigine is e ective in the treatment o absence seizures along with ethosuximide and valproic acid (see below). Lamotrigine also distinguishes itsel by having a relatively avorable adverse e ect prof le as evidenced by lower dropout rates among elderly patients taking lamotrigine in clinical trials. Slow titration (adjusted or P450 enzyme interactions) is required when starting lamotrigine to reduce the risk o Stevens-Johnson syndrome.

Lacosamide

Lacosamide is one o the newest antiepileptic medications

that acts via sodium channel-mediated inhibition. In vitro studies show that lacosamide enhances slow inactivation o

258 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

voltage-gated sodium channels without blocking the channel directly. It may also bind to proteins involved in neuronal growth and NMDA receptor modulation. Lacosamide has ewer drug interactions than the other Na channel agents and provides an additional treatment option or patients with drug-resistant epilepsy.

Drugs That Enhance K Channel-Mediated Inhibition An increase in potassium channel conductance leads to hyperpolarization o the cell membrane and reduced neuronal f ring. Enhancing K channel conductance is predicted to reduce the rapid, repetitive f ring associated with onset o seizure activity and provide another mechanism or treatment. The f rst seizure medication to act via this mechanism was approved in 2011.

Ezogabine (Retigabine)

Ezogabine (adopted as the name in the United States) or retigabine (the international nonproprietary name) is the f rst

o a new class o antiepileptic medications that reduces seizure activity by enhancing potassium channel conductance. Ezogabine acts at the KCNQ2/3 (Kv7.2/7.3) voltage-gated K channel to increase the M-current. It requires requent, three-times-a-day dosing and is metabolized through glucuronidation with a 6–10-hour hal -li e. It has the adverse e ect o urinary retention due to the expression o KCNQ2/3 channels in bladder smooth muscle and has also been associated with blue skin discoloration and retinal abnormalities. As a result, baseline vision testing is required prior to treatment with ezogabine, and discontinuation o the medication is recommended i there is not a marked clinical response.

Drugs That Inhibit Calcium Channels Drugs used to treat epilepsy through inhibition o calcium channels all into two main classes: those that inhibit the T-type calcium channel and those that inhibit the high-voltageactivated (HVA) calcium channel. The T-type calcium channel is depolarized and inactive during the awake state (Fig. 16-5B). In absence (petit mal) seizures, paroxysmal hyperpolarization is thought to activate the channel during the awake state, initiating the spike-andwave discharges characteristic o this seizure type. Thus, drugs inhibiting the T-type calcium channel are specif cally used to treat absence seizures. HVA calcium channels play an important role in controlling the entry o calcium into the presynaptic terminal and there ore help to regulate neurotransmitter release. The HVA calcium channel is ormed by an 1 protein that assembles into the channel pore, and it has several auxiliary subunits. Drugs that inhibit HVA calcium channels tend to have pleiotropic e ects; although they are used primarily or ocal seizures with or without secondary generalization, they can also be used or generalized seizures other than absence seizures.

Ethosuximide

In vitro, ethosuximide has a highly specif c molecular prof le. In experiments on thalamocortical preparations rom rats and hamsters, ethosuximide has been shown to reduce low-threshold T-type currents in a voltage-dependent manner. This inhibition occurs without altering the voltage

dependence or recovery kinetics o the Na channel. Ethosuximide does not have any e ect on GABA-mediated inhibition. Ethosuximide is o ten the f rst-line therapy or uncomplicated absence seizures. Consistent with its molecular prof le as a specif c T-type Ca2 channel blocker, ethosuximide is not e ective in the treatment o ocal or secondary generalized seizures.

Valproic Acid

As is the case or many other AEDs, valproic acid acts pleiotropically in vitro. Similar to phenytoin and carbamazepine, valproic acid slows the rate o Na channel recovery rom the inactivated state. At slightly higher concentrations than those necessary to limit repetitive f ring, valproic acid also limits the activity o the low-threshold T-type calcium channel. A third proposed mechanism o valproic acid action occurs at the level o GABA metabolism. In vitro, valproic acid increases the activity o glutamic acid decarboxylase, the enzyme responsible or GABA synthesis, while it inhibits the activity o enzymes that degrade GABA. Taken together, these e ects are thought to increase the availability o GABA in the synapse and thereby to increase GABAmediated inhibition. Perhaps because o its many potential sites o action, valproic acid is one o the most e ective antiepileptic drugs or the treatment o patients with generalized epilepsy syndromes having mixed seizure types. Valproic acid is also the drug o choice or patients with idiopathic generalized seizures and is used or the treatment o absence seizures that do not respond to ethosuximide. Valproic acid is also commonly used as an alternative to phenytoin and carbamazepine or the treatment o ocal seizures. Valproic acid is associated with a higher risk o major congenital mal ormation than other AEDs, and so it should be used with caution in women o childbearing age, generally a ter other agents have ailed to adequately control seizures and the associated risks have been discussed.

Gabapentin

Gabapentin was one o the f rst AEDs developed using the

concept o “rational drug design.” That is, with the recognition that GABA receptors play an important role in the control o seizure spread, gabapentin was synthesized as a structural analogue o GABA and was predicted to enhance GABA-mediated inhibition. Consistent with this hypothesis, gabapentin has been shown to increase the content o GABA in neurons and glial cells in vitro. However, the main antiseizure e ect o gabapentin appears to be through its inhibition o HVA calcium channels, which results in decreased neurotransmitter release. A main advantage o gabapentin is that, because its structure is similar to that o endogenous amino acids, it has ew interactions with other drugs. On the other hand, gabapentin appears to be less e ective than several other AEDs, and it is not generally used as a f rst-line agent.

Pregabalin

Like gabapentin, pregabalin is structurally related to GABA, but it exerts its main therapeutic e ect through inhibition o HVA calcium channels, reducing the release o several neurotransmitters including glutamate and norepinephrine. It also has e ects on substance P and calcitonin, which may contribute to its varied clinical uses. More potent than

C h a p t e r 1 6 Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System 259

gabapentin, pregabalin is a reasonable adjunctive treatment or ocal seizures. It is particularly use ul in patients with hepatic dys unction, since it is metabolized in the kidney and has ew drug–drug interactions.

Drugs That Enhance GABA-Mediated Inhibition In contrast to Na channel blockers and calcium channel inhibitors, whose mechanistic properties correlate well with their clinical activity, the enhancers o GABA-mediated inhibition have more varied e ects and tend not to be as interchangeable. This is largely because o the diversity o GABAA receptors in the brain. The GABAA receptor channel has f ve subunits, with at least two alternative splice variants o several o the subunits (see Chapter 13). There are at least 10 known subtypes o the GABAA receptor, with varying distributions o these subtypes throughout the brain. Barbiturates and benzodiazepines both increase Cl in ux through GABAA channels, but benzodiazepines act on a specif c subset o GABAA channels, whereas barbiturates appear to act on all GABAA channels. The recently approved drug vigabatrin enhances GABA-mediated activity indirectly via inhibition o GABA metabolism. These di erent mechanisms o action result in distinct clinical prof les. Drugs that nonspecif cally increase GABA content (e.g., through enhancement o synthetic pathways or reduced metabolism o GABA) tend to have a prof le similar to the barbiturates.

Benzodiazepines (Diazepam, Lorazepam, Midazolam, Clonazepam, Clobazam) Benzodiazepines increase the a f nity o GABA or the GABAA receptor and enhance GABAA channel gating in the presence o GABA, and thereby increase Cl in ux through the channel (see Chapter 13). This action has the dual e ect o suppressing the seizure ocus (by raising the threshold o the action potential) and strengthening surround inhibition. Thus, benzodiazepines such as diazepam, lorazepam, and midazolam are well suited or the treatment o ocal and tonic–clonic seizures. The benzodiazepines cause prominent adverse e ects, however, including dizziness, ataxia, and drowsiness. Thus, these drugs are typically used only to abort seizures acutely. Clonazepam is unique among the benzodiazepines in its ability to inhibit T-type Ca2 channel currents in in vitro preparations o thalamocortical circuits. In vivo, clonazepam acts specif cally at GABAA receptors in the reticular nucleus (Fig. 16-5B), augmenting inhibition in these neurons and essentially “turning o ” the nucleus. By this action, clonazepam prevents GABA-mediated hyperpolarization o the thalamus and thereby indirectly inactivates the T-type Ca2 channel, which is the channel thought to be responsible or generating absence seizures (see above). However, as with diazepam, clonazepam use is limited because o its extensive adverse e ects. Clonazepam is the ourth drug o choice in the treatment o absence seizures a ter ethosuximide, valproic acid, and lamotrigine. Clobazam is the most recently approved benzodiazepine, although it has been available or decades outside the United States. It is currently approved or adjunctive treatment o Lennox-Gastaut syndrome, a severe pediatric epilepsy syndrome with onset between 3 and 5 years o age that is characterized by developmental disability and re ractory tonic, atonic, and atypical absence seizures. Like clonazepam, it

has an elimination hal -li e o over 30 hours, so it is taken as a scheduled medication twice daily. Adverse e ects are similar to other benzodiazepines except that clobazam is also associated with an increased risk o serious skin reactions, including Stevens-Johnson syndrome.

Barbiturates (Phenobarbital)

Phenobarbital binds to an allosteric site on the GABAA re-

ceptor and thereby potentiates the action o endogenous GABA by increasing the duration o Cl channel opening. In the presence o phenobarbital, there is a much greater in ux o Cl ions or each activation o the channel (see Chapter 13). Barbiturates also display weak agonist activity at the GABAA channel, perhaps urthering the ability o this drug to increase Cl in ux. This enhancement o GABAmediated inhibition, similar to that o the benzodiazepines, may explain the e ectiveness o phenobarbital in the treatment o ocal seizures and tonic–clonic seizures. In contrast to the benzodiazepines, which are sometimes use ul in treating the spike-and-wave discharges o the absence seizure, the barbiturates may actually exacerbate this type o seizure. This exacerbation may be caused by two actors. First, barbiturates act at all GABAA receptors. Unlike benzodiazepines, which selectively augment GABA inhibition in the reticular nucleus, barbiturates potentiate GABAA receptors in both the reticular nucleus and the thalamic relay cells. Importantly, the latter e ect enhances the T-type calcium currents that are responsible or the absence seizure (Fig. 16-5B). Second, unlike benzodiazepines, which are purely allosteric enhancers o endogenous GABA activity, barbiturates can also act on the GABAA channel in the absence o the endogenous ligand. The latter property may unction to increase nonspecif c activity o the barbiturates. Phenobarbital is used primarily as an alternative drug in the treatment o ocal seizures and tonic–clonic seizures. Because o the pronounced sedative e ects o this drug, its clinical use has been decreasing as more e ective antiepileptic medications have become available.

Vigabatrin

Vigabatrin is a structural analogue o GABA that irreversibly

inhibits the enzyme GABA transaminase, thereby increasing levels o GABA in the brain (see Fig. 13-2). Serious adverse e ects, most notably peripheral visual f eld de ects, limit the clinical utility o vigabatrin. The drug is generally used or in antile spasms and re ractory ocal epilepsy. Patients treated with vigabatrin should undergo baseline and routine visual f eld testing and, even in the absence o visual changes, the drug should be discontinued within a ew months i there is not a marked clinical benef t.

Drugs That Inhibit Glutamate Receptors Glutamate is the principal excitatory neurotransmitter o the CNS (see Chapter 13). Not surprisingly, excessive activation o excitatory glutamatergic synapses is a key component o many orms o seizure activity. Numerous studies using animal models have shown that inhibition o the NMDA and AMPA subtypes o glutamate receptors can inhibit the generation o seizure activity and protect neurons rom seizure-induced injury. However, none o the specif c and potent glutamate receptor antagonists have been routinely used clinically or the treatment o seizures because o unacceptable behavioral adverse e ects.

260 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Felbamate

Felbamate has a variety o actions, including the inhibition

o NMDA receptors. It appears to have some selectivity or NMDA receptors that include the NR2B subunit. Because this receptor subunit is not expressed ubiquitously throughout the brain, NMDA receptor antagonism by elbamate is not as widespread as that with other NMDA receptor antagonists. This relative selectivity may explain why elbamate lacks the behavioral adverse e ects observed with the other agents. Benef ts o elbamate include its potency as an antiepileptic drug and its lack o the sedative e ects common to many other antiepileptic drugs. However, elbamate has been associated with a number o cases o atal aplastic anemia and liver ailure, and its use is now restricted primarily to patients with re ractory epilepsy.

Ruf namide

Ruf namide is approved or the treatment o

ocal seizures and drop attacks in Lennox-Gastaut syndrome (described above). While ruf namide acts predominantly by prolonging sodium channel inactivation, it is structurally unrelated to the other antiepileptic agents with this mechanism o action. At higher doses, it may have an inhibitory e ect on a subset o glutamate receptors (mGluR5 subtype), and it is included here based on that secondary mechanism and because its clinical prof le is most similar to elbamate. Unlike elbamate, however, ruf namide has not been demonstrated to have serious adverse e ects, and it may provide an alternative option or patients with re ractory epilepsy.

Perampanel

Perampanel is a noncompetitive antagonist at the -amino-

3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor, which is an excitatory glutamate receptor thought to play an important role in the generation and spread o seizure activity. While it was hoped that AMPA antagonists may have ewer psychomimetic adverse e ects than NMDA antagonists, perampanel increases the risk o certain psychiatric symptoms, including irritability, aggression, hostility, and homicidal ideation. Perampanel is heavily protein bound and metabolized by the P450 system; it has a long elimination hal -li e, which allows or once-daily dosing.

Drugs with Mechanisms Under Investigation Tiagabine , topiramate , zonisamide , and levetiracetam are ad-

ditional seizure medications whose mechanisms o action are somewhat less certain. Tiagabine is thought to unction by blocking GABA reuptake into presynaptic neurons, while topiramate and zonisamide likely block sodium channel activity but probably have other mechanisms as well given

their broad application or ocal and tonic–clonic seizures. Levetiracetam is believed to modulate vesicle exocytosis by binding to SV2A, a synaptic vesicle protein.

CONCLUSION AND FUTURE DIRECTIONS In recent years, improved understanding o the physiology and pathophysiology o neuronal signaling in the CNS has led to a more thorough understanding o the current antiepileptic drugs (AEDs), as well as the design and discovery o novel agents. Under physiologic conditions, Na channel inactivation and GABA-mediated surround inhibition prevent uncontrolled, rapid spread o electrical activity. There are, however, numerous potential alterations in the brain that can weaken these inhibitory orces, such as damage and degeneration o GABAergic neurons, abnormal ion gradients induced by space-occupying lesions, and gene mutations that alter channel unction. The AEDs described in this chapter restore the inherent inhibitory capacity o the brain. These include drugs such as phenytoin, which increases Na channel inactivation, and clonazepam, which enhances GABA-mediated inhibition. Newer classes o AEDs extend this repertoire by acting through modulation o the Ca2 channel required or neurotransmitter release, modulation o K channel conductance to reduce rapid f ring, and modulation o excitatory receptors such as the NMDA receptor. Despite increased understanding o the mechanisms o certain seizure types, the e f cacy o many o the antiepileptics is only partially explained by their known molecular prof les. Hence, current decisions about therapy are o ten driven by empirical example rather than by known molecular mechanisms. As a better understanding is gained o the role o genetics in not only simple inherited epilepsy but also complicated polygenic cases, the application o a more rational, mechanismbased pharmacology will become increasingly possible.

Suggested Reading Lowenstein DH. Seizures and epilepsy. In: Kasper DL, Fauci AS, Hauser SL, Longo DL, Jameson JL, Loscalzo J, eds. Harrison’s principles of internal medicine. 19th ed. New York: McGraw Hill; 2015:2542–2559. (Discussion of seizure pathophysiology and extensive discussion of clinical uses of antiepileptic drugs.) Shorvon S. Drug treatment o epilepsy in the century o the ILAE: the second 50 years, 1959–2009. Epilepsia 2009;50(Suppl 3):93–130. (A historical perspective cataloging the introduction of each therapeutic agent over time.) Westbrook GL. Seizures and epilepsy. In: Kandel ER, Schwartz JH, Jessell TM, Siegelbaum SA, Hudspeth AJ, eds. Principles of neural science. 5th ed. New York: McGraw-Hill; 2013:1116–1139. (Detailed description of normal electrical signaling and seizure pathophysiology.)

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264 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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General Anesthetic Pharmacology

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Awa wa kee P I = 00.04 004 a tm t 0 1 At 01 Atm m P I = 0 .01 p e uti u ticc INTR RODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 ROD 265– 26 5 26 2666 Effect ctss off Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .(s ubthee raa pe Effects of Abnormal Statess . . . . . . . . . . . . . . . . . . . . . . . . ra nge ) PHA AR RMACODYNAMI ODYNA NAMICS MICS C OF INHALED ANESTHETICS . . . . . . . . 26 265 65 Control of Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2777 The hhee M Minimum Mini Alveolar Al Alve lve veolar e olar Concentration (MAC) (MA ) . . . . . . . . . . . . . 266 0. 00 Th R covery . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Therapeutic annd An A algesicc In Indi dice ces5s 266 10 Re 115 5 0 Thee Me Meye yerr-Ov Overtton Ru Rule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 PHARMACOLO OGY OF GENERAL ANESTHETICS (min) (m AND ADJ UVANTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 PHARMACOKINETICS OF INHALED D AN A ESTHETICS. . . . Time .. Inhaled Anesthetic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 Conceptss from rom Re Resp spir irat ator o y Physiolo logy gy . . . . . . . . . . . . . . . . . . 269 IIntravenous A Anesthetic h i A Age g ntts . . . . . . . . . . . . . . . . . . . . . . . . . 280 280 Local Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2699 Addju juva va ntt Drugs ru . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 Global Equilibrationn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2700 Ballanc nced ed Ane nesthesia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2800 The Uptake Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Equilibration of Alveolar with MECHANISMS OF ACTION OF GENERAL AN NESTH ESTHET ETIC ICS S . . . . . 28 2811 Inspired Partial Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Effects on Ion Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 Equilibration of Tissue with CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 283 Alveolar Partial Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . 272 Su Sugg ggeste gges tedd Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28 2833 The R Th Rate-Limiting Li i i S Stepp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 Applications of the Uptake Model. . . . . . . . . . . . . . . . . . . . . . . 275 Effects of Changes in Ventilationn . . . . . . . . . . . . . . . . . . . . . 275 APPENDIX B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Effects of Changes in Cardiac Outputt . . . . . . . . . . . . . . . . . 276

INTRODUCTION Be ore the discovery o general anesthetics , pain and shock severely limited the possibilities or surgical intervention. Postoperative mortality dropped dramatically ollowing the f rst public demonstration o diethyl ether at Massachusetts General Hospital in 1846. Since then, the administration o agents or the induction and maintenance o anesthesia has become a separate medical specialty. The modern anesthesiologist is responsible or all aspects o patient health during surgery. As part o this process, the anesthesiologist controls the depth o anesthesia and maintains homeostatic equilibrium with an arsenal o inhaled and intravenous anesthetics as well as many adjuvant drugs. General anesthetics induce a generalized, reversible depression o the central nervous system (CNS). Under general anesthesia, there is a lack o perception o all sensations. The anesthetic state includes loss o consciousness, amnesia, and immobility (a lack o response to noxious stimuli) but not necessarily complete analgesia. Other desirable e ects provided by anesthetics or adjuvants during surgery may include muscle relaxation, loss o autonomic re exes, analgesia, and anxiolysis. All o these e ects acilitate sa e and painless completion o the procedure; some e ects are more important in certain types o surgery than others.

For example, abdominal surgery necessitates near-complete relaxation o the abdominal muscles, whereas neurosurgery o ten requires light anesthesia that may be li ted rapidly when the neurosurgeon needs to judge the patient’s ability to respond to commands. This chapter provides a ramework or understanding the pharmacodynamics and pharmacokinetics o general anesthetics in the context o physiologic and pathophysiologic variables. A ter discussing the pharmacology o specif c agents and how a balanced anesthetic approach is achieved, the chapter considers what is currently known about the mechanism o action o general anesthetics.

PHARMACODYNAMICS OF INHALED ANESTHETICS General anesthetics distribute well to all parts o the body, becoming most concentrated in the atty tissues. The CNS is the primary site o action o anesthetics. Most likely, loss o consciousness and amnesia ensue rom supraspinal action (i.e., action in the brainstem, midbrain, and cerebral cortex), while immobility in response to noxious stimuli is caused by depression o both supraspinal and spinal sensory and motor pathways. Di erent sites in the CNS are 265

266 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Ma tth e w is a 7-ye a r-o ld , 20-kg b o y w h o h a s b e e n u n d e rg o in g m u ltid ru g ch e m o th e ra p y o r a g g re s s ive o s te o s a rco m a o h is rig h t e m u r. Th e tim e h a s n o w co m e o r a s u rg ica l re s e ctio n . ■

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■

8:00 PM (n ig h t b e o re th e o p e ra tio n ): Dr. S n o w, th e a n e s th e s io lo g is t, p rovid e s re a s s u ra n ce a n d re vis its th e im p o rta n ce o a s tin g a te r m id n ig h t to p re ve n t a s p ira tio n o ga s tric co n te n ts w h ile u n d e r g e n e ra l a n e s th e s ia . 6:30 AM: Ma tth e w clin g s to h is m o th e r a n d a p p e a rs a n xio u s , ca ch e ctic, a n d in s o m e p a in . His vita l s ig n s a re s ta b le w ith a n e le va te d p u ls e o 120 a n d a b lo o d p re s s u re o 110/75. An o ra l d o s e o m id a zo la m (a b e n zo d ia ze p in e ; s e e Ch a p te r 13, Ph a rm a co lo g y o GABAe rg ic a n d Glu ta m a te rg ic Ne u ro tra n s m is s io n ) is g ive n to re lie ve a n xie ty a n d to a llo w Ma tth e w to s e p a ra te ro m h is p a re n ts . 7:00 AM: Dr. S n o w in je cts a s m a ll a m o u n t o lid o ca in e s u b cu ta n e o u s ly (a lo ca l a n e s th e tic; s e e Ch a p te r 12, Lo ca l An e s th e tic Ph a rm a co lo g y) b e o re in s e rtin g a n in tra ve n o u s ca th e te r (w h ich h e ca re u lly co n ce a ls ro m Ma tth e w u n til th e la s t p o s s ib le m o m e n t). Th ro u g h th e ca th e te r, Dr. S n o w d e live rs a n in u s io n o m o rp h in e s u la te (a n o p io id ; s e e Ch a p te r 18, Ph a rm a co lo g y o An a lg e s ia ) o r a n a lg e s ia . 7:30 AM: Dr. S n o w ra p id ly in d u ce s a n e s th e s ia w ith a n in tra ve n o u s b o lu s o 60 m g (3 m g /kg ) o th io p e n ta l (a b a rb itu ra te ; s e e Ch a p te r 13). With in 45 s e co n d s , Ma tth e w is in a d e e p a n e s th e tic s ta te . Th e d o cto r a d d s a d o s e o in tra ve n o u s s u ccinylch o lin e (a d e p o la rizin g m u s cle re la xa n t; s e e Ch a p te r 10, Ch o lin e rg ic Ph a rm a co lo g y) to a cilita te e n d o tra ch e a l in tu b a tio n , a n d Ma tth e w is p la ce d o n a rtif cia l re s p ira tio n . 7:32 AM: A m ixtu re o in h a le d g e n e ra l a n e s th e tics co n s is tin g o 2% is o u ra n e , 50% n itro u s oxid e ,

di erentially a ected by general anesthetics, giving rise to the classical stages observed with increasing anesthetic depth (Fig. 17-1).

The Minimum Alveolar Concentration (MAC) To control the depth o anesthesia, the anesthesiologist must control rather precisely the level o anesthetic in the CNS. This level is denoted by the partial pressure o anesthetic in the CNS, also called the CNS partial pressure , PCNS. (See Box 17-1 or a discussion o partial pressures versus concentrations and Appendix A or a glossary o abbreviations and symbols.) The anesthesiologist maintains PCNS within the desired range by varying the inspired partial pressure , PI. Because the value o PCNS cannot be monitored directly, it is commonly in erred rom the alveolar partial pressure , Palv. The alveolar partial pressure is a use ul substitute or PCNS, because PCNS tracks Palv with only a small time

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a n d 48% oxyg e n is p rovid e d th ro u g h th e ve n tila to r to m a in ta in th e a n e s th e tic s ta te . 7:50 AM: Ma tth e w s h o w s n o re s p o n s e , e ith e r th ro u g h m ove m e n t o r in cre a s e d s ym p a th e tic to n e (e .g ., in cre a s e d h e a rt ra te , in cre a s e d b lo o d p re s s u re ), to th e f rs t s u rg ica l in cis io n . 8:20 AM: Dr. S n o w n o tice s w ith a s ta rt th a t Ma tth e w ’s p u ls e h a s a lle n to 55 a n d h is b lo o d p re s s u re to 85/45. Be ra tin g h im s e l o r o rg e ttin g to tu rn d o w n th e in s p ire d p a rtia l p re s s u re o th e a n e s th e tic a s its m ixe d ve n o u s p a rtia l p re s s u re in cre a s e d , Dr. S n o w re d u ce s th e in s p ire d is o u ra n e le ve l to 0.8% w h ile ke e p in g th e n itro u s oxid e le ve l a t 50%. With in 15 m in u te s , Ma tth e w ’s p u ls e a n d b lo o d p re s s u re re b o u n d . 12:35 PM: A te r a lo n g s u rg e ry, Dr. S n o w s to p s th e is o u ra n e a n d n itro u s oxid e a n d tu rn s o n p u re oxyg e n o r a e w m in u te s . 12:45 PM: In le s s th a n 10 m in u te s , Ma tth e w is b re a th in g s p o n ta n e o u s ly a n d is a b le to re s p o n d to q u e s tio n s , a lth o u g h h e is s till s o m e w h a t g ro g g y. Ma tth e w ’s p a re n ts a re re lie ve d to f n d h im a wa ke a n d a le rt a te r m o re th a n 5 h o u rs o a n e s th e s ia .

Questions 1 . What determines the rate o induction and recovery rom anesthesia, and how does this di er or children as compared to adults? 2 . Why is it necessary to reduce the inspired partial pressure o isof urane some minutes into the procedure (as Dr. Snow initially neglected to do)? 3 . Why did Dr. Snow give pure oxygen or a ew minutes ollowing the cessation o anesthetic administration? 4 . What are the advantages o using a mixture o two anesthetics (in this example, nitrous oxide and isof urane) instead o just one or the other?

lag (see below). Palv may be measured directly as the partial pressure o anesthetic in the end-tidal exhaled gas, when the dead space no longer contributes to the exhaled gas. The alveolar partial pressure that results in the lightest possible anesthesia is termed the minimum alveolar concentration (MAC). Specif cally, MAC is the alveolar partial pressure that abolishes a movement response to a surgical incision in 50% o patients. The potency o an anesthetic is related inversely to its MAC. I the MAC is small, then the potency is high, and a relatively low partial pressure will be su f cient to cause anesthesia. For example, isof urane — which has a MAC o 0.0114 atm—is much more potent than nitrous oxide —which has a MAC o 1.01 atm (Table 17-1).

Therapeutic and Analgesic Indices Loss o response to extremely noxious stimuli, such as endotracheal intubation, requires a higher partial pressure o

C h a p t e r 1 7 General Anesthetic Pharmacology 267 Awa ke

Awa ke

Ana lge s ia (de pe nds on a ge nt) Amne s ia Euphoria Stage II: Excitement Excite me nt De lirium Comba tive be ha vior

R

e

D

c

e

o

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p

ve

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a

Stage I: Analgesia

Stage III: Surgical Anesthesia Comme nce s urge ry

Uncons cious ne s s Re gula r re s pira tion De cre a s ing e ye move me nt

S urge ry comple te d

Stage IV: Medullary Depression Re s pira tory a rre s t Ca rdia c de pre s s ion a nd a rre s t No e ye move me nt

FIGURE 17-1. The stages o anesthesia. The deepening anesthetic state can be divided into four stages, based on observations with diethyl ether. The analgesia of stage I is variable and depends on the particular anesthetic agent. With fast induction, the patient passes rapidly through the undesirable “excitement” phase (stage II). Surgery is generally undertaken in stage III. The anesthesiologist must take care to avoid stage IV, which begins with respiratory arrest. Cardiac arrest occurs later in stage IV. During recovery from anesthesia, the patient progresses through the stages in reverse.

anesthetic than is required or loss o response to a surgical incision (Fig. 17-2). Still higher partial pressures o anesthetic cause medullary depression. In general, however, anesthetics have steep dose–response curves and low therapeutic indices, def ned as the ratio o LP50 (the partial pressure that is lethal in 50% o subjects) to MAC (which is analogous to ED50; see Chapter 2, Pharmacodynamics). Furthermore, the variability among patients in their response to a given dose o

anesthetic is small. There ore, or all patients, the levels o anesthetic that cause respiratory and cardiac arrest are not much higher than the levels that cause general anesthesia. It should also be noted that no pharmacologic antagonists o general anesthetics exist to counteract inadvertently high levels o anesthetic. Although these disadvantages are partially o set by the ability to control PCNS through control o PI (i.e., the anesthetic can be breathed out), the combination o low therapeutic index and lack o antagonist means that anesthetics are dangerous drugs that demand specialty training or their proper and sa e administration. Pain relie (analgesia) may or may not occur at a partial pressure lower than that required or surgical anesthesia. The partial pressure at which 50% o persons lose nociception is the AP50 (partial pressure that results in analgesia in 50% o patients), and the analgesic index is the ratio o MAC to AP50. A high analgesic index implies that analgesia is induced at a partial pressure o anesthetic signif cantly lower than that required or surgical anesthesia. For example, nitrous oxide has a high analgesic index and is a good analgesic, whereas halothane has a low analgesic index and is a poor analgesic.

The Meyer-Overton Rule The potency o an anesthetic can be predicted rom its physicochemical characteristics. The most reliable predictor has been the anesthetic’s solubility in olive oil (or in another lipophilic solvent, such as octanol), as denoted by the oil/gas partition coe f cient, (oil/gas) (Box 17-2). Specif cally, the potency of an anesthetic increases as its solubility in oil increases. That is, as (oil/gas) increases, MAC decreases. The relationship between MAC and (oil/gas) is such that MAC multiplied by (oil/gas) is nearly constant, independent o the identity o the anesthetic. Because multiplication o the partition coe f cient by the partial pressure yields the concentration o anesthetic (Box 17-2), this is equivalent to saying that, at 1 MAC, the concentration o anesthetic in a lipophilic solvent (such as olive oil) is nearly constant or all anesthetics. Thus, the MAC, which varies with the identity o the anesthetic, is actually the partial pressure required to generate a particular concentration o anesthetic in a lipophilic medium, such as the lipid bilayers

B O X 1 7 - 1 Pa rtia l Pre s s u re Ve rs u s Co n ce n tra tio n The partial pressure of Gas A in a mixture of gases is the portion of the total pressure that is supplied by Gas A. For ideal gases, the partial pressure of Gas A is obtained by multiplying the total pressure by the mole fraction of A in the mixture (i.e., the fraction of molecules in the mixture represented by Gas A). The concentration of Gas A in the mixture ([A]mixture ) is the number of moles of Gas A (nA) divided by the volume (V); [A]mixture can also be obtained from the ideal gas equation by dividing the partial pressure of Gas A (P PA) by the temperature (T) and the universal gas constant (R): [A]mixture

nA / V

PA / RT

Inhaled anesthetics dissolve in the tissues of the body, such as the blood and the brain. The partial pressure of a gas dissolved in a liquid is equal to the partial pressure of free gas in equilibrium with that liquid. For gases, partial pressures are convenient because the partial pressures in all compartments are equal at equilibrium. This is true, independent of whether the compartments contain gas that is in the gaseous (alveoli) or the dissolved (tissues) form. In contrast, the concentrations within different compartments are not equal at equilibrium. To convert the partial pressure of a dissolved gas to its concentration within the solvent, the partial pressure is multiplied by a measure of solubility known as the solvent/gas partition coe f cient. ■

268 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 17-1 Properties o Inhaled Anesthetics SOLVENT/GAS PARTITION COEFFICIENTS ANESTHETIC

MAC (atm)

Nitrous oxide

1.01

Desf urane

0.06

Sevof urane

(oil/gas) (Lgas Ltissue

1

(blood/gas) (Lgas Ltissue 1 atm 1)

1

atm )

1.4

CONCENTRATION IN OIL AT 1 MAC (oil/gas) MAC (Lgas Ltissue 1)

0.47

1.4

19

0.45

1.1

0.02

51

0.65

1.0

Diethyl ether

0.019

65

Enf urane

0.0168

98

1.8

1.6

Isof urane

0.0114

98

1.4

1.1

Halothane

0.0077

224

2.3

1.7

12

1.2

The commonly used inhaled anesthetics are listed in order o increasing potency (or decreasing MAC). Also listed are the important solvent/gas partition coe cients (oil/gas) and (blood/gas). (oil/gas) de nes the potency o the anesthetic (higher is more potent), while (blood/gas) de nes the rate o induction and recovery o anesthesia (lower is aster). The product o (oil/gas) and MAC or these anesthetics has a rather constant value o 1.3 Lgas Ltissue 1 (with a standard deviation o 0.27). This is an illustration o the Meyer-Overton Rule; another illustration o the rule is shown in Figure 17-3. Also note the general trend that anesthetics with larger (oil/gas) tend to have larger (blood/gas); this means that there is requently a trade-o between potency and speed o induction among the inhaled anesthetics. The structures o these agents are shown in Figure 17-14.

in the CNS. This correlation, known as the Meyer-Overton rule , holds over at least f ve orders o magnitude o anesthetic potency (Fig. 17-3). The constant that represents the concentration o anesthetic at 1 MAC is 1.3 liters o gas per liter o oil (Lgas / Loil), or 0.05 M a ter dividing by the volume

Nonre s pons ive to tra pe zius s que e ze

g n i t i

MAC

LP 50

Nonre s pons ive to s kin incis ion

Nonre s pons ive to intuba tion

60

40 Ca rdia c a rre s t (de a th)

20

P

e

r

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80

Equation 17-1

1.3/ (oil /gas)

MAC

100

b

o one mole (see Box 17-2). Thus, i one knows the oil/gas partition coe f cient o an anesthetic, one can estimate its MAC rom the ollowing equation (see also Table 17-1):

0

0.01

0.02

0.03

0.04

0.05

Alve ola r pa rtia l pre s s ure of is oflura ne (a tm) FIGURE 17-2. Isof urane dose–response curves or various endpoints. These curves depict the percentage o patients exhibiting endpoints o nonresponsiveness to a set o stimuli and o cardiac arrest as the alveolar partial pressure o isof urane is increased. Note that the dose–response curves are quite steep, especially or mild stimuli, and that higher partial pressures are required to achieve lack o response to stronger stimuli. In the example shown, lack o response to intubation in 50% o patients requires nearly 0.02 atm isof urane, while lack o response to a squeeze o the trapezius muscle requires only 0.008 atm. The MAC is de ned as the alveolar partial pressure at which 50% o patients do not respond to a skin incision. The therapeutic index is de ned as the lethal pressure (LP50) divided by the MAC. The theoretical curve or cardiac arrest is derived rom a known therapeutic index o about 4 or isof urane. Accordingly, the anesthesiologist must care ully monitor each individual patient to achieve the desired e ect while avoiding cardiac depression.

C h a p t e r 1 7 General Anesthetic Pharmacology 269

B O X 1 7 - 2 Pa rtitio n Co e f cie n ts The solvent/gas partition coe f cient, (solvent/gas), def nes the solubility o a gas in a solvent or, in other words, the extent to which the gas “partitions” between its gaseous state and the solution. More specif cally, (solvent/gas) is the ratio o the amount o gas dissolved in a given volume o solvent to the amount o ree gas that would occupy the same volume o space, all at standard temperature (25°C) and pressure (1.0 atm) (STP). The solvent could be olive oil, blood, or brain tissue, or example. Dissolved amounts o gas are typically given not in terms o moles but in terms o the volume that the gas would occupy at STP in a gaseous state. Recall that, to convert rom moles to liters at STP, one multiplies by the volume o one mole o gas at 25°C and 1.0 atm (i.e., by 24.5 L/mol). Thus, (solvent/gas) is the number o liters o gas that will dissolve in one liter o solvent per atmosphere o partial pressure. [Note that the units o (solvent/gas) are Lgas Lsolvent 1 atm 1, or simply atm 1.] For a particular solvent, a gas with a larger (solvent/gas) is more soluble in that solvent. For example, diethyl ether has a (blood/gas) o about 12 Ldiethyl ether Lblood 1 atm 1, so diethyl ether is relatively soluble in blood. In contrast, nitrous oxide has a (blood/gas) o about 0.47 Lnitrous oxide Lblood 1 atm 1, so nitrous oxide is relatively insoluble in blood (see Table 17-1 and Fig. 17-8 or examples). Likewise, a gas may have di erent solubilities in di erent solvents. Solvents or tissues in which a gas has a high partition coe f cient (high solubility) will dissolve large amounts o the gas at a given partial pressure, resulting in a high concentration o the gas in that solvent or tissue. Thus, large amounts o gas must be trans erred to change the partial pressure by an appreciable amount. In contrast, solvents or tissues in which a gas has a low partition coe f cient (low solubility) will dissolve only small amounts o the gas at a given partial pressure. In this case,

PHARMACOKINETICS OF INHALED ANESTHETICS A cardiopulmonary model o the uptake o anesthetic rom the alveoli into the circulation and the distribution o anesthetic rom the circulation to the tissues allows determination o the rate at which the partial pressure o anesthetic rises within the CNS. The anesthesiologist must navigate the small space between allowing a patient to awaken and causing medullary depression by predicting the e ects o various physiologic responses and disease states on the depth o anesthesia. For example, an understanding o the distribution characteristics o anesthetics enabled Dr. Snow to respond appropriately to Matthew’s hypotension by lowering the PI o isof urane without overcorrecting and causing him to awaken. The anesthesiologist must also be aware o the di erences in the pharmacokinetics o the various anesthetics. The pharmacokinetic characteristics o an ideal general anesthetic would be such that the anesthetic provides a rapid and pleasant induction o surgical anesthesia, ollowed by

trans erring a small amount o the gas will signif cantly change the partial pressure (Fig. 17-8). For any given partial pressure, Henry’s law or dilute solutions allows the concentration o Gas A in a solvent ([A]solution) to be calculated rom (solvent/gas). The partial pressure is multiplied by the partition coe f cient to calculate the concentration in terms o Lgas per Lsolvent. The result is divided by the volume o one mole o gas at 25°C at 1.0 atm (24.5 L/mol) to yield the molar concentration. [A]solution

Psolvent (solvent/gas) {in terms o Lgas /Lsolvent} Psolvent (solvent/gas)/(24.5 L/mol) {in terms o molgas /Lsolvent}

For example, because the (blood/gas) o nitrous oxide is 0.47 Lnitrous oxide Lblood 1 atm 1, i the partial pressure o nitrous oxide in the blood is 0.50 atm, then the concentration is 0.50 atm 0.47 Lnitrous oxide Lblood 1 atm 1 0.24 Lnitrous oxide Lblood 1 or 9.6 mM (a ter dividing by 24.5 L/mol). Also note that doubling the partial pressure will double the concentration. A partition coe f cient can also be def ned or the partitioning o a gas between two solvents. For example, the tissue/blood partition coe f cient, (tissue/blood), is the ratio o the molar concentration o gas in the tissue ([A]tissue ) to the molar concentration o gas in the blood ([A]blood) at equilibrium (note that this coe f cient is unitless). From the previous equation def ning concentration and the act that partial pressures are equal at equilibrium, it ollows that (tissue/blood)

[A]tissue /[A]blood (tissue/gas)/ (blood/gas) ■

a smooth and rapid recovery to a ully unctional and conscious state. The pharmacokinetics o individual agents are discussed below; this section deals with general principles o the uptake model, which uses basic respiratory and cardiovascular physiology to predict the pharmacokinetics o the inhaled anesthetics. As discussed below, the uptake model depends on calculations o the time required or the equilibration o anesthetic partial pressures in the tissues with the inspired anesthetic partial pressure.

Concepts rom Respiratory Physiology Local Equilibration

During general anesthesia, the patient breathes, either spontaneously or via a ventilator, an anesthetic or mixture o anesthetics together with oxygen and/or normal air. Once the anesthetic gas reaches the alveoli, it must di use across the respiratory epithelium into the alveolar capillary bed. According to Fick’s law, the rate o di usion o gas through a sheet o tissue down its partial pressure gradient is proportional to the tissue area and the partial pressure di erence

270 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY 10,000 Thiome thoxyflura ne Me thoxyflura ne Ha lotha ne Is oflura ne Die thyle the r Enflura ne

100

Cyclopropa ne

10

e

n

c

y

(

1

/

a

t

m

)

1,000

P

o

t

Xe non

1

Nitrous oxide

0.1 Nitroge n 0.01 0.01

0.1

1

10

100

1,000 10,000

Oil/ga s pa rtition coe fficie nt FIGURE 17-3. The Meyer-Overton rule. Molecules with a higher oil/gas partition coe f cient [ (oil/gas)] are more potent general anesthetics. This log–log plot shows the very tight correlation between lipid solubility [ (oil/ gas)] and anesthetic potency over f ve orders o magnitude. Note that even such gases as xenon and nitrogen can act as general anesthetics when breathed at su f ciently high partial pressures. The equation describing the line is: Potency (oil/gas) / 1.3. Recall that Potency 1/MAC.

between the two sides and is inversely proportional to the thickness o the sheet: Diffusion rate

D

(A/l )

P

Equation 17-2

where D di usion constant; A sur ace area; l thickness; and P partial pressure di erence. One principle evident rom Fick’s law is that the equalization o the partial pressure o the gas, not its concentration, def nes the approach to equilibrium across a boundary sheet. Thus, at equilibrium (i.e., when the net di usion rate is zero), the partial pressure in the two compartments is the same, even though the concentration in the two compartments may be di erent. With its enormous alveolar sur ace area ( 75 m2, or nearly hal a tennis court) and thin epithelium ( 0.3 m, which is less than 1/20th the diameter o a red blood cell), the lung optimizes the rate o gas di usion. Accordingly, the alveolar partial pressure Palv and the systemic arterial partial pressure Part are nearly the same at all times. (In normal individuals, small amounts o physiologic shunting keep Part slightly lower than Palv.) By using the lungs as an uptake system or inhaled anesthetics, anesthesiologists take advantage o the body’s system or absorbing oxygen. Similarly, the capillary beds in tissues have evolved to deliver oxygen rapidly to all cells in the body. The distances between arterioles are small, and di usion pathways are on the order o one cell diameter. Consequently, the arterial partial pressure o a general anesthetic can equilibrate completely with tissues in the time required or blood to traverse the capillary bed. Likewise, the partial pressure in the postcapillary venules Pvenule equals the partial pressure in the tissue Ptissue.

Another way o stating the above conclusion is that the transfer of anesthetic in both the lungs and the tissues is limited by perfusion rather than diffusion. Because per usion is rate-limiting, increasing the rate o di usion (e.g., by using a lower molecular weight anesthetic) will not, by itsel , increase the rate o induction o anesthesia.

Global Equilibration

I an anesthetic is inspired or a su f ciently long period o time, all compartments in the body will equilibrate to the same partial pressure (equal to PI). This global equilibration may be divided into a series o partial pressure equilibrations between each successive compartment and its incoming ow o anesthetic. In the case o the tissues, the incoming ow is the arterial blood ow, with partial pressure approximately equal to Palv. In the case o the alveoli, the incoming ow is the alveolar ventilation with partial pressure PI. The time constant describes the rate o approach o a compartment’s partial pressure to that o its incoming ow. Specif cally, is the time required or equilibration to be 63% complete. This time constant is convenient because it can be calculated by dividing the compartment’s volume capacity (relative to the delivering medium; see below) by the f ow rate . In other words, once a volume o ow equal to the capacity o a compartment has gone through that compartment, the partial pressure o anesthetic in the compartment (i.e., in the tissues or alveoli) will be 63% o the partial pressure in the incoming ow (i.e., in the arterial blood ow or alveolar ventilation, respectively). Equilibration is 95% complete a ter three time constants. Volume Capacity/Flow Rate P compartment

Pflow [ 1

e

(t/ )

]

Equation 17-3 Equation 17-4

where t elapsed time. These equations describe what should make intuitive sense: equilibration o the partial pressure o the compartment with the incoming ow takes place more quickly (i.e., the time constant is smaller) when the in ow is larger or the compartment capacity is smaller.

The Uptake Model For simplicity, the model o anesthetic uptake and distribution organizes the tissues o the body into groups based on similar characteristics. Each group can be modeled as a container with a particular capacity or anesthetic and a particular level o blood ow delivering anesthetic. An adequate approximation groups the tissues into three main compartments that are per used in parallel (Fig. 17-4). The vessel-rich group (VRG), which consists o the CNS and visceral organs, has a low capacity and high ow. The muscle group (MG), which consists o muscle and skin, has a high capacity and moderate ow. The at group (FG) has a very high capacity and low ow. (A ourth group, the vessel-poor group [VPG], which consists o bone, cartilage, and ligaments, has a negligible capacity and ow, and its omission does not signif cantly a ect the model.) The rate o increase o the partial pressure in the VRG (PVRG) is o the greatest interest because the VRG includes the CNS. The overall equilibration o PVRG with the inspired partial pressure occurs in two steps, either o which may be

C h a p t e r 1 7 General Anesthetic Pharmacology 271 PI

P a lv P a rt

P MVR

Tissue group

% Cardiac output

% Body weight

Vol. cap. for N2O at Palv = 0.8atm

Vol. cap. for halo. at Palv = 0.01atm

VRG: bra in, live r, kidneys

VRG

75%

9%

2.6 L

0.30 L

MG: mus cle, s kin

MG

18%

50%

16 L

3.0 L

FG: fa t

FG

5.5%

19%

12 L

17 L

VP G

1.5%

22%

7.0 L

1.3 L

P VRG

P MG

P FG

VP G: bone, ca rtila ge, liga me nts P VP G

FIGURE 17-4. Distribution of cardiac output and volume capacity for general anesthetics among the major tissue compartments. The tissues of the body can be divided into four groups based on their level of perfusion and their capacity to take up anesthetic. These include the vessel-rich group (VRG), muscle group (MG), fat group (FG), and vessel-poor group (VPG). (The contribution of the VPG is generally ignored in most pharmacokinetic models of anesthesia.) The VRG, which contains the internal organs including the brain, constitutes a small percentage of the total body weight (9%), has the lowest capacity for anesthetic, and receives most of the cardiac output (75%). The high perfusion and low capacity allow PVRG to equilibrate rapidly with Part. Also, the VRG makes the largest contribution to the mixed venous return partial pressure PMVR, which is equal to (0.75 PVRG 0.18 PMG 0.055 PFG 0.015 PVPG). N2O, nitrous oxide; Halo., halothane; Vol. cap., volume capacity.

rate-limiting. First, the alveolar and inspired partial pressures equilibrate (Palv approaches PI, or Palv → PI). Second, PVRG (and specif cally PCNS) equilibrates with the arterial partial pressure (which is essentially equal to the alveolar partial pressure) (PVRG → Part). The discussion will now consider the time constant or each o these two steps and def ne conditions under which one or the other is rate-limiting.

Equilibration of Alveolar with Inspired Partial Pressure

The equilibration o Palv with PI is conceptually the f rst step o the equilibration o PVRG with PI. During induction o anesthesia, PVRG can never be higher than Palv; i Palv rises slowly, then PVRG must also rise slowly. To calculate the time constant or the approach o Palv to PI, {Palv→PI}, the ow rate and volume capacity must be def ned. The delivering medium is ree gas arriving through

the airways, and the compartment is the lung and alveoli. The volume capacity is simply the volume o gas that remains in the lungs a ter normal exhalation, or the functional residual capacity (FRC, typically 3 L or an average adult). Assume initially that the only component o the ow rate is the rate o alveolar ventilation, which delivers the anesthetic (Valv {Tidal Volume Dead Space} Respiratory Rate; or an average adult, Valv {0.5 L 0.125 L} 16 min 1 6 L/min). Then, because { P alv Æ PI }

FRC /Valv

Equation 17-5

a typical value or {Palv→PI} is 3 L / 6 L/min, or 0.5 min— independent o the particular gas being inhaled. In children, the increased alveolar ventilation rate and decreased FRC (smaller lungs) both tend to shorten the time constant and

272 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

to accelerate equilibration between the alveolar and inspired partial pressures. The assumption to this point has been that no uptake o anesthetic into the bloodstream occurs, as would be the case i the solubility o the anesthetic in blood were zero. In practice, at the same time that alveolar ventilation is delivering anesthetic to the alveoli, anesthetic is also being removed rom the alveoli by di usion into the bloodstream. The balance between delivery and removal is analogous to adding water into a leaky bucket (Fig. 17-5). The level o water in the bucket (which represents the alveolar partial pressure) is determined both by the rate at which the water is added (the minute ventilation) and the size o the leak (the rate o anesthetic uptake rom the alveoli into the bloodstream). Increasing anesthetic delivery (e.g., by using a higher ventilation rate or a higher inspired partial pressure) will increase the alveolar partial pressure o the gas, just as adding water aster will increase the level o water in the bucket. Conversely, increasing anesthetic removal (e.g., by increasing the per usion rate or using a more blood-soluble anesthetic) will decrease the alveolar partial pressure o the gas; this is analogous to increasing the leakiness o the bucket. Thus, uptake o anesthetic rom the alveoli into the bloodstream constitutes a negative component to the ow (i.e., a ow out o the lungs), which makes the time constant longer than the theoretical case where {Palv→PI} equals FRC divided by Valv. The magnitude o the increase in the time constant compared to the limiting case depends on the rate o uptake o anesthetic by the blood, with longer {Palv→PI} resulting

rom greater uptake. I one knows the cardiac output (i.e., the volume o blood pumped by the heart in 1 minute) and the value o the instantaneous di erence between the pulmonary arterial partial pressure (which equals the systemic partial pressure o the mixed venous return, PMVR) and the pulmonary venous partial pressure (which equals the systemic arterial partial pressure, Part), then one can calculate the rate o uptake o gas rom the alveoli: Rate of uptake {in Lgas / m i n } (blood/gas)

(Part

PMVR)

CO

Equation 17-6

where CO cardiac output in liters o blood per minute. Equation 17-7 ollows rom Equation 17-6 because the anesthetic concentration [A]blood is equal to (blood/gas) Pblood (see Box 17-2): Rate of uptake

([ A]art

[ A]MVR )

CO

Equation 17-7

I any o the terms in these equations approaches zero, the rate o uptake becomes small, and the delivery o anesthetic by ventilation drives the alveolar partial pressure toward the inspired partial pressure. In other words, equilibration o alveolar with inspired partial pressure is aster (i.e., {Palv→PI} is smaller) with lower blood solubility o the anesthetic [lower (blood/gas)], lower cardiac output, or smaller arterial ( alveolar) to venous partial pressure di erence.

Equilibration of Tissue with Alveolar Partial Pressure

Ve ntila tion brings a ne s the tic into a lve oli

P a lv

The ba la nce be twe e n input a nd output s e ts the leve l of P a lv

Upta ke into bloods tre a m re move s a ne s the tic from a lve oli

FIGURE 17-5. Determinants of the alveolar partial pressure of an inhaled anesthetic. The alveolar partial pressure, represented by the depth o f uid in the bucket, results rom the balance between delivery by ventilation and removal by uptake into the bloodstream. Increased delivery o anesthetic, resulting rom either increased ventilation or an increased inspired partial pressure o anesthetic, raises Palv. In contrast, increased uptake into the bloodstream, caused by a large (blood/gas) or increased cardiac output, lowers Palv.

In addition to the equilibration between Palv and PI, equilibration between Ptissue and Part (which is nearly equal to Palv) must occur or Ptissue to equilibrate with PI. Changes in Palv are transmitted rapidly to systemic arterioles, because equilibration across the pulmonary epithelium is ast and the circulation time rom pulmonary veins to tissue capillaries is generally less than 10 seconds. Thus, the time constant or equilibration between Ptissue and Palv can be approximated as the time constant or equilibration between Ptissue and Part. To calculate the time constant {Ptissue→Part}, one must def ne the capacity o the compartment and the ow rate o the delivering medium. The ow rate is simply the rate at which blood per uses the tissue. Recall that capacity is a volume capacity relative to the delivering medium. Specif cally, the capacity is the volume that the tissue would need to contain all of its gas if the solubility of the gas in the tissue were the same as that in the blood. (This def nition is similar to that o the volume o distribution o a drug; see Chapter 3, Pharmacokinetics): Relative Volume Capacity of Tissue ([A]tissue

Voltissue)/ [A]blood

Equation 17-8

where Voltissue is the volume o tissue. Equation 17-9 ollows rom Equation 17-8 because [A]tissue / [A]blood at equilibrium is equal to (tissue/blood) (see Box 17-2): Relative Volume Capacity of Brain (brain/blood)

Volbrain

Equation 17-9

C h a p t e r 1 7 General Anesthetic Pharmacology 273

{ Pbrain Æ Part }

TABLE 17-2 Tissue/Blood Partition Coe cients

{ Pbrain Æ Part }

TISSUE/BLOOD PARTITION COEFFICIENTS (BRAIN/ BLOOD) (UNITLESS)

(MUSCLE/ BLOOD) (UNITLESS)

(FAT/ BLOOD) (UNITLESS)

(brain /blood) (1.6

Volbrain /Qbrain

1.4 L)/(0.9 L/min)

Equation 17-13

2.5 min

Nitrous oxide

1.1

1.2

2.3

Diethyl ether

2.0

1.3

5

Desf urane

1.3

2.0

27

Enf urane

1.4

1.7

36

Isof urane

1.6

2.9

45

where Volbrain is the volume o the brain and Qbrain is the blood f ow to the brain. Variations in (brain/blood) among the di erent anesthetic agents cause {Pbrain→Part} to range rom 1.5 min or nitrous oxide [ (brain/blood) 1.1] to 2.7 min or diethyl ether [ (brain/blood) 2.0] (Table 17-3). O course, variability in blood f ow to the brain also a ects {Pbrain→Part}. In summary, the time constant for equilibration of the CNS with the alveolar partial pressure is short and relatively independent of the particular anesthetic being used.

Sevof urane

1.7

3.1

48

The Rate-Limiting Step

Halothane

1.9

3.4

51

ANESTHETIC

The tissue/blood partition coe cient describes the comparative solubility o an anesthetic in a tissue compared to blood. (tissue/blood) is obtained rom the ratio o the concentration o anesthetic in the tissue to the concentration in the blood at equilibrium (i.e., when the partial pressure is the same in both tissues). Alternatively, one may calculate (tissue/blood) rom the equation (tissue/blood) (tissue/gas)/ (blood/gas) (see Box 17-2). With very ew minor exceptions, the general trend is ( at/blood) (muscle/blood) (brain/blood). High values o ( at/blood) give the FG a very high capacity or the inhaled anesthetics.

{ Ptissue Æ Palv }

Relative Vol. Cap. of Tissue /Qtissue

Equation 17-10

{Ptissue Æ Part} (tissue/blood)

Voltissue /Qtissue

Equation 17-11

where Qtissue is tissue per usion in L/min. The tissue groups di er greatly in their capacities or anesthetic and in the time constants or their equilibration with arterial (and thus alveolar) partial pressure. With a low (tissue/blood) (Table 17-2) and a small volume ( 6 L), the VRG has a low capacity or anesthetic. The combination o low capacity and high blood f ow (75% o cardiac output) results in a very short equilibration time constant ( {PVRG→Palv}) or the VRG. With a slightly higher (tissue/ blood), a much larger volume ( 33 L), and only moderate blood f ow, the MG has a longer equilibration time constant ( {PMG→Part}). Finally, with an extremely high (tissue/ blood), a large volume, and low blood f ow, the FG has an extremely long equilibration time constant ( {PFG→Part}) (Table 17-3 and Fig. 17-6). Because the anesthesiologist seeks to control PCNS, the time constant or equilibration o the brain partial pressure Pbrain with the arterial partial pressure Part (which is nearly equal to Palv) is o particular interest. The volume o the brain is approximately 1.4 L, the blood f ow to the brain is about 0.9 L/min, and an average (brain/blood) or most anesthetics is about 1.6. Then, because Relative Volume Capacity of Brain (brain / blood)

Volbrain

■

■

Ventilation-limited anesthetics, such as diethyl ether, enf urane , isof urane , and halothane ; and Per usion-limited anesthetics, such as nitrous oxide , desf urane , and sevof urane . Ventilation-limited anesthetics have a long, rate-limiting

Then, using Equation 17-3, we can write { Ptissue Æ Part }

As described above, the equilibration o the CNS with the inspired partial pressure occurs in two steps. Unlike {Pbrain→Part}, which is relatively independent o the particular anesthetic being used, {Palv→PI} varies greatly among di erent anesthetics. On this basis, inhaled anesthetics can be divided into two broad categories:

Equation 17-12

{Palv→PI} because o their high (blood/gas): the high rate o uptake o anesthetic into the bloodstream prevents Palv rom rising rapidly. Thus, the slow and rate-limiting equilibration o alveolar with inspired partial pressure results in slow induction o anesthesia and slow recovery TABLE 17-3 Time Constants or Equilibration o Tissue with Arterial Partial Pressure TIME CONSTANT FOR EQUILIBRATION OF TISSUE WITH ARTERIAL PARTIAL PRESSURE, {Ptissue →Part} ANESTHETIC

VRG (min)

MG (min)

FG (min)

Nitrous oxide

1.5

36

104

Diethyl ether

2.7

39

227

Desf urane

1.7

61

1,223

Enf urane

1.9

51

1,631

Isof urane

2.1

88

2,039

Sevof urane

2.3

94

2,175

Halothane

2.5

103

2,311

The time constants {Ptissue→Part}describe the time or 63% equilibration o the tissue with arterial (and there ore alveolar) partial pressure. Notice the very small time constants or equilibration o the VRG, in contrast to the large time constants or MG equilibration and very large time constants or FG equilibration. For all anesthetics except nitrous oxide, the partial pressure o the FG remains ar below that o the alveolus or even the longest surgical procedures. Conversely, the VRG partial pressure is nearly in equilibrium with the alveolar partial pressure rom almost the start o anesthetic administration. The values in this table were calculated rom the equation {Ptissue→Part} (tissue/blood) Volume o tissue/Blood f ow to tissue.

274 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

d e r i n f r

P f ( s e

v

) /

P

I

Is oflura ne , λ = 1.4

l

63% e quilibra tion

e

u

r

a

0.6

r

s

Ha lotha ne , λ = 2.3

u

s

a

p

s

r

s

e

p

0.4

l

e

40

r

d e

0.8 a

a

e r

60

r

t

i

a

l

p

63% e quilibra tion

t

i

a

n

a

r

s

l

t

i

p

p

a

i

r

De s flura ne , λ = 0.45

o n o c

t

i

80

r

p

i

a

20

p

f

Nitrous oxide , λ = 0.47

i

MG FG

s

s

u

r

e

Alve ola r VRG

0.2

r

%

o

1.0

s

100

Ha lotha ne (P I = 0.01)

p

Nitrous oxide (P I = 0.75)

o

l

a

Ethe r, λ = 12

10

Time (min)

100

1

10

100

A

1

l

ve

0

Time (min)

FIGURE 17-6. Equilibration of the tissue groups with the inspired partial pressure. These curves show, as a function of time, the approach of the partial pressures in the alveoli and in the three major tissue groups toward the inspired partial pressure. The partial pressure in the VRG equilibrates rapidly with the alveolar partial pressure, while the MG equilibrates more slowly, and the FG much more slowly. For a perfusion-limited anesthetic such as nitrous oxide, the alveolar partial pressure rises so quickly that the rate of rise of the VRG partial pressure is as much limited by its rise toward the alveolar partial pressure as by the rise of Palv toward PI. For a ventilation-limited anesthetic such as halothane, the rate at which the partial pressure in the VRG rises is limited not by its approach to the alveolar partial pressure but rather by the rise of the alveolar toward the inspired partial pressure. In other words, the ratelimiting step is the equilibration of the alveolar partial pressure with the inspired partial pressure. The dashed line shows the point at which the partial pressure is 63% of PI, and the time constant for equilibration of each tissue group with PI is approximated by the time at which each curve crosses this line.

rom anesthesia. Accordingly, or these anesthetics, physiologic or pathologic changes that act to increase the rate o rise o the alveolar partial pressure will speed induction. Conversely, because equilibration o the tissue with the arterial partial pressure is not rate-limiting, physiologic or pathologic changes that shorten {PVRG→Part} will have little e ect on induction time (see below). Perfusion-limited anesthetics have a {Palv→PI} that is similar in magnitude to {PVRG→Part} because their (blood/ gas) is low. Induction and recovery occur quickly, and neither {Palv→PI} nor {PVRG→Part} may be clearly rate-limiting. Accordingly, induction time may be a ected by changes in either the rate o rise o alveolar partial pressure or the rate at which PCNS approaches Part (e.g., see the discussion o hyperventilation below). Physiologic changes may alter the balance between {Palv→PI} and {PVRG→Part}. See Figure 17-6 or a graphic comparison o the kinetics o ventilation-limited and per usion-limited anesthetics. The characteristic that distinguishes per usion-limited rom ventilation-limited anesthetics is the blood/gas partition coe f cient, (blood/gas). With the lower (blood/gas) o per usionlimited anesthetics, the bloodstream removes less anesthetic rom the alveoli; thus, the alveolar partial pressure rises more quickly and induction is aster (Fig. 17-7). This is the key point, although the correlation may seem paradoxical at f rst: agents that are less soluble in the blood induce anesthesia faster.

0.0

0

10

20

30

Minute s of a dminis tra tion FIGURE 17-7. Rate of approach of the alveolar toward the inspired partial pressure. For agents with lower (blood/gas), such as nitrous oxide, the alveolar partial pressure approaches the inspired partial pressure quickly, while for agents with higher (blood/gas), such as diethyl ether, the alveolar partial pressure approaches the inspired partial pressure much more slowly. The dashed line shows the point at which Palv /PI 0.63; the time constant {Palv→PI} is approximated by the time at which each curve crosses this line. (blood/gas). Ether, diethyl ether.

To clari y, consider two hypothetical anesthetics that di er solely in (blood/gas) (Fig. 17-8): Anesthetic A has a low (blood/gas), while Anesthetic B has a high (blood/gas). Because Anesthetics A and B are identical in (oil/gas), they have the same MAC. They also have identical (brain/blood), so their {Pbrain→Palv} is the same (see Equations 17-12 and 17-13). To cause anesthesia, both must achieve the same partial pressure in the CNS. At any particular partial pressure, however, the blood and CNS contain more moles o Anesthetic B than Anesthetic A because Anesthetic B is more soluble than Anesthetic A in the blood and CNS. The trans er o a larger number o moles o Anesthetic B out o the lungs slows the rate o rise o Palv, so a longer period is necessary or Anesthetic B than or Anesthetic A to achieve the anesthetic partial pressure in the CNS (Fig. 17-8).a a

In this hypothetical model, one may correctly note that the concentration o Anesthetic B in the CNS as a whole will be higher than that o Anesthetic A at any particular time point. One may, there ore, wonder how Anesthetic B can have a slower induction, i anesthesia results when a particular concentration (0.05 M) is reached at the site o action (see “The Meyer-Overton Rule,” above). At this point, one must recognize that the brain is primarily aqueous, but that anesthetics are likely to have a hydrophobic site o action, and that both Anesthetic A and Anesthetic B must have the same concentration (0.05 M) in the key hydrophobic portions o the brain at their anesthetic partial pressures. However, Anesthetic B, with its larger aqueous solubility [ (blood/gas)], will partition relatively more than Anesthetic A into the aqueous portions o the brain. To provide the higher aqueous concentrations, many more moles o Anesthetic B than Anesthetic A must be trans erred rom the lungs. The overall conclusion still holds i (oil/gas) and thus MAC di er or the two hypothetical anesthetics. Palv or a less blood-soluble agent will rise proportionally aster toward its PI than or a more blood-soluble agent, independent o what that PI is (note that PI will be larger or the less oil-soluble anesthetic). A larger (oil/gas) allows the anesthetic to cause anesthesia at a lower partial pressure but does not a ect the proportional rate at which the partial pressure rises.

C h a p t e r 1 7 General Anesthetic Pharmacology 275 A Initia l P a lv = 0.1 a tm λ (blood/ga s ) = 0.5 Fina l P a lv = P a rt = 0.067 a tm

B Initia l P a lv = 0.1 a tm λ (blood/ga s ) = 11 Fina l P a lv = P a rt = 0.0083 a tm

Ane s the tic Alve olus Ca pilla ry

FIGURE 17-8. Why do anesthetics with lower (blood/gas) have shorter induction times? Consider two equally potent anesthetics inspired at the same partial pressure, PI. Be ore any anesthetic molecules have been taken up rom the alveolus into the blood, the alveolar partial pressure, Palv, o each anesthetic is 0.1 atm. This partial pressure would be represented in the diagram by 12 anesthetic “spheres” in each alveolus. For each anesthetic, equilibration o the partial pressures in the alveolus and the capillary then takes place. For a relatively blood-insoluble agent with (blood/gas) 0.5 (Anesthetic A, which closely resembles nitrous oxide, desf urane, and sevof urane), the trans er o a small amount o anesthetic rom the alveolus signi cantly raises the partial pressure in the capillary. To illustrate, consider a time, tv, when the volume o blood that has f owed past the alveolar wall is equal to the volume o the alveolus. At that time, the concentration in the alveolus will be twice that in the capillary (because (blood/gas) 0.5; see Box 17-2), i.e., our o the “spheres” will have been trans erred rom the alveolus to the capillary and eight “spheres” will remain in the alveolus. The partial pressure in the alveolus will now have dropped to (8/12) 0.1 0.067 atm. This is also the partial pressure in the capillary. In contrast, or a very blood-soluble agent with (blood/gas) 11 (Anesthetic B, which closely resembles diethyl ether), much larger amounts o anesthetic must dissolve in the blood to raise the partial pressure in the capillary. Using the same illustration as above, at tv, 11 o the 12 “spheres” will have been trans erred rom the alveolus to the capillary, and the remaining Palv will be given by (1/12) 0.1 0.0083 atm. Thus, although the inspired partial pressure o the two anesthetics is the same, at time tv, the Palv and Part o Anesthetic A will be eight times higher than that o Anesthetic B. Within approximately 2 minutes (Table 17-3), Pbrain will also reach these values. Thus, the brain partial pressure rises toward the inspired partial pressure much more rapidly or Anesthetic A than or Anesthetic B (i.e., the induction time or Anesthetic A is much shorter than that or Anesthetic B). I the reader is con used by the act that more molecules o Anesthetic B are being carried to the brain, recall that (brain/blood) is 1 or all o the commonly used anesthetics [that is, or each agent, (blood/gas) is approximately equal to (brain/gas); see Table 17-2]. Thus, proportionally, many more molecules o Anesthetic B than Anesthetic A must be delivered to the brain in order to raise the partial pressure o each anesthetic by an equivalent amount. See Boxes 17-1 and 17-2 and Appendix A or de nitions.

Applications of the Uptake Model Throughout the following discussion, it is critical to remember that the primary responsibility of the anesthesiologist is to keep the patient well oxygenated and the vital signs stable while manipulating the inspired partial pressure of anesthetic to maintain the desired depth of anesthesia. Armed with the uptake model, the anesthesiologist can predict the e ects o cardiopulmonary changes and pathologic states on the depth o anesthesia. Changes in ventilation and cardiac output may be caused by the general anesthetic itsel , by the trauma o surgery, or by some other physiologic or pathophysiologic process.

The e ects o changes in both ventilation and cardiac output on PCNS are greatest when the di erence between PI and Palv is greatest; that is, early in the course o anesthesia (Fig. 17-6). To understand this, consider the partial pressure in the mixed venous return (MVR), PMVR, which is a weighted average o the partial pressures in each o the tissue groups, with PVRG making the largest contribution because the VRG receives the majority o the cardiac output (Fig. 17-4). When Palv (and thus PVRG) is much less than PI, PMVR is low, and the bloodstream is capable o carrying large amounts o anesthetic away rom the alveoli to the tissues. Under these conditions, the rate o uptake o anesthetic rom the alveoli into the bloodstream can be greatly modif ed by cardiopulmonary changes, and PCNS can be greatly a ected by changes in ventilation and cardiac output. As each successive tissue group approaches saturation with anesthetic, PMVR approaches PI. When PMVR is nearly equal to PI, the bloodstream cannot remove much anesthetic rom the lungs under any circumstances, and changes in ventilation or cardiac output have little e ect on PCNS. Upon commencement o anesthetic administration, the length o time during which there is a signif cant di erence between PI and Palv increases with (blood/gas). With ventilation-limited anesthetics, such as diethyl ether and halothane, the prolonged time during which Palv lags behind PI allows cardiopulmonary changes to modulate Palv signif cantly, potentially leading to unexpected CNS partial pressures. With per usion-limited anesthetics, such as nitrous oxide, the alveolar partial pressure rises so rapidly that Palv is signif cantly less than PI or only a short time, minimizing the time during which cardiopulmonary changes could have a signif cant e ect on PCNS (Fig. 17-7).

Effects of Changes in Ventilation

Hypoventilation decreases the delivery o anesthetic to the alveoli. Meanwhile, removal o anesthetic rom the alveoli continues provided that cardiac output is maintained. Consequently, the alveolar partial pressure rises more slowly, and {Palv→PI} is prolonged. In other words, hypoventilation slows induction. This e ect is greater with ventilationlimited than with per usion-limited anesthetics (Fig. 17-9A). General anesthetics themselves can cause hypoventilation by depressing the medullary respiratory center. In this manner, anesthetic-induced hypoventilation sets up a benef cial negative eedback loop on the depth o anesthesia. Increasing anesthetic depth leads to medullary depression, which, in turn, depresses respiration. The benef cial e ect o this physiologic response is that the depressed ventilation slows the rate o rise o the alveolar partial pressure, while perusion continues to remove anesthetic rom the lung at the same rate (Fig. 17-5). Thus, Palv alls, and shortly therea ter, the partial pressure o anesthetic in the medulla alls as well. This decrease in PCNS relieves the respiratory depression. In the extreme example o a respiratory arrest, there is no ventilation to deliver anesthetic to the alveoli, but cardiac output continues to distribute anesthetic rom the alveoli and VRG to the MG and FG. In the case o diethyl ether, the decrease in PCNS can be o a su f cient magnitude that spontaneous ventilation resumes. Hyperventilation delivers anesthetic more quickly to the alveoli. This decreases the time constant or equilibration o the alveolar with the inspired partial pressure (recall that {Palv→PI} FRC / Valv, in the limiting case). However, the

276 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Effects of Changes in Cardiac Output

A Ve ntila tion Effe cts 1.0 Nitrous oxide

Ha lotha ne

0.5

P

a

l

v

/

P

I

63% e quilibra tion

Die thyl e the r

0.0 0

20

40

Minute s 2 L/min ve ntila tion

8 L/min ve ntila tion

B Ca rdia c Output Effe cts 1.0 Nitrous oxide

0.5

Ha lotha ne

P

a

l

v

/

P

I

63% e quilibra tion

At anesthetic partial pressures higher than those required to depress the respiratory center, cardiac output alls. When cardiac output alls, the bloodstream removes anesthetic rom the alveoli at a slower rate. Consequently, the alveolar partial pressure rises aster (Fig. 17-9B). Because the alveolar partial pressure equilibrates relatively quickly with the VRG (even at the lower cardiac output), the partial pressure in the CNS also rises more rapidly. In other words, decreased cardiac output speeds induction. This e ect is more marked with ventilation-limited than with per usion-limited anesthetics. Moreover, cardiac depression by anesthetics sets up a harm ul positive eedback loop on the depth o anesthesia. Increasing PCNS depresses cardiac unction, which urther increases Palv, which urther increases PCNS, which urther depresses cardiac unction. I cardiac arrest occurs, then positive measures must be taken to restore the circulation (e.g., cardiopulmonary resuscitation [CPR]) while reducing the alveolar partial pressure through controlled breathing with oxygen. Increased cardiac output increases per usion to the lungs and accelerates equilibration between the alveoli and the tissues. However, because the increased blood ow to the lungs removes anesthetic rom the alveoli at a aster rate, the rate o rise o the alveolar partial pressure is slowed. Thus, increased cardiac output slows induction. This e ect is greater with ventilation-limited than with per usion-limited agents.

Effects of Age Die thyl e the r

0.0 0

20

40

Minute s 2 L/min ca rdia c output

18 L/min ca rdia c output

FIGURE 17-9. Effects of changes in ventilation and cardiac output on the rate at which alveolar partial pressure rises toward inspired partial pressure. The rate of equilibration of the alveolar partial pressure with the inspired partial pressure can be affected by changes in ventilation (A) and cardiac output (B). Increasing ventilation from 2 L/min (dashed lines) to 8 L/min (solid lines) accelerates equilibration. On the other hand, increasing cardiac output from 2 L/min (dashed lines) to 18 L/min (solid lines) slows equilibration. Both effects are much larger for more blood-soluble gases, such as halothane and diethyl ether, which have rather slow induction times. For nitrous oxide, the rate of equilibration is so fast that any changes caused by hyperventilation or decreased cardiac output are small. The dashed horizontal lines represent 63% equilibration of Palv with PI, and the time required for each curve to cross this line represents {Palv→PI}.

hyperventilation-induced hypocapnia may concomitantly decrease cerebral blood ow, increasing {PCNS→Part}. Thus, while the partial pressure in the alveoli rises aster, the rate o equilibration between the CNS and the alveoli could be slower. The net e ect depends on which o these two steps is rate-limiting. For per usion-limited anesthetics such as nitrous oxide, the decrease in cerebral blood ow results in a slower induction. For the most soluble ventilationlimited anesthetics such as diethyl ether, the aster delivery o anesthetic to the alveoli speeds induction. For less soluble ventilation-limited anesthetics such as iso urane, the e ects roughly balance, and induction is not signif cantly a ected.

Relative to their body weight, young children such as Matthew have higher ventilation than do adults. This e ect tends to speed induction. However, young children also have relatively higher cardiac output than do adults; this e ect tends to slow induction. Although one might expect that these e ects would cancel out, two additional actors cause the partial pressure o anesthetic in the mixed venous return to rise more rapidly in children. First, relative to adults, a greater proportion o the blood ow serves the VRG in children, resulting in a higher partial pressure o anesthetic in the mixed venous return early in the course o anesthesia. Second, the lower capacity o the tissues or anesthetic in children relative to adults accelerates the rate at which the tissues become saturated with anesthetic. Both e ects lead to a decreased alveolar-to-venous partial pressure di erence because PMVR rises more rapidly, blunting the removal o anesthetic by the pulmonary circulation and moderating the extent to which cardiac output slows the rise in alveolar partial pressure. Thus, proportional increases in ventilation and cardiac output result in an accelerated rise o alveolar partial pressure and aster induction in children than in adults (Fig. 17-10). Ventilation-limited anesthetics, which are most a ected by cardiopulmonary changes, have a markedly aster induction in children. There ore, care must be taken to guard against the attainment o unexpectedly high (toxic) levels o anesthetic during anesthesia induction in children.

Effects of Abnormal States

In hemorrhagic shock, per usion to the CNS may be maintained in the ace o decreased cardiac output and hyperventilation. The decreased cardiac output and hyperventilation both accelerate the rise in alveolar partial pressure o anesthetic. PMVR also rises aster because o the relatively greater per usion to the VRG, lowering the ability o the pulmonary circulation

C h a p t e r 1 7 General Anesthetic Pharmacology 277

0.9 Childre n (1–5 ye a rs ) 0.8

0.7

P

a

l

v

/

P

I

Adults 0.6

0.5

0.4 0

10

20

30

40

50

60

Minute s of a ne s the s ia FIGURE 17-10. Anesthesia induction in children. Using halothane as an example, the alveolar partial pressure o anesthetic rises more quickly in children than in adults. The aster induction time in children results rom a balance between children’s increased respiration ( avoring aster induction) and increased cardiac output ( avoring slower induction). The time-dependent increase in the mixed venous partial pressure o anesthetic limits anesthetic uptake rom the lungs, dampening the e ect o increased cardiac output on induction time.

to remove anesthetic rom the alveoli and urther accelerating the rise in the alveolar partial pressure. In patients with hemorrhagic shock, the additive combination o these e ects can speed induction to a signif cant degree. In such cases, per usion-limited anesthetics, whose kinetics are not greatly a ected by cardiopulmonary changes, are pre erred over ventilation-limited agents (Fig. 17-9). In ventilation/per usion (V/Q) mismatch (e.g., in chronic obstructive pulmonary disease [COPD]), some alveoli are underventilated and overper used, while others may be adequately ventilated but underper used. Because the alveolar partial pressure o anesthetic rises more slowly in the underventilated alveoli, the anesthetic partial pressure in the arterial blood leaving these alveoli is lower than normal. Conversely, the partial pressure o anesthetic leaving the adequately ventilated but underper used alveoli is higher than normal. Because the ormer (overper used) alveoli contribute a larger percentage to the overall per usion, the weighted average partial pressure o anesthetic in the blood leaving the lung is decreased. Thus, PCNS equilibrates with a lower than normal arterial partial pressure and may not achieve the level required to induce anesthesia. There ore, higher inspired partial pressures are necessary to compensate or the e ects o V/Q mismatch. This e ect is mitigated somewhat with ventilation-limited anesthetics because the partial pressure in the underper used but overventilated alveoli rises much aster than normal. For this reason, per usion-limited anesthetics are most a ected by V/Q mismatch. Based on the principles and examples discussed above and summarized in Table 17-4, it should be possible to make reasonable predictions about the e ect o other changes in cardiopulmonary unction on anesthesia induction.

TABLE 17-4 Summary of the Effects of Physiologic, Pathophysiologic, and Clinical Variables on Rate of Induction of Anesthesia CAUSE FASTER THAN USUAL INDUCTION

CAUSE SLOWER THAN USUAL INDUCTION

Hyperventilation (ventilationlimited anesthetics)

Hyperventilation (per usion-limited anesthetics)

Decreased cardiac output

Hypoventilation

Young age (i.e., children)

Increased cardiac output

Shock

Chronic obstructive pulmonary disease

Thyrotoxicosis

Right-to-le t shunt

Initial PI higher than f nal desired PCNS

—

Based on the uptake model or inhaled anesthetics, the e ect o changes in physiologic variables on the rate o induction can be predicted. Entities in the column on the left speed induction, while entities on the right slow induction, as discussed in the text. Note that the e ect o hyperventilation depends on whether a ventilation-limited or per usion-limited anesthetic is being administered (see text).

Control of Induction

An anesthesiologist can decrease induction time by setting the initial PI higher than the f nal desired PCNS. (This concept is similar to that o a loading dose, which is discussed in Chapter 3.) Because the time constant or equilibration o PCNS with PI does not depend on the absolute level o PI, administration o anesthetic or a given amount o time always results in the same proportional equilibration o PCNS with PI. Consequently, a given absolute PCNS is reached aster when PI is higher because that PCNS is a smaller raction o the higher PI. Dr. Snow took advantage o this concept by starting iso urane at a PI o 0.02 atm, even though the MAC o iso urane is only 0.0114 atm. However, the anesthesiologist must remember to reduce PI as Palv approaches the target value, or, as demonstrated by Dr. Snow, PCNS will equilibrate with this higher PI and cause cardiopulmonary depression (Fig. 17-11).

Recovery It is desirable that recovery rom general anesthesia proceeds quickly, so that patients can maintain their own airways as soon as possible ollowing surgery. In general, the stages o recovery rom anesthesia occur in the opposite sequence rom those o anesthesia induction, including the unpleasant excitement stage (Fig. 17-1). During recovery, the partial pressure o anesthetic in the mixed venous return (PMVR) is the weighted average o the partial pressures in the VRG, MG, and FG, with the VRG making the largest contribution (see Fig. 17-4). Ventilation removes anesthetic rom the bloodstream into the exhaled air, and there ore, increased ventilation always accelerates recovery. As is the case with induction, recovery rom anesthesia with per usion-limited agents is rapid, whereas recovery rom ventilation-limited agents is more prolonged. Recovery di ers rom induction in several important ways, however. First, the anesthesiologist can increase the inspired partial pressure o anesthetic to speed the process o induction, whereas during recovery, the inspired partial

278 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Clinical Status of Patient

0.03 m

)

Re s pira tory de pre s s ion (toxic ra nge )

0.02

s

s

u

r

e

(

A

t

Continuing a t P I = 0.04 Atm

Ane s the s ia (the ra pe utic ra nge )

0.01

De s ire d P bra in for a ne s the s ia

o

l

a

r

p

a

r

t

i

a

l

p

r

e

Va rying P I be twe e n 0.015 a nd 0.02 Atm

P I = 0.01 Atm

A

l

ve

P I = 0.04 a tm

Awa ke (s ubthe ra pe utic ra nge )

0.00 0

5

10

15

Time (min) FIGURE 17-11. Applying overpressure to speed induction. Using halothane as an example, the anesthesiologist can use an initial PI greater than the f nal desired Pbrain to speed induction. I the desired partial pressure o anesthetic in the brain is about 0.013 atm, then the anesthesiologist could initially administer the inspired anesthetic at a higher partial pressure, or example, 0.04 atm. This method is e ective because the time constant or Palv→PI is independent o the absolute value o PI. In other words, i PI is increased, then the ratio Palv /PI will increase proportionally at the same rate, resulting in a greater absolute rise in Palv in a given amount o time. The anesthesiologist must be sure to decrease the inspired partial pressure in a timely manner, however, or the desired PbraIn or anesthesia can be overshot and, instead, partial pressures capable o causing respiratory depression can be reached. On the other hand, i the inspired partial pressure is reduced too rapidly, the patient may awaken as Palv is decreased because o uptake o anesthetic rom the alveoli into the bloodstream (not shown).

pressure cannot be decreased below zero. Second, during induction, all o the tissue compartments start out at the same partial pressure (zero). In contrast, at the start o recovery, the compartments may have very di erent partial pressures depending on the duration o anesthesia and the characteristics o the anesthetic. The VRG quickly equilibrates with the alveolar partial pressure during most surgical procedures, but the MG may or may not equilibrate, and the FG equilibrates so slowly that, in all but the longest procedures, PFG is ar rom equilibrium. Consequently, during recovery, per usion redistributes anesthetic down its partial pressure gradient rom the VRG to the MG and FG as well as to the lung. Because o this redistribution, the initial decrease in alveolar partial pressure during recovery is more rapid than the corresponding increase during induction. This initial decrease in alveolar partial pressure is dominated by the decrease in the VRG partial pressure. When the alveolar pressure alls to the level o the MG, then the decrease in the partial pressure o the MG becomes rate-limiting and likewise subsequently or the FG. I the MG or both the MG and FG are heavily saturated ollowing prolonged administration o anesthetic, then recovery will also be prolonged (Fig. 17-12). Third, although anesthetic is delivered by one route, ventilation, it can be eliminated by both ventilation and metabolism. In most cases, metabolism is not a signif cant route o anesthetic elimination. Halothane is an exception because metabolism may account or 20% o its elimination. Finally, the out ow o high partial pressures o nitrous oxide into the lungs can cause an e ect called diffusion hypoxia . To understand this, it is help ul f rst to understand an e ect on anesthetic induction called the concentration effect. When high partial pressures o nitrous oxide are

administered, the rate o anesthetic uptake by the blood may be quite large, on the order o 1 L/min or a 75% nitrous oxide mixture. The absorbed gas is rapidly replaced by inspired gas owing into the lung, e ectively increasing alveolar ventilation by 1 L/min above the normal minute ventilation and thereby accelerating induction. Di usion hypoxia is conceptually the opposite o the concentration e ect. When anesthesia is terminated, nitrous oxide gas di uses out o the blood into the alveoli at a high rate because o the high partial pressure di erence between these two compartments (recall Fick’s law). This volume o nitrous oxide displaces up to 1 L/min o air that would otherwise have been inhaled. Thus, the alveolar (and arterial) partial pressure o oxygen alls. The decrease is not signif cant or a healthy patient but may be threatening to a compromised patient. To counteract this e ect, pure oxygen is routinely administered or a ew minutes ollowing anesthesia with nitrous oxide, as Dr. Snow did or Matthew.

PHARMACOLOGY OF GENERAL ANESTHETICS AND ADJ UVANTS Inhaled Anesthetic Agents From the preceding analysis, we can distill two physicochemical properties o inhaled anesthetics that predict their behavior. First, the oil/gas partition coe f cient predicts potency; an anesthetic with a higher (oil/gas) is more potent and causes anesthesia at lower partial pressures. Second, the blood/gas partition coe f cient predicts the rate o induction; an anesthetic with a lower (blood/gas) has a shorter induction time. Typically, there is a trade-o between ast

C h a p t e r 1 7 General Anesthetic Pharmacology 279

Nitrous oxide λ (blood/ga s ) = 0.47

1.0

Ha lotha ne λ (blood/ga s ) = 2.3

Me thoxyflura ne λ (blood/ga s ) = 13.0

Minute s of Ane s the s ia

∞

0.5

P

E

/

P

E

0

240 120 60 30 15

0

0

40

80

120

0

40

Time (min)

80

Time (min)

120

0

40

80

120

Time (min)

FIGURE 17-12. Recovery rom inhaled anesthetics. These curves show, as a function of time, the exhaled partial pressure of anesthetic (PE) as a fraction of the exhaled partial pressure at the moment administration of the anesthetic is stopped (PE0). The rate of recovery is inversely proportional to the (blood/ gas) of the anesthetic, because anesthetics with lower (blood/gas) values equilibrate faster between alveolar and inspired partial pressures (the latter being zero after anesthetic administration is stopped). The rate of recovery is also proportional to the duration of anesthesia because the partial pressures of anesthetic in the muscle group and fat group increase with duration. During recovery, anesthetic redistributes from these slowly equilibrating, high-capacity tissues to the vessel-rich group, thus slowing the rate of fall of Pbrain. This effect occurs only with a long duration of anesthesia (see text).

induction and high potency. An anesthetic that has a rapid induction, as denoted by a low (blood/gas), typically has a low potency, represented by a low (oil/gas). Conversely, a very potent anesthetic with a high (oil/gas) typically has a high (blood/gas) and, thus, a long induction time (see Table 17-1). Halothane has a high (oil/gas), providing high potency and, thus, low MAC; however, halothane also has a high (blood/gas), causing slow induction and recovery. The nonirritating smell o halothane makes it use ul in pediatric anesthesia, but sevof urane is increasingly replacing halothane or use in pediatric anesthesia (see below). One disadvantage o halothane is that toxic metabolites can result in atal hepatotoxicity. The incidence o this serious adverse e ect is approximately 1 in 35,000 in adult populations but much lower in pediatric populations; this is another reason or its continuing role in pediatric anesthesia. Another rare but potentially lethal adverse e ect, seen most o ten with halothane but occasionally with the other halogenated anesthetics, is malignant hyperthermia . The susceptibility or this adverse reaction is inherited, typically as an autosomal dominant mutation in the sarcoplasmic reticulum Ca2 channel (also known as the ryanodine receptor). In individuals expressing this mutation, halothane causes uncontrolled calcium e ux rom the sarcoplasmic reticulum, with subsequent tetany and heat production. Malignant hyperthermia is treated with dantrolene , an agent that blocks calcium release rom the sarcoplasmic reticulum. Isof urane and enf urane are somewhat less potent than halothane [they have a lower (oil/gas)], but they equilibrate aster because they have a lower (blood/gas). Enurane is metabolically de uorinated to a greater extent than iso urane and may thus have a higher risk o causing

renal toxicity. It also induces seizure-like activity in the EEG o some patients. Iso urane is probably the most widely used general anesthetic today. Although less potent than iso urane and en urane, diethyl ether is still quite potent, with a rather high (oil/ gas). However, because o its ammability and very slow induction, attributable to its extremely high (blood/gas), this agent is no longer in common use in the United States and Europe. In developing countries, however, its low price and simplicity o application avor its continued use. Nitrous oxide has a very low (blood/gas) and thus equilibrates extremely rapidly. However, its low (oil/gas) results in a very high MAC, close to one atmosphere. Thus, the need to maintain an acceptable partial pressure o oxygen (normally, greater than 0.21 atm) prevents the attainment o ull anesthesia using nitrous oxide alone, and this agent is commonly employed in combination with other agents (see the section “Balanced Anesthesia”). Desf urane and sevof urane are newer anesthetics that, by design, have low (blood/gas); times o equilibration between their alveolar and inspired partial pressures are nearly as short as that o nitrous oxide. Furthermore, they are much more potent than nitrous oxide because their oil/gas partition coe f cients are higher. Thus, these agents o er great improvements over earlier agents. However, des urane is a poor induction agent because its pungency irritates the airway, potentially causing cough or laryngospasm. Sevo urane is sweet-tasting but can be chemically unstable when exposed to some carbon dioxide adsorbents in anesthetic machinery, degrading to an olef nic compound that is potentially nephrotoxic. These disadvantages have been overcome with improved machinery, and sevo urane is gaining in popularity.

280 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Intravenous Anesthetic Agents Intravenous anesthetics, such as barbiturates (see also Chapter 13), allow or rapid induction. Ultrashort-acting barbiturates, such as thiopental, are capable o inducing surgical anesthesia within seconds. As nonvolatile compounds, intravenous agents di er rom inhaled anesthetics in that they cannot be removed rom the body by ventilation. Accordingly, one must take great care during their administration to avoid severe medullary depression that is not easily reversible. The primary method o removal o these agents rom the CNS is by redistribution rom the VRG to the MG and f nally to the FG. Metabolism and/or excretion then slowly decrease the overall body levels o drug (Fig. 17-13). Propo ol is an important intravenous anesthetic prepared in an intralipid ormulation. This agent produces anesthesia at a rate similar to the ultrashort-acting barbiturates. Propo ol is both rapidly redistributed and rapidly metabolized, resulting in a aster recovery than or barbiturates. Propo ol is used both or induction and or maintenance, especially in short day-surgery procedures where its ast elimination avors prompt recovery and early discharge. The intralipid preparation o propo ol can rarely be a source o in ection, and the lipid preparation provides a large caloric source; these considerations can be important in critically ill patients who may receive prolonged propo ol in usions. Etomidate is an imidazole that is used or induction o anesthesia because its kinetics are similar to those o propool. This agent causes minimal cardiopulmonary depression,

100 Blood MG

VRG

60

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Adjuvant Drugs Adjuvant drugs provide additional e ects that are desirable during surgery but are not necessarily provided by the general anesthetics. Benzodiazepines (see Chapter 13), such as diazepam, lorazepam, and midazolam, are o ten given or their anxiolytic and anterograde amnesic properties. These agents are typically administered 15 to 60 minutes be ore the induction o anesthesia to calm the patient and obliterate memory o the induction, although they may also be used or intraoperative sedation. I necessary, benzodiazepine e ects can be reversed with the antagonist f umazenil. Opioids (see Chapter 18) such as morphine and entanyl are used or their ability to produce analgesia. Their action can be reversed by an antagonist such as naltrexone . Opioids are poor amnesics, however, and are typically used in combination with a general anesthetic. The combination o entanyl and droperidol produces both analgesia and amnesia. Together with nitrous oxide, this combination is called neuroleptanesthesia (the pref x “neurolept” is added because droperidol is a butyrophenone antipsychotic related to haloperidol; see Chapter 14, Pharmacology o Dopaminergic Neurotransmission). Nicotinic acetylcholine receptor blockers, such as the competitive antagonists tubocurarine and pancuronium or the depolarizing agonist succinylcholine , are commonly used to achieve muscle relaxation (see Chapter 10). The e ects o the competitive antagonists can be reversed by an acetylcholinesterase inhibitor such as neostigmine .

Balanced Anesthesia

20

0.1

perhaps because o its unique lack o e ect on the sympathetic nervous system. In spite o this advantage, it is seldom used because it inhibits the synthesis o adrenocortical steroids with high potency. Unlike the above agents, ketamine produces dissociative anesthesia, in which the patient seems to be awake but is actually in an analgesic and amnesic state. Ketamine has the unusual property that it increases cardiac output by increasing sympathetic out ow; or this reason, it is occasionally use ul in emergency trauma situations. However, it can also produce unpleasant hallucinations. This agent is rarely used today.

100

Time (min) FIGURE 17-13. Distribution o a bolus o intravenous anesthetic. When a bolus o intravenous anesthetic is administered, it is initially transported through the vascular system to the heart and then distributed to the tissues. The vessel-rich group (VRG) receives the highest percentage o the cardiac output; its anesthetic concentration rises rapidly, reaching a peak within 1 minute. Redistribution o anesthetic to the muscle group (MG) then quickly decreases the anesthetic level in the VRG. Because o very low at group (FG) per usion, redistribution rom the MG to the FG does not occur until much later. Note that rapid redistribution rom the VRG to the MG does not occur i the MG has previously approached saturation through prolonged administration o anesthetic (not shown); this can lead to signif cant toxicity i intravenous barbiturates are administered continuously or long periods o time. Newer agents, such as propo ol, are designed to be eliminated by rapid metabolism and, there ore, can be used sa ely or longer periods o time.

No single drug achieves all o the desired goals o anesthesia. Accordingly, in a method termed balanced anesthesia , several inhaled and/or intravenous drugs are used in combination to produce the anesthetic state. The anesthetic e ects o simultaneously administered general anesthetics are additive. That is, 0.5 MAC o one inhaled anesthetic in combination with 0.5 MAC o another is equivalent in terms o potency to 1 MAC o either anesthetic as a single agent. Using a mixture o inhaled anesthetics allows the two goals o potency and rapid recovery to be achieved. For example, although using nitrous oxide alone is generally impractical because the MAC o this gas is higher than atmospheric pressure, nitrous oxide is desirable or its ast induction and recovery characteristics and its high analgesic index. I nitrous oxide is part o the anesthetic mixture, then the nitrous oxide component o the anesthesia can be rapidly removed by ventilation during recovery or in an emergency situation. Matthew was able to awaken quickly rom anesthesia because nitrous oxide was responsible or about hal o his anesthetic state. He remained groggy because o the

C h a p t e r 1 7 General Anesthetic Pharmacology 281

lingering iso urane. The advantages o using iso urane in combination with nitrous oxide include iso urane’s low cost and its relatively low incidence o adverse e ects (especially hepatic and renal toxicity) as compared to other anesthetics. Dr. Snow’s use o the intravenous agent thiopental in combination with an inhaled anesthetic agent has a similar rationale. Short-acting intravenous agents can be used to induce stage III surgical anesthesia quickly, allowing the patient to pass through the undesirable excitement o stage II rapidly. Subsequently, the anesthetic depth can be maintained with inhaled anesthetics that could be removed by ventilation i necessary. Because intravenous agents act additively with inhaled anesthetics, less than 1 MAC o inhaled anesthetic will be required or as long as the intravenous agent is acting. As another example, the use o high concentrations o opioids in cardiac surgery allows the partial pressure o the inhaled anesthetic to be lowered signif cantly, reducing the risk o cardiovascular and respiratory depression. Finally, balanced anesthesia is clinically use ul because the anesthesiologist has more control i a di erent drug is used to mediate each desired e ect. For example, i the surgeon requires more muscle relaxation, the anesthesiologist can administer more o a neuromuscular blocking agent without having to increase the depth o anesthesia and potentially cause cardiopulmonary depression. Similarly, a bolus o a short-acting opioid can be administered immediately be ore a particularly pain ul surgical maneuver.

MECHANISMS OF ACTION OF GENERAL ANESTHETICS Despite intensive research, the exact mechanism o anesthetic action remains elusive. The unitary hypothesis states that a common mechanism accounts or the action o all anesthetics. Assuming the unitary hypothesis, it is di f cult to imagine a specif c binding site on a protein or receptor molecule capable o accommodating the molecules o disparate sizes and structures that are capable o causing anesthesia (Fig. 17-14). The traditional solution to this contradiction, which ollows rom the empirical Meyer-Overton rule (Fig. 17-3), is the lipid solubility hypothesis . This hypothesis postulates that the hydrophobic site o action o anesthetics is in the lipid bilayer o a cell membrane. According to this hypothesis, general anesthesia results when a su f cient amount o any anesthetic dissolves in the lipid bilayer and a critical (“anesthetic”) concentration is reached. Various distinct lipid theories postulate that the dissolved anesthetics cause perturbations o di erent physical properties o the lipid bilayer, such as uidity. A property that these lipid perturbation models share is that they all cause the bilayer to expand (this is called the critical volume hypothesis ), a act that is consistent with the observation that anesthetized animals can be aroused by raising the pressure, either hydrostatically in amphibians or with the nonanesthetic gas helium in mammals. The lipid solubility hypothesis is remarkably success ul at predicting potency or all the volatile agents and or some o the intravenous agents that have potencies less than 50 M. However, or more potent agents, exceptions become increasingly common. For example, straight-chain alcohols increase in potency as each methylene group is added up to dodecanol, which has an anesthetic potency o 5 M. Straight-chain alcohols with even longer chains lack any anesthetic activity, however, even though their (oil/gas) is higher than that o

the shorter alcohols. Furthermore, R-etomidate, which is used clinically and causes anesthesia at 5 M, is 10 times more potent than its enantiomer, S-etomidate. Such a degree o enantioselectivity is also seen in etomidate’s action at the GABAA receptor, which points to action at a protein binding site. In a genetically engineered “knock-in” mouse, R-etomidate’s potency was reduced ten old by a single mutation in the 3-subunit o the GABAA receptor. The action o volatile and steroid anesthetics, which also act at GABAA receptors, was una ected by this mutation. This f nding suggests that GABAA receptors could have a number o allosteric anesthetic binding sites, each o which binds di erent structural classes o anesthetic.

A Inhaled anesthetics N

N

O

O

HN

Nitrous oxide F F3 C

B Intravenous anesthetics

O HN

F O

* O

P e ntoba rbita l

F

De s flura ne

O CF 3 F3C

HN

O

F

S e voflura ne

S

*

HN O

Thiope nta l O

Die thyl e the r (Ethe r)

OH

Br F3C

* Cl

P ropofol

Ha lotha ne

O Cl Cl

*

F3C

F

* O

F

NH

Is oflura ne Ke ta mine

F

F

F

F O

* Cl

Enflura ne

O O

F

* N N Etomida te

FIGURE 17-14. Structures of general anesthetics. A. Structures of some inhaled anesthetics. B. Structures of some intravenous anesthetics. The extreme variability in the structures of these molecules, all of which are capable of causing general anesthesia, suggests that not all general anesthetics interact with a single receptor site. *Indicates carbons where asymmetry results in enantiomeric structures.

282 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Effects on Ion Channels Current research has ocused on proteins that alter neuronal excitability when acted on by anesthetics. Anesthetics a ect both axonal conduction and synaptic transmission, but only modulation of ligand-gated synaptic transmission occurs at clinically relevant concentrations and is, there ore, likely to be the pharmacologically relevant action. Synaptic action has presynaptic and postsynaptic components that lead to anesthesia, but the postsynaptic actions dominate. The simplest working general model that unites current research is that general anesthetics may act either by enhancing inhibitory ligand-gated ion channels, or by inhibiting excitatory channels, or by a mixture of both effects. Etomidate and ketamine, respectively, provide a clear example o the f rst two actions, with many less potent anesthetics alling into the third class. Excitatory receptors (nicotinic acetylcholine, 5-HT3 and NMDA) are inhibited by anesthetics. The binding o anesthetic to these receptors lowers their maximum activation, without changing the concentration o agonist required to achieve a hal -maximal e ect (EC50) (Fig. 17-15). This action is consistent with noncompetitive inhibition and an allosteric site o action (see also Chapter 2). In contrast, inhibitory receptors (GABAA and glycine) are potentiated by anesthetics. The binding o anesthetic to these receptors decreases the concentration o agonist required to achieve a maximum response, shi ting the activation curves to the le t (lower EC50). The anesthetic stabilizes the open state o the receptor and there ore prolongs the inhibitory current, so the maximum response o ten increases as well (Fig. 17-15). The ligand-gated excitatory and inhibitory ion channels that are a ected by anesthetic action belong to two structural classes. The f rst class is the structurally homologous Cys-loop superfamily o ligand-gated ion channels, which includes the inhibitory GABAA and glycine receptors as well as the excitatory nicotinic and 5-HT3 receptors. The second structural class is the excitatory NMDA glutamate receptor amily (compare Figs. 10-2 and 13-3 with Fig. 13-8). Most progress has been made with the mechanisms o action o

150

Inhibitory s yna ps e with ge ne ra l a ne s the tic

100

e

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75

ve

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e

s

p

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50

R

I binding to any one o these sites could cause anesthesia, the unitary hypothesis would be satisf ed without invoking lipids. On the other hand, nitrous oxide, cyclopropane, xenon, and ketamine, all o which have seen clinical use, have no action on GABAA receptors. Instead, they act on NMDA-type glutamate receptors. Thus, the anesthetic state may be induced by di erent mechanisms. The anesthetic state incorporates many distinct actions o general anesthetics, including sedation, amnesia, antianxiety, and anticonvulsant actions. Do these actions share common sites? A clue to the answer to such questions is provided by another piece o research on knock-in mice. In this work, a mutation was introduced in the 2 subunit o the GABAA receptor at a position homologous to that on the 3 subunit mentioned above. In this case, it was etomidate-induced sedation, not anesthesia, that was attenuated. Thus, di erent GABAA receptor subunits, most likely in di erent neural circuits, may mediate di erent behaviors through homologous but subtly di erent sites. Furthermore, anesthesia induced by NMDA agents di ers in quality rom that induced by volatile agents and is called dissociative anesthesia . Thus, the unitary hypothesis is not generally valid but could still apply to the various components o anesthesia considered separately.

EC 50

25

Excita tory s yna ps e with ge ne ra l a ne s the tic

0 0.01

0.1

1.0

10

100

Re la tive a gonis t conce ntra tion FIGURE 17-15. Actions of anesthetics on ligand-gated ion channels. Anesthetics potentiate the action o endogenous agonists at inhibitory receptors, such as GABAA and glycine receptors, and inhibit the action o endogenous agonists at excitatory receptors, such as nicotinic acetylcholine, 5-HT3, and NMDA glutamate receptors. At GABAA receptors, anesthetics both decrease the EC50 o GABA (i.e., GABA becomes more potent) and increase the maximum response (i.e., GABA becomes more e f cacious). The latter e ect is thought to be due to the ability o anesthetics to stabilize the open state o the receptor channel. At excitatory receptors, anesthetics decrease the maximum response while leaving the EC50 unchanged; these are the pharmacologic hallmarks o noncompetitive inhibition.

anesthetics at the Cys-loop receptors. At the molecular level, direct anesthetic–protein interactions are responsible for the effects of anesthetics on ligand-gated ion channels. Sitedirected mutagenesis, photolabeling, and kinetic studies suggest that inhibition o excitatory acetylcholine receptors occurs at a site in the pore o the ion channel that is on the central axis o symmetry and in contact with the channellining M2 helices o all f ve subunits (see Fig. 10-2 or terminology and structure). However, the site o anesthetic binding to inhibitory GABAA receptors (Fig. 13-3) cannot be in the ion pore because potentiation, not inhibition, is observed at therapeutic concentrations. Indeed, GABAA receptors lack a homologous stretch o hydrophobic amino acids in the M2 helices that line the ion pore in the excitatory receptors. The consensus view is that anesthetics bind in the transmembrane domain o the GABAA receptor to allosterically a ect the channel’s con ormation (and, thus, the equilibria among its open, closed, and desensitized states). Early site-directed mutagenesis studies suggested that volatile anesthetics bind within the our transmembrane helices (intra-subunit sites) o each GABAA receptor subunit. In contrast, recent photolabeling with potent intravenous anesthetics places the binding site between the subunits (inter-subunit sites). Members o the Cys-loop super amily have f ve highly homologous subunits, each with our transmembrane helices. The anesthetic sensitivity of ligand-gated ion channels may vary with their subunit composition. Central GABAA receptors vary in subunit composition; some dozen combinations

C h a p t e r 1 7 General Anesthetic Pharmacology 283

have been established to date (e.g., 1–6 2–3 2, 4 2–3 , ). Reading counterclockwise, the order in 6 2–3 , and which the subunits are arranged around the center o symmetry o each pentameric receptor is / . Thus, there are potentially three distinct intra-subunit anesthetic sites and our distinct inter-subunit anesthetic sites on each distinct GABAA receptor pentamer. O the possible binding sites, only two are established on human GABAA receptors. Etomidate binds in the two – inter aces in the transmembrane domain, some 50 Å below the inter-subunit agonist sites in the same – inter aces in the extracellular domain. A derivative o mephobarbital photolabels the transmembrane domain in both the – inter ace and the – inter ace but not in the etomidate site at the – inter aces. Propo ol binds nonselectively to all our o these sites. Thus, subunitdependent sequence variations within each o the homologous anesthetic binding pockets now provide an explanation or the diversity o general anesthetic structures, and or the selectivity o anesthetic binding, without invoking lipid solubility. O potential clinical signif cance, this conclusion suggests the possibility o designing novel general anesthetics that bind selectively to only some subtypes o GABAA receptors.

CONCLUSION AND FUTURE DIRECTIONS Inhaled and intravenous anesthetics are used to produce the clinical eatures o general anesthesia, including unconsciousness, immobility, and amnesia. The pharmacodynamics o general anesthetics are unique. Anesthetics have steep dose– response curves and low therapeutic indices, and they lack a pharmacologic antagonist. According to the Meyer-Overton rule, the potency o a general anesthetic can be predicted simply rom its oil/gas partition coe f cient. The pharmacokinetics o inhaled anesthetics can be modeled assuming three principal tissue compartments that are per used in parallel. Equilibration o the partial pressure o anesthetic in the CNS with the inspired partial pressure proceeds in two steps: (1) equilibration between the alveolar partial pressure and the inspired partial pressure and (2) equilibration between the CNS partial pressure and the alveolar partial pressure. With ventilation-limited anesthetics, which have a high blood/gas partition coe f cient, the f rst o these steps is slow and rate-limiting. With per usion-limited anesthetics, which have a low blood/gas partition coe f cient,

both steps are rapid and neither is clearly rate-limiting; changes in either can a ect induction time. Recovery rom anesthesia occurs roughly as the reverse o induction, except that redistribution o anesthetic rom the vessel-rich group to the muscle group and at group can also occur. The “ideal” inhaled anesthetic has not yet been ound. Future researchers may attempt to identi y a non ammable anesthetic with high (oil/gas), low (blood/gas), high therapeutic index, good vapor pressure, and ew or no signif cant adverse e ects. Currently, the combined use o adjuvants and balanced anesthesia with multiple inhaled and/or intravenous anesthetics achieves all o the goals o general anesthesia, including ast induction and a state o analgesia, amnesia, and muscle relaxation. The exact mechanism o action o general anesthetics remains a mystery. Although the site o action was ormerly thought to reside in lipid bilayers, direct interactions with several ligand-gated ion channels—specif cally, members o the Cys-loop super amily and the NMDA glutamate receptor amily—now seem to be more likely. More research is required to elucidate the mechanisms o action o general anesthetics. Once discovered, these mechanisms could shed light on such ar-reaching issues as the generation o consciousness itsel .

Suggested Reading Campagna JA, Miller KW, Forman SA. The mechanisms o volatile anesthetic actions. N Engl J Med 2003;348:2110–2124. (Reviews how general anesthetics act.) Eger EI. Uptake and distribution. In: Miller RD, ed. Anesthesia. Philadelphia: Churchill Livingstone; 2000:74–95. (Pharmacokinetics and uptake o inhaled anesthetics.) Forman SA. Monod-Wyman-Changeux allosteric mechanisms o action and the pharmacology o etomidate. Curr Opin Anaesthesiol 2012;25:411–418. (Reviews allosteric models o anesthetic action, with specif c re erence to etomidate.) Rudolph U, Antkowiak B. Molecular and neuronal substrates or general anesthetics. Nat Rev Neurosci 2004;5:709–720. (A short review with good diagrams.) Various authors. Molecular and cellular mechanisms o anaesthesia. In: Can J Anesth 2011; Feb issue. (This special issue is a compilation o detailed reviews relating to all major current theories on the mechanism o action o general anesthetics. An update will appear in mid-2016 in Anesth. Analg.) Wiklund RA, Rosenbaum SH. Anesthesiology. N Engl J Med 1997;337: 1132–1151, 1215–1219. (Two-part review covers many aspects o the modern practice o anesthesiology.) Winter PM, Miller JN. Anesthesiology. Sci Am 1985;252:124–131. (A good account o the clinical approach o the anesthesiologist.)

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C h a p t e r 1 7 General Anesthetic Pharmacology 285

A p p e n d ix A Abbreviations and Symbols PI inspired partial pr preessu essure PE exhaled ed partial pressure Palv alveolar partial pressure l Partt arterial paart rtia iall pr preessuure Ptissue partial pre ress ssure in a tissue Pven partial pressure in a venule venule PMVR mixed venous partial pressure Psolven partial pressuree in a solvent lventt PCNS par arti tial al pre ress ssur uree in the central nervous system PVRG partial pressure in the vessell ri rich ch gro roup upp (oil il//gas) partition coe f cient def ningg so solu lubi bili lity ty o a gas in a lippopphiili licc so solv lven entt suchh as oil (blood/gas) partition coe f cient def ning solubility o a gas in blood (tissue/gas) partition coe f cient def ning solubility o a gas in a tissue (tissue/blood) partition coe f cient describing ratio o solubility in tissue to solubility in blood (tissue/gas) / (blood/gas) time constant or 63% equilibration {Palv→PI} time constant or 63% equilibration o Palv with PI {Ptissue→Palv} time constant or 63% equilibration o Ptissue with Palv

286

[A] concenttra rati tion on o gas A, in te term rmss o either Lgas as/Lsolvent oorr mo mol/ l/Lsolvent CNS centr tral al nervous system VRG G vessel-rich group (includes CNS, live ver, kidney) MG M G mus uscl cle gr g oup (includes muscle, sk skiin) FG at group (includdes adi dipo pose se tisssu sue) VPG vessel-poor group (incllud udes bone, carti tila lage ge, ligaments, tendons) FRC unctionall residual i cap apac acit ityy o lung Valv alve lveollar ventilation CO C O car ardi diac ac outpu p t Q per usion rate Voltissue volume o tissue MAC minimum (or median) alveolar concentration P50 alveolar partial pressure su f cient or immobility in 50% o patients MAC AP50 alveolar partial pressure su f cient to cause analgesia in 50% o patients LP50 alveolar partial pressure su f cient to cause death in 50% o subjects EC50 concentration o agonist required to activate 50% o channels

A p p e n d ix B Equations GAS CONCENTRATIONS In an ideal gas mix ixtu ture re: [A]mixxtur nA / V PA / RT turee {inn te {i terms o mol o /L} In solution (Henry’ss la l w): [A]solution Psollven (ssolvent/gas) e {in termss o Lgas/Lsolvent} ventt [A]sooolluti Psolventt (sol olve v nt/gas) / 24.5 {in terrms o mol/ ution on Lsolve vent} {where nA moles o ga gass A, V tot otal al vol olum ume, e, PA part pa rtia iall prres e sure o A, R universal gas con o stant, T temperaturee in degrees Kelvin}

MEYER-OVERTON RULE MAC ≈ 1.3 / (o ( il/ggas))

FICK’S S LAW FOR DIFFUSION ACROSS A BOUNDARY Rate o di usion D (A / l) P {where D Di usion constant; A Sur ace area; l Thickness; P Partial pressure di erence}

EQUILIBRATION TIME E CONSTANTS (FOR 63% EQUIILIBRATION) Volumee Ca Capa p city / Flow Raate { titissue→Palv}≈ {P } {Ptitisssue su →Part} Vol olum umee Capaaci city ty o Ti Tiss ssue / Tissue Blood Fl Flow ow (tissue/ e/bl bloo ood) Volume o Tis issu suee / Ti Tiss ssue ue Bloodd Flo low w {Pbrain→Part} (bbra rain in/b /blo l od) Volume o Bra rain in / Blood Flow Fl ow to Brain Pcontainer Pf oow [1 e (t/ )] nerr

VOLUME CAPACITY Volum lume me Ca Capa paci pa city ty ([A]com Volum l me o compar partme tment ntt ccom ompa part rtment) / [A]medium {at equilibrium} (compartment/medium)) Vol olum umee o Com ompa part rtme ment nt

MIXED VENOUS PARTIAL PARTIALPRESSURE PRESSURE PMVR

0.75 PVRG

0.18 PMG

0.055 PFG

0.015 PVPG

ALVEOLAR CAPILLARY RATE OF UPTAKE Rate o uptake ([[A]artt [A]MVR) CO {in Lgas/min} Rate o uptake (blood/gas) (Partt PMVR) CO {where CO cardiac output}

287

Ce nt ntra ra l p e rcee p ti tion on (o pio p io id ds )

C o r te Cor texx

18 Pharmacology of Analgesia Tha la mus m us Ro b e rt S. Griffin a n d Cliffo rd J . Wo oTh lf a la Re laa y a nd n d d e s ce ndi ding ng g modu mo o dula la ti tion oMorp on INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288–289 orphi hine, Coddeine, ein aand Derivativess PHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2888 Sensory Transd sduc uc tion: i Excitation o Pri rim mary A erent Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Conduction rom the Periphery to the Spinaa l Co Cord rd. . . . . . . . . 291 Transmission in the Dorsal Hor ornn o the Spinal Cord. . . . . . . . 291 Descc en endi ding ng a nd Loc all Inhibitory Regulation in the Spinal i Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 PATHOPHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Clinical Nociceptive Pain . . . . . . . . . . . . . .Bra .a. ins . . .te . . . . . . 2933 B.ra Br i.ns t.e. m Periph p eral al Sen ensi siti tiza zati tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 Central Sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Neuropathic Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 96 Migraine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 297 Opioid Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 Mechanism o Action and Major Adverse E ects . . . . . . . 297

INTRODUCTION Everyone has experienced pain in response to an intense or noxious stimulus. This physiologic “ouch” pain helps us to avoid potential damage by acting as an early warning or protective signal. Pain can, however, also be incapacitating, as a ter trauma, during recovery rom surgery, or in association with medical conditions that are characterized by inf ammation, such as rheumatoid arthritis. Under circumstances where tissue injury and inf ammation are present, noxious stimuli elicit more severe pain than normal because o increases in the excitability o the somatosensory system, and stimuli that would not normally cause pain become pain ul. In addition, nerve injury produced by disease or trauma—as in amputation, HIV in ection, varicella-zoster (VZV) in ection, cytotoxic treatment, and diabetes mellitus—evokes pain that persists long a ter the initiating cause has disappeared. In these conditions, pathologic and sometimes irreversible alterations in the structure and unction o the nervous system lead to severe and intractable pain. For such patients, the pain is the pathology rather than a physiologic de ense mechanism. Finally, there are patients who experience considerable pain in the absence o noxious stimuli, inf ammation, or lesions to the nervous system. This dys unctional pain, as in tensiontype headache, bromyalgia, or irritable bowel syndrome, results rom an abnormal unction o the nervous system. 288

. . . . . . . . . . . . . . . . . . 2988 Synthetic S Sy ynthetic th ti Ago A Agonists oni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2999 Partia Part Pa rtia ial a l and a d Mixed an Mi Agonists on s . . . . . . . . . . . . . . . . . . . . . . . . . . 300 Opiio Op ioid idd Recep epto ep torr A to Anta tago goni go onist istss . . . . . . . . . . . . . . . . . . . . . . . . . 300 Nonsteroid No o dall A Anti-Inff am a matory m Dru ruggs a nd d Nonop ono iooid i A Anaa lg l esics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 Generall Fe Feat e aturess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ea Specif c Agents gen entss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Anntideprres e saa nt n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 A tiep An eppil ilep epticc Dr Drug ugs ug g an nd An Antiarrrhhythmics . . . . . . . . . . . . . . . . . 301 NMDA NM A Recc ep epto torr Antagonist ist stss . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3022 A rene Ad nergic Agonnists is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Migraine Therapy appy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 CONCLUSION ONC AND ND FUT TUR URE E DI DIR RECT RE R CTIO IONS NS . . . . . . . . . . . . . . . . . . 30 3022 Suggested S t dR Reading di . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .303

These categories o pain—physiologic, inf ammatory, neuropathic, and dys unctional—are produced by di erent mechanisms. Ideally, treatment should be targeted at the speci c mechanisms that produce pain rather than at suppressing the symptom o pain. That said, many o the currently available pharmacologic agents relieve pain by suppressing the symptom. The mechanisms o action o drugs that relieve pain involve inter erence with the response o primary sensory neurons to somatic or visceral sensory stimuli, inhibition o the relaying o pain in ormation to the brain, and blockade o the perceptual response to a pain ul stimulus. In this chapter, the discussion o pain and analgesic pharmacology begins by describing the mechanisms by which noxious stimuli lead to the perception o pain. The chapter continues by considering the processes responsible or the heightened pain sensitivity that occurs in response to inf ammation and lesions o the nervous system. The discussion concludes by describing the mechanisms o action o the major drug classes used or clinical pain relie .

PHYSIOLOGY Pain is the end perceptual consequence o the neural pro-

cessing o particular sensory in ormation. The initial stimulus usually arises in the periphery and is trans erred under

C h a p t e r 1 8 Pharmacology o Analgesia

J D, a 15-ye a r-o ld b o y, is s e ve re ly b u rn e d w h ile e s ca p in g ro m a b u ild in g f re . Th e e xte n s ive b u rn s in clu d e f rs t- a n d s e co n d -d e g re e b u rn s co ve rin g m u ch o h is b o d y a n d a lo ca l, u ll-th ickn e s s b u rn o n h is rig h t o re a rm . He re a ch e s th e e m e rg e n cy d e p a rtm e n t in s e ve re p a in a n d is tre a te d w ith in tra ve n o u s m o rp h in e in in cre a s in g q u a n titie s u n til h e re p o rts th a t th e p a in h a s s u b s id e d . Th is d o s e o m o rp h in e is th e n m a in ta in e d . Th e n e xt d a y, h e h a s s u rg ica l d e b rid e m e n t o h is b u rn w o u n d s a n d a s kin g ra t to h is rig h t o re a rm . Du rin g th e o p e ra tio n , a n a n e s th e s io lo g is t p rovid e s a co n tin u o u s in tra ve n o u s in u s io n o re m i e n ta n il, w ith a b o lu s d o s e o m o rp h in e a d d e d n e a r th e e n d o th e o p e ra tio n . At th e e n d o th e o p e ra tio n , a n d o r 4 d a ys th e re a te r, J D re ce ive s in tra ve n o u s m o rp h in e th ro u g h a p a tie n tco n tro lle d a n a lg e s ia d e vice . As th e b u rn s h e a l, th e m o rp h in e d o s e is ta p e re d a n d e ve n tu a lly re p la ce d w ith a n o ra l o xyco d o n e /a ce ta m in o p h e n co m b in a tio n ta b le t. Th re e m o n th s la te r, J D re p o rts s e ve re lo s s o s e n s a tio n to to u ch in th e a re a o th e s kin g ra t. He a ls o d e s crib e s a p e rs is te n t tin g lin g s e n s a tio n in th is a re a , w ith o cca s io n a l b u rs ts o s h a rp ,

multiple controls through sensory relays in the central nervous system (CNS) to the cortex. This system can be use ully analyzed in terms o the sites o action at which drugs intervene to produce analgesia. First, transduction o intense external, noxious stimuli depolarizes the peripheral terminals o “high-threshold” primary sensory neurons. The primary sensory neurons, called nociceptors because they respond to noxious stimuli, are high-threshold because they require a strong, potentially tissue-damaging stimulus to depolarize their terminals. The resulting action potentials are conducted to the CNS by the axons o the primary a erent sensory neurons, running f rst in peripheral nerves and then in dorsal roots, which then synapse on neurons in the dorsal horn o the spinal cord. The secondary projection neurons transmit in ormation to the brainstem and thalamus, which then relay signals to the cortex, hypothalamus, and limbic system. Transmission is modulated at all levels o the nervous system by remote and local circuit inhibitory and excitatory interneurons (Fig. 18-1).

kn i e -like p a in . A te r re e rra l to a p a in clin ic, J D is p re s crib e d o ra l ga b a p e n tin , w h ich p a rtia lly re d u ce s h is s ym p to m s . Ho w e ve r, h e re p o rts to th e p a in clin ic a g a in 2 m o n th s la te r, s till in s e ve re p a in . At th is tim e , a m itrip tylin e is a d d e d to th e ga b a p e n tin , a n d th e p a in is u rth e r re lie ve d . Th re e ye a rs la te r, J D’s lin g e rin g p a in h a s re s o lve d a n d h e n o lo n g e r re q u ire s m e d ica tio n , b u t th e la ck o o re a rm s e n s a tio n p e rs is ts .

Questions 1 . What mechanisms produced and sustained the pain that lasted rom J D’s exposure to the f re until his initial treatment? 2 . What was the rationale or the sequence o medications used during the skin debridement operation? 3 . Explain the mechanisms that could produce spontaneous pain in the region o the ull-thickness burn months to years a ter healing o the skin and the rationale or using gabapentin to treat J D’s chronic pain. 4 . Why was morphine tapered gradually and replaced with a combination oxycodone/acetaminophen tablet?

Ce ntra l pe rce ption

Tha la mus

Re la y a nd de s ce nding modula tion

Bra ins te m

Sensory Transduction: Excitation of Primary Afferent Neurons The peripheral terminals o primary a erent somatic and visceral sensory nociceptor f bers respond to thermal, mechanical, and chemical stimuli (Fig. 18-2). Highly specialized ion channels/receptors undergo con ormational changes FIGURE 18-1. Overview of the nociceptive circuit. Activation o the peripheral terminal by a noxious stimulus leads to the generation o action potentials, which are conducted to the dorsal horn o the spinal cord. Neurotransmission in the dorsal horn relays the signal to CNS neurons, which send the signal to the brain. This circuit is also subject to descending modulatory control.

289

Tra ns mis s ion S pina l cord

Conduction

P e riphe ra l s timulus

S igna l tra ns duction

Corte x

290 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Chemical

A AS IC, P 2X, P 2Y, B1, B2 re ce ptors

Mechanical Me cha c nos e ns itive ion cha nne ls

Na +/Ca 2+ influx

Ge ne ra tor pote ntia l (me mbra ne de pola riza tion)

Re a ch volta ge -ga te d s odium cha nne l thre s hold

Action potential

Thermal TRP T V1, TRP V2 re ce ptors

FIGURE 18-2. Peripheral transduction. A thermal, chemical, or mechanical sensory event activates a speci c peripheral receptor, leading to ion inf ux and depolarization o the peripheral terminal. Thermal stimuli activate the transient receptor potential (TRP) vanilloid receptor 1 (TRPV1), or the TRP vanilloid receptor-like protein 1 (TRPV2), both o which are heat-sensitive cation channels. Chemical stimuli can activate acid-sensitive ion channels (ASIC), ATPsensitive P2X or P2Y channels, or kinin-sensitive B1 or B2 receptors. Mechanical stimuli can also lead to ion inf ux and depolarization, but the molecular identity o the relevant channels is not certain. In each case, the generator potential induced by the nociceptive signal leads to action potential production i the threshold or activation o the voltage-gated sodium channel is reached.

in response to one or more o these stimuli and thereby mediate the depolarization (generator potential) necessary to initiate an action potential. The requency and duration o the action potentials in the activated ber trans er in ormation to the CNS about the onset, intensity, and duration o the stimulus, while the spatial localization o the ber’s central terminal in the dorsal horn o the spinal cord encodes the site o the stimulus. Thermal pain sensitivity depends on distinct populations o primary sensory neurons: some become active at cold temperatures ( 16°C), whereas others respond to heat. Heat pain-sensing neurons produce action potentials at temperatures higher than 42°C. Responses to noxious heat involve thermosensitive nonselective cation channels, particularly TRPV1, which is a member o the transient receptor potential (TRP) amily o ion channels. This channel becomes active in response to low extracellular pH, vanilloid chemical ligands such as capsaicin (the pungent ingredient in chili peppers), or heat in excess o 42°C. In addition to TRPV1, other TRP channels with varying activation temperatures are involved. TRPV heat-sensitive ion channels represent targets or the development o new drugs to inter ere with peripheral heat sensation, although this may also inter ere with body temperature regulation. In JD’s case, the initial experience o pain was mediated by heat activation o thermosensitive high-threshold peripheral neurons expressing TRPV1. Cool is detected by the TRP channel TRPM8 and intense cold may be detected by TRPA1. TRPM8 is also activated by menthol and TRPA1 by allyl isothiocyanate, the pungent ingredient in mustard and wasabi. Similarly, a speci c subpopulation o primary a erent terminals (the high-threshold mechanonociceptors) is excited by relatively intense mechanical stimuli, such as a pinch or a pinprick. The mechanonociceptor or innocuous tactile stimulation is a very large channel called piezo 2, while

the transducer or noxious mechanotransduction has not yet been identi ed. The peripheral terminals o nociceptor neurons respond not only to thermal and mechanical stimuli but also to multiple chemical signals. Some chemical agents directly excite peripheral terminals (chemical activators ), whereas others increase the sensitivity o the peripheral terminals (sensitizing agents ). Most known chemical ligands that evoke a somatosensory response are associated with cell injury or inf ammation. These chemical ligands include protons, potassium ions, ATP, amines, prostanoids, cytokines, chemokines, nerve growth actor, and bradykinin. For example, cardiac angina is a nociceptive event that involves activation o visceral chemotransducers in nociceptor neurons innervating the heart. These chemotransducers are activated by protons that are released by inadequately per used myocardial tissue. Several di erent types o chemical stimuli can excite nociceptor neurons (Table 18-1). Low extracellular pH, which occurs in ischemia and inf ammation, produces depolarizing cation inf ux through TRPV1 and likely also via acidsensitive ion channels (ASICs). Elevated extracellular ATP TABLE 18-1 Chemosensitive Transduction Receptors Expressed by Nociceptor Neurons NOCICEPTIVE STIMULUS

RECEPTORS

TYPE OF RECEPTOR

Low pH (H )

ASIC

pH-gated ion channel

ATP

P2X P2Y

Ligand-gated ion channel G protein-coupled receptor

Kinin peptides

B1 B2

Gq protein-coupled receptor Gq protein-coupled receptor

C h a p t e r 1 8 Pharmacology of Analgesia

concentration also signals cell injury, because cell rupture releases millimolar concentrations o ATP into the extracellular space (where the ATP concentration is normally very low). Two major classes o ATP receptor include the P2X ligandgated channels and the P2Y G protein-coupled ATP receptors. Kinins are a third set o chemical stimuli that excite the peripheral terminals o sensory neurons. Kinin peptides are produced rom kininogens by the action o kallikrein serine proteases; this process usually occurs in the setting o inf ammation and tissue damage. Kinins act by stimulating bradykinin B1 and B2 receptors. The B2 receptor is constitutively expressed throughout the nervous system, while expression o the B1 receptor is induced in response to bacterial lipopolysaccharide, inf ammatory cytokines, and peripheral nerve injury. Both kinin receptors are G protein-coupled and increase intracellular calcium by production o inositol 1,4,5-trisphosphate. Activation o the B2 receptor also leads to the ormation o prostaglandins E2 and I2. In the introductory case, as the heat sensation was ollowed by burn injury, these chemical mediators likely urther contributed to JD’s pain. Bacterial pathogens can also directly activate nociceptors via ormylated peptides acting on G protein-coupled receptors and via secretion o toxins such as alpha hemolysin, which is a channel-like protein that binds to certain nociceptors and thereby contributes to the pain o bacterial in ection.

Conduction rom the Periphery to the Spinal Cord The axons o primary a erent neurons conduct in ormation rom the peripheral terminal to the CNS. These neurons can be classi ed into three major groups according to their conduction velocity and caliber; these groups also have distinct stimulus sensitivities and distinct central termination patterns. The rst group (A ) consists o rapidly conducting bers that respond with a low stimulus threshold to mechanical stimuli and are activated by light touch, vibration, or movement o hairs. A - bers synapse on CNS neurons located in the dorsal horn o the spinal cord and in dorsal column nuclei o the brainstem. The second population (A ) includes bers that conduct with intermediate velocity and respond to cold, heat, or low- or high-intensity mechanical stimuli. The third group (C-f bers ) conduct slowly, synapse in the spinal cord, and typically respond multimodally; they are capable o producing action potentials in response to heat, warmth, intense and innocuous mechanical stimuli, or chemical irritants (polymodal nociceptors, tactile detectors, and itch-provoking pruriceptors). Some C- ber a erents (re erred to as silent or sleeping nociceptor bers) cannot be activated normally but become responsive only during inf ammation. A - and C- bers terminate in the most supercial laminae o the dorsal horn (lamina I and II). For conduction to occur, voltage-gated sodium channels must convert depolarization o the peripheral terminal into an action potential. Six types o voltage-gated sodium channels are expressed in primary a erent neurons, o which three, Nav1.7, Nav1.8, and Nav1.9, are expressed uniquely in primary a erents. Gain-o - unction mutations in Nav1.7 produce hyperexcitability o nociceptors and thereby contribute to primary erythromelalgia, an inherited condition associated with severe burning pain that is either spontaneous or provoked in response to mild thermal stimuli. Loss-o - unction

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mutations in Nav1.7 result in congenital insensitivity to pain, highlighting the critical role o this channel in nociception and its potential attractiveness as a target or analgesics. Nav1.8 and Nav1.9 are selectively expressed in small-caliber neurons, most o which respond only to high-threshold peripheral stimuli (nociceptors). These two channel types also have higher activation thresholds and inactivate more slowly than other neuronal voltage-gated sodium channels. Because o their speci c expression pattern in pain bers, selective sodium channel blockers represent uture pharmacologic targets o particular interest, especially i they produce a usedependent block. Currently, the topical or regional use o nonselective, sodium channel-blocking local anesthetic agents is a mainstay or the treatment o acute postoperative and procedural pain (see Chapter 12, Local Anesthetic Pharmacology). Sodium channel-blocking antiepileptic and antiarrhythmic drugs (see Chapter 16, Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System, and Chapter 24, Pharmacology o Cardiac Rhythm, respectively) are also used or certain neuropathic pain conditions, particularly trigeminal neuralgia.

Transmission in the Dorsal Horn o the Spinal Cord Action potentials generated in primary a erents induce neurotransmitter release upon reaching their central axon terminals in the dorsal horn o the spinal cord. N-type voltage-gated calcium channels have a substantial role in controlling neurotransmitter release rom synaptic vesicles. Gabapentin and pregabalin are antiepileptic drugs that act on the alpha 2 delta calcium channel subunit. Via a mechanism that is not completely understood but involves disruption o calcium channel tra cking to the membrane, these agents likely modulate CNS transmission o nociceptive in ormation. Although their e ectiveness is limited, both agents are widely used in the treatment o chronic neuropathic pain because o their generally avorable adverse e ect pro le. A naturally occurring snail poison, omega-conotoxin, acts as a selective N-type calcium channel blocker; a synthetic mimic o this peptide, ziconotide, is currently used to treat severe pain conditions. However, such calcium channel blockers also alter the unction o sympathetic neurons (producing hypotension) and many central neurons (a ecting cognitive unction). Thus, the use o ziconotide is limited to the highly specialized scenario o intrathecal administration because it is necessary to limit the drug’s e ects to the spinal cord. Synaptic transmission takes place between C- ber primary a erents and secondary projection neurons in the dorsal horn. This transmission has ast and slow components (Fig. 18-3). Acting on ionotropic AMPA and NMDA receptors, glutamate mediates ast excitatory transmission between primary and secondary sensory neurons. Acting on metabotropic mGluR receptors, glutamate also mediates a slow synaptic modulatory response. Neuropeptides , such as the tachykinins substance P and calcitonin gene-related peptide (CGRP), as well as other synaptic neuromodulators , including the neurotrophin brain-derived neurotrophic actor (BDNF), are co-released with glutamate and also produce slow synaptic e ects by acting on metabotropic G protein-coupled receptors and receptor tyrosine kinases. The presence o these co-released peptides allows considerable use-dependent unctional plasticity o

292 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Descending and Local Inhibitory Regulation in the Spinal Cord Synaptic transmission in the spinal cord is regulated by the actions of both local inhibitory interneurons and projections that descend from the brainstem to the dorsal horn. Because these systems can limit transfer of incoming sensory information to the brain, they represent an important site for pharmacologic intervention. The major inhibitory neurotransmitters in the dorsal horn of the spinal cord are opioid peptides, norepinephrine , serotonin (5-HT), glycine , and GABA (Fig. 18-4). The physiology of GABA receptors is discussed

Primary sensory neuron central terminal Action pote ntia l Glu Ca 2+

Ca lcium influx S yna ptic ve s icle re le a s e

Glu

Ne urope ptide s CGRP S ubs ta nce P

Na + 2+ Ca

Glu

Na +

Primary sensory neuron central terminal

Glu

Glu

NMDA-R

Glu AMPA-R

Ra pid Na influx

Volta ge -de pe nde nt Na + a nd Ca 2+ influx +

mGluR

Ca 2+

GABA

NK1 CGRP -R Ca lcium influx

S low modula tory re s pons e

Re a ch volta ge -ga te d Na + cha nne l thre s hold

GABAB

S yna ptic ve s icle Glu re le a s e

µ Endorphins Enke pha lins Ne urope ptide s CGRP S ubs ta nce P

P os ts yna ptic de pola riza tion Secondary relay neuron (pos ts yna ptic me mbra ne )

Nore pine phrine

α2

Action pote ntia l

Na + 2+ Ca

Glu

Glu

Na +

α2 NMDA-R

Glu

mGluR

Action potential

NK1

CGRP -R

GABAB

Volta ge -de pe nde nt + 2+ Ra pid Na + Na a nd Ca influx influx

AMPA-R

FIGURE 18-3. Neurotransmission in the spinal cord dorsal horn. An incoming action potential rom the periphery activates presynaptic voltagegated calcium channels, leading to calcium inf ux and subsequent synaptic vesicle release. The released neurotransmitters (i.e., glutamate and neuropeptides, such as calcitonin gene-related peptide [CGRP] and substance P) act on postsynaptic receptors. Stimulation o ionotropic glutamate receptors leads to ast postsynaptic depolarization, while activation o other modulatory receptors mediates slow depolarization. Postsynaptic depolarization, i su cient, leads to action potential production (signal generation) in the secondary relay neuron.

pain transmission. The physiologic function of the neuropeptides in synaptic transmission involves signaling responses to stimuli of particularly high intensity, because release of neuropeptide-containing synaptic vesicles requires higher frequency and longer lasting action potential trains than release of glutamate-containing vesicles. New strategies to target CGRP and its actions are being developed, particularly for migraine.

GABAA

Cl- conducta nce Cl-

Cl-

Secondary relay neuron (pos ts yna ptic me mbra ne )

µ K+

K+ conducta nce K+

P os ts yna ptic hype rpola riza tion Volta ge -ga te d Na + cha nne ls re a ching thre s hold

Action potential generation

FIGURE 18-4. Inhibitory regulation of neurotransmission. Norepinephrine, GABA, and opioids, released by descending and/or local circuit inhibitory neurons, act both presynaptically and postsynaptically to inhibit neurotransmission. Presynaptic inhibition is mediated via reduced activity o voltagegated calcium channels, whereas postsynaptic inhibition is mediated primarily by enhanced chloride inf ux and potassium e f ux.

C h a p t e r 1 8 Pharmacology of Analgesia

in Chapter 13, Pharmacology o GABAergic and Glutamatergic Neurotransmission. The opioid peptides inhibit synaptic transmission and are released at several CNS sites in response to noxious stimuli. All endogenous opioid peptides, which include -endorphin, the enkephalins , and the dynorphins , share the N-terminal sequence Tyr-Gly-Gly-Phe-Met/Leu. The opioids are proteolytically released rom the larger precursor proteins proopiomelanocortin, proenkephalin, and prodynorphin. Opioid receptors all into three classes, designated , , and , all o which are seven-transmembrane G protein-coupled receptors. The -opioid receptors mediate morphine-induced analgesia. This conclusion is based on the observation that the -opioid receptor knockout mouse exhibits neither analgesia nor adverse e ects in response to morphine administration. The endogenous opioid peptides are receptor-selective: the dynorphins act primarily on receptors, while both enkephalins and -endorphin act on and receptors. The physiologic role o the endogenous opioid peptides remains poorly understood, although they may mediate reward e ects— or example, a ter sunburn. The e ects o opioid receptor signaling include reduced presynaptic calcium conductance, enhanced postsynaptic potassium conductance, and reduced adenylyl cyclase activity. The rst unction impedes presynaptic neurotransmitter release; the second reduces postsynaptic neuronal responses to excitatory neurotransmitters; the physiologic role o the third remains unknown. Opioids produce analgesia because o their action in the brain, brainstem, spinal cord, and peripheral terminals o primary a erent neurons. In the brain, opioids alter mood, produce sedation, and reduce the emotional reaction to pain. In the brainstem, opioids increase the activity o cells that provide descending inhibitory innervation to the spinal cord; here, opioids also produce nausea and respiratory depression. Spinal opioids inhibit synaptic vesicle release rom primary a erents and hyperpolarize postsynaptic neurons (see above). Evidence also exists that peripheral opioid receptor stimulation reduces the activation o primary a erents and modulates immune cell activity. Action o opioids at these serially located sites is thought to have a synergistic e ect to inhibit in ormation f ow rom the periphery to the brain. Norepinephrine is released by projections that descend rom the brainstem to the spinal cord. The 2-adrenergic receptor, a seven-transmembrane G protein-coupled receptor (see Chapter 11, Adrenergic Pharmacology), is the primary receptor or norepinephrine in the spinal cord. As with opioid receptor activation, 2-adrenergic receptor activation inhibits presynaptic voltage-gated calcium channels, opens postsynaptic potassium channels, and inhibits adenylyl cyclase. Because 2-adrenergic receptors are expressed both presynaptically and postsynaptically, spinal norepinephrine release can both reduce presynaptic vesicle release and decrease postsynaptic excitation. The 2-adrenergic receptor agonist clonidine is sometimes used to treat pain, although this application is limited by adverse e ects that include sedation and postural hypotension. Serotonin is also released in the spinal cord by projections descending rom the brainstem. This neurotransmitter acts on several receptor subtypes that mediate both excitatory and inhibitory e ects on nociception. The 5-HT3 ligand-gated channel may be responsible or the excitatory actions o serotonin in the spinal cord; several o the 5-HT G protein-coupled receptors may mediate the inhibitory actions o 5-HT. Given this

293

complexity, the mechanism o the analgesic e ect o serotonin is not ully understood. Selective serotonin reuptake inhibitors have been tested in the treatment o pain but have generally had little bene cial e ect. Selective norepinephrine (NE) reuptake inhibitors do have analgesic action, as do dual NE/5-HT reuptake inhibitors such as duloxetine — this agent is used to treat many chronic pain conditions. Tramadol, a weak centrally acting opioid, also has monoaminergic actions and is widely used to treat mild pain. Its relatively weak e cacy as a single agent is increased when combined with acetaminophen, and its lack o abuse potential makes the drug attractive to prescribers. Tapentadol is a newer drug with similar dual action, although it is a more potent -opioid receptor agonist than tramadol. Other compounds also have regulatory roles in the spinal cord. The cannabinoid receptors and the endogenous cannabinoids have recently become a ocus or research on pain regulation. There are two cannabinoid receptors, both o which are G protein-coupled: CB1, expressed in the brain, spinal cord, and sensory neurons; and CB2, largely expressed in nonneural tissues, especially immune cells including microglia. Several endogenous cannabinoids have been identi ed, including members o the anandamide and 2-arachidonylglycerol (2AG) amilies. A combination o anecdotal evidence and clinical trial data suggests that cannabinoids may have an analgesic e ect in patients with AIDS neuropathy or multiple sclerosis. Selective cannabinoid pathway agents under development, such as CB1 or CB2 agonists or inhibitors o endocannabinoid metabolism, may prove use ul or pain management.

PATHOPHYSIOLOGY The pain processing circuit described above is responsible or producing acute nociceptive pain, a physiologic, adaptive sensation elicited only by noxious stimuli that acts as a warning or protective signal. There are some clinical situations, such as acute trauma, labor, or surgery, in which it is necessary to control nociceptive pain. In these cases, the pain pathway can be interrupted either by blocking transmission with local anesthetics (see Chapter 12) or by administering high-dose opioids. The opioids may be rapidly acting, such as remifentanil or intraoperative use, or more slowly acting, such as morphine ; administered perioperatively, morphine retains activity or postoperative pain control. Both peripheral inf ammation and nervous system damage produce pain that is characterized by hypersensitivity to noxious and innocuous stimuli and by spontaneous pain that arises in the absence o any obvious stimulus. Understanding the mechanisms responsible or these types o clinical pain will acilitate both the appropriate use o currently available drugs and the development o novel therapeutic agents.

Clinical Nociceptive Pain The ideal treatment o pain would be based on identi ying and targeting the precise pain mechanisms operative in a particular patient and on normalizing abnormal pain sensitivity. Clinical pain syndromes may involve a combination o mechanisms, however, and there are ew diagnostic tools to identi y which speci c mechanisms are responsible in a particular patient. Chronic pain conditions can be complicated to treat, and e ective treatment usually demands multiple drugs (polypharmacy) to obtain the optimal therapeutic e ect while

294 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

reducing adverse e ects. Chronic inf ammatory pain conditions require the use o drugs that reduce the inf ammatory response; such drugs may both correct the underlying inf ammatory condition (disease-modi ying therapy) and reduce the pain. For example, the nonsteroidal anti-inf ammatory drugs (NSAIDs) (see Chapter 43, Pharmacology o Eicosanoids) are the rst line o treatment or rheumatoid arthritis. By reducing inf ammation, this intervention can decrease the release o chemical ligands that sensitize peripheral nerve terminals and thereby prevent peripheral sensitization (see below). Other disease-modi ying anti-inf ammatory treatments that may also reduce pain include cytokine inhibitors, sequestering agents such as TNF- inhibitors, and immunosuppressants, as well as anti-nerve growth actor (NGF) monoclonal antibodies that are now under development. The major agents used to treat most noninf ammatory neuropathic or dys unctional pain conditions are generally not disease-modi ying because the underlying disease processes are either not known (e.g., bromyalgia) or re ractory to currently available treatments (e.g., neuropathic pain). Neuropathic pain associated with peripheral nervous tissue damage, spinal cord injury, or stroke commonly requires the use o several agents to alleviate pain symptoms. In nonmalignant pain, opioids have generally been used as a matter o last resort because o their adverse e ects and because o the potential or the development o tolerance and physical dependence (see Chapter 19, Pharmacology o Drugs o Abuse). However, in recent years, opioids have increasingly been used or the management o chronic noncancer pain, albeit with the risks o producing drug-seeking behavior and creating opportunity or diversion o the drugs or illicit use. The U.S. Centers or Disease Control and Prevention (CDC) reported that opioid

analgesics had a role in 71.3% o the 22,767 deaths due to prescription drug overdose occurring in the U.S. in 2013. Severe acute pain due to trauma, surgery, or inf ammation is usually treated with opioids and NSAIDs. The many available opioid agents permit a great deal o f exibility in both potency and duration o action. Remi entanil, a highpotency opioid, is cleared over the course o a ew minutes, while methadone a ter prolonged use may be cleared over the course o days. Combinations o intermediate- and longacting opioids may be used to tailor an analgesic regimen or pain that f uctuates in intensity over days, hours, or minutes. Remi entanil was administered during JD’s surgical debridement or optimal control o intraoperative pain, ollowed by a morphine bolus and in usion or postoperative pain control. The nausea and sedation produced in many patients by opioids are potential problems when these agents are used or day-surgery cases. Acute inf ammatory pain conditions, such as pancreatitis, are o ten treated with morphine. Gout, a second example o an acute inf ammatory condition producing severe pain, is usually treated with indomethacin (an NSAID) to reduce the pain rapidly, and more speci c disease-modi ying agents are used to correct the underlying disorder over the longer term (see Chapter 49, Integrative Inf ammation Pharmacology: Gout).

Peripheral Sensitization Several peripheral stimuli can cause primary a erent neurons to lower their activation thresholds and increase their responsiveness (Fig. 18-5). These alterations, which constitute peripheral sensitization, can result in allodynia , in which normally innocuous stimuli are perceived as pain ul, and hyperalgesia ,

Mechanical Chemical

P

Na +/Ca 2+ influx

Thermal

Ge ne ra tor pote ntia l (me mbra ne de pola riza tion)

Re a ch volta ge -ga te d s odium cha nne l thre s hold

Action potential

P KC a ctiva tion Sensitizing agents

P KA a ctiva tion Tyr

TrkA

P

Na +

FIGURE 18-5. Peripheral sensitization. Peripherally released sensitizing agents activate signal transduction that can increase sensitivity o the peripheral nerve terminal. Mechanisms mediating increased sensitivity include (1) enhancement o ion inf ux in response to a noxious stimulus and (2) reduction o the activation threshold o the voltage-gated sodium channels responsible or initiating and propagating action potentials. In the example shown, a sensitizing agent activates one o three types o cell sur ace receptor, or example, a G protein-coupled receptor. This receptor initiates two parallel signaling cascades. One branch activates the phospholipase C (PLC) pathway, which results in increased release o calcium rom intracellular stores and in activation o protein kinase C (PKC). Both o these e ects increase ion inf ux— or example, through a TRPV1 receptor (blue)—in response to a nociceptive stimulus. The second branch o the signaling cascade activates adenylyl cyclase (AC), leading to increased ormation o cAMP, activation o protein kinase A (PKA), and sodium channel phosphorylation. Both signaling cascades serve to increase the likelihood o action potential initiation and propagation. Nerve growth actor (NGF) is the ligand or the neurotrophic tyrosine kinase receptor amily member TrkA. See text or urther details.

C h a p t e r 1 8 Pharmacology of Analgesia

in which high-intensity stimuli are perceived as more pain ul and longer lasting than usual at the site o injury (zone o primary hyperalgesia). Some inf ammatory mediators released by injured (ATP) or immune (IL-1 ) cells can directly activate nociceptors to signal to the CNS the presence o tissue injury and thereby evoke pain. The mechanisms responsible or primary hyperalgesia involve both direct changes in transduction and indirect changes induced by the release o e ector molecules. An example o altered transduction is the change in heat activation o the TRPV1 receptor due to posttranslational modi cations and altered membrane tra cking ollowing activation o PKC and PI3K signaling pathways, which reduce the activation threshold o the receptor so that it can be activated by warm stimuli (38°–40°C) that are normally not pain ul. The major known e ectors that produce peripheral sensitization are the inf ammatory mediators bradykinin, the cytokine IL-1 , protons, histamine, prostaglandin E2, and NGF. Prostaglandin E2 acts on EP receptors, o which there are our types, while NGF acts on the neurotrophic tyrosine kinase receptor amily member TrkA. The actions o histamine are more prominent on the subset o sensory neurons that contribute to itch. Sensitizing chemical mediators act on G protein-coupled receptors or receptor tyrosine kinases expressed on the peripheral terminals o nociceptor neurons. Activation o phospholipase C, phospholipase A2, and adenylyl cyclase occurs in response to the activation o G protein-coupled receptors, such as those or bradykinin, prostaglandin E2, and adenosine. In turn, these signaling enzymes generate mediators that activate protein kinase A (PKA) or protein kinase C (PKC). Protein kinase A phosphorylates the voltage-gated sodium channel Nav1.8, resulting in both a decrease in its activation threshold and an increase in the current passed when the channel opens. Protein kinase C phosphorylates TRPV1, thus reducing its activation threshold and thereby increasing the response o peripheral terminals to heat stimuli (see above). In addition to the enhancement o peripheral response caused by an outside event that produces inf ammation, the peripheral terminals themselves can contribute to inf ammation (the neurogenic component o inf ammation). Depolarization and chemical stimuli induce the release o neuropeptides, such as substance P and CGRP, rom the peripheral terminals o primary a erents. The released neuropeptides produce vasodilation and increase capillary permeability, contributing to the wheal-and-f are response to tissue injury. In addition, neuropeptides can induce the release o histamine and TNF- rom inf ammatory cells. The recruitment and activation o granulocytes, as well as the increase in local capillary diameter and permeability to plasma, result in a local inf ammatory response at the site o the excited peripheral terminal. This response contributes to atopic dermatitis and psoriasis. Peripheral sensitization is an important target or clinical pain pharmacology. The NSAIDs are the most widely used pain treatment. By inhibiting the activity o cyclooxygenase enzymes, NSAIDs reduce prostaglandin production and, hence, the local inf ammatory response and peripheral sensitization. There are two iso orms o cyclooxygenase: COX-1 and COX-2 (see Chapter 43). The ormer is constitutively active and is important in a variety o physiologic unctions, such as maintenance o gastric mucosal integrity and normal platelet unction. COX-2 is selectively up-regulated at the site o inf ammation, primarily in macrophages, in response to local secretion o cytokines, particularly IL-1 and TNF- acting via the transcription actor NF- B.

295

Selective inhibitors o COX-2, such as celecoxib, rofecoxib, and valdecoxib, were developed in an attempt to control inf ammatory pain while decreasing some o the adverse e ects o the nonselective NSAIDs, such as gastrointestinal bleeding. However, large postmarketing trials have revealed an increased incidence o serious cardiovascular e ects associated with COX-2 inhibitor therapy, including an increased risk o myocardial in arction. This has led to the withdrawal o most COX-2 selective inhibitors, with celecoxib alone remaining on the market in the United States. In addition to the cyclooxygenases, the transduction molecules, signaling intermediates, and sodium channels expressed at peripheral terminals may all represent targets or the development o new analgesic drugs that reduce peripheral pain hypersensitivity. In the case o JD, peripheral sensitization was induced at the burn site. The high-intensity stimulus led to neurogenic inf ammation. The associated tissue damage urther potentiated inf ammatory mediator release, leading to the activation o second messenger cascades that heightened peripheral terminal excitability over time.

Central Sensitization Hyperalgesia and allodynia requently extend beyond the primary area o inf ammation and tissue damage. Pain hypersensitivity in this region, described as the area o secondary hyperalgesia and/or allodynia, depends on alterations in sensory processing in the dorsal horn o the spinal cord. These alterations, which are a orm o neuronal plasticity termed central sensitization, occur when repetitive, usually high-intensity, synaptic transmission activates intracellular signal transduction cascades in dorsal horn neurons that enhance the response to subsequent stimuli. Several o the postsynaptic receptors expressed by dorsal horn neurons are involved in the induction o central sensitization (Fig. 18-6). These receptors include AMPA, NMDA, and metabotropic glutamate receptors, as well the substance P (neurokinin) receptor NK1 and the BDNF (neurotrophin) receptor TrkB. Upon activation o metabotropic receptors or calcium inf ux through NMDA channels, intracellular protein kinases are activated, such as PKC, calcium/calmodulin kinase (CAMK II), and extracellular signal-related protein kinase (ERK). In turn, these e ectors can alter the unction o existing membrane proteins by post-translational processing, usually by phosphorylation. For example, phosphorylated NMDA receptors open more rapidly and or longer periods in response to glutamate. Phosphorylation o AMPA receptors results in their translocation rom cytosolic stores to the membrane, thus increasing synaptic e cacy. Activation o ERKs leads to a reduction in potassium channel activity in dorsal horn neurons; the decreased potassium current increases neuronal excitability. Most o ten, central sensitization slowly subsides a ter the inducing stimulus ceases. However, chronic injury or inf ammation can produce a state o central sensitization that persists over time. NMDA receptor blockade can prevent both the induction and maintenance o central sensitization. For example, NMDA receptor blockade instituted preoperatively has been shown to reduce pain experienced postoperatively. A component o postoperative pain is likely attributable to NMDA receptor-dependent central sensitization associated with the intense peripheral stimulation that occurs during surgery. The NMDA receptor blocker ketamine can be used to oppose

296 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

This e ect urther enhanced the lingering pain he elt at the burn site, and it produced pain surrounding the burn site, outside the primary area o tissue damage and inf ammation.

Primary sensory neuron central terminal

Neuropathic Pain

Action pote ntia l

Glu Ca 2+

Ca lcium influx Glu

S yna ptic ve s icle re le a s e

BDNF S ubs ta nce P Ca 2+ Mg 2+ Na

Glu

+

Glu

Glu

NMDA-R P

mGluR

AMPA-R P

Ca 2+ Na

NK1

P Tyr

Tyr

P

+

TrkB

Initia l de pola riza tion

Kina s e a ctiva tion (P KC, CAMK II, ERK)

P hos phoryla tion of pos ts yna ptic prote ins

P hos phoryla tion of ge ne re gula tory prote ins

Secondary relay neuron (pos ts yna ptic me mbra ne )

Alte re d ge ne e xpre s s ion

Short term sensitization

Long term sensitization

FIGURE 18-6. Central sensitization. Sustained or intense activation o central transmission can lead to postsynaptic calcium inf ux, primarily through NMDA receptors. Together with a variety o neuromodulatory signals, calcium inf ux activates signal transduction cascades that can enhance both short-term and long-term excitability o the synapse.

activation o sensitized NMDA receptors. NMDA receptors are widely expressed, however, and NMDA blockers produce signi cant psychotropic e ects, including amnesia and hallucinations. Protein kinase C or ERK is an alternative target. Although many o the signaling proteins involved in dorsal horn sensitization are expressed in all cells, it may be possible to target treatment to the spinal cord by intrathecal or epidural injection. Pregabalin and gabapentin reduce central sensitization by reducing transmitter release, as does morphine . Duloxetine enhances the inhibitor e ects o amines on spinal cord neurons and also reduces central sensitization. The intense peripheral activation produced by JD’s burn injury also led to the development o central sensitization.

The mechanisms responsible or the persistent pain that can occur ollowing nerve injury involve both unctional and structural alterations in the nervous system and occur in both primary a erent neurons and the CNS (Fig. 18-7). In the periphery, changes in the physiology and transcriptional pro le o primary a erent sensory neurons occur a ter nerve damage, contributing to neuropathic pain. These alterations are induced by combinations o positive signals, such as inf ammatory cytokines released by macrophages and Schwann cells, and negative signals, such as the loss o peripheral support rom neurotrophic actors. In addition, the expression pattern o sodium channels changes in injured primary sensory neurons: Nav1.8 and Nav1.9 are down-regulated, while Nav1.3, which is normally not detectable in primary sensory neurons, is up-regulated. Nav1.3 channels exhibit accelerated recovery rom inactivation and are thought to contribute to neuropathic pain by enhancing cellular excitability su ciently to generate ectopic action potential activity. The contribution o sodium channels to some orms o neuropathic pain is supported by the e ectiveness o sodium channel blockers, such as carbamazepine and oxcarbazepine , in treating trigeminal neuralgia. Nerve damage also promotes reorganization o synaptic connection patterns within the dorsal horn o the spinal cord. Peripheral nerve injury leads to a regenerative response. Because primarily C- bers are lost upon the withdrawal o peripheral trophic support, regenerating central terminals o A - bers are ree to invade the area normally occupied by the central terminals o C- bers. Another structural change is an excitotoxic loss o inhibitory neurons in the dorsal horn a ter peripheral nerve injury. The loss o inhibition (disinhibition) contributes to the heightened pain sensitivity, and augmenting GABAergic or glycinergic inhibition can be an e ective strategy or treating neuropathic pain. A combination o these mechanisms would have been involved in maintaining JD’s pain over the years ollowing his operation. Neuroprotective treatment designed to prevent transsynaptic neurodegeneration could represent an opportunity or a disease-modi ying approach to neuropathic pain, particularly when the time o nerve damage can be identi ed (e.g., a ter surgery). It may be possible to use neurotrophic actors to treat both the transcriptional changes and some o the structural alterations that contribute to neuropathic pain, as well as the changes in immune cells that occur locally at the site o nerve injury and in the CNS. A highly selective angiotensin II type 2 receptor antagonist has recently been shown to be e ective in postherpetic neuralgia; the mechanism o action involves targeting abnormal excitability and sprouting in injured sensory neurons produced by activation o p38 MAPK.

Migraine Migraine headache is a disorder consisting o headache attacks that last or up to 3 days, typically associated with light and sound avoidance and nausea. Some migraines are accompanied by aura, in which transient neurologic symptoms are associated with the migraine. It is thought that the pathophysiology o migraine comprises several events.

C h a p t e r 1 8 Pharmacology o Analgesia

297

To bra in Dors a l root ga nglion

Alte re d ge ne e xpre s s ion a nd s e ns itivity

Dors a l horn

S chwa nn ce ll re a ction, infla mma tory ce ll infiltra tion, cytokine a nd growth fa ctor s e cre tion Fre e ne rve e ndings

Ne urotrophic s upport

Los s of ne urotrophic s upport

S ite of a xona l injury S pina l cord

FIGURE 18-7. Schematization of neuropathic pain. Nerve injury results in a combination o negative signals and positive signals that alter the physiology o the nociceptive system. The loss o neurotrophic support alters gene expression in the injured nerve ber, whereas the release o inf ammatory cytokines alters gene expression in both the injured and adjacent uninjured nerve bers. These changes in gene expression can lead to altered sensitivity and activity o nociceptive bers and, thus, to the continued perception o injury that is characteristic o neuropathic pain.

First, be ore the headache occurs, a region o neural activation ollowed by inactivation travels across the cortex. This phenomenon is termed cortical spreading depression and is correlated with the sensory disturbances o the migraine aura such as scotoma (visual eld disturbances). Second, release o multiple neuropeptides, possibly evoked by the cortical excitation, occurs in the dural vasculature. Third, trigeminal a erents rom the dural vasculature are activated and sensitized by the local release o neuropeptides and inf ammatory mediators. Fourth, the high degree o activity in trigeminal a erent high-threshold bers produces central sensitization, leading to secondary hyperalgesia and tactile allodynia. Thus, a migraine attack can be considered the acute mani estation o abnormal intermittent peripheral and central excitability. Evidence rom a rare autosomal dominant disorder, amilial hemiplegic migraine (FHM), may shed light on the mechanisms o migraine in general. This disorder consists o migraine attacks with a particular aura characterized by unilateral motor paralysis. Three genes have been associated with FHM: CACNA1A, ATP1A2, and SCNA1. CACNA1A encodes a Cav2.1 voltage-gated calcium channel subunit. In animal models, Cav2.1 gain-o - unction mutations cause increased presynaptic calcium and increased glutamate release, which may help explain the trigger or cortical spreading depression. ATP1A2 encodes a subunit o the Na /K ATPase, which is critical or the maintenance o neuronal membrane potential and which produces the Na gradient needed or glutamate transport. SCNA1 encodes a voltagegated sodium channel subunit that is involved in action potential conduction. Whether the more common orms o migraine are associated with similar changes in these genes remains unknown.

PHARMACOLOGIC CLASSES AND AGENTS Several drug classes are widely used or pain relie . These include opioid receptor agonists , NSAIDs (see Chapter 43),

tricyclic antidepressants (see Chapter 15, Pharmacology

o Serotonergic and Central Adrenergic Neurotransmission), antiepileptic drugs (sodium channel blockers) (see Chapter 16), NMDA receptor antagonists (see Chapter 13), and adrenergic agonists . In addition, 5-HT1 receptor agonists have speci c applications in the acute treatment o migraine.

Opioid Receptor Agonists Opioid receptor agonists are the primary drug class used in the acute management o moderate to severe pain. The naturally occurring opioid agonist morphine has the greatest historical importance and remains in wide use, but synthetic and semisynthetic opioids add pharmacokinetic versatility. Opioids have long been used to treat acute and cancerrelated pain, but in recent years, they have become one component o the management o chronic noncancer pain as well.

Mechanism of Action and Major Adverse Effects

Opioid receptor agonists produce analgesia and other e ects by acting on -opioid receptors (Fig. 18-8). Sites o analgesic action include the brain, brainstem, spinal cord, and primary a erent peripheral terminals, as described previously. The principal mechanism o opioid action, as discussed in greater detail above, is via G protein-coupled signaling that inhibits neurotransmission via decreased presynaptic calcium inf ux and/or increased postsynaptic potassium inf ux at either spinal or supraspinal sites. Opioids produce a wide array o adverse e ects. These e ects are qualitatively similar across opioids but may vary in intensity. The major dose-limiting adverse e ects are sedation and respiratory suppression. By acting on the medullary respiratory control center, opioids blunt the respiratory response to carbon dioxide and can cause periods o apnea. Importantly, the respiratory e ects o opioids interact with other stimuli: pain ul or other arousing stimuli can promote ventilation, while natural sleep synergizes with opioids to

298 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Primary sensory neuron central terminal Action pote ntia l

µ-agonist Glu

µ

Ca 2+

Ca lcium influx

Glu

S yna ptic ve s icle re le a s e Ne urope ptide s CGRP S ubs ta nce P K+

Na +

Des cending modula tion or exogenous drug

µ-agonist

(opioid, e nke pha lin, e ndorphin)

K+ cha nne l

µ-opioid

Glu

re ce ptor

AMPA-R K+ Na +

K+ conductance

P os ts yna ptic hype rpola riza tion

Secondary relay neuron (pos ts yna ptic me mbra ne )

Opioid use is o ten associated with the development o tolerance , in which repeated use o a constant dose o a drug results in a decreased therapeutic e ect (see Chapter 19). The molecular mechanisms responsible or tolerance remain a matter o debate and may involve a combination o gene regulation and post-translational modif cation o opioid receptor activity. Clinically, the development o tolerance requires either a change o analgesic drug or an increase in the dose or requency o administration o the drug to maintain analgesia. Physical dependence can also occur, such that abrupt cessation o treatment results in a characteristic withdrawal syndrome. Addiction, in which physical dependence is accompanied by drug abuse or drug-seeking behavior, is a potential adverse e ect o opioid administration. The incidence and prevalence o opioid addiction in patients receiving opioids or therapeutic reasons remain unknown but are not negligible. Balancing the risk o opioid addiction against the undertreatment o pain is a complex issue in pain management and a topic o considerable debate. Several strategies are being investigated to reduce abuse potential, including mechanisms to prevent the disruption o slowrelease opioid ormulations (see next section), combinations that include both an opioid agonist and an opioid antagonist, and prodrugs that are slowly metabolized to the active opioid agonist. In JD’s case, intravenous morphine was tapered and replaced with a combination oral analgesic to prevent the onset o opioid withdrawal symptoms.

Volta ge -ga te d Na + cha nne ls re a ching thre s hold

Action potential generation

FIGURE 18-8. Mechanism of action of -opioid receptor agonists in the spinal cord. Activation o both presynaptic and postsynaptic -opioid receptors by descending and local circuit inhibitory neurons inhibits central relaying o nociceptive stimuli. In the presynaptic terminal, -opioid receptor activation decreases Ca 2 inf ux in response to an incoming action potential. Postsynaptic -opioid receptor activation increases K conductance and thereby decreases the postsynaptic response to excitatory neurotransmission.

suppress ventilation. Opioids stimulate receptors in the medullary chemoreceptor zone and the gastrointestinal tract, leading to nausea, vomiting, and constipation. In the genitourinary system, opioids can cause urinary urgency and urinary retention. In the central nervous system, opioids can cause sedation, con usion, dizziness, euphoria, and myoclonus. It has recently become apparent that excessive use o opioids can lead to a paradoxical opioid-induced hyperalgesia. In the cardiovascular system, opioids can reduce sympathetic tone and lead to orthostatic hypotension. Opioids can also cause bradycardia. Respiratory e ects are o ten the major, dose-limiting adverse e ect o opioids.

Morphine, Codeine, and Derivatives

Morphine, codeine (methylmorphine), and their semisynthetic derivatives are the most widely used opioids or control o pain outside o the context o anesthesia or procedural sedation. Morphine is metabolized in the liver, and its f rstpass metabolism reduces its oral bioavailability. In the liver, morphine undergoes glucuronidation at either the 3 position (morphine-3-glucuronide; M3G) or the 6 position (M6G). While M3G is inactive, M6G has analgesic activity. M6G is excreted by the kidney, and its accumulation in patients with chronic kidney disease may contribute to opioid toxicity. Morphine does not undergo metabolism by the cytochrome P450 system and has relatively ew interactions with other drugs. Hydromorphone is a widely used morphine derivative with similar properties to morphine but approximately 5 to 10 times higher potency. Like morphine, hydromorphone undergoes glucuronidation but not cytochrome P450-based metabolism and has ew interactions with other drugs. Codeine, like morphine, is a naturally occurring opioid receptor agonist. Codeine is commonly used or its antitussive (i.e., cough-suppressing) and antidiarrheal e ects because it has considerably higher oral bioavailability than morphine; it is also used in conditions or which a low-potency oral opioid analgesic is pre erred. The analgesic action o codeine results largely rom its hepatic demethylation to morphine, which has substantially greater -agonist activity. Genetic polymorphisms in the cytochrome P450 enzymes CYP2D6 and CYP3A4, which are responsible or demethylation o codeine, may determine interindividual variation in response to codeine treatment. In some cases, most notably in children, ultrarapid metabolism o codeine to morphine has resulted in accidental death due to unanticipated opioid overdose. The semisynthetic compounds oxycodone and hydrocodone are more potent analogues o codeine that are also orally available and are widely used, o ten in combination

C h a p t e r 1 8 Pharmacology of Analgesia

with acetaminophen. Oxycodone is hepatically metabolized via the cytochrome P450 system to the highly potent opioid agonist oxymorphone and the less potent metabolite noroxycodone. Hydrocodone is metabolized via the hepatic cytochrome P450 system to the active metabolite hydromorphone. For these two drugs, the primary drug is likely the agent responsible or the therapeutic e ect, while metabolic products may a ect drug interactions and interindividual variation in drug response. Several di erent routes are available or administering morphine. Controlled-release oral preparations are marketed to reduce the number o daily doses required or analgesia. These ormulations allow drug release over the course o 12–24 hours. Un ortunately, sustained-release ormulations have been associated with a high abuse potential, especially when they are illegally re ormulated to deliver the entire dose at once rather than over the course o hours. Abusers o these ormulations seek a “high” rom a rapid increase in plasma levels (see Chapter 19). As a result, several o the sustained-release opioids have been re ormulated into abuse-deterrent pills that are di f cult to crush or dissolve. Intravenous opioids, most commonly hydromorphone or morphine, may be administered in patient-controlled analgesia devices, which are now used to control a multitude o pain states, primarily in inpatient settings. Epidural or intrathecal morphine can produce highly e ective analgesia by achieving locally high concentrations in the dorsal horn o the spinal cord. Neuraxial administration o the drug results in a much longer duration o action than does parenteral administration because o the time required or the relatively hydrophilic morphine to di use out o the CNS into the systemic circulation.

Synthetic Agonists

All o the opioids thus ar discussed (morphine, codeine, hydromorphone, oxymorphone, oxycodone, and hydrocodone) are naturally occurring or semisynthetic and all into the chemical class o phenanthrenes. The two major classes o synthetic -receptor agonists are the diphenylheptane (methadone) and phenylpiperidine ( entanyl, su entanil, al entanil, remi entanil, and meperidine) classes. Tramadol and tapentadol are classif ed separately and a ect several biochemical pathways in addition to the -opioid pathway. Methadone is well known or its use in opioid addiction treatment but is also used or the treatment o pain. Methadone has a hal -li e in the range o 25–35 hours, is more lipophilic than morphine, distributes highly into tissues, and binds to plasma proteins. Methadone undergoes extensive cytochrome P450-mediated metabolism and as a result is subject to numerous drug–drug interactions. Due to methadone’s long duration o action, it is o ten used to achieve sustained relie o chronic pain. The hal -li e o methadone lengthens with repeated administration as its distribution and clearance mechanisms become progressively saturated. As a result, patients initiating methadone therapy are at risk or delayed respiratory depression a ter tolerating a starting drug regimen, and multiple daily dosing at a f xed dose rate can result in “dose stacking” and a progressively increasing drug level with time. Methadone also causes dose-related prolongation o the QT interval and has been associated with torsades de pointes, a orm o ventricular tachycardia. In addition to its primary, high-potency action as a -opioid agonist, methadone has a low-potency e ect

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as an NMDA receptor antagonist. It is not clear whether the NMDA antagonism o methadone is a clinically important drug e ect. Fentanyl is a short-acting synthetic opioid agonist that is 75–100 times more potent than morphine and has an elimination hal -li e comparable to that o morphine. Sufentanil, which is even more potent than entanyl, and alfentanil, which is less potent, are structurally related to entanyl. Fentanyl is most widely used or intraoperative and periprocedural analgesia due to its high potency and rapid onset o action. Interestingly, entanyl’s analgesic e ect is limited by redistribution to inactive tissue stores, which occurs over the course o minutes a ter a single, low-to-moderate bolus dose. With prolonged in usion, the e ective analgesic (and respiratory suppressing) duration o action o entanyl then progressively lengthens until the time required to eliminate entanyl may be measured in hours rather than minutes. This prolonged duration o action is due to redistribution o entanyl out o inactive tissue stores to active sites a ter the entanyl in usion is terminated. The same phenomenon occurs with al entanil and su entanil, although there is less prolongation o the elimination time than with entanyl. Because o its high lipophilicity, entanyl is bioavailable via several routes. For example, entanyl has been ormulated as a lozenge or buccal transmucosal administration, which is particularly valuable or avoiding parenteral treatment in pediatric patients. Fentanyl can also be administered transdermally in the orm o a patch that releases the drug slowly over time to provide long-acting systemic analgesia. Remifentanil, the most recently developed phenylpiperidine, exhibits distinct pharmacokinetic behavior. Remi entanil contains a methyl ester moiety that is essential or activity but that is also the substrate or the action o numerous nonspecif c tissue esterases. Thus, it has unusually rapid metabolism and elimination, and the drug e ect has an approximately 5-minute hal -li e. Administered as a continuous in usion during anesthesia, remi entanil permits precise matching o the drug dose to the clinical response (see Chapter 17, General Anesthetic Pharmacology). However, i any postoperative pain is anticipated, the rapid termination o action demands that the use o remi entanil during anesthesia be coupled with the administration o a longer acting drug to maintain analgesia postoperatively. In the introductory case, remi entanil was used or intraoperative analgesia during the skin debridement procedure to ensure that JD did not experience pain during surgery. Morphine was added be ore the end o the operation to provide postoperative pain coverage. Because o remi entanil’s short hal -li e, pain associated with surgical tissue damage would have returned immediately a ter surgery i morphine had not been added. Meperidine is a phenylpiperidine -agonist with analgesic e f cacy similar to morphine, but lower potency: 75–100 mg o meperidine is equivalent to 10 mg o morphine. Unlike other opioids, meperidine causes mydriasis rather than miosis. The toxic meperidine metabolite normeperidine can cause increased CNS excitability and seizures. Normeperidine is excreted by the kidneys, and its elimination hal li e is longer than that o meperidine; there ore, meperidine toxicity is a particular problem with repeated dosing o the drug or in patients with acute or chronic kidney disease. In addition, meperidine has a serotonergic e ect and, in combination with MAO inhibitors or selective serotonin reuptake inhibitors, can precipitate serotonin syndrome.

300 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Partial and Mixed Agonists

In addition to the ull -opioid receptor agonists described earlier, several drugs have been developed as partial and mixed - or -agonists. These include buprenorphine, a partial -agonist, and nalbuphine, a -agonist with -antagonist activity. Buprenorphine is widely used in opioid addiction treatment or either maintenance or detoxi cation and may also be used or pain treatment. Buprenorphine is highly potent (approximately 30- old more potent than morphine) and has a prolonged duration o analgesic action (on the order o several hours). It is administered parenterally, sublingually, or transdermally. As a partial agonist, buprenorphine binds to the -opioid receptor with high binding a nity but only moderate receptor activation (see Chapter 2, Pharmacodynamics). As a result, there is a ceiling or both its analgesic and adverse opioid e ects; as the buprenorphine dose increases, -opioid receptors eventually become saturated with bound but relatively inactive partial agonist. Because o buprenorphine’s high -receptor binding a nity, patients treated with high-dose buprenorphine may be re ractory to the e ects o other opioids and to the e ects o opioid reversal agents. Patients chronically exposed to other opioids should discontinue the other opioid prior to initiating buprenorphine therapy, because buprenorphine exposure can induce acute opioid withdrawal in opioid-dependent individuals. Nalbuphine is an opioid agonist/antagonist with analgesic e ects that are thought to be mediated through the -opioid receptor. Nalbuphine is commonly used in clinical practice to reverse the pruritus induced by neuraxial opioid administration.

Opioid Receptor Antagonists

-Opioid receptor antagonists are used to reverse li ethreatening adverse e ects o opioid administration, speci cally respiratory depression. Naloxone , one such antagonist, is a synthetic derivative o oxymorphone that is administered parenterally. Because the hal -li e o naloxone is shorter than that o morphine, it is not sa e to leave the patient unattended immediately a ter success ul treatment o an episode o respiratory depression with naloxone; monitoring can be relaxed only when it is certain that morphine no longer remains in the system. The orally administered antagonist naltrexone is primarily used in outpatient settings, typically or detoxi cation o individuals addicted to opioids (see Chapter 19). Combinations o opioid agonists and antagonists are being developed to reduce illicit drug use. Antagonists restricted to the periphery, such as alvimopan and methylnaltrexone , have been developed to reduce postoperative ileus and to ameliorate the gastrointestinal e ects o chronic opioid use.

Nonsteroidal Anti-Inf ammatory Drugs and Nonopioid Analgesics General Features

Nonsteroidal anti-inf ammatory drugs (NSAIDs) inhibit the activity o cyclooxygenase enzymes (COX-1 and COX-2) that are required or the production o prostaglandins (see Chapter 43). NSAIDs a ect pain pathways in at least three di erent ways. First, prostaglandins reduce the activation threshold at the peripheral terminals o primary a erent nociceptor neurons (Fig. 18-9). By reducing prostaglandin synthesis, NSAIDs decrease inf ammatory hyperalgesia and allodynia. Second, NSAIDs decrease the recruitment o

Peripheral inflammation

Central cytokine release

COX-2 upre gula tion in infla mma tory ce lls

COX-2 upre gula tion in dors a l horn ne urons a nd s upporting ce lls

Acetaminophen Celecoxib NSAIDs P ros ta gla ndin production

P ros ta gla ndin production

Cons titutive COX-1 Action a t pe riphe ra l te rmina l P GE 2 re ce ptors

Action a t P GE 2 re ce ptors on dors a l horn ne urons

P e riphe ra l s e ns itiza tion

Enha nce d de pola riza tion of s e conda ry s e ns ory ne urons

FIGURE 18-9. Mechanism o analgesic action o cyclooxygenase inhibitors. Inf ammatory states are o ten associated with the production o prostaglandins, which are important mediators o both peripheral (left) and central (right) pain sensitization. In the periphery, prostaglandins produced by inf ammatory cells sensitize peripheral nerve terminal prostaglandin (EP) receptors, making them more responsive to a pain ul stimulus. In central pain pathways, cytokines released in response to inf ammation induce prostaglandin production in the dorsal horn o the spinal cord. These prostaglandins sensitize secondary nociceptive neurons and thereby increase the perception o pain. Nonsteroidal anti-inf ammatory drugs (NSAIDs) block peripheral and central sensitization mediated by prostanoids that are released in inf ammation; NSAIDs also reduce the extent o inf ammation.

leukocytes and, thereby, the production o leukocyte-derived inf ammatory mediators. Third, NSAIDs that cross the blood–brain barrier prevent the generation o prostaglandins that act as pain-producing neuromodulators in the spinal cord dorsal horn. Acetaminophen and NSAIDs act through mechanisms that do not involve the -opioid receptor, and NSAID– opioid and acetaminophen–opioid combinations can act synergistically to reduce pain. NSAIDs and COX-2 inhibitors act both peripherally and centrally, whereas acetaminophen acts only centrally. Preclinical data suggest that, while the acute action o NSAIDs is peripheral, much o their analgesic e ect derives rom their central action to prevent a PGE2-induced reduction in glycinergic inhibition. Like the opioids, nonselective COX-inhibiting NSAIDs have some deleterious adverse e ects, particularly injury to the gastric mucosa and the kidneys. It had been thought that the anti-inf ammatory and analgesic e ects o the NSAIDs are primarily attributable to inhibition o COX-2, an inducible enzyme active in inf ammatory states, whereas the adverse e ects o the NSAIDs are primarily attributable to inhibition o COX-1, a constitutive enzyme responsible or the production o prostanoids involved in physiologic tissue maintenance and vascular regulation. However, this view may be an oversimpli cation, because COX-2 may be induced to support COX-1 activity in the setting o gastric mucosal injury, and COX-1 may produce prostaglandins in tandem

C h a p t e r 1 8 Pharmacology of Analgesia

with COX-2 in inf ammatory states. There is also concern that COX-2 inhibition may promote thrombosis and reduce or delay wound healing.

Specif c Agents

The major classes o NSAIDs include the salicylates (aspirin or acetylsalicylate ), propionic acid derivatives (ibupro en), indole acetic acid derivatives (indomethacin), phenylacetic acid derivatives (diclo enac ), and oxicam derivatives (piroxicam) (see Fig. 43-8). The para-aminophenols (acetaminophen) are a related class o compounds with analgesic and antipyretic activity but not anti-inf ammatory activity. The COX-2 selective inhibitors celecoxib, ro ecoxib, and valdecoxib were designed to produce analgesia equivalent to that o the NSAIDs while decreasing the adverse e ects associated with chronic NSAID use. However, both ro ecoxib and valdecoxib have been withdrawn rom the market because o an increased risk o adverse cardiovascular e ects and skin reactions. Representative agents are discussed here; urther in ormation on their anti-inf ammatory uses and adverse e ects is discussed in Chapter 43. Acetylsalicylic acid (aspirin) acts by covalently acetylating the cyclooxygenase active site in both COX-1 and COX-2. Aspirin is rapidly absorbed and distributed throughout the body. Chronic aspirin use can produce gastric irritation and erosion, hemorrhage, vomiting, and renal tubular necrosis. These concerns limit the use ulness o aspirin primarily to acute pain settings. The coxibs are COX-2 selective enzyme inhibitors. Currently, only celecoxib remains in clinical use in the United States. This class o drugs was originally reserved or patients who required NSAIDs but were at high risk or developing gastrointestinal (GI), renal, or hematologic adverse e ects, although there is no clinical evidence that celecoxib reduces the risk o adverse GI e ects. The widely used compound ibupro en is a derivative o propionic acid. Used primarily or its analgesic and antiinf ammatory e ects, ibupro en is also an antipyretic, and it has a lower incidence o adverse e ects than aspirin. Another common propionic acid derivative is naproxen. Compared to ibupro en, naproxen is more potent and has a longer hal -li e; there ore, it can be administered less requently with equivalent analgesic e cacy. Its adverse e ect pro le is similar to ibupro en, and it is generally well tolerated. As with all NSAIDs, ibupro en and naproxen can cause GI complications ranging rom dyspepsia to gastric bleeding. The phenylacetic acid derivatives diclo enac and ketorolac are used to treat moderate to severe pain. Ketorolac can be administered orally or parenterally, while diclo enac is available in oral ormulations. Both agents carry a risk o severe adverse e ects, including anaphylaxis, acute renal ailure, Stevens-Johnson syndrome (a di use li e-threatening rash involving the skin and mucous membranes), and gastrointestinal bleeding. Ketorolac is valuable or short-term pain control when avoidance o adverse opioid e ects is desirable, or example, in day-surgery patients. Topical ormulations o these drugs may have some utility. Acetaminophen (paracetamol) pre erentially reduces central prostaglandin synthesis by an uncertain mechanism; as a result, the drug produces analgesia and antipyresis but has little anti-inf ammatory e cacy. Acetaminophen is requently combined with weak opioids or the treatment o moderate pain, and preparations are available eaturing

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acetaminophen combined with codeine, hydrocodone, oxycodone, pentazocine, or propoxyphene. A ter deacetylation to its primary amine, acetaminophen is conjugated to arachidonic acid by atty acid amide hydrolase in the brain and spinal cord; the product o this reaction, N-arachidonoylphenolamine, may inhibit COX-1 and COX-2 in the CNS. N-arachidonoylphenolamine is an endogenous cannabinoid and an agonist at TRPV1 receptors, suggesting that direct or indirect activation o TRPV1 and/or cannabinoid CB1 receptors could also be involved in the mechanism o acetaminophen action. A major concern with acetaminophen use is its low therapeutic index; overdose can result in liver ailure (see Chapter 6, Drug Toxicity).

Antidepressants Drugs originally developed to treat depression are widely used as adjuvant therapy in pain management, particularly or treatment o chronic pain conditions. Although patients with chronic pain commonly experience depression, and reducing depression may improve quality o li e, antidepressants have an analgesic action distinct rom their antidepressant e ect. Based on results rom animal models, the analgesic action appears to be mediated mainly in the spinal cord and to involve the reduction o central sensitization. It is thought that tricyclic antidepressants produce analgesia both by blocking sodium channels and by increasing the activity o antinociceptive noradrenergic and serotonergic projections descending rom the brain to the spinal cord. In general, the least selective agents (i.e., those with the broadest neurochemical e ects), such as the tricyclics amitriptyline , nortriptyline , and imipramine , have been more e ective than the selective serotonin reuptake inhibitors (SSRIs) paroxetine , f uoxetine , and citalopram. The use o these drugs in mood disorders is discussed in Chapter 15. Venla axine and duloxetine are dual norepinephrine/ serotonin (NE/5-HT) reuptake inhibitors with actions as both antidepressants and analgesics. These agents are used in the treatment o neuropathic pain and bromyalgia. Duloxetine has a balanced action on NE and 5-HT reuptake and a weak action on dopamine reuptake as well. Although SSRIs have minimal analgesic action by themselves, inhibition o the serotonin reuptake transporter appears to produce some analgesic e ect when NE reuptake is also blocked.

Antiepileptic Drugs and Antiarrhythmics Some therapeutic agents used to control the excessive cellular excitability that leads to seizures (see Chapter 16) or cardiac arrhythmias (see Chapter 24) can also be used to manage the symptoms o some chronic pain conditions. In the search or drugs that produce analgesia, several o these agents have been tested on the basis o their ability to reduce neuronal excitability. O these, the most widely used are the antiepileptic drugs gabapentin and pregabalin. Gabapentin is widely used or the management o chronic pain. It was originally developed as a structural analogue o GABA, but it does not bind to GABA receptors and does not a ect the metabolism or reuptake o GABA. Gabapentin binds to the 2 subunit o voltage-dependent calcium channels and reduces the tra cking o the channel to the membrane. Randomized clinical trials in diabetic neuropathy and trigeminal neuralgia show that gabapentin is superior to placebo in reducing subjectively reported pain.

302 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Gabapentin also has some e f cacy in reducing postoperative pain. Gabapentin is associated with several adverse e ects, particularly dizziness, somnolence, con usion, and ataxia. In the introductory case, gabapentin reduced JD’s spontaneous paroxysmal pain, probably by decreasing aberrant neuronal excitability and synaptic transmission. One problem with gabapentin is that its oral bioavailability is not predictable or linear. Some patients require 10 times as much drug as others to achieve a similar e ect; this may be related to variable GI absorption. A newer antiepileptic drug with a similar structure is pregabalin; this substituted GABA analogue is more potent, has a aster onset o action, and has more predictable bioavailability than gabapentin. Pregabalin produces an analgesic e ect similar to that o gabapentin in patients with neuropathic pain and f bromyalgia, and the two drugs have similar adverse CNS e ects. Pregabalin also produces a mild euphoric e ect in some patients. Because o its increased potency, it is claimed that dose-related adverse e ects may be lower with pregabalin than with gabapentin. Carbamazepine acts to block sodium channels; the drug is used primarily to treat trigeminal neuralgia, but it has a relatively high adverse e ect prof le. Oxcarbazepine is a close structural derivative o carbamazepine with an additional oxygen atom decorating the benzylcarboxamide group. This di erence alters metabolism o the drug in the liver. More importantly, it reduces the risk o aplastic anemia, which is a serious adverse e ect occasionally associated with carbamazepine. Lidocaine , a use-dependent sodium channel blocker, is typically used as a local anesthetic or regional anesthesia (see Chapter 12); this drug is also used topically in patches or patients with cutaneous pain, or example, patients with postherpetic neuralgia. In some instances, lidocaine administered intravenously can be valuable or blunting the autonomic response to short-term, high-intensity pain ul stimuli, such as the placement o cranial pins or head positioning in neurosurgical procedures.

NMDA Receptor Antagonists Because o the critical role o NMDA receptors in the induction and maintenance o central sensitization, NMDA receptor antagonists are currently under investigation or use in pain treatment. Two currently available analgesic drugs act as antagonists at the NMDA receptor: the anesthetic ketamine and the antitussive dextromethorphan. Ketamine is widely used at high doses as an anesthetic induction agent and at lower doses or procedural sedation and or periprocedural pain relie . It can also be o utility in the periprocedural management o highly opioid-tolerant patients. Since ketamine has no -agonist properties, it is not a respiratory depressant; as such, it may be use ul in circumstances in which analgesia is needed without concomitant respiratory compromise. Ketamine use is severely limited by its psychomimetic e ects, which contribute to its abuse liability; ketamine can also suppress cardiac contractility while stimulating sympathetic activity.

Adrenergic Agonists Stimulation o 2-adrenergic receptors in the dorsal horn o the spinal cord produces an anti-nociceptive state. There ore, 2-adrenergic agonists may have therapeutic utility as analgesics. The 2-agonist clonidine has been used systemically,

epidurally, intrathecally, and topically, and appears to produce analgesia in both acute and chronic pain states. However, clonidine also causes postural hypotension, which limits its use ulness in pain control.

Migraine Therapy The treatment o migraine pain has eatures distinct rom the treatment o other pain conditions. In many but not all patients, an e ective treatment or migraine is the triptan class o serotonin receptor agonists; the best-studied example is sumatriptan. The triptans are selective or the 5-HT1B and 5-HT1D receptor subtypes o the 5-HT1 amily, one o the seven amilies o serotonin receptor (see Chapter 15). 5-HT1B receptors are located on vascular endothelial cells, smooth muscle cells, and neurons, including trigeminal nerves. 5-HT1D receptors are present on the trigeminal nerves that innervate meningeal blood vessels. Triptans reduce both sensory activation in the periphery and nociceptive transmission in the brainstem trigeminal nucleus, where they diminish central sensitization. The triptans also cause vasoconstriction, opposing the vasodilation thought to be involved in the pathophysiology o migraine attacks. It remains unclear whether the vasoconstriction is help ul in producing the antimigraine actions o these drugs, however. Furthermore, as a result o this vasoconstrictive e ect, the triptans can be dangerous in patients with coronary heart disease. The triptans can reduce the pain and other symptoms associated with acute migraine attack and have replaced the vasoconstrictive agent ergotamine in the treatment o migraine. Sumatriptan can be administered subcutaneously, orally, or by nasal inhalation; the nasal ormulation may have an improved therapeutic index. Several other orally administered agents in the triptan class are also available, including zolmitriptan, naratriptan, and rizatriptan (see Drug Summary Table). NSAIDs, opioids, ca eine, and antiemetics also have activity and some utility or treatment o acute migraine headaches. For example, a combination o indomethacin, prochlorperazine, and ca eine may have e ectiveness similar to triptans in the treatment o migraine attacks. During an attack, migraine patients o ten experience gastric stasis that can reduce the bioavailability o oral medications. CGRP receptor antagonists are promising candidates or migraine therapy. Although the triptans are relatively e ective in ameliorating the acute symptoms o migraine, other classes o drugs are used to reduce the requency o attacks. Several drug classes are used or migraine prophylaxis, including -adrenergic blockers, valproic acid, serotonin antagonists, and calcium channel blockers. These agents are generally chosen based on the severity and requency o the migraine attacks, the cost o the drug, and the adverse e ects o the drug in the context o the individual patient. None has been shown to have a high level o e f cacy, and new drugs need to be developed or more e ective migraine prophylaxis.

CONCLUSION AND FUTURE DIRECTIONS Because o the limited e f cacy o any single drug, it is common in clinical practice to use a polypharmacy approach to manage pain. In combination, several drugs that are only

C h a p t e r 1 8 Pharmacology o Analgesia

moderately e ective as single agents can have additive or supra-additive e ects. This is largely a consequence o the multiple processing events and mechanisms responsible or producing pain; intervention at several steps may be required to achieve adequate analgesia (Fig. 18-10). Because many Ce ntra l pe rce ption (opioids)

Corte x

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FIGURE 18-10. Summary of the sites of action of the major drug classes used for pain management. Analgesics target various steps in pain perception, rom the initiation o a pain stimulus to the central perception o that pain. NSAIDs modulate the initial membrane depolarization (signal transduction) in response to a peripheral stimulus. Sodium channel blockers decrease action potential conduction in nociceptive f bers. Opioids, antidepressants, NSAIDs, antiepileptic drugs (anticonvulsants), and 2-adrenergic agonists all modulate transmission o pain sensation in the spinal cord by decreasing the signal relayed rom peripheral to central pain pathways. Opioids also modulate the central perception o pain ul stimuli. The multiple sites o action o analgesics allow a combination drug approach to be used in pain management. For example, moderate pain is o ten treated with combinations o opioids and NSAIDs. Because these drugs have di erent mechanisms and sites o action, the combination o the drugs is more e ective than one drug alone.

303

drugs used to treat pain are also active systemically and/ or in parts o the nervous system that are not related to somatic sensation, analgesics can produce deleterious adverse e ects. One approach to limiting toxicity is to use localized (nonsystemic) orms o drug delivery. In particular, epidural and topical delivery limit exposure to the drug to a local site o action. Many o the opioids are short-acting and must be administered requently to patients in severe pain. Modes o drug delivery have also been developed to optimize the pharmacokinetics o the short-acting opioids; these methods include transdermal and buccal dosage orms, patientcontrolled analgesia devices, and controlled-release oral preparations. Patient-controlled devices ensure that patients do not su er pain because o waning drug e ects, and instrumental controls can e ectively prevent overdose. At the present time, however, patient-controlled technologies are suitable only or inpatient treatment. Most o the currently available analgesics have been identif ed by empirical observation (opioids, NSAIDs, and local anesthetics) or serendipity (antiepileptic drugs). Now that the mechanisms responsible or pain are being explored at a molecular level, many new targets are being revealed that are likely to lead to new and di erent classes o analgesics. It is hoped that drugs active at these targets will achieve higher e f cacy and have ewer adverse e ects than current therapies. E ective pain management approaches must not rely only on pharmacologic intervention; physical therapy and rehabilitation and, in some situations, surgical approaches may also have a role. The growing complexity o pain management has spawned specialized pain services or inpatient pain control, as well as pain clinics and centers or the outpatient management o chronic pain.

Acknowledgment The authors thank Salahadin Abdi, Rami Burstein, and Carl Rosow or their valuable comments.

Suggested Reading Chou R, Turner JA, Devine EB, et al. The e ectiveness and risks o long term opioid therapy or chronic pain: a systemic review or a National Institutes o Health Pathways to Prevention Workshop. Ann Intern Med 2015;162:276–286. (A systematic review of randomized controlled trials using opioids to treat chronic pain.) Costigan M, Scholz J, Wool CJ. Neuropathic pain: a maladaptive response o the nervous system to damage. Annu Rev Neurosci 2009;32:1–32. (Overview of mechanisms of neuropathic pain.) Finnerup NB, Attal N, Haroutounian S, et al. Pharmacotherapy or neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol 2015;14:162–173. (Clinical approach to management of neuropathic pain.) Rosow CE, Dershwitz M. Pharmacology o opioid analgesics. In: Longnecker D, Brown DL, Newman MF, Zapol WM, eds. Anesthesiology. New York: McGraw Hill; 2008. (Detailed review of opioid pharmacology.) von Hehn CA, Baron R, Wool CJ. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 2012;73:638–652. Waxman SG, Merkles IS, Gerrits MM, et al. Sodium channel genes in painrelated disorders: phenotype-genotype associations and recommendations or clinical use. Lancet Neurol 2014;13:1152–1160. (Reviews channelopathies that produce pain and their treatment.) Wool CJ. Central sensitization: implications or the diagnosis and treatment o pain. Pain 2011;152(3)(Suppl):S2–S15.

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C h a p t e r 1 8 Pharmacology of Analgesia 307

P re ffro o ntta l c ort co rtee x rt

Nic o tine

DA A AC h

19

Ve nntr nttra ra l ttee gme ggm m e nta n ttaa l a re nt re a

Nicotin n ic ACh ree cee pt p tor or

Acc NA NA

Pharmacology of Drugs of Abuse Opio ids Op

Pe t e r R. M a rt in a n d S a ch in Pa t e l

µ -opi o pioi oid d re cee pt ptor o

Cannab abiino ids

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INTRODUCTION RODUCTION CTI CTIO TIO O & CASE E . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308–309 Alcoho hol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 NMD NMDA A-R R NMD DA Nicc otine Ni t and Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 DEFIINITIONS NS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3088 Cocaiinee an andd Am A phetamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 MECH M CHAN N NIS NIIS SMS MS OF F TO TOLE TOLER T OLERANCE, LER L LE ERA ANCE, CE DEPENDENCE, DEPEND D AN AND j . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 pptor Marijuana WIT WI W THDRAWAL AWAL AW L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . µ . .-o .opioi .p. .oid . .d. .re . . ce . e311 Otherr Abused Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Toolerance nce ce . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 A COMPLICATION ONS S OF Dependence Depende ependence ce and nd Withdraa wa wall . . . . . . . . . . . . . . . . . . . . . . . . . . 313 MEDICAL A US USE E DI DIS SORDERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 id ids SUBSTANCE MEC ME ECH CHAN ANIIS IS SMS SM MS OF M F SUBSTANCE USE D DISORDERS DISO ISO SORD ORDE R E . . . . . . . Opio . . . . o315 TREA EATM TME MENTS FOR SUBSTANCE USE DISORDERS . . . . . . . . . . 326 N c o Substa NA Learning Le earning rnin ningg aand an nd Development Devel eloopme ment Substanc e Us Substance U e Disord Dis der erss . . . 31 3155 TR Detoxif x cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Variables a ri riab able les ess A eecting ctin ct ingg th thee Deve Dee velopment velo ve lopm lo pment pmen pm entt o Substanc en Substance bstan sta Use Sel -Help H and Mutual al Sup uppo port rt Pro rogr gram amss . . . . . . . . . . . . . . . . . 327 Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 Disorders Disorder Pharmacologic m Treatment o Substance Person ssooonal onality liit ity ty Ch Characteristic aracteristics and an Co-Occu Co-Occurring Co-Oc Co-Occ O Oc Occurr Alc o h o l Roolee o Pe Use s Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 Disorders rd rde inn Sub Substance S ance Us Use Diso iso sorder eers er rss . . . . . . . . . . Co . . . .c.aine . ine 319 PCP PC P U AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 329 DRU UGS OF ABUSE A E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 CONCLUSION Canna abino bino o id ids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 S Opioids Suggested uggest s tedd R Reading e adi ding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .331 Benzodiazepines and Barbiturates . . . . . . . . . . . . . . . . . . . . . . 322

INTRODUCTION This chapter considers pharmacologic agents implicated in substance use disorders and the relevant brain processes involved in the clinical progression o these disorders. While the pharmacology o these agents is important to understanding their e ects on behavior and their abuse liability, personality characteristics, as well as the presence o co-occurring psychiatric and medical conditions, may also contribute to the risk o developing substance use disorders. Understanding substance use disorders as biopsychosocial syndromes, rather than simply the pharmacologic consequences o chronic alcohol/drug use, has led to recognition o the central role that learning plays in substance use disorders and the potential or an integrated pharmacopsychosocial approach to treatment. Most individuals with substance use disorders also have a second diagnosable psychiatric condition, but it is not easy to determine whether psychiatric symptoms are a cause or consequence o alcohol/drug use. For example, although alcohol is widely used to sel -medicate depression and/or anxiety, it may be di cult to determine whether such psychiatric symptoms in alcoholics are the cause o drinking or its e ect, because the actions o alcohol per se, as well as alcohol withdrawal and dependence, can also result in signi cant anxiety or depression. Genetic determinants o the psychopharmacologic actions o abused drugs are increasingly recognized. Nonetheless, environmental variables have a signi cant inf uence on 308

the development o substance use. For example, societal attitudes toward substance use o ten inf uence the likelihood that a substance will be taken in the rst place. The availability and cost o a substance are also a ected by its legal and tax status. The availability o other, nondrug alternatives may be a key actor in determining the likelihood that substance use disorders emerge or a given agent. This chapter describes the mechanisms o action o selected representative substances o abuse, and the mechanisms o other important substances o abuse are summarized in Table 19-1. Since addiction is a disorder o brain reward pathways, learning, and motivated behavior, the use o medications in an integrated pharmacopsychosocial approach to treat substance use disorders is also discussed.

DEFINITIONS The empirically based nomenclature promulgated by the American Psychiatric Association (APA) in the Diagnostic and Statistical Manual (DSM, Box 19-1) de nes substance use disorder (used interchangeably in this chapter with addiction) as “a problematic pattern o use” leading to “signi cant impairment or distress.” This de nition avoids value judgment and is generalizable across cultures. Psychosocial eatures o substance use disorders are similar or diverse psychopharmacologic agents with abuse liability and are likely more important in the development and maintenance o pathologic drug use than the unique pharmacologic pro le

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

CA, a 33-ye a r-o ld m a n , is b ro u g ht to the e m e rg e n cy d e p a rtm e n t w ith ch ills , s e ve re n a u s e a , vo m itin g , d iarrh e a, m u s cle a ch e s , a n d a n xie ty. Mr. A e xp la in s th a t h e is cu rre n tly s h o o tin g m o rp h in e o r h e ro iin a b o u t 3 d a ys p e r w e e k a n d u s in g m a riju a n a o r co ca in e “ wh e n e ve r.” He e e ls h e wa n ts to d ie . On e xa m in a tio n , h e h a s e n la rg e d p u p ils , te m p e ra tu re is 103°F, b lo o d p re s s u re is 170/95 m m Hg , a n d h e a rt ra te is 108 be a ts /m in. He is irrita ble a n d h a s a b do m in a l cra m p in g , hyp e ra lg e s ia , a n d p h o to p h o b ia . Mr. A is g ive n clo nid in e , w ith lo p e ra m id e , ib u p ro e n , a n d p ro m e th a zin e a s n e e d e d o r d ia rrh e a , p a in , a n d n a u se a/vom itin g, resp ectively. The s everity o withd ra wal d o e s n o t d im in is h s ig n if ca n tly u n til h e is g ive n s u b lin g u a l b u p ren o rp h in e /n a loxo n e e ve ry 8 h o u rs . Ove r th e n e xt w e e k, Mr. A’s b u p re n o rp h in e /n a loxo n e is titra te d to a o n ce -d a ily d o s e , w ith p ro g re s s ive d im in u tio n o w ith d ra wa l s ym p to m s a n d d ru g cra vin g . Mr. A is dis ch a rg e d to a 28-d a y in te n s ive o u tp a tie n t tre a tm e n t p ro g ra m d u rin g w h ich h e co n tin u e s b u p re n o rp h in e /n a loxo n e . He a g re e s to a tte n d d a ily m u tu a l s u p p o rt s e l -h e lp m e e tin g s (Alco h o lics o r Narco tics Anonym o us ), wh ere he tells th e tale o h is ad dictio n . He began d iet pills in h is teen s for weig h t co n tro l an d drin king as a resp o ns e to phys ical ab u se by his alcoholic fathe r. He was pres cribed acetam ino p h e n a n d co d e in e fo r p a in a fte r m in o r s u rg e ry; th e re a fte r, h e s ta rte d “ s tre e t” p a in p ills a n d “ d o cto r sh o pp in g” for back pa in and d epres sio n , an d h e even h a d h e a lthy te e th e xtra cte d to g e t o p io id s fro m th e de ntis t. He was distraught when a clos e rela tive died, a n d h e s w itch e d to in tra ve n o u s o p io id s , in clu d in g fentanyl pa tches th at h e ate o r in je cted . He ha d th ree inp atient treatm ents, always relapsin g after d ischarge

o any given drug. Diagnostic eatures are conceptualized as clinical clusters “o cognitive, behavioral, and physiological symptoms indicating that the individual continues using the substance despite signif cant substance-related problems:” loss o control, salience to the behavioral repertoire, and neuroadaptation. However, the undamental element is drugseeking, the sine qua non o addiction. In the introductory case, Mr. A elt that he had little else than drugs in his li e and could not stop using without help, and it is likely that he was addicted to opioids (and to the other drugs he was using). The terms tolerance, dependence, and withdrawal may be def ned based on clinically apparent physiologic changes as well as more subtle alterations in brain reward neurocircuitry. Tolerance re ers to the decreased e ect o a substance that develops with continued use, in other words, the dose–response curve shi ts to the right as larger doses are needed to produce the same response, as happened in the case o Mr. A. The drug toxicity prof le and the drug lethality prof le o ten do not shi t in the same way or to the same degree as the psychopharmacologic e ect(s) or which the drug is primarily sel administered. Thus, when taking heroin, it is likely that Mr. A experienced adverse e ects o constipation and pupillary

309

to increas ingly higher drug doses. He also tried m etha d o n e m a in te n a n ce tre a tm e n t fo r 6 m o n th s w ith som e bene t but was unable to com pletely stop using “street drugs” and “never felt norm al on m ethadone.” Mr. A continu es to be ab s tine nt rom illicit o pioids a ter 10 years o n sublingu al bu prenorphine/naloxone. He vis its h is p s ych ia tris t m o n th ly o r p s ych o th e ra p y a n d a d h e re s to h is tre a tm e n t co n tra ct o m o n th ly a tte n d a n ce a t g ro u p th e ra p y a n d w e e kly Na rco tics An o n ym o u s (NA) o r Alco h o lics An o n ym o u s (AA) m e e tin g s . He co n s is te n tly p rovid e s d ru g - re e u rin e te s ts . He h a s b e e n p ro m o te d a t wo rk, h a s n eg o tia te d a lo an o r p u rch a s e o a h o u s e, a n d s e e m s re la tively co n te n t h a vin g ga in e d co n s id e ra b le u n d e rs ta n d in g about m anagem ent o his drug use disorder. Recently, h e ha s be gu n dis cu s s ing ta pe ring a nd e ven tu a lly d is co n tin u in g b u p re n o rp h in e /n a loxo n e m ain te n a n ce .

Questions 1 . What caused Mr. A’s physical symptoms and signs (i.e., chills, nausea, vomiting, fever, enlarged pupils, and hypertension) on his initial visit to the emergency department? 2 . How could Mr. A’s pain be managed if he were to need surgery while taking buprenorphine/naloxone? 3 . How can mutual support programs such as AA or NA help treat addiction, and how should such programs be complemented by medical oversight? 4 . What was the rationale for initiating clonidine treatment for Mr. A? 5 . Why did Mr. A’s initial symptoms and long-term opioid craving abate with the combination of buprenorphine/ naloxone? How long should maintenance be continued?

constriction at a dose insu f cient to get “high.” Additionally, since the brain respiratory centers o ten do not develop tolerance to increased doses o heroin, a lethal overdose is more likely as a person takes higher doses o this drug. The opposite e ect, termed sensitization (also called inverse tolerance ), re ers to a shi t o the dose–response curve to the le t, so that repeated administrations of a drug result in a greater effect of a given dose, and a lower dose is required to achieve the same e ect. Interestingly, tolerance and sensitization to di erent pharmacologic actions o a drug may occur concurrently. Thus, upon repeated administration o central nervous system (CNS) depressants like alcohol, stimulant e ects (e.g., disinhibition) demonstrate sensitization, whereas depressant actions (e.g., sleep) acquire tolerance. Dependence can be def ned only indirectly by (1) tolerance, (2) the emergence o a withdrawal syndrome upon drug discontinuation or administration o a specif c antagonist, (3) drug “craving,” or (4) drug-seeking behavior mani ested as a result o conditioned stimuli a ter withdrawal has abated. In Mr. A’s case, his initial symptoms in the emergency department were caused by heroin withdrawal, a mani estation o physical dependence that could be alleviated by any

310 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

TABLE 19-1 Major Drugs of Abuse DRUG CLASS

EXAMPLES

RECEPTOR (ACTION)

CLINICAL SIGNS

NOTES

Opioids

Morphine Heroin Codeine Oxycodone

-Opioid (agonist)

Euphoria, followed by sedation, respiratory depression

Used therapeutically as analgesics (except for heroin) Prescription opioid abuse is rapidly increasing

Benzodiazepines

Triazolam Lorazepam Diazepam

GABAA (modulator)

Sedation, respiratory depression

Used therapeutically as anxiolytics, sedatives; risk of overdose death in combination with alcohol or opioids

Barbiturates

Phenobarbital Pentobarbital

GABAA (modulator, weak agonist)

Sedation, respiratory depression

Used therapeutically as anxiolytics and sedatives; greater danger of death from overdose than benzodiazepines

Alcohol

Ethanol

GABAA (modulator), NMDA (antagonist)

Intoxication, sedation, memory loss

Legal in many countries and often used in conjunction with other psychoactive agents

Nicotine

Tobacco

Nicotinic ACh (agonist)

Alertness, muscle relaxation

Legal in many countries and often complicates other drug use disorders

Psychostimulants

Cocaine Amphetamine

Dopamine, adrenergic, serotonin (reuptake inhibitor)

Euphoria, alertness, hypertension, paranoia

Amphetamines also reverse the reuptake transporter and release neurotransmitter from synaptic vesicles into the cytoplasm

Caffeine

Coffee Soft drinks

Adenosine (antagonist)

Alertness, tremulousness

Generally legal, addiction rare

Cannabinoids

Cannabis

CB1, CB2 (agonist)

Changes in mood, hunger, giddiness

Controversial recent legalization in many states of US

Phencyclidine (PCP)

N/A

NMDA (antagonist)

Hallucinations, hostile behavior

Effects may be confused with psychosis

Phenylethylamines

MDMA (Ecstasy), MDA

Serotonin, dopamine, adrenergic (reuptake inhibitors, multiple actions)

Euphoria, alertness, hypertension, hallucinations

Structurally related to amphetamines, effects similar to psychedelic agents, may cause lasting injury to serotonergic neurons

Psychedelic agents

LSD DMT Psilocybin

5-HT2 (partial agonist)

Hallucinations

Flashbacks may relate to overlearning of traumatic experience with intoxication

Inhalants

Toluene Amyl nitrate Nitrous oxide

Unknown

Dizziness, intoxication

May result in lasting brain damage

MDMA, methylenedioxymethamphetamine; MDA, methylenedioxyamphetamine; LSD, lysergic acid diethylamide; DMT, dimethyltryptamine; GABA, -aminobutyric acid; ACh, acetylcholine; CB, cannabinoid; 5-HT, serotonin; NMDA, N-methyl-D-aspartate.

-opioid receptor agonist. Physical dependence is sometimes distinguished rom psychological dependence , or the continued craving or drug and proclivity to return to out-o control opioid use even a ter acute withdrawal symptoms have abated. Physical dependence results rom many o the same mechanisms that produce tolerance. As with tolerance, homeostatic set-points are altered to compensate or the presence o the drug. I drug use is discontinued, the altered set-points produce e ects opposite to those mani ested in the presence o the drug. The altered set-points also activate autonomic nervous system stress responses, which partially explains the benef ts o drugs such as clonidine in treating withdrawal. For example, abrupt withdrawal rom a CNS depressant involves hyperarousal, whereas withdrawal rom a stimulant involves depression and lethargy; discontinuing a drug o either class results in a nonspecif c increase in autonomic activity. Psychological dependence involves resetting the reward system o the brain as a result o repeated drug use. Thus, even a ter

drug use has ceased, brain reward mechanisms may be altered so that a ective and neuroendocrine disturbances and drug craving persist and the individual is prone to relapse. There is signif cant neurobiological overlap between “psychological” and “physical” components o dependence, and some have questioned the real utility o such a distinction. In any event, the recently published DSM-5 eschews the term “dependence” because the clinical and pharmacologic meanings o this term can be con using. The newly adopted diagnostic term is “substance use disorder,” which is meant unequivocally to serve as a clinical construct, whereas dependence retains its pharmacologic meaning only (Box 19-1). Tolerance and dependence typically coexist, but the presence o either does not necessarily imply pathologic drug use. For example, a patient given an opioid or surgical pain will likely develop tolerance to the drug and require progressively larger doses or analgesia; moreover, should drug administration cease or an opioid antagonist be given,

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311

B O X 1 9 - 1 Crite ria fo r S u b s ta n ce Us e Dis o rd e rs (Ad d ictio n ) fro m th e Dia g n o s tic a n d S ta tis tica l Ma n u a l o f Me n ta l Dis o rd e rs , Fifth Ed itio n (DS M-5) A problematic pattern o substance use leading to clinically signif cant impairment or distress, as mani ested by at least two o the ollowing, occurring within a 12-month period: 1. The substance is o ten taken in larger amounts or over a longer period than was intended. 2. There is a persistent desire or unsuccess ul e orts to cut down or control substance use. 3. A great deal o time is spent in activities necessary to obtain the substance (e.g., visiting multiple doctors or driving long distances), to use the substance (e.g., chainsmoking), or to recover rom its e ects. 4. Craving, or a strong desire or urge to use the substance. 5. Recurrent substance use resulting in a ailure to ulf ll major role obligations at work, school, or home. 6. Continued substance use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the e ects o the substance. 7. Important social, occupational, or recreational activities are given up or reduced because o substance use.

8. Recurrent substance use in situations in which it is physically hazardous. 9. Substance use is continued despite knowledge o having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the substance (e.g., current cocaine use despite recognition o cocaineinduced depression, or continued drinking despite recognition that an ulcer was made worse by alcohol consumption). 10. Tolerance, as def ned by either o the ollowing: a. A need or markedly increased amounts o the substance to achieve intoxication or desired e ect. b. A markedly diminished e ect with continued use o the same amount o substance. 11. Withdrawal, as mani ested by either o the ollowing: a. The characteristic withdrawal syndrome or the substance (as def ned by the APA criteria or withdrawal or a specif c substance). b. The same (or closely related) substance is taken to relieve or avoid withdrawal symptoms. ■

The presence o 2–3 o the above symptoms is specif ed as “Mild” substance use disorder; 4–5 symptoms, “Moderate” substance use disorder; and 6 or more symptoms, “Severe” substance use disorder. Reprinted with permission rom the American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Arlington, VA: American Psychiatric Association; 2013

the patient will likely develop a withdrawal syndrome. However, it will likely be possible to taper and eventually eliminate the analgesic once the surgical basis o pain abates. In this case, i the patient does not mani est drug-seeking behavior, a pathologic condition (substance use disorder) is not present despite the presence o tolerance and dependence. This point underlines the vital role o physicians in (1) e ectively treating postsurgical pain without providing open-ended opioid prescriptions and (2) directly addressing drug-seeking should it arise.

MECHANISMS OF TOLERANCE, DEPENDENCE, AND WITHDRAWAL Tolerance Acquired tolerance results when repeated administration o a

drug shi ts the dose–response curve o the drug to the right, so that a larger dose o the drug is required to produce the same e ect. Innate tolerance re ers to preexisting interindividual variations in sensitivity to the drug (i.e., variations that are present be ore the rst administration o the drug). Innate di erences in sensitivity can arise rom genetic variation o receptors at which the drug acts or di erences among individuals in drug absorption, metabolism, or excretion. As with any multi actorial trait, genetic variability is strongly inf uenced by the environment. An example o innate tolerance is observed with alcohol: those with low innate sensitivity as young adults are at higher risk or alcoholism later in li e. Acquired tolerance includes pharmacokinetic, pharmacodynamic, and learned components. Pharmacokinetic tolerance

develops when the capacity to metabolize or excrete the drug increases as a result o drug exposure. Increased metabolism is typically attributable to induction o metabolic enzymes such as the cytochrome P450s (see Chapter 4, Drug Metabolism). In such cases, pharmacokinetic tolerance results in a lower concentration o drug at its site o action or any given dose. Pharmacodynamic tolerance is caused by neuronal adaptations resulting in reduced response to the same concentration o drug at its site o action in the nervous system. Short-term exposure to a drug can induce neuroadaptive changes in neurotransmitter release and clearance rom the synapse, a decrease in the number o neurotransmitter receptors, altered conductance o ion channels, or modi ed signal transduction (Fig. 19-1). Longer term administration o the drug can cause neuroadaptive changes in the expression o genes relevant to the pharmacologic action o the drug; these changes are closely linked with adaptations in the brain that are thought to be involved in learning and memory ormation (Fig. 19-2). Indeed, persistent adaptations to drug use both modi y existing synapses and create new synapses, e ectively “rewiring” the brain. Such long-lasting molecular and cellular adaptations are likely to explain the cravings and relapses that can occur long a ter drug use has been discontinued. Pharmacodynamic tolerance is closely related to another orm o tolerance, termed learned tolerance . In behavioral tolerance , a orm o learned tolerance, drug use results in compensatory changes in behavior that are not directly related to the pharmacologic action o the drug but rather to accommodation to drug e ects through learning acquired while the person is in the intoxicated “state” or in the environment in which the intoxication occurred. Conditioned tolerance

312 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY Acute use

Withdrawal

A

GABAA-R(α 4 ) GABAA-R

Cl-

Cl-

Cl-

Cl-

Ca 2+

Ca 2+

Ca 2+

Ca 2+ Cl-

-

Cl

Alcohol

Cl-

P

Ca 2+

Ca 2+

P

Ca 2+ Ca 2+

NMDA-R(NR2B)

P

NMDA-R

De cre a s e d e xcita bility

Incre a s e d e xcita bility

Anxiolys is / s e da tion

Anxie ty

Coma

S e izure s

B

Acute use

Withdrawal

GABA Morphine αi β

γ

GDP

Neuroadaptation

γ

K+

Time x Dos e

K+cha nne l

Dependence

AC AC

PKA PKA

CREB

Ge ne tra ns cription

C

Arous a l/ple a s ure

Fa tigue /dys phoria

Dopa mine re ce ptor Dopa mine

Acute use

Withdrawal

Cocaine Amphetamine

De cre a s e d e xcita bility

Incre a s e d e xcita bility

De cre a s e d ne urotra ns mitte r re le a s e

Incre a s e d ne urotra ns mitte r re le a s e

Euphoria

Withdra wa l s ymptoms (dys phoria , la crima tion, dia rrhe a )

Neuroadaptation Time x Dos e DA tra ns porte r Dopa mine D2 re ce ptor DA

DA

L-DOPA

L-DOPA

Tyrosine hydroxylase

Tyrosine hydroxylase

Tyros ine

Neuroadaptation Time x Dos e

Dependence

Tyros ine

αi GDP

AC

µ-opioid re ce ptor

β

Dependence

P

Re ce ptor de gra da tion K+

K+ cha nne l (clos e d)

C h a p t e r 1 9 Pharmacology o Drugs o Abuse

313

FIGURE 19-1. Mechanisms of acute drug action for depressants, opioids, and psychostimulants and development of neuroadaptation and dependence in response to chronic drug use. A. Alcohol modulates the major inhibitory and excitatory neurotransmitter systems o the brain via e ects on GABAA and NMDA receptors (GABAA-R and NMDA-R), respectively. Alcohol is a positive allosteric modulator o GABAA receptors. Alcohol increases chloride conductance through GABAA receptors, resulting in cellular hyperpolarization. Alcohol also decreases calcium conductance through NMDA receptors, urther decreasing cellular excitation. These dual actions on GABAA and NMDA receptors contribute to alcohol’s anxiolytic, sedative, and CNS-depressant e ects. Molecular adaptations to chronic alcohol exposure include (1) internalization and decreased sur ace expression o “normal” 1 subunit-containing GABAA receptors, (2) increased sur ace expression o “low alcohol sensitivity” 4 subunit-containing GABAA receptors, and (3) increased phosphorylation o NMDA receptors containing “high conductance” NR2B subunits. Thus, neuroadaptation results in tolerance to the acute depressant e ects o alcohol and occurs concomitantly with dependence. During withdrawal (i.e., in the dependent state but in the absence o alcohol), these adaptations result in generalized hyperexcitability o neurons. CNS excitation is expressed as anxiety, insomnia, delirium, and potentially seizures. B. Opioids activate -opioid receptors located on synaptic nerve terminals. Acute activation o -opioid receptors results in G protein-dependent activation o potassium channels and inhibition o adenylyl cyclase activity. These e ects result in cellular hyperpolarization and decreased GABA release rom the nerve terminal; the decreased GABA release results in disinhibition o ventral tegmental area (VTA) dopamine neurons. Molecular adaptations to chronic -opioid receptor stimulation include (1) increased -opioid receptor phosphorylation, resulting in receptor internalization and degradation; (2) decreased e f cacy o -opioid signal transduction; and (3) hyperactivation o adenylyl cyclase signaling, leading to enhanced GABA release and to increased gene transcription via activation o transcription actors including cyclic AMP response element binding protein (CREB). Thus, neuroadaptation results in tolerance to the euphoric e ects o opioids. During withdrawal (i.e., in the dependent state but in the absence o opioid), the enhanced GABA release rom inhibitory interneurons results in inhibition o VTA dopamine neurons, dysphoria, and anhedonia. C. Acute cocaine exposure inhibits dopamine reuptake transporters (DAT), resulting in increased synaptic dopamine levels and increased postsynaptic dopamine receptor activation at synapses in the nucleus accumbens; in turn, these e ects cause eelings o euphoria and increased energy. Increased extrasynaptic dopamine also results in D2 autoreceptor activation, which decreases dopamine synthesis. Amphetamine both releases vesicular transmitter stores into the cytoplasm and inhibits neurotransmitter reuptake into vesicles; these combined actions cause neurotransmitter concentrations to increase in the synaptic cle t. During chronic psychostimulant exposure, DAT expression increases, the number o postsynaptic dopamine receptors decreases, and presynaptic dopamine is depleted. Thus, neuroadaptation results in tolerance to the euphoric e ects o psychostimulants. During withdrawal (i.e., in the dependent state but in the absence o psychostimulant), the decreased synaptic levels o dopamine that result rom reduced dopamine synthesis and increased clearance through DAT cause decreased activation o postsynaptic dopamine receptors and eelings o dysphoria, atigue, and anhedonia.

occurs when environmental cues associated with exposure to a drug induce preemptive, ref exive compensatory changes, called a conditioned opponent response . This mechanism o conditioning is an unconscious phenomenon but is o ten the basis or relapse in addicts. For example, seeing paraphernalia associated with use o a drug such as cocaine (which produces tachycardia) may elicit preemptive bradycardia and, thus, craving or the drug.

Dependence and Withdrawal Dependence is typically associated with tolerance, and it results rom mechanisms closely related to those that produce pharmacodynamic and learned tolerance. Substance use disorders are clinical mani estations o dependence and result rom the need or the drug to be present in the brain to maintain “near-normal” unctioning. I the drug is eliminated rom the body so that it no longer occupies its site o action, the adaptations that produced dependence are unmasked and mani ested as an acute withdrawal syndrome that lasts until the system re-equilibrates to the absence o drug (days). Subsequently, a protracted withdrawal syndrome , characterized by craving or the drug (i.e., an intense preoccupation with obtaining the drug), may emerge and continue inde nitely (years). Protracted withdrawal is also associated with subtle dysregulation o learning, drives/motivations, reward, and the potential or relapse. This syndrome should be distinguished rom premorbid risk actors or addiction that do not resolve with abstinence and rom brain injury that is sustained as a result o drug use. Like tolerance, dependence is associated with changes in cellular signaling pathways (Fig. 19-1). For example, upregulation o the cAMP pathway by a drug contributes to acute withdrawal upon discontinuation o the drug because up-regulated adenylyl cyclase causes a “supranormal” response in neurons when physiologic levels o neurotransmitter stimulate the cAMP-coupled receptor. Conversely, a drug

that produces dependence by decreasing receptor number or receptor sensitivity renders the down-regulated receptors understimulated by physiologic levels o neurotransmitter a ter drug discontinuation. The e ects o alcohol illustrate that excitatory and inhibitory mechanisms can act in a synergistic ashion on opposing neurotransmitter systems. Acute alcohol intake causes sedation by acilitating the inhibitory activity o GABA at its receptors and inhibiting the excitatory activity o glutamate at its receptors. Over time, the GABA receptors are downregulated and their subunit structure is modi ed through a variety o molecular mechanisms, thus decreasing the level o inhibition to counter the sedative e ects o alcohol. Simultaneously, the NMDA receptors are up-regulated, also decreasing the level o inhibition due to alcohol. I the alcohol is abruptly removed, the decreased GABAergic inhibition and enhanced glutamatergic excitation result in a state o central nervous system hyperactivity, which causes the signs and symptoms o alcohol withdrawal. The balance between these inhibitory (GABAergic) and excitatory (glutamatergic) pathways may explain the alternating sedation and hyperactivity characteristic o alcohol intoxication and withdrawal, respectively. Because dependence can occur without tolerance and vice versa, it is clear that learning-related changes, not necessarily due to the pharmacologic actions o a drug, are also involved. In the 1950s, Olds and Milner implanted electrodes in various regions o the rat brain to systematically determine which neuroanatomic areas could rein orce sel -stimulation. (Sel -stimulation consisted o a short pulse o nondestructive electric current that was delivered in the brain at the site o the electrode upon the animal’s pressing o a lever.) The medial forebrain bundle and ventral tegmental area (VTA) in the midbrain were ound to be particularly e ective sites. These sites have been termed pleasure centers, or the oci o reward in the brain. A subset o dopaminergic neurons projects directly rom the VTA to the nucleus accumbens (NAc) via the

314 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY From corte x S e ns ory cue s (a ction pote ntia l)

A

S e ns ory cue s (a ction pote ntia l)

B

C

Gluta ma te NMDA-R Dopa mine re ce ptor Na tura l re wa rd

Neuroadaptation Na +

Ca 2+

Na +

Ca 2+

non-NMDA-R Ca 2+ Na +

Time x Dos e

Incre a s e in gluta ma te re ce ptors

Dependence

Drug of a bus e

Ca 2+ Na +

Incre a s e in s tructura l prote ins

From VTA

Dopa mine

Ca MKII

NAc

Ca MKII

NAc Ge ne tra ns cription

Reward learning

NAc Ge ne tra ns cription

Out-of-control drug use

Dependence

P e riod of a bs tine nce

D Relapse mechanisms Sensory cues

1

2

Ca 2+ Na +

3

Ca 2+ Na +

Stress

Ca 2+ Na +

Drug re-exposure

NAc

Ce llula r e xcita tion

Relapse

NAc

Ce llula r e xcita tion

Relapse

NAc

Ce llula r e xcita tion

Relapse

FIGURE 19-2. Synaptic changes linking environmental stimuli, drug effects, and reward learning in drug dependence and mechanisms of relapse after abstinence. A. Natural rewards such as ood or sex increase dopamine release in the nucleus accumbens (NAc) and give rise to reward learning that links relevant environmental stimuli (sensory cues) with concurrent rewarding elements by altering neural circuitry in associative areas o the brain. Spiny neurons within the NAc receive glutamatergic inputs rom the cortex that relay sensory cue in ormation and dopaminergic inputs rom the ventral tegmental area (VTA). The glutamatergic inputs act via NMDA receptors (permeable to calcium) and non-NMDA receptors (permeable to sodium). Coincident release o dopamine and glutamate results in potentiation o NMDA signaling, activation o calcium-calmodulin dependent kinase (CaMKII), and ultimately alterations in transcription o structural protein genes and glutamate receptor genes. These synaptic changes are thought to underlie reward learning. B. Drugs o abuse induce amplif ed dopamine release and activate the same synaptic adaptations as natural rein orcers. Thus, drugs o abuse are thought to “hijack” evolutionary brain reward learning systems in a manner that leads to out-o -control drug use. C. A ter chronic drug use, synaptic adaptations result in “potentiated synapses.” This potentiation is mediated via increased dendritic spine size, increased structural protein expression, and increased glutamate receptor sur ace expression; all o these adaptations occur in response to long-term transcriptional changes. D. A ter a period o abstinence rom drug use, multiple mechanisms can induce relapse to drug-taking behavior. 1. Stress can trigger relapse by increased dopamine release. In this potentiated state, dopamine can trigger cellular excitation and trigger relapse behaviors. 2. Exposure to drug-related sensory cues can trigger relapse via increased glutamate release, and the increased sur ace expression o glutamate receptors can lead to cellular excitation and relapse. 3. Exposure to small amounts o drug can reactivate relapse to drug sel -administration in this potentiated state, since the amplif ed dopamine release can trigger cellular excitation.

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

315

FIGURE 19-3. Integration of brain behavioral systems via conPrefrontal cortex Exe cutive function Cognitive control Hippocampus NAc

Conte xt / Me mory

Orbitofrontal cortex J udgme nt De cis ion ma king

Amygdala S tre s s / Anxie ty

Locus ceruleus Arous a l / Nove lty

nections to the mesolimbic dopamine pathway. Noradrenergic neurons originating in the locus ceruleus (black) relay information regarding novelty and arousal to dopaminergic neurons in the ventral tegmental area (VTA). The VTA projects to the nucleus accumbens (NAc) and cortex (red ). Multiple inputs from the brain modify VTA output: glutamatergic input from the prefrontal cortex relays executive function and cognitive control; excitatory input from the amygdala signals stress and anxiety; and glutamatergic input from the hippocampus conveys contextual information and past experiences (blue). Together, these multiple inputs modify signaling in the mesolimbic dopamine pathway and modulate the perception of pleasure.

Ventral tegmental area

medial orebrain bundle. It is believed that these neurons are crucial or the brain reward pathway, which rein orces motivated behavior and acilitates learning and memory via links to the hippocampus, amygdala, and pre rontal cortex. Severing this pathway, or blocking dopamine receptors in the NAc with a dopamine receptor antagonist (such as haloperidol; see Chapter 14, Pharmacology o Dopaminergic Neurotransmission), decreases electrical sel -stimulation o the VTA. Moreover, release o dopamine in the NAc can be detected in vivo using the technique o microdialysis, whereby a cannula is inserted into a specif c brain region in order to determine the concentrations o neurotransmitters. These measurements show that increases in concentrations o dopamine are associated with drug sel -administration by laboratory animals and that dopaminergic synapses in the NAc are active during electrical stimulation o the brain reward pathway, supporting the hypothesis that NAc dopamine is necessary or reward. Drugs capable o causing dependence are readily sel -administered by animals directly into the VTA, NAc, or the cortical or subcortical areas that innervate these two areas, o ten at the cost even o eating ood (Fig. 19-3). Although the dopaminergic pathway mediates reward, dopamine may also increase the salience o stimuli, alert the organism to the importance o stimuli, and guide motor activity to seek rewarding stimuli. As discussed above, the dopamine pathway is activated by all drugs of abuse. Importantly, behaviors that are necessary or survival o the species (e.g., eeding, reproduction, and exploration) also result in dopamine release in the NAc but to a much smaller degree, suggesting that drugs o abuse may pharmacologically “hijack” the normal evolutionary unctions o reward pathways. With repeated experiences via conditioning (i.e., the association o an element o the environment with the reward through rewiring o brain circuits), this dopamine pathway is also activated during anticipation o the reward, as can be demonstrated in humans using unctional neuroimaging techniques such as positron emission tomography (PET) and unctional magnetic resonance imaging ( MRI) when addicts are exposed to drug-related sensory cues. Although the dopaminergic neurons that link the VTA and the NAc serve as the f nal common pathway o reward, these neurons receive inputs rom

a number o brain regions (cortex, hippocampus, thalamus, amygdala, and raphe nuclei) that modi y reward and thereby mediate reward-associated learning (Figs. 19-3 and 19-4). Since withdrawal rom certain drugs o abuse can be aversive, avoiding acute withdrawal was or many years thought to be the primary motivation or continued abuse. However, this explanation is not consistent with the observations that the e ects o addiction are elt long a ter the physical symptoms o withdrawal have abated; withdrawal can occur without concomitant drug-seeking, as is o ten the case a ter treatment or acute pain; and drugs such as stimulants, hallucinogens, and cannabinoids cause signif cant dependence without a striking acute withdrawal syndrome. Years a ter an addict has discontinued use o a substance, he or she can experience intense cravings and, thus, is prone to relapse . The likelihood o relapse is especially strong in situations in which individuals simultaneously encounter both stress and the context in which the drug was previously used. In part, this is due to the interplay between reward and memory circuitry in the brain that, under normal circumstances, assigns emotional value to certain memories. Hence, the motivational underpinnings o drug-seeking are tied to both socioenvironmental stimuli and subjective e ects o the drug, each o which can have both rewarding and aversive linkages with previous experiences via learning. This is a more complex explanation than the “simple” avoidance o acute withdrawal.

MECHANISMS OF SUBSTANCE USE DISORDERS The drug-seeking activity characteristic o substance use disorders results rom the interplay o learning, reward mechanisms, and individual propensity toward the development o addiction.

Learning and Development of Substance Use Disorders Recognition that chronic drug sel -administration results in long-lasting changes in the experience o reward has led to

316 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

P re fronta l corte x

Nicotine

DA ACh

Ve ntra l te gme nta l a re a

Nicotinic ACh re ce ptor

NAc

Cannabinoids CB1 re ce ptor C

Opioids µ-opioid re ce ptor

GABA

Corte x

NMDA-R µ-opioid re ce ptor Opioids NAc Alcohol PCP

Cocaine

Cannabinoids DAT CB1 re ce ptor

DA

FIGURE 19-4. The mesolimbic dopamine pathway: a f nal common substrate or the rewarding actions o drugs. All drugs of abuse activate the mesolimbic dopamine pathway, which comprises ventral tegmental area (VTA) dopamine neurons that project to the nucleus accumbens (NAc). Different interneurons interact with VTA neurons and NAc neurons to modulate mesolimbic neurotransmission. Nicotine interacts with excitatory nicotinic cholinergic receptors located on VTA dopamine neuron cell bodies to enhance dopamine release in the NAc. Cocaine acts predominantly at the dopamine nerve terminal to inhibit reuptake of dopamine via the dopamine transporter (DAT), thus increasing synaptic levels of dopamine that can impinge on the NAc. Amphetamine also acts at the dopamine nerve terminal to facilitate release of dopamine-containing vesicles and possibly to enhance reverse transport of dopamine through DAT (not shown). Both cannabinoids and opioids decrease GABA release from local inhibitory interneurons in the VTA, resulting in disinhibition of dopamine neuron activity and increased dopaminergic neurotransmission. Cannabinoids and opioids can also act within the NAc. Alcohol, other CNS depressants, and phencyclidine [PCP] act on NMDA receptors (NMDA-R) to reduce glutamatergic neurotransmission in the NAc. The effects of alcohol on dopaminergic neurons in the VTA appear to be both excitatory and inhibitory and are the subject of active investigation (not shown).

our understanding that the relevant neural circuits can never return to their predrug state. The term allostasis describes this enduring, progressively evolving adaptive process in brain reward pathways upon repeated exposure to abused drugs. Allostasis means that the baseline to which the brain returns upon discontinuing drug use can change even a ter acute withdrawal has abated. (This is in contrast to homeostasis, which is def ned as the process whereby a system repeatedly re-equilibrates to the same baseline.) Accordingly, even when the drug is no longer present in the brain, the addict cannot experience positive emotions in the way he or she did prior to beginning drug use (termed anhedonia); the unsuccess ul attempt to recapture the previous “near-normal”

state uels drug-seeking. Human and animal studies have ound evidence or long-term neuroadaptation in altered neurotransmitter levels (e.g., dopamine and serotonin depletion a ter chronic alcohol or stimulant use), changes in neurotransmitter receptors, altered signal transduction pathways, changes in gene expression, and altered synaptic conf guration and unction. Clinically, abstinent patients report not only craving but also dysphoria, sleep disturbances, and increased stress reactivity (e.g., panic attacks), which can last or weeks, months, or years a ter detoxif cation. A common misconception is that addicts are pleasure seekers and that their ocus on drugs represents withdrawal rom li e into irresponsible hedonism. Current thinking about

C h a p t e r 1 9 Pharmacology o Drugs o Abuse

addiction recognizes the heterogeneity o the addictive process. For some individuals, reward actors (positive reinforcement) may predominate, and getting high or eeling euphoric motivates drug use. For others, relie actors (negative reinforcement) predominate, such as drinking to reduce stress or to reduce the dysphoria o protracted withdrawal. A large proportion o addicts sel -medicate to reduce distress associated with co-occurring psychiatric and medical disorders. Furthermore, the motivations to use early in the course o substance use disorder may di er substantially rom motivations as the illness progresses (Fig. 19-5). As a result o allostasis, positive rein orcement is rare in the later stages o the illness. For example, drinking in one’s teens to relieve shyness may progress to drinking or euphoria and disinhibition. Ultimately, a ter years o drinking to intoxication, the middle-aged person may drink to prevent withdrawal-associated depression and anxiety, or perhaps to alleviate chronic pain. Drug use in each o these situations is linked via learning to elements o the environment associated with drug use or to memories and emotions, each o which can trigger craving and drug-seeking. The essence o substance use disorder is drug-seeking behavior, whereby an individual cannot control the urge to obtain and use a psychoactive substance despite recognized negative consequences and at the exclusion o other needs that typi-

Socio-environmental stimuli P e e r group Drug pa ra phe rna lia

Discriminative stimuli S ubje ctive e ffe cts of drug Drug ta s te , s me ll, a ppe a ra nce

Initiation of drug use

Chronic drug dependence

Reinforcers

Reinforcers

Euphoria Be ha viora l a ctiva tion Nove lty Anxiolys is Ana lge s ia

Continued drug seeking

Aversive effects S e da tion Acute withdra wa l (ha ngove r) Na us e a Le ga l proble ms

S ocia l inte ra ction P re ve ntion of withdra wa l

Aversive effects Cessation of drug

Orga nic dis e a s e S ocie ta l s tigma Le ga l proble ms

FIGURE 19-5. Clinical determinants of drug-seeking change throughout the life course of addiction. The motivational underpinnings o drug-seeking are determined by socioenvironmental stimuli paired with subjective e ects o the drug. Rein orcers o drug sel -administration result in continued drug use, whereas aversive drug e ects contribute to cessation o drug sel -administration: whether an individual continues to use is a unction o whether rein orcing or aversive e ects predominate under the circumstances. Brain reward pathways are modif ed during the course o repeated drug sel -administration, such that rein orcing and aversive e ects are o ten di erent when drug use f rst begins compared to later in the course when drug sel -administration may have become repetitive and out-o -control. Ultimately, whether addiction progresses or the addictive disorder can be success ully arrested is determined by learning-related modif cation o rein orcing and aversive e ects o drugs using pharmacopsychosocial interventions.

317

cally constitute a balanced li e. Studies in laboratory animals suggest that drug-seeking behavior is the result o dys unctional “reward learning” (i.e., the processes that guide the organism to ul ll needs or goals have gone awry). Thus, i the organism initiates an action that results in a goal or “reward” (e.g., sel -administration o a psychoactive agent), and i the organism “learns” that its action resulted in the reward, the likelihood o engaging in that behavior is enhanced. For example, i a person uses cocaine or the rst time and nds it pleasurable or that it alleviates depressive symptoms rom which the individual is su ering, obtaining and using cocaine are rein orced. The intense experience o cocaine, relative to natural rewards such as ood and sex, results in a pre erential expenditure o energy to obtain cocaine over other rewards. Thus, cocaine has e ectively “hijacked” reward-learning systems, biasing uture behavior in avor o obtaining cocaine over natural rewards. Reexposure to environmental or a ective states that are associated with cocaine use serve as cues to increase drug-seeking behavior. For example, reexposure to drug paraphernalia can induce intense craving, drug-seeking behavior, and relapse in cocaine addicts.

Variables Affecting the Development of Substance Use Disorders The development o substance use disorder is dependent on the nature o the drug; genetic, acquired, psychological, and social traits o the drug user; and environmental actors. The ability o a drug to activate reward mechanisms is strongly correlated with its ability to cause addiction. Pharmacokinetic properties o the drug can signi cantly inf uence its e ects on the brain. In general, the more rapid the rise in drug concentrations at the target neurons, the greater the activation o reward pathways. For example, many drugs o abuse are highly lipophilic and can easily permeate the blood–brain barrier. In addition, direct injection or rapid absorption o drug through a large sur ace area (e.g., through the lungs via smoking) is more highly reinorcing than slower absorption through the intestinal or nasal mucosa. Furthermore, rapidly eliminated drugs are more addictive than slowly eliminated drugs, since slow clearance o a drug maintains the drug concentration at the site o action or a longer duration, diminishing the severity o acute withdrawal. The importance o pharmacokinetic e ects is demonstrated by the potential or abuse o various orms o cocaine (Fig. 19-6), and these principles are readily applicable to other drugs o abuse. The use o coca leaves as a chew or in teas is widely practiced among people living in the Andean mountains: this has a relatively low potential or addiction because o the slow rate o rise and low peak concentration o drug attained by absorption through the buccal or intestinal mucosa. The rapid absorption o extracted cocaine through the nasal mucosa is substantially more rein orcing. The most rein orcing and addictive orms o cocaine are intravenous injections and inhalation o smoked reebase (crack cocaine), both o which result in a very rapid rise in plasma concentration and a high peak concentration o drug. Di erent people react di erently to drugs. Some individuals use a drug once and never use it again; others use a drug repeatedly in moderate amounts without developing a drug use disorder; in others, the rst use o a drug produces such an intense e ect that the likelihood o addiction

318 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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Time (minute s a fte r dos e ) FIGURE 19-6. Plasma cocaine concentrations and levels of intoxication as a function of route of administration of the drug. The pharmacokinetics (A) and pharmacodynamics (B) of cocaine are highly dependent on the route of administration of the drug. Intravenous (IV) cocaine and smoked freebase cocaine are associated with very rapid attainment of peak plasma drug concentrations (A) and high levels of intoxication (B). In contrast, the nasal and oral routes of administration are associated with a slower rise in plasma drug concentrations (A) and lower levels of intoxication (B). Because of the very rapid rise in plasma drug concentration and very high intoxication levels, intravenous and smoked cocaine carry a higher risk of addiction than cocaine taken nasally or orally.

is high. The actors that make individuals more or less vulnerable to addiction upon exposure to a given drug are o continued research interest. A variety o predisposing or protective genetic, acquired, psychosocial, and environmental actors have been identi ed, but—as expected in a complex, multi actorial illness—individually each can explain only a relatively small component o the risk or addiction. Individual actors include (1) resistance or sensitivity to the acute e ects o a given drug, (2) di erences in drug metabolism, (3) the potential or neuroadaptive changes with chronic drug exposure, (4) personality traits and co-occurring psychiatric and medical disorders that incline an individual to drug use, and (5) susceptibility o the individual to brain injury associated with drug use that may modi y drug e ects. Genetic inf uences have been best studied in individuals with alcohol use disorder. Heritability estimates suggest that genetic actors account or 50–60% o the variance associated with alcohol use disorder, but the speci c determinant(s) that lead to alcoholism in an individual are not known. In act, many individuals whose amily history highly predisposes

them to alcohol use disorder do not develop the disorder. Alcohol use disorder may present as complex phenotypes determined by multiple genes, environmental exposures throughout the li espan, gene–environment interactions, gene–behavior interactions, and gene–gene interactions. The best known examples o candidate genes that alter risk or alcohol use disorder are the alcohol metabolism genes, including those encoding the alcohol dehydrogenases ADH1B*2, ADH2, and ADH3 that metabolize alcohol more rapidly and those encoding certain aldehyde dehydrogenases (particularly ALDH2*2). Polymorphisms in these genes alter enzymatic activity and increase the levels o acetaldehyde, which causes aversive symptoms that may act as a deterrent to drinking alcohol and to the development o alcohol use disorder. Sensitivity to alcohol is also a physiologically based trait inf uenced by genetic inheritance. Low sensitivity to alcohol (high innate tolerance ) is associated with an increased risk or developing alcoholism. Schuckit and colleagues have ound evidence or genetic linkage o the “low level o response”

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

phenotype to the same region on chromosome 1 that is linked to the “alcohol use disorder” phenotype. However, subjective response to alcohol is a complex trait a ected by several neurotransmitter systems. For example, individuals with the alcohol dependence-associated GABRA2 allele have a blunted subjective response to alcohol, and individuals carrying the Asp40 variant o the -opioid receptor or those with a certain single nucleotide polymorphism o the cannabinoid receptor appear to have an enhanced euphoric response to alcohol.

Role of Personality Characteristics and Co-Occurring Disorders in Substance Use Disorders The clinical characterization o individuals who develop a substance use disorder has been most extensively studied or alcoholism. The Cloninger classi cation o alcohol use disorder subtypes relates genetic and neurobiological di erences to the age o alcoholism onset and to personality traits. Type 1 (“late” onset) alcohol use disorder is characterized by alcohol-related problems beginning a ter 25 years o age, less antisocial behavior, in requent spontaneous drinking or loss o control, and guilt and concern about one’s alcoholism. Type 1 alcoholics are low in thrill-seeking, are harmavoidant, and are dependent on approval rom others. In contrast, type 2 alcohol use disorder is characterized by early onset o alcohol-related problems (be ore age 25), antisocial behavior, requent spontaneous alcohol-seeking and loss o control, and little concern about the consequences o one’s drinking or its e ects on others. Genetic predispositions to late-onset alcohol use disorder are signi cantly inf uenced by precipitating environmental actors, whereas genetic predispositions to early-onset alcohol use disorder are less inf uenced by the environment. The Lesch classi cation envisions our alcoholism subtypes: type 1 exhibits withdrawal symptoms, including alcohol-related delirium and seizures, relatively early in the drinking history; type 2 exhibits anxiety related to premorbid conf icts; type 3 is characterized by associated mood disorders; and type 4 has premorbid cerebral injuries and associated social problems. Alcohol use disorder subtypes are now being examined as predictors o response to medications used or the treatment o alcoholism. For example, early-onset alcoholics may worsen their drinking and impulsive behavior in response to a selective serotonin reuptake inhibitor (SSRI), whereas late-onset alcoholics may improve with an SSRI. According to a major epidemiologic survey in the United States, the odds o having a mental disorder are three times greater i an individual also has a drug use disorder than i the individual has no drug use disorder. In decreasing order o association, these psychiatric diagnoses include bipolar disorder, antisocial personality disorder, schizophrenia, major depressive disorder, and anxiety disorders. Drug use disorders occur at higher rates in those with alcohol use disorder, and alcoholism is more prevalent among individuals with addiction to other drugs. The association between psychiatric disorders and drug use disorders has led to theories o common pathogenesis and treatment strategies. For example, individuals with major depressive disorder are two to three times more likely to have a drug use disorder throughout their li etime than those without depression, and exacerbations o mood symptoms are prime precipitants o relapse to drug use (and vice versa). O note, these associations seem to be

319

generic with respect to which drugs are abused, suggesting that such abuse is related more to availability than to a speci c pharmacologic mechanism o action. Physical disability and pain associated with medical illness or traumatic injury can greatly enhance the risk o a co-occurring drug use disorder. Moreover, drug use not only complicates certain medical conditions, but or many o these illnesses (e.g., cirrhosis or traumatic brain injury due to motor vehicle accidents), alcohol and drug use should also be considered a signi cant causal actor. Similarly, increased pain perception is now understood to be a requent complication o chronic opioid administration (opioid hyperalgesia ). Thus, many pain physicians no longer advise long-term use o opioid analgesics or treatment o chronic (nonterminal) pain, recognizing that detoxi ying a patient rom chronic opioid use can o ten result in a pre erable outcome to continuing to increase the opioid dose. In conclusion, substance use disorders are not only illnesses in their own right but also common consequences o many psychiatric and medical conditions that, in turn, are urther exacerbated by continued substance use.

DRUGS OF ABUSE Many psychoactive substances have abuse potential through their activation o inputs in brain reward pathways. It is vital to understand the unique pharmacology o each agent to appropriately address overdose complications, metabolic consequences, and organ toxicity associated with a speci c substance use disorder. Several drugs with the potential to cause addiction are readily available and widely used, and they exact an enormous toll on public health (e.g., alcohol, nicotine). Other drugs are commonly prescribed or accepted medical purposes, and their mechanisms o action have been discussed in detail in previous chapters (e.g., opioids, barbiturates, benzodiazepines, stimulants). These drugs represent a signi cant cause o iatrogenic dependence in patients, and prescription drug abuse represents perhaps the astest growing US drug problem. In recent years, deaths associated with prescription drug use have surpassed deaths due to motor vehicle accidents in many parts o the country. Other commonly abused drugs are not generally prescribed in medical practice and are typically available only rom illicit sources (e.g., cocaine, heroin). Finally, some drugs a ect receptors that are actively being pursued as potential targets or therapeutic intervention, and it is controversial whether or how they should be regulated (e.g., cannabis, nicotine).

Opioids Opioid alkaloids have been used medically or centuries or analgesia, treatment o diarrhea and cough, and sleep induction. Central e ects o opioids are biphasic, with behavioral activation at low doses and sedation at higher doses. These drugs depress respiration, and death rom opioid overdose is invariably due to respiratory arrest. The -opioid receptor appears to be the most important subtype or the rein orcing actions o opioids. Addicts describe an intense euphoric eeling (“rush”) that lasts or less than a minute upon the intravenous injection o heroin and that seems to be the reason or abuse. There appear to be two pathways by which opioids interact with the brain reward system. One site o action lies in the ventral tegmental area, where GABAergic interneurons tonically inhibit the dopaminergic neurons responsible or activating the brain reward pathway in the nucleus accumbens.

320 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

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FIGURE 19-7. Role of opioids in the brain reward pathway. A. GABAergic neurons tonically inhibit the dopaminergic neurons that originate in the ventral tegmental area and are responsible for reward. These GABAergic neurons can be inhibited by endogenous enkephalins, which locally modulate the release of neurotransmitter at the GABAergic nerve terminal. B. Administration of exogenous opioids results in decreased GABA release and disinhibition of the dopaminergic reward neurons. The increased release of dopamine in the nucleus accumbens signals a strong reward.

These GABAergic interneurons can be inhibited by endogenous enkephalins, which bind to -opioid receptors on the GABAergic terminals. Because exogenous opioids such as morphine also bind to and activate -opioid receptors (see Chapter 18, Pharmacology o Analgesia), exogenously administered opioids can activate the brain reward pathway by disinhibiting dopaminergic neurons in the ventral tegmental area (Fig. 19-4, Fig. 19-7). The second pathway is localized in the nucleus accumbens. Opioids acting in this region may inhibit GABAergic neurons that project back to the ventral tegmental area, perhaps as part o an inhibitory eedback loop. The relative importance o these two pathways is still being debated. As the case o Mr. A illustrates, opioid use disorder can lead to signif cant alterations in these reward pathways that mani est as opioid craving and a high probability o relapse long a ter the physical symptoms o withdrawal have abated. The partial agonist buprenorphine binds to and modulates activation o -opioid receptor-mediated reward circuits and can greatly diminish craving or opioids, as the case o Mr. A demonstrates (Fig. 19-8). The multiple inputs into brain reward circuits underline the potential or co-occurrence o addiction to opioids and

to other pharmacologically disparate drugs o abuse (crossdependence ). For example, sel -administration o opioids together with other psychoactive drugs, such as the cocaine/ heroin combination “speedball,” is used to enhance reward (Fig. 19-4); this combination also augments the risk o abuse and death due to overdose. In addition, open-ended opioid prescriptions a ter surgery may precipitate relapse to another drug o abuse to which the person was previously addicted and rom which abstinence has been achieved, even i the individual has never previously had an opioid use disorder. However, the potential or addiction should not deter physicians rom prescribing a medication or legitimate medical purposes. Un ortunately, opioids are o ten under-prescribed or the treatment o pain because tolerance—mani ested as a request or increasingly higher doses o drug—is mistaken or an opioid use disorder. Tolerance is an expected e ect o the drug, and physicians should be prepared to increase the dose, i necessary, to control the patient’s pain. Because o the high potential or withdrawal symptoms upon discontinuation o an opioid, physicians should also be care ul to taper the opioid dose and to explain to the patient the rationale or the taper. Finally, drug-addicted patients who must undergo surgery or require analgesia or other reasons should be treated with su f cient medication to attain analgesia, and they may need considerably higher doses due to preexisting tolerance to opioids. This can be a common problem when patients are chronically taking buprenorphine. Buprenorphine may partially block the e ects o opioid analgesics because it is a partial agonist at opioid receptors, and a patient may require much higher opioid doses than usual to attain adequate analgesia. Nevertheless, whenever opioids are used, there needs to be a clear understanding o exactly how the decision to discontinue the medication will be made, and treatment should be determined by the physiologic basis o the expected pain rather than allowed to continue indef nitely. Although all opioids have the potential to cause tolerance and dependence, certain opioids are more rein orcing and more likely to cause drug-seeking. Opioids associated with the astest rise in brain concentration o drug, including those injected intravenously, have the highest likelihood or abuse. Abuse o the drug oxycodone (sold as slow-release OxyContin®), which is commonly prescribed or moderate or severe pain, has received much publicity because o misuse and cases o iatrogenic addiction when patients take the medication “as prescribed.” Experienced addicts have learned that the oral tablets o oxycodone can be broken up, dissolved, and injected. This orm o administration results in a much more rapid rise in plasma (and hence brain) concentration o drug, a more intense eeling o euphoria, and a greater abuse liability compared to the prescribed, slow-release oral orm o the drug. Analogously, although heroin and morphine are close structural analogues (heroin is deacetylated to 6-monoacetylmorphine, and morphine is acetylated to the same compound), heroin is signif cantly more hydrophobic than morphine. Because o this property, intravenously administered heroin crosses the blood–brain barrier more rapidly than morphine. The more rapid increase in brain concentration o heroin produces a sharper “high,” which explains why heroin is typically pre erred over morphine as a drug o abuse. The rapid rise in brain concentration o heroin, together with the uncertain dose and potentially toxic impurities in heroin preparations available “on the street,” account or the substantial mortality by respiratory arrest due to heroin overdose.

C h a p t e r 1 9 Pharmacology o Drugs o Abuse

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FIGURE 19-8. Partial agonists in the treatment of addiction. A. Full agonists at -opioid receptors, such as morphine, produce maximal signal transduction (100%). Partial agonists, such as buprenorphine, produce reduced signal transduction ( 50% o a ull agonist). Antagonists, such as naloxone, do not stimulate signal transduction. B. Both buprenorphine and naloxone have very high binding a f nities or -opioid receptors compared to morphine. Consequently, when -opioid receptors are ully occupied by an agonist like morphine, both naloxone and buprenorphine displace morphine rom the receptor and lead to withdrawal. C. Upon morphine binding to -opioid receptors, intracellular signaling leads to inhibition o adenylyl cyclase activity and a decrease in cyclic AMP (cAMP) production. Upon removal o morphine rom -opioid receptors, either by discontinuing morphine or by administration o an antagonist or partial agonist (withdrawal), the inhibition o adenylyl cyclase is released. The resulting large increase in cAMP production causes withdrawal symptoms, such as diarrhea, hyperalgesia, tachypnea, and photophobia. The use o a partial agonist, buprenorphine, can alleviate these withdrawal symptoms by “partial” activation o -opioid receptors. In addition, binding o the high-a f nity buprenorphine molecule to -opioid receptors prevents lower a f nity ull agonists, such as morphine, rom binding to and activating the receptor. Thus, the antagonist property o buprenorphine prevents the “high” associated with morphine use but also alleviates craving and drug-seeking behavior. D. Nicotine activates nicotinic acetylcholine receptors (nAChR), causing neuronal excitation. Nicotine withdrawal causes a rapid decrease in nAChR activity and a withdrawal syndrome associated with intense craving. Treatment with the partial nAChR agonist varenicline results in partial activation o nAChR and alleviation o withdrawal symptoms, but this activation is insu f cient to cause dependence or a “high.” Importantly, binding o the high-a f nity varenicline molecule to nAChRs prevents the lower a f nity nicotine molecule rom binding to and activating the receptor. Thus, varenicline can prevent the subjective “high” associated with nicotine use.

322 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

FIGURE 19-9. Pharmacokinetic determinants of CNS depressant-induced A

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withdrawal severity. A. Due to rapid elimination o alcohol and alprazolam, plasma levels all rapidly a ter cessation o drug use. Plasma levels o diazepam, which has a long elimination hal -li e, decline at a slower rate. Furthermore, the e ective biological hal -li e o diazepam is longer still because the active metabolites desmethyldiazepam (which has an even longer elimination hal -li e) and oxazepam are ormed via the metabolism o diazepam. B. The onset, severity, and duration o the CNS-depressant withdrawal syndrome are directly related to the rate o elimination o the drug and thus the rate o removal o the drug rom its target receptor. Alprazolam and alcohol withdrawal are more rapid in onset, o greater severity, and o relatively limited duration compared to diazepam withdrawal. C. Treatment o CNS-depressant withdrawal is aimed at maintaining occupancy o the target receptor or a su f ciently long period o time that the system can re-equilibrate and thereby minimize the risk o severe withdrawal symptoms. This is accomplished by using a cross-tolerant drug (i.e., another CNS depressant) with a relatively slower rate o removal rom the target receptor than the abused drug. Administration o diazepam to treat alcohol withdrawal illustrates this point. Although plasma levels o alcohol drop rapidly, administration o diazepam results in continued occupancy and activation o receptor sites (such as the GABAA receptor) or a much longer period o time and throughout the period o highest risk or withdrawal-related seizures. D. The more gradual reduction in receptor occupancy a ter diazepam administration reduces the severity o alcohol withdrawal symptoms, prevents seizures, and reduces the morbidity and mortality rom alcohol withdrawal. E. In addition to its slower elimination compared to alcohol, diazepam has higher e f cacy at GABAA receptors than alcohol, resulting in enhanced GABAA receptor activation. This property holds even when the receptor is in a desensitized state due to chronic alcohol consumption. Thus, the combination o the slower elimination and the higher e f cacy o diazepam compared to alcohol makes it the medication o choice or the treatment o alcohol withdrawal.

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Benzodiazepines and barbiturates are two o the major classes o sedative and hypnotic agents. Benzodiazepines are widely prescribed or management o patients with anxiety and insomnia. Barbiturates have a narrower therapeutic window than benzodiazepines and are used less requently. For both o these drug classes, euphoric eelings are o ten reported in the early stage o intoxication and typically are the expressed reason or drug sel -administration. Anxiolytic and tension-reducing properties may also contribute to the rein orcing actions and abuse potential o these drugs. All sedative-hypnotics can cause substance use disorder, but the risk o abuse can be limited i they are used judiciously in a time-limited ashion. Benzodiazepines and barbiturates increase the e f ciency o GABAergic pathways, and chronic use can induce down-regulation o these pathways by neuroadaptation. One possible mechanism o down-regulation is uncoupling o the benzodiazepine site rom the GABA site on GABAA receptors (see Chapter 13, Pharmacology o GABAergic and Glutamatergic Neurotransmission). Thus, the binding o benzodiazepines to GABAA receptors would remain unchanged, but the drug would have little or no potentiating e ect on the binding o GABA to the receptor. Down-regulation o inhibitory GABAergic pathways would be expected to leave the brain “underinhibited,” increasing the possibility o seizures and delirium upon abrupt withdrawal o the benzodiazepine or barbiturate (see Chapter 16, Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System). Associated central sympathetic hyperactivity can lead to physical symptoms such as anxiety, sleep disturbance, and dizziness and to emotional concomitants such as ear and panic. Because the central nervous system depressant actions o barbiturates are more widespread than those o the GABAA-specif c benzodiazepines (Fig. 19-9), barbiturate dependence is associated

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C h a p t e r 1 9 Pharmacology of Drugs of Abuse

with a more severe and potentially dangerous withdrawal syndrome than benzodiazepine dependence. Within a given class o sedative-hypnotics, the onset, amplitude, and duration o the withdrawal syndrome are determined by the rate o elimination o the drug and its active metabolites. For example, among the barbiturates and benzodiazepines, withdrawal usually begins within 12 hours a ter drug discontinuation and is most severe or rapidly eliminated compounds (e.g., amobarbital and alprazolam); withdrawal may be delayed or several days and is less severe or slowly eliminated compounds (e.g., phenobarbital, diazepam, and clonazepam) (Fig. 19-9). Co-occurring benzodiazepine and/or barbiturate use disorder and alcohol use disorder are particularly prevalent due to the similarity o these drugs’ e ects on GABAergic neurotransmission (Fig. 19-9). Benzodiazepines (not barbiturates) are the accepted treatment or alcohol withdrawal; these drugs are e f cacious in alleviating “rough spots” when alcoholics cannot drink, and the e ects o alcohol are greatly accentuated by benzodiazepines (or barbiturates). Benzodiazepines are almost never associated with mortality due to overdose when used alone; combined with alcohol, however, they can be atal because o synergistic depression o cardiorespiratory centers. Benzodiazepines and opioids can sometimes be coprescribed under conditions when pain is associated with signif cant anxiety. This combination can also be atal due to synergistic e ects on respiration; in act, even the relatively sa e partial agonist buprenorphine can cause respiratory arrest when combined with benzodiazepines. Physicians may try to limit use o these dangerous combinations, but some drug-seeking patients may resort to obtaining prescriptions rom multiple physicians or even orging prescriptions, especially in cases where the underlying condition has been suboptimally managed. Nonetheless, under-medication o pain must be avoided, and benzodiazepines should only be used over the short term or treatment o alcohol withdrawal or signif cant anxiety. Another serious concern is the misuse o prescription opioids (or, less commonly, benzodiazepines or barbiturates) by health pro essionals. For at least two reasons, health pro essionals who misuse prescription medication are at greater risk or developing substance use disorder. First, they have more ready access to prescription medication. Second, they may mistakenly believe that, because they understand a drug’s e ects, they will be able to control its use more easily.

Alcohol Alcoholic beverages are readily available at a ordable cost with minimal legal restriction. Alcohol use disorder stands as the most prevalent drug problem in the United States. Early in intoxication, CNS stimulation and euphoria result rom depression o inhibitory control, and aspects o discrimination, memory, and insight are impaired. As blood levels rise, judgment, emotional control, and motor coordination su er. Traumatic injuries sustained while intoxicated are likely the most common public health problem associated with alcohol abuse. Respiratory depression and death can result rom overdose, and the most serious consequences occur when alcohol is combined with other psychoactive agents. Ethanol a ects GABAA receptors, NMDA glutamate receptors, and cannabinoid receptors. Although the specif c sites o action are unknown, GABAA channels are believed to mediate the anxiolytic and sedative e ects o alcohol, as well as the e ects o alcohol on motor coordination, tolerance, dependence, and sel -administration. Alcohol increases

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GABA-mediated chloride conductance and enhances hyperpolarization o the neuron. Its mechanisms o dependence are likely similar to those o other sedative-hypnotic drugs a ecting GABA neurotransmission. In severity and time course, the symptoms o alcohol withdrawal lie between those o short-acting barbiturates and intermediate-acting benzodiazepines. Evidence also points to a role or NMDA receptors in the development o tolerance and dependence to alcohol, and NMDA receptors also have a role in the alcohol withdrawal syndrome. Specif cally, alcohol inhibits subtypes o NMDA receptors that seem to be capable o long-term potentiation. The rewarding e ects o alcohol may also be mediated in part by indirect activation o cannabinoid receptors . Endogenous cannabinoids are “retrograde” neuromodulators that act as a eedback mechanism to enhance dopaminergic activity in the mesolimbic reward pathway (Fig. 19-10; see also Fig. 19-4). Endocannabinoid signaling has been implicated in reward learning, appetite regulation, mood regulation, pain modulation, and cognition. Thus, although GABAA receptors have a vital role in mediating the e ects o alcohol, the ability o alcohol to interact with a number o di erent receptor types suggests that our understanding o its mechanisms o action remains incomplete.

Nicotine and Tobacco Smoking, or the combustion o tobacco or the purpose o nicotine sel -administration, represents a major source o preventable medical morbidity and mortality. Nicotine activates nicotinic acetylcholine receptors that are located centrally, peripherally, and at the neuromuscular junction. Cholinergic neurons arising rom the laterodorsal tegmental area (near the border o the midbrain and pons) activate nicotinic and muscarinic acetylcholine receptors on dopaminergic neurons in the ventral tegmental area; stimulation o these nicotinic receptors by nicotine activates the dopaminergic brain reward pathway (Fig. 19-4). In addition, activation o presynaptic nicotinic receptors on dopaminergic axon terminals acilitates the release o dopamine. These strong and direct e ects on the mesolimbic reward pathway, combined with the inhalational route o administration and short hal -li e o nicotine, explain the high addiction potential o nicotine and hence o cigarettes and other orms o tobacco. Activation o central nicotinic receptors also produces anxiolytic e ects, increases arousal, and suppresses appetite, while activation o peripheral nicotinic receptors increases blood pressure and stimulates smooth muscle contraction. A strong and spontaneous withdrawal syndrome is associated with the decreases in plasma levels o nicotine that occur upon cessation o smoking. The major symptoms include irritability, anxiety, autonomic arousal, and intense craving and associated drug-seeking behavior. These symptoms are readily relieved by smoking, and given the widespread availability o tobacco products, it is easy to see why smoking is so recalcitrant to treatment. Because smoking can alleviate a number o symptoms associated with depression and anxiety, it is commonly associated with the use o other drugs and with mental disorders.

Cocaine and Amphetamine Cocaine is isolated rom the South American shrub Erythrox-

ylon coca and has been used as a local anesthetic since 1884.

324 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

A

B

GABA te rmina l

GABA te rmina l Rimona ba nt

2-AG

CB1 re ce ptor

GABA Action pote ntia l

DAGL

Cl-

GABAA-R

Enhanced excitation of VTA dopaminergic neuron

Action pote ntia l

DAGL

Cl-

Increased inhibition of VTA dopaminergic neuron

FIGURE 19-10. Endogenous cannabinoid neurotransmission in the mesolimbic dopamine pathway. A. Endogenous cannabinoids are a class of lipid neurotransmitters that act as “retrograde signals” to inhibit release of other neurotransmitters. Here, activation of dopaminergic neurons in the ventral tegmental area (VTA) results in rapid synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via the activity of diacylglycerol lipase (DAGL). 2-AG then activates CB1 cannabinoid receptors located on presynaptic GABAergic terminals. Activation of CB1 receptors causes a transient decrease in vesicular release of GABA on a time scale of seconds to minutes. This results in “feed-forward” enhancement of VTA dopaminergic neuron activity and could contribute to drug-seeking behavior. Thus, endocannabinoids can modulate VTA dopaminergic neuronal activity by inhibiting GABAergic (inhibitory) inputs to the VTA. Activation of VTA dopaminergic neurons in response to environmental cues associated with drug use can often trigger relapse (see Fig. 19-2). B. The CB1 receptor antagonist rimonabant has been shown to inhibit cue-induced relapse in preclinical studies. A putative mechanism of action of rimonabant involves blockade of CB1 receptors on presynaptic GABAergic terminals in the VTA, which would sustain high levels of GABA and thus inhibit VTA dopaminergic neuron activity in response to drug-associated cues, and possibly reduce relapse.

Amphetamine and congeners are used clinically as nasal

decongestants, analeptics, antidepressants, and diet pills and or treatment o attention-def cit hyperactivity disorder (ADHD). Cocaine and many amphetamine-related drugs have substantial abuse liability; hence, other medications with lower risk prof les have taken their place or many o their uses. Nevertheless, these drugs are widely available by prescription and through illicit sources. They are highly reinorcing because o the pro ound sense o well-being, energy, and optimism associated with stimulant intoxication; however, this state can rapidly progress to psychomotor agitation, severe paranoia, and even psychosis due to augmented dopamine neurotransmission. The initial euphoric e ects o cocaine appear to be more pronounced than those o amphetamine, while amphetamine intoxication ar outlasts that o cocaine. Elevated mood is o ten ollowed by listlessness, drowsiness, and depressed mood upon the withdrawal o stimulants. Appetite suppression can be ollowed by ravenous hunger. Stimulants are almost always taken with another drug o abuse, most commonly alcohol, since the other drug accentuates the “high” and alleviates the sleeplessness and sense o being “wired” (Fig. 19-4). By blocking or reversing the direction o the neurotransmitter transporters that mediate reuptake o the monoamines dopamine, norepinephrine, and serotonin into presynaptic terminals, cocaine and amphetamine potentiate dopaminergic, adrenergic, and serotonergic neurotransmission. Cocaine is most potent at blocking the dopamine transporter

(DAT), although higher concentrations block the serotonin

and norepinephrine transporters as well (SERT and NET, respectively). Recall that the tricyclic antidepressants (TCAs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and selective serotonin reuptake inhibitors (SSRIs) unction in a similar manner, blocking reuptake o norepinephrine and serotonin (TCAs and SNRIs) or serotonin alone (SSRIs) into presynaptic neurons. Amphetamine reverses the direction o all three monoamine transporters, although this drug is more e ective at the norepinephrine transporter. Amphetamine also releases vesicular transmitter stores into the cytoplasm; these combined actions cause the catecholamine neurotransmitter to be transported into, rather than out o , the extracellular space. By these actions, cocaine and amphetamine increase the concentration o monoamine neurotransmitters in the extracellular space, potentiating neurotransmission (Fig. 19-1). Although cocaine and amphetamine act on monoaminergic neurons throughout the body, it is the action o these drugs on neurons in two major centers in the brain that likely governs their potential or abuse. The f rst set o neurons, in the locus ceruleus in the pons, sends ascending adrenergic projections throughout the hypothalamus, thalamus, cerebral cortex, and cerebellum and descending projections to the medulla and spinal cord. These projections maintain alertness and responsiveness to unexpected stimuli (see Chapter 11, Adrenergic Pharmacology). Thus, drugs such as cocaine and amphetamine, which potentiate the actions o norepinephrine by inhibiting neurotransmitter reuptake,

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

produce enhanced arousal and vigilance and are called psychostimulants . The second major site at which cocaine and amphetamine act is on midbrain dopaminergic neurons, the axons o which terminate in the nucleus accumbens, striatum, and cortex (Fig. 19-4). As discussed above, these dopaminergic terminals in the nucleus accumbens are a critical component o the brain’s reward pathway. It was long believed that the psychostimulants do not cause signi cant withdrawal and that behaviors to seek these drugs rarely attain levels that are out o control. However, cocaine use can be associated with withdrawal symptoms such as bradycardia, sleepiness, and atigue. Withdrawal rom cocaine or amphetamine also produces psychological symptoms, such as dysphoria and anhedonia (an inability to experience pleasure), that are opposite to the euphoria experienced immediately a ter administration o the drug. Many o these symptoms are not strictly attributable to withdrawal because they cannot be alleviated by the administration o more cocaine or amphetamine. In act, symptoms o withdrawal can appear even when psychostimulant levels in the plasma are high. This phenomenon occurs both because o allostasis o reward pathways (discussed earlier) and because these drugs cause tachyphylaxis , an acute process in which the target tissue becomes less and less responsive to constant concentrations o a drug. In the case o cocaine and amphetamine, tachyphylaxis may be caused by depletion o the neurotransmitter. Because the drugs block presynaptic neurotransmitter reuptake, the elevated levels o neurotransmitter in the extracellular space eed back to inhibit its synthesis, and neurotransmitter stores in the presynaptic terminal are progressively depleted. The combination o tachyphylaxis and allostasis makes discontinuation o stimulants particularly di cult or addicts, both in the short and long term.

Marijuana Cannabinoids are compounds derived rom Cannabis sativa

(marijuana). The primary psychoactive component o marijuana is 9-tetrahydrocannabinol (THC), which is a partial agonist at the G protein-coupled type-1 cannabinoid receptor (CB1). The endogenous ligand o the CB1 receptor is the arachidonic acid derivative anandamide , which is representative o a class o endocannabinoid “retrograde” neuromodulators that act as a eedback mechanism to reduce neuronal excitation (Fig. 19-10). Since blockade o CB1 receptors by the antagonist rimonabant eliminates the e ects o smoked marijuana in humans, the subjective e ects o marijuana are thought to be mediated by the CB1 receptor. The CB1 receptor is widely distributed within the pre rontal cortex, hippocampus, amygdala, basal ganglia, and cerebellum. In rats, the administration o natural and synthetic cannabinoids causes dopamine release in the nucleus accumbens o the brain reward pathway. Endogenous cannabinoids appear to modulate a variety o appetitive (rein orcing and consumptive) behaviors including eating, smoking, and alcohol drinking. Cannabinoid use causes a prompt and generalized “high” characterized by euphoria, laughter, giddiness, and depersonalization. A ter 1–2 hours, cognitive unctions such as memory, reaction time, coordination, and alertness are compromised, and the user has di culty concentrating. This e ect corresponds to a “mellowing” phase, which results in relaxation and even sleep. High doses o marijuana can cause anxiety, overt panic reactions, perceptual distortions, impairments in

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reality testing, and, rarely, overt psychosis in susceptible individuals. Synthetic cannabinoids (colloquially, K2 or Spice) that have recently become available “on the street” seem particularly prone to present with f orid mani estations, which are potentially misdiagnosed as primary psychotic disorders. Overt panic reactions are the most common reason cited or stopping marijuana use. Tolerance to marijuana occurs via down-regulation o CB1 receptor expression and post-translational modi cations that reduce signal transduction e ciency. Withdrawal rom marijuana is generally mild due to its high volume o distribution and long elimination hal -li e. Withdrawal symptoms can include insomnia, loss o appetite, irritability, and anxiety, perhaps due to activation o central corticotropin-releasing actor (CRF) systems, particularly in the amygdala. Blockade o CB1 receptors by rimonabant can precipitate a withdrawal syndrome in chronic users.

Other Abused Drugs Phencyclidine (PCP) was developed as a dissociative anes-

thetic but is no longer used because o behavioral toxicity. PCP blocks NMDA glutamate receptors, which mediate excitatory synaptic transmission and are involved in synaptic plasticity and memory. By inter ering with these processes, PCP produces complex e ects such as anesthesia, delirium, hallucinations, intense paranoia, and amnesia. Methylenedioxymethamphetamine (MDMA), known colloquially as Ecstasy, is one in the class o phenylethylamine hallucinogenics that un ortunately has been alsely advertised by some as a “sa e” drug. Although it is chemically related to methamphetamine and has similar dopaminergic e ects, the primary e ect o MDMA is on serotonergic neurotransmission. MDMA causes serotonin release into the extracellular space, inhibition o serotonin synthesis, and block o serotonin reuptake. Together, these complex actions o MDMA increase serotonin in the extracellular space while depleting presynaptic stores o the neurotransmitter. The drug causes a central stimulant e ect like cocaine and amphetamine but, unlike those drugs, it also has hallucinogenic properties. Like cocaine and amphetamine, MDMA a ects the brain reward pathway through dopaminergic stimulation. MDMA may be neurotoxic to a subpopulation o serotonergic neurons when the drug is administered repeatedly or in large amounts. Caffeine and the related methylxanthines theophylline and theobromine are ubiquitous drugs ound in co ee, tea, cola, “energy” drinks, chocolate, and many prescribed and overthe-counter medications. Methylxanthines act by blocking adenosine receptors that are expressed presynaptically on many neurons, including dopaminergic and adrenergic neurons. Because activation o adenosine receptors inhibits dopamine and norepinephrine release, competitive antagonism o the receptors by ca eine increases dopamine and norepinephrine release and, thus, acts as a stimulant. Ca eine may also block adenosine receptors on cortical neurons and thereby disinhibit these neurons. Because CNS adenosine is a natural promoter o sleep and drowsiness, ca eine’s blocking o adenosine receptors has alerting e ects and improves per ormance in a variety o circumstances but can also produce insomnia. Symptoms o withdrawal rom ca eine can include lethargy, irritability, and a characteristic headache, but addiction, although documented, is rare. Ca eine withdrawal symptoms are commonly observed in even low to moderate users o ca eine, but these typically resolve without treatment.

326 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

Inhalants are volatile organic compounds that are inhaled

(sometimes called hu f ng) or their psychoactive e ects. The typical user o inhalants is a male teenager. Inhalants include organic solvents such as gasoline, toluene, ethyl ether, f uorocarbons, and volatile nitrates, including nitrous oxide and butyl nitrate. Inhalants are readily available in many households and workplaces. At low doses, inhalants produce mood changes and ataxia; at high doses, they may produce dissociative states and hallucinations. Dangers o organic solvent use include su ocation and organ damage, especially hepatotoxicity and neurotoxicity in the central and peripheral nervous systems. Cardiac arrhythmias and sudden death can occur. Inhaled nitrates can produce hypotension and methemoglobinemia. Hydrocarbon inhalants do not appear to act at a speci c receptor but rather to disrupt cell unctions by binding nonspeci cally to hydrophobic sites on receptors, signal transduction proteins, and other macromolecules. Nitrates, however, act at speci c receptors or nitric oxide, a small-molecule neuromodulator (see Chapter 22, Pharmacology o Vascular Tone).

MEDICAL COMPLICATIONS OF SUBSTANCE USE DISORDERS Individuals with substance use disorders typically present to a physician complaining o the indirect e ects o drug/alcohol sel -administration. These can include amily disruptions and emotional trauma, legal problems and physical injury, sel neglect (e.g., malnutrition, harm rom adulterants mixed with drugs, in ection rom needle administration), inappropriate use o prescribed medication (e.g., analgesics, anxiolytics), and lack o adherence with medical regimens or coexisting illnesses. These e ects are clearly not speci c to the pharmacologic actions o any given drug but are the consequence o out-o -control, o ten sel -destructive behaviors that inter ere with a balanced li e because the reward and salience o drug use supersede that o other elements o the environment. Less commonly, patients seek medical care or acute and chronic direct pharmacologic and toxic actions o the substance(s) o abuse. Given the multiplicity o drugs, the means by which they are obtained, and the variety o routes o administration, complications may also be secondary to tissue toxicity and induced metabolic changes. Adequate treatment o the medical complications related to substance use disorders requires knowledge o a given substance’s pharmacologic actions. Many patients with substance use disorder use more than one drug or alcohol. Pharmacodynamic and pharmacokinetic e ects o having more than one substance use disorder are o ten di cult to predict rom the actions o each individual agent. For example, research has revealed a potentially dangerous interaction between cocaine and alcohol. When taken together, the two drugs are converted to cocaethylene . Cocaethylene has a longer duration o action in the brain and is more toxic than either drug alone. The vast majority o individuals with substance use disorders also smoke cigarettes and, despite attaining abstinence rom their “drug o choice,” the eventual cause o death is o ten related to complications o cigarettes (e.g., cancer, cardiovascular disease). Alcohol use disorder is associated with widespread toxicity. Alcoholic cardiomyopathy can result in a li e-threatening decrease in le t ventricular unction. Ethanol is directly toxic to heart muscle cells, a ecting contractility o the myocytes and inhibiting the repair o injury to these cells. The mechanism o myocyte damage may relate to the overproduction o

oxygen-containing molecules (reactive oxygen species) secondary to alcohol metabolism, with damage to the plasma membrane o the myocyte. Nutritional de ciencies o watersoluble vitamins such as thiamine may also be involved. With moderate drinking, there is typically an increase in systolic blood pressure. Alcohol withdrawal also plays a role in hypertension because sympathetic activity is increased during withdrawal. Stress appears to cause a greater rise in blood pressure in drinkers than in nondrinkers. There appears to be a protective e ect o drinking on coronary artery disease, at least in older individuals and those otherwise at risk or coronary disease. The so-called J-shaped mortality curve shows that these populations have decreased mortality with low to moderate drinking (generally 0.5–2 drinks/day) and increased mortality with heavy drinking. The mechanism o this protection involves bene cial e ects o ethanol on lipoprotein metabolism and thrombosis: ethanol increases high-density lipoprotein (HDL) levels in a dose-dependent manner in low to moderate drinkers, and ethanol inhibits platelet aggregation and lowers plasma brinogen levels. Chronic alcoholism has other signi cant medical complications. Metabolic consequences o alcohol use disorder include gout, hyperlipidemia and atty liver, and hypoglycemia. Chronic alcoholics can develop obesity when the high caloric content o alcohol is added to normal ood intake; when ood intake is limited and/or malabsorption is present, weight loss with mineral and electrolyte imbalances and vitamin de ciencies can result. Alcohol toxicity can lead to pancreatic insu ciency and diabetes. The gastrointestinal system is requently a ected by chronic alcohol consumption, resulting in esophagitis, gastritis or ulcer, pancreatitis, and alcoholic hepatitis and cirrhosis. E ects o alcohol on the cytochrome P450 system alter drug and carcinogen metabolism, accounting or signi cant drug interactions and increased cancer incidence in chronic alcoholics. Alcohol increases the release o ACTH, glucocorticoids, and catecholamines and inhibits testosterone synthesis and the release o ADH and oxytocin. Neurologic complications o chronic alcoholism include dementia, amnestic disorder, cerebellar degeneration, and neuropathy, due to both direct neurotoxicity and thiamine de ciency. Finally, alcohol consumption during pregnancy has widespread teratogenic consequences, termed fetal alcohol spectrum disorder. Pharmacologic consequences o psychostimulant abuse relate to speci c e ects o these drugs on the nervous and cardiovascular systems. Potentiation o norepinephrine neurotransmission increases heart rate and blood pressure. Cocaine, in particular, can cause vasospasm leading to stroke, cerebrovasculitis, myocardial in arction, and aortic dissection. The inhibition o cardiac and CNS sodium channels by cocaine can cause arrhythmias and seizures. Psychostimulants can reset temperature regulation, causing hyperpyrexia and associated rhabdomyolysis. Cocaine and amphetamine can also cause involuntary movements through their action on the basal ganglia.

TREATMENTS FOR SUBSTANCE USE DISORDERS Despite the high prevalence o alcohol and drug problems in medical practice (10–15% in ambulatory care, 30–50% in emergency departments, and 30–60% in general hospital settings), the diagnosis is o ten overlooked. As is the case with other stigmatized diseases, specialized services are o ten

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

inaccessible. Recent health legislation in the United States promises parity or medical and mental disorders (including alcohol and drug problems) and more widespread availability o addiction treatment. Treatments or substance use disorders can be divided into two broad approaches: pharmacologic and psychosocial. Traditionally, pharmacologic treatments or substance use disorder have ocused on acute detoxi cation to relieve the withdrawal symptoms that accompany the cessation o drug use. It has been increasingly recognized, however, that detoxi cation alone does not a ect the long-term course o substance use disorder. Based on this understanding, new pharmacologic agents are being developed to speci cally treat the chronic condition o substance use disorder by diminishing craving, to prevent relapse when the patient has attained abstinence, and to reduce harm ul alcohol and drug use. These agents are summarized in the Drug Summary Table at the end o this chapter. Attention is also being directed toward treatment o co-occurring psychiatric disorders that may contribute to drug relapse. Thus, substance use disorder is now considered a chronic medical condition, and treatment must include li elong management. Psychosocial treatment approaches— or example, counseling techniques such as cognitive-behavioral therapy and an emphasis on wellness such as exercise and mindulness/relaxation techniques—have been e ective when used alone or in combination with pharmacologic treatment. O ten, the integrated use o both pharmacologic and psychosocial approaches increases the positive outcomes o treatment. In addition, participation in mutual support sel help programs (e.g., Alcoholics Anonymous) o ten improves outcomes, either utilized alone or when sel -help messages are incorporated into psychiatric treatment programs. These psychosocial strategies speci cally address the role o social learning and motivation in the pathogenesis o substance use disorders. Although counseling typically ocuses on an individual patient’s psychological needs, e ective treatment must also address the underlying social actors that impede long-term recovery, such as unemployment, housing, amily disruption, and lack o access to health care. Treatment outcomes in substance use disorders are comparable to those in other chronic diseases, such as diabetes, hypertension, and asthma. Although some treatments are more e ective in some patients than in others, the best predictor o positive outcomes is participation in treatment.

Detoxif cation The rst step in the treatment o substance use disorder is detoxif cation. The goals o detoxi cation are to allow the body to adapt to the absence o drug or alcohol, to diagnose and manage medical and psychiatric complications o substance use disorder, and to prepare the patient or long-term rehabilitation. Although detoxi cation may be achieved technically within a ew days, protracted withdrawal symptoms such as anxiety and insomnia may persist and require prolonged attention. Psychosocial counseling should begin early in detoxi cation and proceed with more intensity a ter detoxi cation. For example, Mr. A completed a 28-day intensive outpatient rehabilitation program a ter acute detoxi cation. The mani estations o drug withdrawal depend on the class o drug abused and can range rom mild dysphoria to li e-threatening seizures. The most commonly employed

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strategies or alleviating withdrawal are to taper the dose o the drug slowly or to use a long-acting drug in the same class that demonstrates cross-tolerance . For example, a common treatment or nicotine withdrawal is the administration o nicotine via a sustained-release transdermal patch or via a chewing gum. The dose is tapered slowly to allow the patient to avoid many o the unpleasant e ects o nicotine withdrawal. Another example is the administration and tapering o the long-acting opioid methadone or the treatment o opioid withdrawal. Buprenorphine can also be used or opioid withdrawal treatment; however, care must be taken to ensure that the patient is actually in withdrawal prior to starting buprenorphine, because administration o this partial agonist can precipitate or worsen withdrawal i opioid receptors are still occupied by the opioid o abuse (Fig. 19-8). Withdrawal symptoms rom alcohol, benzodiazepines, and barbiturates can be severe and, in some cases, even li e-threatening. In alcohol withdrawal, administration o a long-acting benzodiazepine (such as diazepam) is indicated to prevent withdrawal seizures (Fig. 19-9). Withdrawal rom benzodiazepines is accomplished with either a loading dose o phenobarbital, which has a very long elimination hal -li e, or tapering doses o a longer acting benzodiazepine. Withdrawal rom barbiturates should be managed only with phenobarbital. Other antiepileptic medications also suppress CNS hyperactivity due to withdrawal rom CNS depressants and can be e cacious in alcohol and benzodiazepine withdrawal (but not barbiturate withdrawal). Detoxi cation can also be accomplished by using medications rom a di erent class to block the signs and symptoms o withdrawal. For example, 2-adrenergic agonists such as clonidine and lo exidine can block sympathetic hyperactivity, which is a mani estation o withdrawal rom all drugs o abuse. 2-Receptors inhibit noradrenergic outf ow rom neurons in the brain to the periphery and modulate the activity o cells in the gut responsible or f uid absorption and intestinal motility; by these two mechanisms, 2-agonists partially block opioid withdrawal symptoms. Clonidine also diminishes symptoms o withdrawal rom nicotine and several other drugs. However, such a strategy is not recommended to treat withdrawal rom central nervous system depressants because it does not adequately prevent withdrawal seizures.

Sel -Help and Mutual Support Programs As the case o Mr. A illustrates, the risk o relapse a ter detoxi cation is high, and long-term management o addiction is needed to achieve continued sobriety. While not acceptable or help ul to all patients, sel -help and mutual support programs have played a prominent role in success ul recovery or millions o individuals. These approaches are modeled a ter Alcoholics Anonymous (AA). Foremost is the understanding that the problem is drinking and, there ore, the ocus is on acquiring strategies to prevent a relapse. AA and related programs such as Narcotics Anonymous (NA) and Cocaine Anonymous (CA) provide community support groups and mentoring. The presence o such help mitigates the sense o alienation and loneliness o ten elt by addicts. Participation is ree and readily available. Related mutual support groups such as Al-Anon or spouses and Alateen or teenage amily members provide important support or recovery. The mechanisms by which AA-related programs provide bene t are not ully understood but may reside in power ul social learning e ects that can modi y the incentive

328 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

salience o drugs o abuse. Most physicians now recognize that these programs can be use ul and complementary to the medical treatment o substance use disorders. Moderation management, another therapeutic stance toward alcoholism, emphasizes moderation rather than abstinence. This strategy is ine ective in individuals with alcohol use disorder, who (by de nition) can no longer control their drinking, and thus is recommended only in “problem drinkers”—patients who sometimes overindulge but have not yet lost control over their drinking.

Pharmacologic Treatment o Substance Use Disorders The recognition that addiction is caused by undamental changes in brain reward pathways indicates that pharmacotherapy could have an important role in the management o substance use disorders. To date, several pharmacologic strategies have been employed. The rst o these strategies is the chronic administration o an agent that causes aversive e ects when the drug o abuse is used. For example, disulf ram inhibits aldehyde dehydrogenase, a critical enzyme in the alcohol metabolism pathway. In an individual who ingests ethanol while taking disul ram, alcohol dehydrogenase oxidizes the ethanol to acetaldehyde, but disul ram prevents aldehyde dehydrogenase rom metabolizing the acetaldehyde. There ore, this toxic metabolite accumulates in the blood. Acetaldehyde causes a number o aversive symptoms, including acial f ushing, headache, nausea, vomiting, weakness, orthostatic hypotension, and respiratory di culty. These symptoms can last rom 30 minutes to several hours and are ollowed by exhaustion and atigue. The aversive e ects o alcohol consumption in the presence o disul ram are intended as a deterrent to urther drinking. Un ortunately, the e ectiveness o disul ram is limited by ailures in adherence and by substantial toxicity. A second strategy used to treat addiction is to block the e ects o the drug o abuse. Naltrexone is an opioid antagonist that competitively blocks the binding o opioids to the opioid receptor. Thus, a patient who injects an opioid, such as heroin, while taking naltrexone will not experience the “high” that normally accompanies drug use. Studies have shown that naltrexone also acts as an opioid inhibitor in the brain reward pathway. Thus, the e ects o a drug such as ethanol, which releases endogenous opioids that cause disinhibition (or stimulation) o mesolimbic dopamine, share a nal common reward pathway involving the opioid receptor and dopamine and are there ore also inhibited by naltrexone. For this reason, naltrexone has been used to treat alcohol use disorder. Placebo-controlled clinical trials have generally shown e cacy o naltrexone compared to placebo, particularly in reducing relapse to heavy drinking. Naltrexone should not be administered when there are traces o exogenous opioids in the system, because antagonism o remaining drug by naltrexone can lead to the development or exacerbation o opioid withdrawal symptoms. Although naltrexone can e ectively prevent the “high” associated with opioids, it does not alleviate cravings or withdrawal e ects, and there is a relatively high likelihood o nonadherence. There ore, naltrexone has been e ective only in individuals addicted to opioids or alcohol who have a high motivation to stay drug- ree or who have supervised administration. An injectable long-acting naltrexone preparation has been

approved by the US Food and Drug Administration (FDA) or the treatment o alcohol use disorder. Sustained-release naltrexone is injected intramuscularly once a month, and it has been demonstrated to reduce heavy alcohol consumption and increase alcohol abstinence. This ormulation is also bene cial in opioid use disorder, especially in those with low adherence to treatment. A third pharmacologic approach is the use o a long-acting agonist or medication maintenance. Methadone , as discussed earlier, is a long-acting opioid agonist. Because it is taken orally, it is less likely to produce the sharp increases in plasma levels required to elicit a “high” such as that accompanying the injection o heroin or other opioids. Methadone also has a long hal -li e compared to heroin or morphine. Thus, oncedaily administration o methadone produces plasma opioid levels that remain relatively constant over time and, thereore, mitigate cravings and prevent the emergence o withdrawal signs and symptoms. Moreover, methadone produces cross-tolerance to other opioids, so that a patient who injects heroin or another opioid while taking methadone experiences a reduced e ect o the injected drug. However, methadone has signi cant abuse liability, and there is a risk o death by overdose when methadone is combined with another opioid or CNS depressant. For these reasons, methadone should be dispensed or opioid maintenance treatment only under controlled circumstances in government-licensed programs. Conceptually similar to substitution treatments or opioid use disorder, nicotine replacement therapy is o ten the rst line o treatment or nicotine use disorder. Nicotine replacement is available in the orm o chewable gum, lozenge, transdermal patch, smokeless inhaler, or the recently popularized electronic nicotine delivery system (“e-cigarettes”). These orms o nicotine replacement curb cravings and withdrawal symptoms caused by decreases in plasma nicotine levels a ter cessation o smoking. All orms o nicotine replacement therapy are more e ective than placebo or smoking cessation, with the important bene t o avoiding exposure to toxic products o tobacco pyrolysis. Based on the observations that antagonist (e.g., naltrexone)based treatment o opioid use disorder su ers rom poor adherence and that ull agonists with advantageous pharmacokinetic properties (e.g., methadone) can nevertheless be diverted rom medical care and abused, partial agonist medications have been developed or the treatment o opioid use disorder. The partial agonist action o buprenorphine at -opioid receptors alleviates withdrawal symptoms associated with decreases in plasma levels o abused opioids, and reduces opioid cravings by increasing mesolimbic dopaminergic neurotransmission (Fig. 19-8). Thus, buprenorphine not only acilitates opioid detoxi cation but also can be employed or maintenance treatment. Since it is not a ull agonist, buprenorphine carries a low risk o overdose; since it antagonizes the rein orcing e ects o ull opioid agonists such as heroin, it reduces the likelihood o relapse. Because o its partial agonist properties and relatively long hal -li e (compared to most abused opioids), withdrawal rom buprenorphine per se is mild. To minimize abuse in the outpatient setting, buprenorphine is usually administered daily or on alternate days as a sublingual preparation (Suboxone ® ) that also contains the opioid antagonist naloxone. I Suboxone® is diverted and administered parenterally, the naloxone antagonizes the agonist e ects o buprenorphine; when administered sublingually, the naloxone is not bioavailable and the

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

ull e ects o buprenorphine are experienced. Outpatient use o buprenorphine will likely replace methadone-based treatment programs or opioid use disorder in all but the most severely addicted patients. The nicotinic receptor partial agonist varenicline has recently been demonstrated to acilitate smoking cessation in large-scale clinical trials. Varenicline is a partial agonist at the 4 2 nicotinic acetylcholine receptor subtype; hence, this agent has an analogous mechanism o action or treatment o nicotine dependence as that o buprenorphine or opioid dependence (Fig. 19-8). The partial agonist e ect o varenicline increases mesolimbic dopaminergic neurotransmission and thus both reduces withdrawal symptoms and diminishes the nicotine cravings that can lead to relapse. Varenicline also acts as a pharmacologic antagonist at nicotinic receptors in the presence o the ull agonist nicotine, thus mitigating the dopamine-enhancing e ects (and addictive potential) o nicotine. Importantly, varenicline administration has been associated with neuropsychiatric adverse e ects including emotional lability and acute psychosis, leading to an FDA warning regarding the use o varenicline in patients with psychiatric disorders; however, most recent evidence has not supported these concerns. A ourth approach is to utilize medications to prevent the long-term dysphoria and dys unctional reward mechanisms (allostasis) that are common in addicts who are newly abstinent. For example, one o the consequences o longterm alcohol consumption is a hyperactive glutamatergic system that persists even a ter alcohol consumption ceases. Acamprosate , which modulates glutamate hyperactivity to reestablish a more normal state, has been e cacious in preventing relapse to alcohol drinking in some but not all studies and has been approved or the treatment o alcohol use disorder. Recent comparisons o naltrexone and acamprosate with and without cognitive therapy have shown only naltrexone to be signi cantly more e cacious than placebo, however. The antiepileptic medication topiramate , which inhibits the AMPA/kainate class o glutamate receptors, signi cantly reduced alcohol drinking in a double-blind, placebo-controlled study. Importantly, topiramate treatment response in patients with alcohol use disorder has recently been ound to be moderated by a polymorphism in GRIK1, which encodes the kainate GluK1 receptor subunit. Together with other antiepileptic drugs, topiramate is being studied in larger clinical trials, but none o these drugs is currently FDA-approved or the treatment o alcohol use disorder. The antidepressant bupropion inhibits reuptake o dopamine and norepinephrine and has demonstrated e cacy in smoking cessation. The mechanism o action o bupropion may be related to increased dopaminergic neurotransmission in the mesolimbic reward pathway, thus bu ering nicotine withdrawal-induced cravings. Bupropion lowers the seizure threshold, suggesting that this treatment may not be appropriate or patients with underlying seizure disorders or those who abuse drugs that are associated with intoxication- or withdrawal-induced seizures. A th approach is to speci cally treat co-occurring psychiatric symptoms that are highly prevalent in individuals diagnosed with substance use disorders. Depressed and anxious mood, mood instability, and psychotic symptoms are o ten observed in abstinent patients. A meta-analysis o antidepressant treatment o patients with substance use disorders ound that these medications are not e ective unless the

329

patients are diagnosed with a co-occurring major depression. In act, there is some evidence that selective serotonin reuptake inhibitors (SSRIs) may cause early-onset, antisocial alcoholics to become worse and drink more alcohol than those receiving a placebo. Also, the high co-occurrence o bipolar spectrum disorders in those with substance use disorders suggests that depressive symptoms in these patients o ten do not warrant antidepressant treatment but rather mood stabilization. Treatment o abstinent addicts with bipolar or psychotic disorders, using mood stabilizers and antipsychotics, is generally viewed as bene cial. Nevertheless, most clinicians recognize that it may be di cult to accurately diagnose and treat co-occurring psychiatric disorders i individuals are actively using alcohol or other drugs. In contrast to the various pharmacologic treatments available or alcohol and opioid use disorders, there is a paucity o current treatments or cocaine and amphetamine use disorders, and none is approved by the FDA. Several trials have attempted to use antidepressants, such as the tricyclic antidepressant desipramine or the selective serotonin reuptake inhibitor f uoxetine . Desipramine acts by blocking monoamine reuptake (especially norepinephrine reuptake), whereas f uoxetine inhibits serotonin reuptake. Both agents have been shown to reduce drug craving, but un ortunately, neither has been shown to prevent cocaine use. There is recent evidence that disul ram (see above) may have some e ectiveness in the treatment o cocaine dependence. In addition to its inhibition o aldehyde dehydrogenase, disul ram inhibits dopamine -hydroxylase and can increase brain dopamine levels, possibly counteracting the dopamine-depleting e ects o chronic cocaine use. Because cocaine sensitization involves glutamate, antiepileptics (e.g., carbamazepine, oxcarbazepine, and topiramate) are also being studied or e cacy in the treatment o cocaine dependence.

CONCLUSION AND FUTURE DIRECTIONS This chapter has discussed the major causes o substance use disorders. Substance use disorder is de ned as a problematic pattern o drug use associated with context-induced craving and drug-seeking, especially under situations o stress, that leads to clinically signi cant impairment or distress. Substance use disorder is caused by an allostatic adaptation to the presence o the drug in brain reward pathways. Although each drug has its own molecular and cellular mechanism o action that may account or drug toxicity, all abused drugs speci cally a ect the mesolimbic dopamine brain reward pathway. This chapter has also discussed the major treatments or substance use disorders, including the pharmacologic prevention and treatment o withdrawal symptoms, the long-term psychosocial management o addiction, and newer pharmacologic treatments that, when integrated with psychosocial approaches, promote long-lasting sobriety. Together, these addiction treatments achieve outcomes approximating those o other long-term chronic medical disorders, such as atherosclerosis, hypertension, and diabetes. New directions in addiction research are ocused on the pharmacologic modulation o brain reward, stress responses, and learning-related neural processes. In addition, these approaches are complemented by basic and clinical studies o the neurobiology o learning and memory and the

330 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY A

modif cation o these processes through psychosocial treatments. Current approaches to cocaine use disorder provide two specif c examples. First, drugs that specif cally interact with di erent dopamine receptor subtypes have been explored, investigating the hypotheses that a D1-specif c agonist or D4-specif c antagonist could suppress drug cravings, and that a D2-specif c antagonist could prevent the rein orcing e ects o cocaine. Second, researchers have recently completed clinical trials o a cocaine vaccine, under the theory that cocaine will be less rein orcing in vaccinated persons who are exposed to the drug (Fig. 19-11). I success ul, this approach could be extended to other drugs o abuse. (Trials o an analogous anti-nicotine vaccine are also orthcoming.) However, vaccinated individuals may switch to other drugs o abuse or which they have not produced antibodies, and hence, this is not likely to be a totally satis actory approach. Broader and more promising are e orts to develop pharmacologic treatments or addiction aimed at (1) modulating the chemical mediators o synaptic plasticity that underlie reward learning and memory (Fig. 19-2) and (2) modi ying the negative a ective states and stress responses, the previously mentioned “allostatic load,” associated with chronic drug abuse. These approaches address shared brain mechanisms o addiction to all drugs o abuse. For example, a ailure o the pre rontal cortex to control drug-seeking behaviors has been linked to glutamatergic dys unction in reward pathways, which may be amenable to new glutamate- and neuroplasticity-based pharmacotherapies. Another approach targets neural systems mediating behavioral stress responses; or example, an antagonist at the neurokinin-1 receptor, which is expressed in brain areas involved in stress responses and drug reward, has been shown in preliminary studies to suppress alcohol cravings, improve well-being, and attenuate the cortisol stress response in abstinent alcoholics. Preclinical studies have shown that CRF antagonists may block stress-induced reinstatement o drug use in animal models o substance use disorder (Fig. 19-12). Endocannabinoid signaling has also been implicated in a variety o physiologic

Dopamine

Cocaine

Dopa mine D1 re ce ptor

Drug e ffe ct

Dopa mine D2 re ce ptor

DA tra ns porte r

Anti-cocaine antibody

B

Re duce d drug e ffe ct

FIGURE 19-11. Vaccine approach to treat cocaine use disorder. A. Cocaine inhibits the dopamine transporter (DAT), thereby preventing dopamine reuptake and allowing excessive stimulation of postsynaptic dopamine receptors (D1 and D2). B. After treatment with a “cocaine vaccine,” antibodies are generated that bind to exogenous cocaine once it enters the bloodstream. The antibody-bound cocaine is incapable of binding to and inhibiting DAT, and the rewarding effects of the drug are reduced.

A

B CRF re ce ptor a nta gonis t

CRF CRF re ce ptor

Gluta ma te

Gluta ma te re ce ptor

Ce llula r excita tion

Excita tion re duce d Anxie ty Autonomic hype ra rous a l Withdra wa l

Re duce d withdra wa l-a s s ocia te d a nxie ty

Figure 19-12. Corticotropin-releasing factor (CRF) antagonist approach to reduce alcohol/drug withdrawal and relapse. A. As a result of allostatic changes associated with chronic alcohol/drug use, when use is discontinued, a small subset of extended amygdala neurons (red ) locally release CRF, which activates glutamatergic neurotransmission via stimulation of CRF receptors on nearby amygdala neurons, contributing to anxiety and negative affect. This state is important in driving continued drug-seeking behavior (relapse). B. CRF receptor antagonist blocks CRF activation of extended amygdala neurons during alcohol/drug discontinuation. This approach has been suggested to mitigate aversive affective concomitants of drug withdrawal that lead to relapse-associated behaviors.

C h a p t e r 1 9 Pharmacology of Drugs of Abuse

unctions including reward learning, appetite, mood, pain, and cognition (Fig. 19-10). Elucidation o endocannabinoid signaling as a pro-hedonic system involving CB1 receptor activation led to f ndings that the CB1 cannabinoid receptor antagonist rimonabant was e ective in obesity treatment, and this drug is now being investigated as a treatment or drug addiction. Rimonabant has not received FDA approval because it is associated with signif cant psychiatric adverse e ects, but this approach remains a promising direction or uture research.

Acknowledgment We thank David C. Lewis, Robert M. Swi t, Joshua M. Galanter, and Alan A. Wartenberg or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Alcoholics Anonymous. www.aa.org. (Excellent information on Alcoholics Anonymous.) Camí J, Farré M. Mechanisms o disease: drug addiction. N Engl J Med 2003;349:975–986. (Current understanding of neural mechanisms leading to addiction.) Dani JA, Harris RA. Nicotine addiction and comorbidity with alcohol abuse and mental illness. Nat Neurosci 2005;8:1465–1470. (Examines the

331

interface between the neuropharmacologic underpinnings of nicotine addiction and psychiatric disorders, especially alcoholism.) Goldman D, Oroszi G, Ducci F. The genetics o addictions: uncovering the genes. Nat Rev Genet 2005;6:521–532. (A review that examines how heritable factors operate in causation of drug use disorders.) Goldstein RZ, Craig AD, Bechara A, et al. The neurocircuitry o impaired insight in drug addiction. Trends Cogn Sci 2009;13:372–380. (Discusses current understanding of lack of insight and awareness in addiction.) Kalivas PW. The glutamate homeostasis hypothesis o addiction. Nat Rev Neurosci 2009;10:561–572. (Review that links learning mechanisms to reward through the glutamatergic system.) Koob GF, Le Moal M. Neurobiological mechanisms or opponent motivational processes in addiction. Philos Trans R Soc B Biol Sci 2008;363: 3113–3123. (Reviews relationships between stress and reward pathways.) McLellan AT, Lewis DC, O’Brien CP, Kleber HD. Drug dependence, a chronic medical illness: implications or treatment, insurance, and outcomes evaluation. JAMA 2000;284:1689–1695. (Seminal analysis of the status of drug use disorders in the health care system.) Nestler EJ. Transcriptional mechanisms o addiction: role o delta-FosB. Philos Trans R Soc B Biol Sci 2008;363:3245–3255. (Reviews the role of gene regulation as a unitary neurobiological mechanism in reward and stress responses.) Substance Abuse and Mental Health Services Administration. www.samhsa .gov. (Contains a wealth of information about prevention and treatment and co-occurring diagnoses; also access to listings of evidence-based treatment practices.) Volkow ND, Baler RD, Goldstein RZ. Addiction: pulling at the neural threads o social behaviors. Neuron 2011;69:599–602. (Review of interface between medical and societal/legal aspects of addiction.)

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334 PRINCIPLES OF CENTRAL NERVOUS SYSTEM PHARMACOLOGY

III Principles of Cardiovascular Pharmacology

Acee tyl C oA + Ace toa cee tyl Ac t CoA

S tatins HM MG CoA Co oA

20

HMG HM G Co CoA A re ductaa s e M va lonn a te Me t

In a s e d LDL Incre rree ce ptor expre s s ion

5-pyrop ophh os phomevva lon lona te

Pharmacology of Cholesterol and Lipoprotein Metabolism Is ope ntylpyrophoss pphh a te

3,3-dime thh yl ylaa llylpyrrop opho hos p ha te

Iss opp re no noid ids

Ge ra nylpp yr y opho op hoss phh a te

Tib o r I. Kris ko , Eh rin J . Arm s t ro n g , a n d David E. Co h e n

Faa rne s ylp ylp p yr y ophos op pha te INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336–33 INT 3377 PATHOPHYSIS qua lee ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 Hypercholesterolemia o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3477 BIOCHEMISTRY AND PHYSIOLOGY Y OF CHOLESTEROL AN ND Hypertriglyceridemia d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 LIPOPROTEIN META ABO BOLI LISM SM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 L nos te rol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3448 La Mixed Hyperlipidemia Mee ta tabo boli lism li sm m of ApoB ApoB-Containing Lipoproteins . . . . . . . . . . . . 338 Disorders of HDL L Met etab abol oliis m . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Assembly o ApoB-Contain i ing ing Li Lipo popr prot otei e ns . . . . . . . . . . . . 338 Second ndar daryy Hyperlipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Intr In trav avas ascu cula larr Me Meta tabo boli lism o ApoB-Containing Chole Chol Ch olee s te roll Lipoproteinss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 349 Rece Re cept ptor or-Med Mediiatedd Clearance o ApoB-Containingg Inhibitors of Cholesterol Synthesis . . . . . . . . . . . . . . . . . . . . . . 350 Chole Inhibitors s te rol of VLDL Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 Lipoproteinss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 34 C Formation and Clearance o LDL Particless . . . . . . . . . . . . . 342 Inhibitors of Bile Acid Absorption . . . . . . . . . . . . . . . . . . . . . . . 352 HDL Metabolism and Reverse Cholesterol Transport . . . . . . 342 Inhibitors of Cholesterol Absorption . . . . . . . . . . . . . . . . . . . . . 352 HDL Formationn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Fibrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Intravascular Maturation o HDL L . . . . . . . . . . . . . . . . . . . . . 344 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 HDL-Mediated Cholesterol E f ux rom Cells. . . . . . . . . . . . 344 Omega-3 Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 Delivery o HDL Cholesterol to the Liverr . . . . . . . . . . . . . . . 344 PCSK9 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Biliary Lipid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 355 Cholesterol Balance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .355

INTRODUCTION Lipids are insoluble or sparingly soluble molecules that are essential or membrane biogenesis and maintenance o membrane integrity. They also serve as energy sources, hormone precursors, and signaling molecules. In order to acilitate transport through the relatively aqueous environment o the blood, nonpolar lipids, such as cholesteryl esters and triglycerides, are packaged within lipoproteins. Increased concentrations o certain lipoproteins in the circulation are associated strongly with atherosclerosis. Much o the prevalence o cardiovascular disease (CVD), the leading cause o death in the United States and most Western countries, can be attributed to elevated blood concentrations o cholesterol-rich low-density lipoprotein (LDL) particles as well as lipoproteins that are rich in triglycerides. Epidemiologically, decreased concentrations o high-density lipoproteins (HDL) also predispose to atherosclerotic disease. The major contributors to lipoprotein abnormalities appear to be Western diets combined with sedentary li estyles, but a limited number o genetic causes o hyperlipidemia have also been identif ed. The role o genetics in common orms o hyperlipidemia is the subject o intense study utilizing cutting-edge genomic 336

approaches. It is apparent that genes modi y both the sensitivity o individuals to adverse dietary habits and li estyles and the response o individuals to lipid-lowering therapies. This chapter highlights the biochemistry and physiology o cholesterol and lipoproteins, with an emphasis on the role o lipoproteins in atherogenesis, and the pharmacologic interventions that can ameliorate hyperlipidemia. Abundant clinical outcomes data have proven that morbidity and mortality rom cardiovascular disease can be reduced by the use o lipid-lowering drugs.

BIOCHEMISTRY AND PHYSIOLOGY OF CHOLESTEROL AND LIPOPROTEIN METABOLISM Lipoproteins are macromolecular aggregates that transport triglycerides and cholesterol in the blood. Circulating lipoproteins can be di erentiated on the basis o density, size, and protein content (Table 20-1). As a general rule, larger, less dense lipoproteins have a higher percentage composition o lipids; chylomicrons are the largest and least dense lipoprotein subclass, whereas HDLs are the smallest lipoproteins, containing the lowest lipid content and the highest proportion o protein.

C h a p t e r 2 0 Pharmacology of Cholesterol and Lipoprotein Metabolism 337

J ake P, a 29-year-old construction worker, m a kes an ap pointm ent to s ee Dr. Cus h. J a ke co m p lain s o h a rd , e le va te d s w ellin g s a ro u n d h is Ach ille s te n d o n th a t s ee m to rub cons ta ntly a ga ins t his con s tru ctio n b ooo ts . J a ke h a d b e e n h e s ita n t to s e e th e d o cto r (h is las t a pp ointm e n t wa s 10 ye a rs ag o ), bu t he rem em bers that his dad, who died at age 42 o a h e a rt a tta ck, h a d s im ilar s w e llin gs . On exa m in ation , Dr. Cu sh recogn izes the Achilles swellings as xanthom a s (lipid depos its ); the phys ica l e xa m is oth erwis e with in n orm al lim its. J ake co m m en ts th at his d ie t is quite “ atty,” including three to our donuts each day and requent ham burgers. Dr. Cush explains that the xanth om as on J ake’s eet are the result o cholesteryl ester deposition, probably rom high cholesterol levels in his blood. Dr. Cush orders a asting plasm a chole s te ro l le vel a n d reco m m e n d s th a t J ake re d u ce h is in ta ke o o od s h ig h in s a tu ra te d a t a n d ch o les tero l an d increase his intake o po ultry, f s h, who le cereal grains, ruits, and vegetables. J ake has gained about 15 pounds since he was 19 and has a sm all paunch. Dr. Cush recom m ends regular exercise and weight loss. Results o the blood test reveal a total plasm a chole s te ro l co n cen tratio n o 315 m g /d L (n o rm al, 200), with elevated LDL cholesterol o 250 m g/dL (desirable, 100), low HDL o 35 m g/dL (norm al, 35 to 100), and norm al concentrations o triglycerides and very-lowdensity lipoprotein (VLDL). Based on these test results, h is ag e, the Achilles h e el xan th om a s , and a pos itive a m ily h is to ry o r an ea rly m yo ca rd ia l in a rctio n , Dr. Cush tells J ake that he likely has an inherited disorder

o cholesterol m etabolism known as heterozygous am ilial hypercholesterolem ia. Th is disease p uts J ake at very high risk or early atherosclerosis and m yocardial in arction. The low HDL cholestero l level also contributes to his increased risk o cardiovascular disease. Dr. Cu s h te lls J a ke th a t a g g re s s ive lo w e rin g o ch o le s te ro l le ve ls ca n a m e lio ra te m a ny o th e d is e a s e s e q u e la e . In a d d itio n to th e d ie ta ry ch a n g e s , Dr. Cu s h p re s crib e s a s ta tin to h e lp re d u ce J a ke’s ch o le s te ro l. A s ta rtin g d o s e o a s ta tin re d u ce s h is LDL b y 45% to 138 m g /d L, w h ile h is HDL in cre a s e s s lig h tly. Dr. Cu s h th e n in cre a s e s th e s ta tin d o s e , a n d th is p ro d u ce s a n a d d itio n a l 12% re d u ctio n in LDL. Be ca u s e LDL h a s s till n o t re a ch e d 100 m g /d L, a n d HDL re m a in s lo w , Dr. Cu s h a d d s th e ch o le s te ro l a b s o rp tio n in h ib ito r e ze tim ib e a s w e ll a s e xte n d e d re le a s e n ia cin . A te r th e s e m o d if ca tio n s , J a ke’s LDL d ro p s b e lo w 100, a n d h is HDL in cre a s e s to 45 m g /d L. J a ke e xp e rie n ce s cu ta n e o u s u s h in g d u rin g th e f rs t e w m o n th s o n ia cin tre a tm e n t, b u t a te r th a t p e rio d , h e h a s o n ly o cca s io n a l u s h in g e p is o d e s .

Questions 1 . How do high cholesterol levels predispose to cardiovascular disease? 2 . What is the etiology of familial hypercholesterolemia? 3 . How do statins, ezetimibe, niacin, lomitapide, mipomersen, and PCSK9 inhibitors act pharmacologically? 4 . What are the major adverse effects of concomitant statin and niacin therapy about which J ake should be aware?

TABLE 20-1 Characteristics o Plasma Lipoproteins CM

VLDL

IDL

LDL

HDL

0.95–1.006

1.006–1.019

1.019–1.063

1.063–1.210

Density (g/mL)

0.95

Diameter (nm)

75–1,200

30–80

25–35

18–25

5–12

Total lipid (% wt)

98

90

82

75

67

Protein

2

10

18

25

33

Triglycerides

83

50

31

9

8

Unesterif ed cholesterol and cholesteryl esters

8

22

29

45

30

Phospholipids (% wt lipid)

7

18

22

21

29

None

Pre-

Composition, % dry weight

Electrophoretic mobilitya Plasma hal -li e Major apolipoproteins a

1 hour B48, AI, AIV, E, CI, CII, CIII

30-60 minutes B100, E, CI, CII, CIII

or Pre30 minutes B100, E, CI, CII, CIII

2–4 days

2–5 days

B100

AI, AII, CI, CII, CIII, E

Electrophoretic mobility of lipoprotein particles is designated relative to migration of plasma - and -globulins. CM, chylomicron; VLDL, very-low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein

338 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Core : Triglyce ride a nd chole s te ryl e s te rs

Apolipoprote in B100 Apolipoprote in C

Fre e chole s te rol Apolipoprote in E P hos pholipid monola ye r

and lipoproteins that are involved primarily in cholesterol transport (HDL and the remnants o apoB-containing lipoproteins). HDL also serves as a reservoir or exchangeable apolipoproteins in the plasma, including apoAI, apoCII, and apoE. The ollowing discussion presents each lipoprotein class in the context o its unction.

Metabolism of ApoB-Containing Lipoproteins The primary unction o apoB-containing lipoproteins is to deliver atty acids in the orm o triglycerides to muscle tissue or use in ATP biogenesis and to adipose tissue or storage. Chylomicrons are ormed in the intestine and transport dietary triglycerides, whereas VLDL particles are ormed in the liver and transport triglycerides that are synthesized endogenously. The metabolic li espan o apoB-containing lipoproteins can be divided into three phases: assembly, intravascular metabolism, and receptor-mediated clearance. This is a convenient categorization because pharmacologic agents are available that in uence each phase.

Assembly of ApoB-Containing Lipoproteins FIGURE 20-1. Structure of lipoprotein particles. Lipoproteins are spherical particles (5– 1,000 nm in diameter) that transport hydrophobic molecules, principally cholesterol and triglycerides, as well as at-soluble vitamins. The sur ace o the particle is composed o a monolayer o phospholipid and unesterif ed cholesterol molecules. These polar lipids orm a coating that shields a hydrophobic core o nonpolar triglyceride and cholesteryl esters rom interacting with the aqueous environment o plasma. Lipoproteins contain amphipathic apolipoproteins (also called apoproteins) that associate with the sur ace lipids and hydrophobic core. Apolipoproteins provide structural stability to the lipoprotein particle and act as ligands or specif c cell sur ace receptors or as co actors or enzymatic reactions. In the example shown, a very-low-density lipoprotein (VLDL) particle contains apolipoprotein E, apolipoprotein B100, and apolipoproteins CI, CII, and CIII (shown here as apolipoprotein C).

Structurally, lipoproteins are microscopic spherical particles ranging rom 5 to 1,000 nm in diameter. Each lipoprotein particle consists o a monolayer o polar, amphipathic lipids that surrounds a hydrophobic core. Each lipoprotein particle also contains one or more types o apolipoprotein (Fig. 20-1). The polar lipids that comprise the sur ace coat are unesterif ed cholesterol and phospholipid molecules arranged in a monolayer. The hydrophobic core o a lipoprotein contains cholesteryl esters (cholesterol molecules linked by an ester bond to a atty acid) and triglycerides (three atty acids esterif ed to a glycerol molecule). Apolipoproteins (also re erred to as apoproteins) are amphipathic proteins that intercalate into the sur ace coat o lipoproteins. In addition to stabilizing the structure o lipoproteins, apolipoproteins engage in biological unctions. They may act as ligands or lipoprotein receptors or may activate enzymatic activities in the plasma. The apolipoprotein composition determines the metabolic ate o the lipoprotein. For example, each LDL particle contains one apolipoprotein B (apoB) 100 molecule, which is a ligand or the low-density lipoprotein receptor (discussed below); in turn, binding o LDL to the LDL receptor promotes cholesterol uptake into cells. From a metabolic perspective, lipoprotein particles can be divided into lipoproteins that participate in the delivery o triglyceride molecules to muscle and at tissue (the apoB-containing lipoproteins, chylomicrons, and VLDL)

The cellular mechanisms by which chylomicrons and VLDL are assembled are quite similar. Regulation o the assembly process depends on the availability o apoB and triglycerides, as well as the activity o microsomal triglyceride-transfer protein (MTP). The gene that encodes apoB is transcribed principally in the intestine and the liver. Apart rom this tissue-specif c expression, there is little transcriptional regulation o the apoB gene. In contrast, a key regulatory event that di erentiates chylomicron metabolism rom VLDL metabolism is the editing o apoB mRNA (Fig. 20-2). Within enterocytes but not hepatocytes, a protein named apoB editing complex-1 (apobec-1) is expressed. This protein constitutes the catalytic subunit o the apoB editing complex, which deaminates a cytosine at position 6666 o the apoB mRNA molecule. Deamination converts the cytosine apoB ge ne Transcription

Live r a nd inte s tine

a poB mRNA

Editing

S ma ll inte s tine

No editing

Live r

S top codon mRNA Translation P rote in a poB48

a poB100

FIGURE 20-2. Editing of apoB mRNA. The apoB gene, with exons represented by rectangles and introns by lines, is transcribed in both the intestine and the liver. In the intestine, but not the liver, a protein complex containing apobec-1 modif es a single nucleotide in the apoB mRNA. As a result, the codon containing this nucleotide is converted to a premature stop codon, as indicated by the red X. The protein that is synthesized in the intestine (apoB48) is only 48% as long as the ull-length protein that is synthesized in the liver (apoB100).

C h a p t e r 2 0 Pharmacology o Cholesterol and Lipoprotein Metabolism 339

to uridine. As a result, the codon containing this nucleotide is converted rom glutamine to a premature stop codon. When translated, the intestinal orm apoB48 is 48% as long as the ulllength protein that is expressed in the liver and re erred to as apoB100. As a consequence, chylomicrons, the apoB-containing lipoprotein produced by the intestine, contain apoB48, whereas VLDL particles produced by the liver contain apoB100. Figure 20-3 illustrates the cellular mechanisms by which apoB-containing lipoproteins are assembled and secreted. As the apoB protein is synthesized by ribosomes, it crosses into the endoplasmic reticulum. Within the endoplasmic reticulum, triglyceride molecules are added co-translationally to the elongating apoB protein (i.e., apoB is lipidated) by the action o a co actor protein, MTP. Once apoB has been ully synthesized, the nascent lipoprotein is enlarged in the Golgi apparatus; during this process, MTP adds additional triglycerides to the core o the particle. By unclear mechanisms, cholesteryl esters are also added to the core. Each lipoprotein particle assembled by this process contains a single molecule o apoB. Because the triglyceride component o chylomicrons originates primarily rom the diet (Fig. 20-4), the assembly, secretion, and metabolism o chylomicrons are collectively re erred to as the exogenous pathway o lipoprotein metabolism. During digestion, cholesteryl esters and triglycerides in ood are hydrolyzed to orm unesterif ed cholesterol, ree atty acids, and monoglycerides. Bile acids, phospholipids, and cholesterol are secreted by the liver into bile and stored in the gallbladder during asting as micelles and vesicles, which are macromolecular lipid aggregates that orm due to the detergent properties o bile acid molecules. The stimulus o eating a meal promotes

Ente rocyte or he pa tocyte Cytos ol a poB mRNA

Chole s te ryl e s te r

Ribos ome MTP

MTP

Chylomicron (e nte rocyte ) or VLDL (he pa tocyte )

ApoB

Endopla s mic re ticulum Triglyce ride

Fa tty a cid Bile s a lt Apica l me mbra ne

Ba s ola te ra l me mbra ne

Ezetimibe Mice lle NP C1L1

Chole s te rol ACAT

Chole s te ryl e s te r ABCG5/G8

Monoglyce ride

DGAT Triglyce ride id Fa tty a cid

FIGURE 20-4. Absorption of cholesterol and triglycerides. Exogenous

Mipomersen

Lipidation

Monoglyce ride Chole s te rol P hos pholipid

Lomitapide

FIGURE 20-3. Assembly and secretion of apolipoprotein B-containing lipoproteins. Chylomicrons and VLDL particles are assembled and secreted by similar mechanisms in the enterocyte and hepatocyte, respectively. The apoB mRNA (i.e., apoB48 or apoB100 mRNA) is translated by ribosomes to yield a protein that enters the lumen o the endoplasmic reticulum. I triglycerides are available, the apoB protein is lipidated by the action o microsomal triglyceride-trans er protein (MTP) in two distinct steps, accumulating triglyceride as well as cholesteryl ester molecules. The resulting chylomicron or VLDL particle is secreted by exocytosis into the lymphatics by enterocytes or into the plasma by hepatocytes. In the absence o triglycerides, the apoB protein is degraded (not shown). Sortilin (not shown) regulates the intracellular tra f cking o VLDL particles and can direct apoB to lysosomal-dependent degradation, thereby decreasing its secretion. Mipomersen inhibits translation o apoB by binding to apoB mRNA. Lomitapide inhibits lipidation o apoB by binding to MTP.

cholesterol and triglycerides are simultaneously absorbed rom the intestinal lumen by di erent mechanisms. Cholesterol is taken up rom micelles through a regulatory channel named NPC1L1. A raction o the cholesterol is pumped back into the lumen by ABCG5/G8, a heterodimeric ATP-dependent plasma membrane protein. The remainder o the cholesterol is converted to cholesteryl esters by ACAT. Triglycerides are taken up as atty acids and monoglycerides, which are re-esterif ed to triglycerides by DGAT. Ezetimibe inhibits cholesterol uptake through NPC1L1.

emptying o gallbladder bile into the small intestine, where the micelles and vesicles solubilize the digested lipids. Lipid absorption into enterocytes o the duodenum and jejunum is acilitated mainly by micelles. Long-chain atty acids and monoglycerides are taken up separately into the enterocyte by carrier-mediated transport and then re-esterif ed to orm triglycerides by the enzyme diacylglycerol acyltransferase (DGAT). By contrast, medium-chain atty acids are absorbed directly into the portal blood and metabolized by the liver. Dietary and biliary cholesterol rom micelles enter the enterocyte via a protein channel named Niemann-Pick C1-like 1 protein (NPC1L1). Some o this cholesterol is immediately pumped back into the intestinal lumen by the ATP-dependent action o a heterodimeric protein, ABCG5/ABCG8 (ABCG5/G8). The raction o cholesterol that remains is esterif ed to a long-chain atty acid by acetyl-CoA:cholesterol acyltransferase (ACAT). Once triglycerides and cholesteryl esters are packaged together with apoB48, apoA1 is added as an additional structural apolipoprotein and the chylomicron particle is exocytosed

340 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

into the lymphatics or transport to the circulation via the thoracic duct. The plasma concentration o triglyceride-rich chylomicrons varies in proportion to dietary at intake. Very-low-density lipoproteins (VLDL) contain triglycerides that are assembled by the liver using plasma atty acids derived rom adipose tissue or synthesized de novo. For this reason, the assembly, secretion, and metabolism o VLDL are o ten re erred to as the endogenous pathway o lipoprotein metabolism. Hepatocytes synthesize triglycerides in response to increased ree atty acid ux to the liver. This typically occurs in response to asting, thereby ensuring a continuous supply o atty acids or delivery to muscle in the absence o triglycerides rom the diet. Interestingly, dietary saturated ats as well as carbohydrates also stimulate the synthesis o triglycerides within the liver. By cellular mechanisms that are similar to those that produce chylomicrons (Fig. 20-3), MTP in hepatocytes lipidates apoB100 to orm nascent VLDL particles. Under the continued in uence o MTP, the nascent VLDL particles coalesce with larger triglyceride droplets and are secreted directly into the circulation. VLDL particles may also acquire apoE, apoCI, apoCII, and apoCIII within the hepatocyte prior to secretion. However, these apolipoproteins may also be trans erred to VLDL rom HDL in the circulation.

The synthesis o apoB48 in the intestine and apoB100 in the liver is constitutive. This permits the immediate production o chylomicrons and VLDL particles when triglyceride molecules are available. In the absence o triglycerides, such as in enterocytes during asting, apoB is degraded by a variety o cellular mechanisms. Recent studies have revealed a role or sortilin in the cellular tra f cking o VLDL particles. Sortilin is encoded by the Sort1 gene, and genomewide association studies (GWAS) have shown that Sort1 is associated with reduced levels o LDL cholesterol (LDL-C). Sortilin acilitates the post-translational degradation o apoB by a lysosome-dependent mechanism.

Intravascular Metabolism of ApoB-Containing Lipoproteins

Within the circulation, chylomicrons and VLDL particles must be activated in order to target triglyceride delivery to muscle and adipose tissue (Fig. 20-5). Activation requires the addition o an optimal complement o apoCII molecules, which occurs by aqueous trans er o apoCII rom HDL particles. Because there is an inherent delay in the trans er o apoCII to chylomicrons and VLDL particles, there is time or widespread circulation o triglyceride-rich particles throughout the body.

HDL

a poA-I a poE

a poC-II

Live r

Inte s tine s a poC-II

a poB100

a poC-II

a poB48

VLDL

Chylomicron

Plasma

Mus cle or a dipos e tis s ue

Mus cle or a dipos e tis s ue

a poC-II

a poB48 a poC-II

Fa tty a cids

VLDL Lipoprotein lipase

Ca pilla ry e ndothe lium

a poB100

Fa tty a cids

Chylomicron Ca pilla ry e ndothe lium

Lipoprotein lipase

FIGURE 20-5. Intravascular metabolism of apoB-containing lipoproteins. After secretion, chylomicrons and VLDLparticles are activated for lipolysis when they encounter HDL particles in the plasma and acquire the exchangeable apolipoprotein apoCII. When chylomicrons and VLDL circulate into capillaries of muscle or adipose tissue, apoCII promotes binding of the particle to lipoprotein lipase, which is bound to the surface of endothelial cells. Lipoprotein lipase mediates hydrolysis of triglycerides, but not cholesteryl esters, from the core of the lipoprotein particle. The resulting fatty acids are taken up into muscle or adipose tissue.

C h a p t e r 2 0 Pharmacology o Cholesterol and Lipoprotein Metabolism 341

Lipoprotein lipase (LPL) is a lipolytic enzyme expressed on

the endothelial sur ace o capillaries in muscle and at tissue. LPL is a glycoprotein that is synthesized by myocytes and adipocytes and transported to the endothelial cell sur ace by a specif c glycosylphosphatidylinositol (GPI)-linked protein, GPIHBP1. On the endothelial cell membrane, GPIHBP1 also serves to anchor LPL in place. Once chylomicrons and VLDL particles acquire apoCII, they can bind to LPL, which hydrolyzes triglycerides rom the core o the lipoprotein (Fig. 20-5). LPL-mediated lipolysis liberates ree atty acids and glycerol. The ree atty acids are then taken up by the neighboring parenchymal cells. The expression level and intrinsic activity o LPL in muscle and adipose tissue are regulated according to the ed/ asting state, allowing the body to direct the delivery o atty acids pre erentially to muscle during asting and to adipose a ter a meal. The rate o lipolysis o chylomicron and VLDL triglycerides is also controlled by apoCIII, which is an inhibitor o LPL activity. LPL inhibition by apoCIII may be A

Triglyceride-depleted VLDL

Triglyceride-depleted chylomicron

Triglyce ride

Receptor-Mediated Clearance of ApoB-Containing Lipoproteins As LPL continues to hydrolyze triglycerides rom chylomicrons and VLDL, the particles become progressively depleted o triglycerides and relatively enriched in cholesterol. Once approximately 50% o the triglycerides have been removed, the particles lose their a f nity or LPL and dissociate rom the enzyme. The exchangeable apolipoproteins apoAI and apoCII (as well as apoCI and apoCIII) are then trans erred to HDL in exchange or apoE (Fig. 20-6A), which serves as a high-a f nity ligand or receptor-mediated clearance o the particles. Upon acquiring apoE, the particles are termed chylomicron or VLDL remnants . Remnants o chylomicrons and VLDL are taken up by the liver in a three-step process (Fig. 20-6B). The f rst step is B

a poB48

He pa tic s inus oida l e ndothe lium

a poB100

a poC-II

an additional mechanism promoting widespread distribution o triglyceride-rich particles in the circulation.

Chylomicron or VLDL remnant

a poE

a poC-II

HS P G

a poB48 Chole s te ryl e s te r a poB48

HDL Sequestration a poE a poE

Hepatic lipase

a poA-I

HDL

Fa tty a cid

Space of Disse

a poB48

Lipolysis apoC-II a poA-I

a poE

VLDL remnant (IDL)

Chylomicron remnant a poB100

LDL-R

LRP

HS P G

Uptake He pa tocyte a poE

a poE a poB48

FIGURE 20-6. Formation and hepatic uptake of remnant particles. A. Upon completion o hydrolysis, chylomicrons and VLDL lose a f nity or lipoprotein lipase. When an HDL particle is encountered, apoCII is trans erred back to HDL particles in exchange or apoE. The resulting particles are chylomicron and VLDL remnants. B. The activity o lipoprotein lipase results in remnant lipoprotein particles that are small enough to enter the space o Disse. Remnant lipoproteins are sequestered in the space o Disse by binding to high-molecular-weight heparan sul ate proteoglycan (HSPG) molecules. This is ollowed by the action o hepatic lipase, which promotes lipolysis o some residual triglycerides in the core o the remnant lipoproteins and the release o atty acids. Uptake o remnant lipoprotein particles into hepatocytes is mediated by the LDL receptor (LDL-R), the LDL receptor-related protein (LRP), a complex ormed between LRP and HSPG, or HSPG alone.

342 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

sequestration o the particles within the space of Disse between the enestrated endothelium o the liver sinusoids and the sinusoidal (basolateral) plasma membrane o the hepatocytes. Sequestration requires that the remnant particles become small enough during lipolysis to f t between the endothelial cells. Once in the space o Disse, remnants are bound and sequestered by large heparan sulfate proteoglycans . The next step is particle remodeling within the space o Disse by the action o hepatic lipase , a lipolytic enzyme that is similar to LPL but is expressed by hepatocytes. Hepatic lipase appears to optimize the triglyceride content o remnant particles so that they can be cleared e f ciently by receptor-mediated mechanisms. The f nal phase o remnant clearance is receptor-mediated particle uptake. This is accomplished by one o our pathways. At the sinusoidal hepatocyte plasma membrane, remnant particles may be bound and taken up by the LDLreceptor, the LDLreceptor-related protein (LRP), or heparan sul ate proteoglycans. A ourth pathway is mediated by the combined activities o LRP and heparan sul ate proteoglycans. These redundant mechanisms allow or e f cient particle clearance, so that the hal -li e o remnants in the plasma is approximately 30 minutes.

Formation and Clearance of LDL Particles

ApoB48-containing chylomicron remnants are completely cleared rom the plasma. By contrast, the presence o apoB100 alters the metabolism o VLDL remnants so that only approximately 50% are cleared by the pathways or remnant particles. The di erence is mani ested during the metabolism o the remnant particles by LPL. VLDL remnants are avidly metabolized by LPL, becoming an increment smaller, relatively more def cient in triglycerides, and relatively enriched in cholesteryl esters. When converted to remnants ollowing exchange o apolipoproteins with HDL, these more dense particles are called intermediate-density lipoproteins (IDL). Because IDL contain apoE, a raction o these particles (approximately 50%) may be cleared by the liver via remnant receptor pathways (Fig. 20-6). However, the remainder are converted to LDL by hepatic lipase, which urther hydrolyzes triglycerides in the core o IDL. The urther reduction in size o the particle results in the trans er o apoE to HDL. As a consequence, LDL is a distinct, cholesteryl ester-enriched lipoprotein with apoB100 as its only apolipoprotein (Fig. 20-7A). The LDL receptor is the only receptor capable o clearing signif cant amounts o LDL rom the plasma. The LDL receptor is expressed on the sur ace o hepatocytes, macrophages, lymphocytes, adrenocortical cells, gonadal cells, and smooth muscle cells. Due to the lack o apoE, LDL particles are relatively weak ligands or the LDL receptor. As a result, the hal -li e o LDL in the circulation is markedly prolonged (2–4 days). This explains why LDL cholesterol accounts or approximately 65–75% o total plasma cholesterol. The uptake o LDL-C by the LDL receptor also appears to be promoted by sortilin (Fig. 20-7B). Interaction o apoB100 with the LDL receptor acilitates receptor-mediated endocytosis o LDL particles and subsequent vesicle usion with lysosomes (Fig. 20-7B). The LDL receptor is recycled to the cell sur ace, while the cholesteryl esters and triglycerides within the LDL particle are hydrolyzed by lysosomal acid lipase (LAL) to release unesterif ed cholesterol and atty acids. These hydrolysis products a ect three major homeostatic pathways. First, intracellular cholesterol inhibits HMG-CoA reductase, the

enzyme that catalyzes the rate-limiting step in de novo cholesterol synthesis. Second, cholesterol activates ACAT to increase esterif cation and storage o cholesterol in the cell. Third, LDL receptor expression is down-regulated, reducing urther uptake o cholesterol into the cells. The majority o LDL receptors (70%) are expressed on the sur ace o hepatocytes. As a result, the liver is primarily responsible or the removal o LDL particles rom the circulation. Proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a plasma protein that regulates LDL receptor activity. PCSK9 is synthesized as a 72-kDa proPCSK9 proprotein that is autocatalytically cleaved in the endoplasmic reticulum to orm the mature protein. It then enters the secretory pathway, and with the assistance o sortilin in the transGolgi network, it is secreted into the plasma. PCSK9 then binds to the epidermal growth actor-like repeat A (EGFA) moti o the LDL receptor. This complex is targeted to lysosomes or degradation (Fig. 20-7B). Gain-o - unction mutations in PCSK9 result in marked elevations in LDL-C, whereas loss-o - unction mutations reduce LDL-C. LDL particles that are not taken up by LDL receptorexpressing tissues may migrate into the intima o blood vessels and bind to proteoglycans (Fig. 20-8). There, they are subject to oxidation or nonenzymatic glycosylation. Oxidation o LDL results in lipid peroxidation and may create reactive aldehyde intermediates that ragment apoB100. The modif ed LDL is internalized by scavenger receptors (e.g., SR-A), which are expressed predominantly by mononuclear phagocytic cells. Unlike the LDL receptor, scavenger receptors are not down-regulated when the phagocytic cells begin to accumulate cholesterol. As a result, the continued accumulation o oxidized LDL in macrophages can lead to foam cell ormation (cholesterol-rich macrophages). These oam cells may undergo apoptotic or necrotic death, releasing ree radicals and proteolytic enzymes. Oxidized LDL also causes up-regulation o cytokine production, impairs endothelial unction, and increases expression o endothelial adhesion molecules. All o these e ects increase the local in ammatory response and promote atherosclerosis. Foam cells are a major constituent o atherosclerotic lesions, and excessive oam cell death can destabilize atherosclerotic plaques. This is attributable in part to the liberation o matrix metalloproteinases. Because plaque rupture is the main cause o acute ischemic cardiovascular events, particularly heart attacks and strokes, high plasma levels of LDL are a major risk factor for the development of atherosclerosis and subsequent cardiovascular disease. This is why Jake’s doctor became concerned when he discovered that Jake had very high plasma concentrations o LDL.

HDL Metabolism and Reverse Cholesterol Transport Virtually all cells in the body are capable o synthesizing all the cholesterol they require. However, only the liver has the capacity to eliminate cholesterol, and it does so by secreting unesterif ed cholesterol into the bile or by converting cholesterol to bile acids. As noted above, HDL serves as a reservoir or exchangeable apolipoproteins or the metabolism o apoB-containing lipoproteins. HDL also plays a key role in cholesterol homeostasis by removing excess cholesterol rom cells and transporting it in plasma to the liver. This process is o ten re erred to as reverse cholesterol

C h a p t e r 2 0 Pharmacology o Cholesterol and Lipoprotein Metabolism 343 Hepatic lipase

A

a poC-II a poB100

Liver

a poB100

Fa tty a cid a poE

IDL

LDL a poE a poC-II

a poA-I

HDL a poB100

B LDL

Chole s te ryl linole a te

P CS K9

LDL binding to LDL re ce ptor

LDL-R S ortilin Inte rna liza tion LDL re ce ptor re cycling

Lys os ome

P rote olys is

Hydrolys is by LAL

Amino a cids

1

HMG CoA re ducta s e

FIGURE 20-7. Formation and clearance of LDL particles. A. Formation

Chole s te rol

ER

2

ACAT

3

LDL re ce ptors

Chole s te ryl ole a te

transport (Fig. 20-9A). The major apolipoproteins o HDL

are apoAI and apoAII. ApoAI, the main structural determinant o HDL, participates in the ormation o the particle and its interaction with its receptor, scavenger receptor class B, type I (SR-BI). The unction o apoAII is not well understood—it appears to play a role in maintaining the structural integrity o HDL.

HDL Formation

HDL ormation occurs mainly in the liver, although a small percentage is contributed by the small intestine. The earliest

o LDL occurs when IDL particles interact with hepatic lipase to become denser and cholesteryl ester-enriched. As a result, both apoE and apoCII lose a f nity or the particle and are trans erred to HDL, leaving only apoB100. B. Binding o apolipoprotein B100 to the LDL receptor (LDL-R) sortilin complex on hepatocytes or other cell types promotes LDL internalization into endocytic vesicles and usion o the vesicles with lysosomes. LDLreceptors are recycled to the cell sur ace unless bound to PCSK9, whereas lipoprotein particles are proteolyzed to amino acids ( rom apoB100 and PCSK9-bound LDL-R) and ree cholesterol and atty acids ( rom the action o lysosomal acid lipase [LAL] on cholesteryl esters and triglycerides). Intracellular ree cholesterol has three regulatory e ects on the cell. First, cholesterol decreases the activity o HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Second, cholesterol activates acetyl-CoA:cholesterol acyltrans erase (ACAT), an enzyme that esterif es ree cholesterol into cholesteryl esters or intracellular storage or export. Third, cholesterol inhibits the transcription o the gene encoding the LDL receptor and thereby decreases urther uptake o cholesterol by the cell.

events occur when lipid-poor apoAI is secreted by the liver or intestine or dissociates rom lipoprotein particles in the plasma. These amphipathic apoAI molecules interact with ABCA1, which is localized in the sinusoidal membrane o the hepatocyte or the basolateral membrane o the enterocyte. ABCA1 incorporates a small amount o membrane phospholipid and unesterif ed cholesterol into the apoAI molecule. The resulting small, disk-shaped particle, which consists mainly o phospholipid and apolipoprotein AI, is re erred to as nascent or pre- -HDL, due to its characteristic migration on agarose gels.

344 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Vessel lumen

Circula ting monocyte s

Na tive LDL

Endothe lia l injury

Endothe lia l dys function

thereby maintains the appropriate sur ace concentration or the shrinking core. By trans erring phospholipids to the surace o HDL, PLTP also replaces the molecules that are consumed by the LCAT reaction. This allows the core o HDL to continue to enlarge.

HDL-Mediated Cholesterol E f ux rom Cells B S R-A

A Re s ide nt monocyte ma cropha ge

Ce ll-me dia te d oxida tion

D

E

C

Foa m ce ll Oxidize d LDL

Foa m ce ll ne cros is

Subendothelial space

FIGURE 20-8. LDL and atherosclerosis. Elevated LDL is a major risk factor for the development of atherosclerosis. Native LDL that migrates into the subendothelial space can undergo chemical transformation to oxidized LDL via lipid peroxidation and fragmentation of apoB100. Oxidized LDL has a number of deleterious effects on vascular function. Oxidized LDL promotes monocyte chemotaxis into the subendothelial space (A) and inhibits monocyte egress from that space (B). Resident monocyte–macrophages bind to oxidized LDL via a scavenger receptor (SR-A), resulting in the formation of lipid-laden foam cells (C). Oxidized LDL can directly injure endothelial cells and cause endothelial dysfunction (D). Continued accumulation of oxidized LDL in foam cells can also cause foam cell necrosis, with release of numerous proteolytic enzymes that can damage the intima (E).

Intravascular Maturation o HDL

Because disk-shaped pre- -HDL particles are relatively ine f cient at removing excess cholesterol rom cell membranes, these particles must mature into spherical particles in the plasma. HDL maturation occurs as a result o the activity o two distinct circulating proteins (Fig. 20-9A, B). Lecithin:cholesterol acyltransferase (LCAT) binds pre erentially to disk-shaped HDL and converts cholesterol molecules within the particle to cholesteryl esters. This is accomplished by transesterif cation o a atty acid rom a phosphatidylcholine molecule on the sur ace o the HDL to the hydroxyl group o a cholesterol molecule. The reaction also creates a lysophosphatidylcholine molecule, which dissociates rom the particle and binds to serum albumin. Because they are highly insoluble, cholesteryl esters migrate into the core o the HDL particle. The development o a hydrophobic core converts the pre- -HDL to a spherical -HDL particle. The second important protein that contributes to HDL maturation in the plasma is phospholipid transfer protein (PLTP). PLTP trans ers phospholipids rom the sur ace coat o apoB-containing remnant particles to the sur ace coat o HDL. During LPL-mediated lipolysis o apoB-containing lipoproteins, the particles become smaller as triglycerides are removed rom the core. This leaves a relative excess o phospholipids on the sur ace o the particle. Because phospholipids are highly insoluble and cannot otherwise dissociate rom a particle, PLTP removes excess phospholipids and

Cellular cholesterol e ux is the mechanism by which excess insoluble cholesterol molecules are removed rom cells. This occurs when unesterif ed cholesterol is trans erred rom the plasma membrane o cells to an HDL particle. The mechanism o cholesterol e ux varies depending on the cell type and the type o HDL particle. Lipid-poor pre- HDL particles, apoAI, and apoAII can promote cholesterol e ux by interacting with ABCA1. This process is not only important in HDL ormation by the liver but is also a mechanism or removing excess cholesterol rom cells within the subendothelial space and or protecting macrophages rom cholesterol-induced cytotoxicity. Spherical HDL very e f ciently stimulates cholesterol e ux by several di erent mechanisms. First, the interaction o apoAI on HDL with SR-BI on the plasma membrane promotes cholesterol e ux. Second, macrophages express not only ABCA1 and SR-BI but also ABCG1, which also mediates cholesterol e ux to spherical HDL. Finally, spherical HDL particles may promote cholesterol e ux in the absence o binding to a specif c cell sur ace protein. Although cholesterol has very low monomeric solubility, it can dissociate in appreciable amounts and di use short distances through the plasma to acceptor particles that are enriched with phospholipids on their sur aces. Quantitatively, e f ux to spherical HDL particles accounts or most o the removal o excess cholesterol rom cells. This capacity o HDL to remove cellular cholesterol is enhanced by the activities o LCAT and PLTP, which prevent the sur ace coat o the particle rom becoming saturated with cholesterol.

Delivery o HDL Cholesterol to the Liver

When mature HDL particles circulate to the liver, they interact with SR-BI, the principal HDL receptor (Fig. 20-9A). SR-BI is highly expressed on the sinusoidal plasma membranes o hepatocytes. In contrast to its action on most nonhepatic cells, where SR-BI mediates e f ux o excess cholesterol rom the membrane, SR-BI in the liver promotes selective uptake o lipids. In this process, the cholesterol and cholesteryl esters o HDL particles are taken up into the hepatocyte in the absence o uptake o apolipoproteins. During SR-BI–mediated selective lipid uptake, apoAI is liberated to participate in pre- -HDL ormation. The “li espan” o an HDL particle is 2–5 days, suggesting that each apoAI molecule can participate in many cycles o reverse cholesterol transport. Among the nonhepatic tissues that express high levels o SR-BI are the adrenal glands and gonads, presumably re ecting the requirement o these organs or cholesterol to support steroidogenesis. Delivery o cholesterol rom extrahepatic tissues to the liver is optimized by two additional proteins: cholesterol ester transfer protein (CETP) and hepatic lipase. CETP is a plasma protein that trans ers cholesteryl esters rom mature spherical HDL to the cores o remnant lipoproteins in exchange or a triglyceride molecule, which is inserted into the core o the HDL particle (Fig. 20-9B). This process allows the body to utilize remnant particles that have completed

C h a p t e r 2 0 Pharmacology o Cholesterol and Lipoprotein Metabolism 345

A

LCAT

Liver

PLTP

Chole s te rol P hos pholipid Chole s te ryl e s te r

Pre-β-HDL ABCA1

S R-BI

Fa tty a cid

a poA-I

Plasma

α-HDL Tissues Chole s te rol

Hepatic lipase

ABCG1

S R-BI

CETP LCAT PLTP

ABCA1

B Plasma

Chylomicron or VLDL remnant

Albumin

Lys o-P C LCAT

Cell

PLTP

CETP

PC

Lys o-P C

LCAT

Chole s te rol

α-HDL

Chole s te ryl e s te r

FIGURE 20-9. Reverse cholesterol transport. A. The process o reverse cholesterol transport begins when apoAI is secreted rom the liver. ApoAI in plasma interacts with ATP binding cassette protein AI (ABCA1), which incorporates a small amount o phospholipid and unesterif ed cholesterol rom hepatocyte plasma membranes to orm a discoidal-shaped pre- -HDL particle. Due to the activity o lecithin:cholesterol acyltrans erase (LCAT) in plasma, pre- -HDL particles mature to orm spherical -HDL. Spherical -HDL particles unction to accept excess unesterif ed cholesterol rom the plasma membranes o cells in a wide variety o tissues. The unesterif ed cholesterol is trans erred rom the cell to nearby HDLparticles by interactions with ABCA1, ABCG1, and SR-BI, as well as by aqueous di usion through the plasma. As explained in panel B, LCAT and phospholipid trans er protein (PLTP) increase the capacity o HDL to accept unesterif ed cholesterol molecules rom cells by allowing or expansion o the core and the sur ace coat o the particle. Cholesteryl ester trans er protein (CETP) removes cholesteryl ester molecules rom HDL and replaces them with triglycerides rom remnant particles. HDL particles interact with scavenger receptor, class B type I (SR-BI), which mediates selective hepatic uptake o cholesterol and cholesteryl esters, but not apoAI. This process is acilitated when hepatic lipase hydrolyzes triglycerides rom the core o the particle. The remaining apoAI molecules may begin the cycle o reverse cholesterol transport again. B. LCAT, PLTP, and CETP promote the removal o excess cholesterol rom the plasma membranes o cells. LCAT removes a atty acid rom a phosphatidylcholine molecule in the sur ace coat o - (or pre- -) HDL and esterif es an unesterif ed cholesterol molecule on the sur ace o the particle. The resulting lysophosphatidylcholine (lyso-PC) becomes bound to albumin in the plasma, whereas the cholesteryl ester migrates spontaneously into the core o the lipoprotein particle. The unesterif ed cholesterol molecules that are consumed by LCAT are replaced by unesterif ed cholesterol rom cells. HDL phospholipids that are consumed by LCAT action are replaced with excess phospholipids rom remnant particles by the activity o PLTP. As described in panel A, CETP increases the e f ciency o cholesterol transport to the liver by exchanging cholesteryl ester molecules in -HDL or triglycerides in VLDL remnants. Unlike phospholipids, triglycerides, and cholesteryl esters (which require transport proteins), unesterif ed cholesterol and lyso-PC can di use over short distances in the plasma.

346 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

their unction o triglyceride transport or purposes o transporting cholesterol to the liver. Removal o cholesteryl ester molecules rom HDL appears to serve two unctions. First, it urther increases the capacity o HDL to take on additional cholesterol molecules rom cells. Second, it makes the process o selective uptake by SR-BI more e f cient. This is because hydrolysis o triglycerides by hepatic lipase on the hepatocyte sur ace acilitates the activity o SR-BI (Fig. 20-9A). As noted above, reverse cholesterol transport is the overall process by which HDL removes cholesterol rom macrophages and other extrahepatic tissues and returns it to the liver. The concept that increased plasma concentrations o HDL cholesterol may re ect increased rates o reverse cholesterol transport provides a possible explanation or the inverse relationship between plasma HDL levels and risk o cardiovascular disease. HDL particles also exert direct benef cial e ects on vascular tissue, including enhancement o antioxidant enzyme activities that inhibit oxidation o LDL. HDL also inhibits the expression o in ammatory mediators (e.g., intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]) by vascular cells. Increased understanding o HDL metabolism may lead to the development o novel biochemical targets or increasing reverse cholesterol transport in order to slow or even reverse the progression o atherosclerosis.

A

HO

Cholesterol 7α-hydroxylase

COO -

(live r) HO

HO

Chole s te rol

OH

Bile a cid (Chola te )

B

Blood

S inus oida l me mbra ne

Bile

ABCG5/G8

Chole s te rol Mice lle

Hepatocyte ABCB11

Bile a cid Bile s a lt ABCB4

P hos pholipid

Ca na licula r me mbra ne

Biliary Lipid Secretion Once cholesterol is delivered to the liver by the process o reverse cholesterol transport, it is eliminated by biliary secretion. An essential step occurs when a raction o the cholesterol is converted to bile acids (Fig. 20-10A). Cholesterol 7 -hydroxylase (CYP7A1), an enzyme expressed only in hepatocytes, catalyzes the rate-limiting step in the catabolism o cholesterol to bile acids. Bile acids, unlike cholesterol, are highly soluble in water. Moreover, bile acids are biological detergents that promote the ormation o micelles (Fig. 20-10B). These macromolecular aggregates, which are rich in phospholipids derived rom hepatocyte membranes, solubilize cholesterol in bile or transport rom the liver to the small intestine. In this way, micelles serve as a unctional counterpart to HDL particles in plasma. Bile ormation begins when bile acids are pumped into bile by the action o a canalicular membrane transport pump known as ABCB11 (Fig. 20-10B). In turn, these bile acids stimulate the biliary secretion o phospholipids and cholesterol. Phospholipid and cholesterol secretion are mediated by two additional transporters: ABCB4 or phospholipids and a heterodimer o ABCG5 and ABCG8 or cholesterol. Large amounts o bile acids, phospholipids, and cholesterol are secreted into bile at approximate rates o 24, 11, and 1.2 grams each day, respectively. These molecules comprise the biliary lipids, which are stored in the gallbladder during asting. The stimulus o a atty meal leads to gallbladder contraction, which propels its contents into the small intestine. As described above, bile acilitates the digestion and absorption o ats, in addition to promoting the elimination o endogenous cholesterol.

Cholesterol Balance Because cholesterol is converted by the liver to bile acids and secreted unmodif ed into bile, overall cholesterol balance

FIGURE 20-10. Biliary lipid secretion. A. Within hepatocytes, a portion of cholesterol is converted to bile acids. This process is rate-limited by cholesterol 7 -hydroxylase, which is expressed only in hepatocytes. Cholate is the most abundant bile acid synthesized by the human liver. B. Within the canalicular (apical) membranes, an ATP-dependent pump ABCB11 drives the secretion of bile acids out of the cell against a concentration gradient. Bile acids then stimulate the activities of two other proteins, ABCB4 and a heterodimer of ABCG5 and ABCG8 (ABCG5/G8), to secrete phospholipids and cholesterol, respectively, into bile. Within bile, the interactions among bile acids, phospholipids, and cholesterol result in the formation of micelles.

depends on the disposition o both cholesterol and bile acids. Most bile acid molecules are not lost in the eces a ter participating in cholesterol transport and at digestion; instead, they are taken up and recycled by high-a f nity transport proteins in the distal ileum. Bile acids enter the portal circulation and are transported back to the liver, where they are cleared rom the blood by hepatocytes with high f rst-pass e f ciency. Bile acids are then re-secreted into bile. The process o recycling bile acids between the liver and intestine is re erred to as enterohepatic circulation. The enterohepatic circulation is highly e f cient, allowing 5% o secreted bile acids to be lost in the eces. However, because bile acids are secreted in such large amounts, the small ractional loss o bile acids amounts to about 0.4 grams per day. Considering that cholesterol is the substrate or bile acid synthesis, ecal bile acids represent a source o cholesterol loss rom the body. Sensitive nuclear hormone receptors within the liver are capable o detecting the rate o loss o bile acids into the eces. These receptors tightly regulate transcription o bile acid synthetic genes. As a result, the liver synthesizes precisely the amount o bile acids that is su f cient to replace what is lost in the eces.

C h a p t e r 2 0 Pharmacology of Cholesterol and Lipoprotein Metabolism 347

In addition to the 1.2 grams o cholesterol that are secreted into bile each day, the average American diet contributes approximately 0.4 grams each day to intestinal cholesterol. There ore, dietary cholesterol represents only a minor raction (25%) o the total (i.e., biliary and dietary) cholesterol that passes through the intestine. The extent to which intestinal cholesterol is absorbed appears to be genetically regulated. Each individual absorbs a f xed percentage o intestinal cholesterol. In the population, percentages range rom as low as 20% to more than 80%. For example, when an average individual absorbs 50% o intestinal cholesterol, this will amount to hal o the 1.6 grams (i.e., 1.2 grams o biliary cholesterol plus 0.4 grams o dietary cholesterol), and the other hal (0.8 grams) will be lost in the eces. Combined with a loss o 0.4 grams per day o cholesterol in the orm o ecal bile acids, this yields a total cholesterol loss rom the body o 1.2 grams each day. Taking into account intestinal absorption o dietary cholesterol and reabsorption o biliary cholesterol, total body cholesterol synthesis is approximately 0.8 grams per day (i.e., cholesterol synthesis ecal loss o cholesterol bile acids dietary cholesterol intake). Thus, the amount o endogenous cholesterol synthesis is about two old greater than the amount consumed in the average diet.

PATHOPHYSIOLOGY Numerous studies have demonstrated a def nitive link between elevated plasma lipid concentrations and the risk o cardiovascular disease. Increased risk o cardiovascular mortality is most closely linked to elevated levels o LDL cholesterol and decreased levels o HDL cholesterol. In addition, hypertriglyceridemia represents an independent risk actor. The risk is urther increased when hypertriglyceridemia is associated with low HDL-cholesterol concentrations, even i LDL-cholesterol concentrations are normal. From a clinical perspective, the dyslipidemias can be divided into hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, and disorders o HDL metabolism. The causes o hyperlipidemia are multi actorial. These include well-def ned monogenic diseases and the contributions o genetic polymorphisms, as well as less well-def ned gene– environment interactions. For many individuals, elevated cholesterol may be the consequence o a diet high in saturated at and cholesterol superimposed on a susceptible genetic prof le. The ollowing section describes the major genetic predispositions or hyperlipidemia. This is ollowed by a brie overview o the secondary causes o hyperlipidemia. It is important to appreciate that the decision to treat elevated cholesterol concentrations is based on estimations o the risk o cardiovascular disease. Current clinical practice does not incorporate genetic causes o hyperlipidemia into these calculations. As common genetic predispositions to dyslipidemia and the contributions o these predispositions to cardiovascular disease become better understood, lipid-lowering therapies may one day be tailored toward individual genetic susceptibilities.

Hypercholesterolemia Isolated hypercholesterolemia is characterized by elevated levels o total plasma cholesterol and LDL cholesterol, with normal concentrations o triglycerides. The causes o primary hypercholesterolemia are amilial hypercholesterolemia, amilial de ective apoB100, gain-o - unction mutations in PCSK9, amilial combined hyperlipidemia (FCHL), and, most commonly, polygenic hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal domi-

nant disease involving de ects in the LDL receptor. Mutations in the gene encoding the LDL receptor result in one o our molecular de ects: lack o receptor synthesis, ailure to reach the plasma membrane, de ective LDL binding, and ailure to internalize bound LDL particles. Heterozygous individuals (1 in 500 in the United States) have elevated total plasma cholesterol concentrations rom birth throughout li e, with adult levels averaging 275–500 mg/dL (normal, 200 mg/dL). Clinical eatures include tendon xanthomas (caused by intracellular and extracellular accumulation o cholesterol) and arcus corneae (deposition o cholesterol in the cornea). Homozygous FH is a much more severe but rare disorder (1 in 1 million in the United States) that is characterized by the absence o unctional LDL receptors. This leads to very high plasma cholesterol concentrations (700–1,200 mg/dL) and cardiovascular disease that presents clinically prior to the age o 20. Heterozygotes or FH respond well to statins and other LDL-lowering drugs that up-regulate LDL receptor density on the cell sur ace. In the introductory case, Jake was most likely heterozygous or FH. Because homozygotes lack unctional LDL receptors, until recently, the only e ective treatment has been plasmapheresis with immunoadsorption o LDL particles. However, the development o molecules that inhibit PCSK9, MTP, or apoB synthesis now show promise toward a complementary treatment to reduce the severe elevations in LDL-C observed in patients with homozygous FH. An autosomal recessive orm o hypercholesterolemia has also been described in which a de ective molecular adaptor protein that participates in LDL receptor internalization leads to a phenotype similar to that o FH. Familial defective apoB100 is an autosomal dominant disorder in which mutations in the apoB100 protein lead to decreased a f nity o the LDL particle or LDL receptors. Due to decreased catabolism o LDL, cholesterol concentrations in amilial de ective apoB100 can be similar to those in patients with FH. Gain-o - unction mutations in PCSK9 have been identif ed in amilies with clinical eatures similar to FH; the pathophysiology o this disorder re ects increased PCSK9 unction and decreased LDL receptor expression on cell suraces. Familial combined hyperlipidemia is characterized by di erent combinations o hyperlipidemia in di erent amilies (see below); one presentation is elevated LDL cholesterol. Polygenic hypercholesterolemia is a general term that has been used to categorize the majority o patients with hypercholesterolemia who have no def ned genetic cause or the disorder. Polygenic hypercholesterolemia may be the result o complex gene–environment interactions, multiple uncharacterized genetic susceptibilities, or variant LDL particles such as small dense LDL and lipoprotein(a) [Lp(a)]. Further research into genetic predispositions or hypercholesterolemia will be necessary in order to identi y clear etiologies or the majority o patients with hypercholesterolemia.

Hypertriglyceridemia Primary hypertriglyceridemia is characterized by high plasma triglyceride concentrations (200–500 mg/dL or higher; normal, 150 mg/dL), when measured ollowing an overnight ast. Three major etiologies o hypertriglyceridemia have been identif ed: amilial hypertriglyceridemia, amilial lipoprotein lipase (LPL) def ciency, and apoCII def ciency. Familial combined hyperlipidemia can also present with isolated hypertriglyceridemia. More commonly, hypertriglyceridemia

348 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

develops with age, weight gain, obesity, and diabetes and is an important component o the metabolic syndrome. Familial hypertriglyceridemia is a common autosomal dominant disorder characterized by hypertriglyceridemia with normal LDL-cholesterol concentrations. HDL cholesterol is o ten reduced. Although the underlying de ect in this disorder is unknown, it is hypothesized to be a de ect in bile acid metabolism, leading to increased hepatic production o triglyceride-rich VLDL. A strong amily history o premature coronary heart disease is usually absent. Management is generally with exercise and diet. I that approach is unsuccess ul at reducing triglyceride concentrations below 500 mg/dL, a f brate should be considered. Drug therapy should be initiated i triglycerides exceed 1,000 mg/dL. Familial lipoprotein lipase def ciency is an autosomal recessive disorder caused by the absence o active LPL. This condition may be diagnosed by testing the plasma or lipase activity ollowing an in usion o heparin, which competes or binding sites on endothelial cells and dislodges LPL molecules into the plasma. Patients with LPL def ciency exhibit pro ound hypertriglyceridemia, which is characterized by elevated chylomicrons during in ancy and impaired removal o VLDL later in li e. Inants or young adults may present with pancreatitis, eruptive xanthomas, hepatomegaly, and splenomegaly attributable to the accumulation o lipid-laden oam cells. Treatment consists o a at- ree diet and avoidance o substances that increase VLDL production by the liver, such as alcohol and glucocorticoids. ApoCII def ciency is a rare genetic disorder with presentation and treatment similar to amilial lipoprotein lipase def ciency. It is caused by def ciency o apoCII, a co actor protein o LPL. It may be distinguished rom LPL def ciency by demonstrating that the triglyceride levels o patients are reduced ollowing in usion o plasma that contains normal apoCII; this does not occur in patients with amilial LPL def ciency. It is now appreciated that mutations in apoAV can present with chylomicronemia and severe hypertriglyceridemia, consistent with an apparent role or apoAV in acilitating the interaction between apoCII and LPL.

Mixed Hyperlipidemia Patients with mixed hyperlipidemia exhibit complex lipid prof les that may consist o elevated total cholesterol, LDL cholesterol, and triglyceride concentrations. HDL cholesterol is o ten reduced. Etiologies o mixed hyperlipidemia include amilial combined hyperlipidemia (FCHL), dysbetalipoproteinemia , and lysosomal acid lipase def ciency (LAL-D). FCHL is a common disease associated with moderately elevated concentrations o asting triglycerides and total cholesterol and reduced concentrations o HDL cholesterol. These patients o ten present with other eatures o the metabolic syndrome , including abdominal obesity, glucose intolerance, and hypertension. The molecular de ects are still under investigation. Current hypotheses ocus on insulin resistance, which leads to increased lipolysis in at tissue. Fatty acids liberated rom at tissue return to the liver, where they are reassembled into triglycerides. The increase in triglycerides increases the production o VLDL particles, which leads to an increase in apoB-containing lipoproteins in the plasma. In part because o the complex phenotypes o FCHL, the underlying genetic de ects have remained elusive. Faith ul adherence to dietary modif cation may be an e ective means o controlling FCHL. However, drug treatment is o ten required, and statins are commonly utilized. Combination therapy that includes addition o a f brate or niacin may be

necessary to normalize triglyceride and LDL-cholesterol concentrations, as well as to increase HDL cholesterol. Dysbetalipoproteinemia is a disorder characterized by increased cholesterol-rich chylomicrons and IDL-like particles. These f ndings are the result o accumulated chylomicron and VLDL remnants, leading to both hypertriglyceridemia and hypercholesterolemia. ApoE has three iso orms (E2, E3, and E4) in humans, and apoE2 has been implicated in the disease. Chylomicrons and VLDL particles in patients with the homozygous apoE2/apoE2 phenotype have reduced a f nity or their lipoprotein receptors, leading to accumulation o remnant particles in the plasma. Although the apoE iso orm is present at birth, symptoms generally present in adult males and in postmenopausal emales. The mechanism underlying this delay in expression o the phenotype is unknown, and additional metabolic actors (e.g., obesity, diabetes, or hypothyroidism) may be required to unmask the disorder. Dysbetalipoproteinemia can be managed by decreased intake o at and cholesterol, along with weight reduction and omission o alcohol intake. In addition, niacin and f brates are e ective pharmacologic therapies. Lysosomal acid lipase def ciency (LAL-D) is a rare lysosomal storage disorder caused by mutations in the LIPA gene, which encodes lysosomal acid lipase. This mutation leads to def ciency o the enzyme, with a corresponding reduction in the ability o LDL-C to be processed normally by hepatocytes. The result is hepatic steatosis and dyslipidemia, with elevated LDL-C, elevated triglycerides, and reduced HDL-C. Clinically, LAL-D is re erred to as Wolman disease in in ants and children or as cholesterol ester storage disease (CESD) in adults. Patients with LAL-D develop early atherosclerosis and progressive liver disease, with associated high rates o mortality at a young age.

Disorders o HDL Metabolism Decreased HDL cholesterol is an independent risk actor or development o atherosclerosis and cardiovascular disease. Numerous rare genetic de ects in HDL metabolism have been identif ed, including de ects in apoAI, ABCA1, and LCAT. Each o these de ects results in decreased levels o HDL, or which no e ective treatments are currently available. More commonly, low HDL is associated with visceral obesity and insulin resistance. Elevated concentrations o HDL occur in the setting o aerobic activity, alcohol consumption, estrogen use, and corticosteroid therapy. Recently, reductions in CETP activity have been characterized as a relatively common genetic cause o increased HDL levels. The increased plasma HDL concentration associated with decreased CETP activity has been attributed to a decrease in the trans er o cholesterol rom HDL to remnant particles. Although it might be assumed that the increased HDL levels would be cardioprotective, this is not always observed. Decreased CETP activity may increase the risk o atherogenesis in some cases, whereas in others, it appears to be cardioprotective. Additional research will be necessary be ore the role o CETP polymorphisms in lipid metabolism and cardiovascular disease risk can be identif ed. Genetic variations in hepatic lipase and endothelial lipase can also lead to increased HDL.

Secondary Hyperlipidemia In addition to the genetic causes o primary dyslipidemia described above, a number o secondary actors can lead to

C h a p t e r 2 0 Pharmacology of Cholesterol and Lipoprotein Metabolism 349

TABLE 20-2 Secondary Causes of Hyperlipidemia HYPERTRIGLYCERIDEMIA

HYPERCHOLESTEROLEMIA

Diabetes mellitus

Hypothyroidism

Chronic renal failure

Nephrotic syndrome

Hypothyroidism

Anorexia nervosa

Glycogen storage disease

Acute intermittent porphyria

Stress

Cholestasis

Sepsis

Obstructive liver disease

Alcohol excess

Corticosteroid treatment

Lipodystrophy

Protease inhibitor therapy

Pregnancy Oral estrogen replacement therapy Antihypertensive drugs: betablockers, diuretics Glucocorticoid treatment Protease inhibitor therapy Acute hepatitis Systemic lupus erythematosus Numerous secondary causes of hyperlipidemia exist; screening for the presence of these underlying factors should be performed before initiating pharmacologic therapy for a dyslipidemia. These lists are not exhaustive.

hyperlipidemia (Table 20-2). For example, alcohol intake increases the synthesis o atty acids, which are then esterif ed to glycerol to orm triglycerides. There ore, excess alcohol consumption can result in increased VLDL production. Hypertriglyceridemia in type 2 diabetes mellitus results rom

increased VLDL synthesis and secretion and rom reduced chylomicron and VLDL catabolism by LPL. Furthermore, apoCIII levels are increased in association with insulin resistance, and this reduces the catabolism o chylomicrons and VLDL particles. Hypothyroidism is an important and common cause o secondary hyperlipidemia. Any patient with a lipid disorder should be screened or hypothyroidism.

PHARMACOLOGIC CLASSES AND AGENTS The decision to treat dyslipidemia is largely dependent on the calculated cardiovascular risk. A number o clinical algorithms exist or determining initiation o therapy. Goals or lipid lowering were established in the 2001 National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines, which were updated in 2004 based on the results o several additional large, randomized clinical trials. These guidelines provide target LDL levels based on 10-year risk o death rom cardiovascular disease (Table 20-3) and have been generally adopted in clinical practice. In 2013, the American College o Cardiology and American Heart Association (ACC/AHA) published new guidelines on the treatment o cholesterol. These guidelines no longer utilize baseline LDL-C as an indication to initiate treatment, nor do they establish LDL-C goals o treatment. Instead, the new guidelines def ne our discrete “statin benef t groups” (Table 20-4). Both sets o guidelines emphasize the importance o therapeutic li estyle changes (TLCs), which include reduction o dietary saturated at and cholesterol intake, weight reduction, increased physical activity, avoidance o tobacco products, and, possibly, stress reduction. Success ul dietary therapy can reduce total cholesterol by up to about 25%, depending on adherence and the metabolic basis or elevated cholesterol concentrations. I this approach is unsuccess ul or insu f cient to normalize lipid levels, drug therapy is generally recommended. Five well-established classes o drugs are available or pharmacologic modif cation o lipid metabolism. Three o these classes (inhibitors

TABLE 20-3 Updated National Cholesterol Education Program Adult Treatment Panel III Guidelines ATP 2004 UPDATE: LDL-C THERAPY BY RISK CATEGORIES BASED ON RECENT CLINICAL TRIAL EVIDENCE RISK CATEGORY

LDL-C GOAL

INITIATE THERAPEUTIC LIFESTYLE CHANGES

CONSIDER DRUG THERAPY

High risk: CHD or CHD risk equivalents (10-year risk 20%)

100 mg/dL; optional goal 70 mg/dL

100 mg/dL

100 mg/dL

Moderately high risk: 2 risk factors (10-year risk 10–20%)

130 mg/dL

130 mg/dL

130 mg/dL (consider drug options if 100–129 mg/dL)

Moderate risk: 2 risk factors (10-year risk 10%)

130 mg/dL

130 mg/dL

160 mg/dL

Low risk: 0–1 risk factor

160 mg/dL

160 mg/dL

190 mg/dL (consider drug options if 160–189 mg/dL)

Adapted with permission from Grundy SM, Cleeman J I, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J Am Coll Cardiol 2004;44:720–732. More information about lipid management guidelines and details about calculation of cardiovascular risk are available at: https://www.nhlbi.nih.gov/health-pro/guidelines/ in-develop/cholesterol-in-adults. LDL-C, low-density lipoprotein cholesterol; CHD, coronary heart disease.

350 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

TABLE 20-4 2013 American College o Cardiology/American Heart Association Guideline on the Treatment o Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults Statin Therapy or Individuals at Increased ASCVD Risk Based on Recent Clinical Trial Evidence STEP 1: For adults, check baseline asting lipids, counsel on therapeutic li estyle changes, and assign to a statin benef t group: Clinical ASCVD

Initiate statin therapy at moderate intensity (age (age 75).

LDL 190 mg/dL

Initiate statin therapy at high intensity.

Diabetes type I/II

Initiate statin therapy at moderate intensity (high intensity if 10-year ASCVD risk 7.5%).

7.5% estimated 10-year ASCVD risk

75) or high intensity

Moderate- to high-intensity statin therapy.

STEP 2: Reassess or adherence, response to treatment; consider checking LDL-C and other biomarkers as indicated. Adapted with permission from Stone NJ , Robinson J G, Lichtenstein AH, et al. 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults. Circulation 2014;129:S1–S45. (New clinical guidelines or cholesterol-lowering therapy based on the def nition o our “statin benef t groups.”) More information about lipid management guidelines and details about calculation of cardiovascular risk are available at https://my.americanheart.org/professional /StatementsGuidelines/PreventionGuidelines/Prevention-Guidelines_UCM_457698_SubHomePage.jsp. Moderate-intensity statin therapy: daily dose lowers LDL-C by 30% to 50%. High-intensity statin therapy: daily dose lowers LDL-C by 50% or greater. ASCVD, atherosclerotic cardiovascular disease; LDL-C, low-density lipoprotein cholesterol.

o cholesterol synthesis, bile acid sequestrants, and cholesterol absorption inhibitors) have relatively well-def ned e ects on lipid metabolism. While the overall e ects o the other two classes (f brates and niacin) are clear, their molecular mechanisms o actions are diverse and remain subjects o active investigation. The inhibitors o cholesterol synthesis (i.e., HMG-CoA reductase inhibitors, also known as statins) are the most important class due to their welldemonstrated e f cacy in reducing cardiovascular morbidity and mortality. However, agents in each o the other classes act as important adjunctive therapies and may be the agents o choice or patients with certain specif c causes o dyslipidemia. The newest therapies or treating rare diseases and individuals with inadequate response to maximum medical management include VLDL secretion inhibitors and PCSK9 inhibitors.

Inhibitors o Cholesterol Synthesis Statins competitively inhibit the activity o HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Inhibition o this enzyme results in a transient, modest decrease in cellular cholesterol concentration (Fig. 20-11). The decrease in cholesterol concentration activates a cellular signaling cascade culminating in the activation o sterol regulatory element binding protein 2 (SREBP2), a transcription actor that up-regulates expression o the gene encoding the LDL receptor. Increased LDL receptor expression causes increased uptake o plasma LDL and consequently decreases plasma LDL-cholesterol concentration. Approximately 70% o LDL receptors are expressed by hepatocytes, with the remainder expressed by a variety o cell types in the body. Statins have been shown in numerous clinical trials to reduce mortality signif cantly a ter a myocardial in arction. This is re erred to as secondary prevention. Recent studies have also concluded that lowering o LDL with statins can decrease mortality even in the absence o overt

cardiovascular disease, which is called primary prevention. Despite these convincing percentage risk reductions in both secondary and primary prevention trials, it should be noted that statin use is associated with a greater absolute risk reduction in secondary prevention; the reason may be that patients in this treatment group have a higher absolute risk o death and there ore display the greatest benef t rom statins. It is also important to note that statins have proven to be e ective in reducing cardiovascular disease risk or highrisk patients (e.g., diabetic patients) with average, or even below average, LDL-cholesterol levels. The magnitude o LDL-cholesterol lowering depends on the e f cacy and dose o the statin that is administered. In general, statins reduce LDL-cholesterol concentrations by up to about 60%. Statins increase HDL-cholesterol concentrations by an average o 10% and reduce triglyceride concentrations by up to about 40%, depending on statin dose and degree o hypertriglyceridemia. The e ect o statins on triglyceride levels is mediated by decreased VLDL production and increased clearance o remnant lipoproteins by the liver. The dose–response relationship o statins is nonlinear: the largest e ect occurs with the starting dose. Each subsequent doubling o the dose produces, on average, an additional 6% LDL reduction. This is sometimes re erred to as the “rule o 6s.” In addition to reducing LDL-cholesterol concentrations, statins have a number o other pharmacologic consequences. These are collectively re erred to as pleiotropic effects, which include decreased in ammation, reversal o endothelial dysunction, decreased thrombosis, and improved stability o atherosclerotic plaques. Evidence or diminished in ammation with statin therapy includes decreases in acute-phase reactants, which are plasma proteins that are increased during in ammatory states and may play a role in the destabilization o atherosclerotic plaques. The best characterized acutephase reactant is C-reactive protein (CRP). Importantly, a recent large randomized clinical trial has shown that, among patients with a moderate risk o developing cardiovascular

C h a p t e r 2 0 Pharmacology of Cholesterol and Lipoprotein Metabolism 351 Incre a s e d LDL-R e xpre s s ion a nd upta ke of pla s ma LDL

Ace tyl CoA + Ace toa ce tyl CoA

Statins HMG CoA HMG CoA reductase Me va lona te

Incre a s e d LDL re ce ptor e xpre s s ion

5-pyrophos phome va lona te Is ope ntylpyrophos pha te 3,3-dime thyla llylpyrophos pha te Is opre noids Ge ra nylpyrophos pha te Fa rne s ylpyrophos pha te S qua le ne La nos te rol Chole s te rol Chole s te rol

P rote a s e a ctiva tion S REBP (ina ctive )

S REBP (a ctive )

Nucle us LDL-R ge ne

S RE

FIGURE 20-11. Mechanism o LDL lowering by statins. Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. Decreased cellular cholesterol concentrations lead to protease activation and cleavage of the sterol regulatory element binding protein (SREBP), which is a transcription factor that normally resides in the cytoplasm. The cleaved SREBP diffuses into the nucleus, where it binds to sterol response elements (SRE), leading to up-regulation of LDLreceptor gene transcription. This leads to increased cellular LDLreceptor expression. This promotes uptake of LDLparticles and results in reduced LDL-cholesterol concentrations in the plasma.

disease and with elevated baseline CRP levels, use o a statin reduces cardiovascular morbidity and mortality, even when the patients do not have elevated LDL-cholesterol concentrations. Evidence or reversal o endothelial dys unction with statin therapy includes an improved vasodilatory response o endothelium to nitric oxide. Improved vasodilation could

help prevent ischemia. Evidence or decreased thrombosis with statin therapy includes a decrease in prothrombin activation and a decrease in tissue actor production. Because thrombosis is at the root o most acute coronary syndromes, its reduction could contribute to the survival benef t o statins. Finally, plaque stability is enhanced with statin therapy because the f brous cap that overlies the lipid-rich plaque becomes thicker. This e ect may be attributable to decreased macrophage inf ltration and inhibition o vascular smooth muscle proli eration. It is important to emphasize that most o these pleiotropic e ects o statins have been demonstrated only in vitro or in animal models, and their relevance in humans is unclear. Clinical data indicate that the reductions in cardiovascular morbidity and mortality due to statins are primarily attributable to the lowering o LDLcholesterol concentrations in the plasma. Seven statins—lovastatin, pravastatin, simvastatin, f uvastatin, atorvastatin, rosuvastatin, and pitavastatin—are currently approved or use in hypercholesterolemia and mixed hyperlipidemia. They are considered f rst-line therapy or increased LDL levels, and their use is supported by numerous trials showing that statins decrease both cardiovascularrelated and total mortality. Stroke is also reduced. All o the statins are believed to act by the same mechanism. The main di erences are attributable to potency and pharmacokinetic parameters. Among the statins, uvastatin is the least potent, and atorvastatin and rosuvastatin are the most potent. Beyond their capacity to reduce LDL-cholesterol concentrations, the clinical relevance o these potency di erences has not been determined. The pharmacokinetic di erences among the statins result rom di erential cytochrome P450 metabolism. Lovastatin, simvastatin, and atorvastatin are metabolized by CYP3A4, whereas other cytochrome P450-mediated pathways metabolize uvastatin and pitavastatin. Pravastatin and rosuvastatin are not metabolized via the cytochrome P450 pathway. As explained below, the pathways o statin metabolism have important implications or drug interactions. Statins are generally well tolerated; the incidence o adverse e ects is lower with statins than with any o the other lipid-lowering drug classes. The main adverse e ect is myopathy and/or myositis with rhabdomyolysis. The latter is a very rare complication that occurs primarily at high doses o the most potent statins. There ore, plasma creatine kinase levels (a marker o muscle injury) are not use ul or routine monitoring o statin-treated patients. Certain patients who have inherited a molecular variant o an organic anion transporter responsible or statin uptake may be at higher risk o developing statin-induced myopathy (see Chapter 7, Pharmacogenomics). High-potency statins can also cause increases in serum transaminase levels (i.e., alanine transaminase [ALT] and aspartate transaminase [AST]). In the vast majority o cases, these commonly observed elevations in ALT and AST most likely re ect an adaptive response o the liver to changes in cholesterol homeostasis. True hepatotoxicity is indicated by ALT and AST elevations that are accompanied by elevations in serum bilirubin concentrations. I a statin alone is insu f cient to lower LDL to target levels, the statin can be used e ectively in combination with other agents. The combination o a statin with a bile acid sequestrant or cholesterol absorption inhibitor results in additive LDL decreases and is not associated with signif cant drug interactions. The combination o niacin and

352 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

a statin may be most use ul in patients with high levels o LDL cholesterol and low levels o HDL cholesterol. However, because co-administration o niacin and a statin could slightly increase the risk o myopathy, such patients should be closely monitored or the development o adverse e ects. Fibrates and statins have also been reported to be e f cacious in combination. However, certain f brates inhibit both the transport o statins into the liver and the glucuronidation o statins in the liver, thereby decreasing statin clearance. These agents may there ore raise the plasma statin concentration and increase the risk o rhabdomyolysis. This e ect has been documented or gemf brozil but does not occur with enof brate . Finally, in patients who require LDL lowering and are taking drugs that are metabolized by cytochrome P450—such as certain antibiotics, calcium channel blockers, war arin, and protease inhibitors (see Chapter 4, Drug Metabolism)—a statin that is not metabolized by P450 enzymes is pre erable.

Inhibitors o VLDL Secretion Currently approved inhibitors o VLDL secretion act by two di erent mechanisms (Fig. 20-3). Lomitapide is a small molecule that inhibits lipid trans er by binding to MTP, while mipomersen is a synthetic single-strand antisense oligonucleotide that binds to the apoB100 mRNA and thereby reduces apoB protein levels. The net e ect o each drug is to reduce VLDL secretion. Lomitapide is approved or use in patients with homozygous amilial hypercholesterolemia (HoFH). At therapeutic doses, lomitapide reduces LDL-C by 30–50% in these patients. Adverse e ects include gastrointestinal distress due to at malabsorption, reductions in plasma vitamin E levels, and transaminase elevations that are correlated with increased hepatic at content. Transaminase elevations have not been associated with parallel increases in plasma bilirubin concentrations and generally normalize with continued lomitapide treatment. Mipomersen is indicated or the treatment o patients with homozygous FH who are already prescribed maximal medical therapy. Adverse e ects may include injection site reactions, u-like symptoms, increases in C-reactive protein, and increased transaminases. Similar to lomitapide, the liver test abnormalities likely correspond to an increase in liver at content. This increased at content appears to remain stable over 1 year o treatment and is reversible with cessation o therapy.

Inhibitors o Bile Acid Absorption The bile acid sequestrants are cationic polymer resins that bind noncovalently to negatively charged bile acids in the small intestine. The resin–bile acid complex cannot be reabsorbed in the distal ileum and is excreted in the stool. Decreased bile acid reabsorption by the ileum partially interrupts enterohepatic bile acid circulation, causing hepatocytes to up-regulate 7 -hydroxylase, the rate-limiting enzyme in bile acid synthesis (Fig. 20-10A). The increase in bile acid synthesis decreases hepatocyte cholesterol concentration, leading to increased expression o the LDL receptor and enhanced LDL clearance rom the circulation. The e ectiveness o bile acid sequestrants in clearing LDL rom the plasma is partially o set by concomitant up-regulation o hepatic cholesterol and triglyceride synthesis, which

stimulates the production o VLDL particles by the liver. As a result, bile acid sequestrants may also raise triglyceride levels and should be used with caution in patients with hypertriglyceridemia. The three available bile acid sequestrants are cholestyramine , colesevelam, and colestipol. These drugs possess similar e f cacy, causing up to 28% reductions in LDL levels at therapeutic concentrations. In order to maximize the binding o these agents to bile acids, drug administration is timed so that the drugs are present in the small intestine a ter a meal (i.e., a ter gallbladder emptying). Because bile acid sequestrants are not absorbed systemically, they have little potential or serious toxicity. However, signif cant bloating and dyspepsia o ten limit patient adherence. Bile acid sequestrants can decrease absorption o at-soluble vitamins, and bleeding due to vitamin K def ciency has occasionally been reported. They can also bind certain co-administered drugs, such as digoxin and war arin, and thereby lower the bioavailability o the co-administered agents. This interaction can be eliminated by administering the bile acid sequestrant at least 1 hour be ore or 4 hours a ter other drugs. Colesevelam is more selective and appears to avoid this problem. Because o the demonstrated clinical e f cacy and tolerability o statins, bile acid sequestrants have been relegated to second-line agents or lipid reduction. Currently, bile acid sequestrants are used mainly or treatment o hypercholesterolemia in young ( 25 years old) patients and in patients or whom statins alone do not provide su f cient plasma LDL reduction. Some experts pre er bile acid sequestrants or young patients (such as patients with amilial hypercholesterolemia) because these agents are not absorbed and are generally considered sa e or long-term use. However, other experts pre er to use a statin or initial therapy in children.

Inhibitors o Cholesterol Absorption Cholesterol absorption inhibitors reduce cholesterol absorption by the small intestine. Although this involves reduced absorption o dietary cholesterol, the more important e ect is reduced reabsorption o biliary cholesterol, which comprises the majority o intestinal cholesterol. Whereas statins and bile acid sequestrants reduce LDL cholesterol principally by increasing LDL clearance via the LDL receptor, inhibitors o cholesterol absorption also appear to reduce LDL cholesterol by inhibiting hepatic production o VLDL. The two available cholesterol absorption inhibitors are plant sterols and ezetimibe . Plant sterols and stanols are naturally present in vegetables and ruits, and they may be consumed in larger amounts rom nutritional supplements. Plant sterols and stanols are similar in molecular structure to cholesterol but are substantially more hydrophobic. As a result, plant sterols and stanols displace cholesterol rom micelles, increasing the excretion o cholesterol in the stool. The plant sterols and stanols are themselves poorly absorbed. Based on their mechanism o action, gram quantities o plant sterols and stanols are required to reduce plasma LDL-cholesterol concentrations by approximately 15%. Because an average diet contains 200–400 mg o plant sterols and stanols, these molecules must be highly enriched in dietary supplements (to approximately 2 grams) in order to be e ective. Ezetimibe decreases cholesterol transport rom micelles into enterocytes by selectively inhibiting cholesterol uptake through the brush border protein NPC1L1 (Fig. 20-4).

C h a p t e r 2 0 Pharmacology o Cholesterol and Lipoprotein Metabolism 353

At therapeutic concentrations, ezetimibe reduces intestinal cholesterol absorption by about 50%, without reducing the absorption o triglycerides or at-soluble vitamins. The end result o reduced cholesterol absorption, achieved by either plant sterols and stanols or ezetimibe, is a decrease in LDL-cholesterol concentrations in the plasma. A reduction in cholesterol absorption presumably decreases the cholesterol content o chylomicrons and there ore decreases the transport o cholesterol rom the intestine to the liver. Within the liver, cholesterol derived rom chylomicron remnants contributes to the cholesterol that is packaged into VLDL particles. Thereore, inhibiting cholesterol absorption can reduce cholesterol incorporation into VLDL and decrease LDL-cholesterol concentrations in the plasma. Importantly, reduced hepatic cholesterol content also leads to up-regulation o the LDL receptor, which contributes to the mechanism o LDL lowering by cholesterol absorption inhibitors. A single daily dose o ezetimibe lowers LDL-cholesterol concentrations by up to about 20%. Ezetimibe also lowers triglyceride concentrations by about 8% and elevates HDL cholesterol to a small extent (approximately 3%). Ezetimibe is particularly e ective in combination with a statin, or the ollowing reason. The reduction in hepatic cholesterol content due to inhibition o cholesterol absorption leads to a compensatory increase in hepatic cholesterol synthesis that partially o sets the benef ts o reducing absorption. By combining ezetimibe with a statin, the compensatory increase in hepatic cholesterol synthesis is prevented. This approach reduces LDL-cholesterol concentrations by an additional 15% compared with the e ect o the statin alone. The e ect is similar throughout the statin dose range. Unlike bile acid sequestrants (which are not absorbed), ezetimibe is rapidly absorbed by the enterocyte and extensively glucuronidated, so that systemic concentrations o both unmodif ed and glucuronidated orms can be measured. Ezetimibe undergoes enterohepatic circulation up to several times each day in conjunction with meals. Cholesterol absorption inhibitors have exhibited good sa ety prof les, with ew i any adverse e ects. Ezetimibe can increase plasma concentrations o cyclosporine, which should be monitored whenever these two drugs are co-administered.

Fibrates Fibrates bind to and activate peroxisome proli eratoractivated receptor (PPAR ), a nuclear receptor expressed in hepatocytes, skeletal muscle, macrophages, and the heart. Upon binding o f brate, PPAR heterodimerizes with the retinoid X receptor (RXR). This heterodimer binds to peroxisome proli erator response elements (PPREs) in the promoter regions o specif c genes, activating transcription o these genes and thereby increasing protein expression. Activation o PPAR by f brates results in numerous changes in lipid metabolism that act collectively to decrease plasma triglyceride levels and increase plasma HDL (Fig. 20-12). The decrease in plasma triglyceride levels is caused in part by increased muscle expression o lipoprotein lipase, decreased hepatic expression o apolipoprotein CIII, and increased hepatic oxidation o atty acids. The increased muscle expression o LPL results in increased uptake o triglyceride-rich lipoproteins, with a resultant decrease in plasma triglyceride levels. Because apoCIII normally unctions to inhibit interaction o triglyceride-rich lipoproteins with their receptors, the decrease in hepatic production o apoCIII may potentiate the increased LPL activity.

Fibrates

P PAR α a ctiva tion

a poA-I, a poA-II s ynthe s is in he pa tocyte s

a poC-III s ynthe s is in he pa tocyte s a nd Lipoprote in lipa s e e xpre s s ion in mus cle va s cula r be ds

Fa tty a cid oxida tion in he pa tocyte s

Fa tty a cid upta ke in mus cle ce lls a nd Fa tty a cid oxida tion in mus cle ce lls

Triglyce ride s ynthe s is

P la s ma HDL

P la s ma triglyce ride s

FIGURE 20-12. In uence o f brates on lipid metabolism. Fibrates have several benef cial e ects on lipid metabolism, all o which appear to be secondary to the activation o the transcription actor PPAR . PPAR activation by f brates increases hepatic synthesis o apoAI and apoAII, which leads to increased plasma HDL-cholesterol concentrations. PPAR activation also down-regulates hepatic synthesis o apoCIII and increases lipoprotein lipase expression in muscle vascular beds. The decrease in apoCIII, an inhibitor o lipoprotein lipase, combines with the increase in lipoprotein lipase expression to increase atty acid uptake and atty acid oxidation in muscle cells. PPAR also increases atty acid oxidation in hepatocytes. The combined e ects o these metabolic changes are decreased plasma triglyceride concentrations and increased plasma HDLcholesterol. Because o decreased hepatic atty acid and triglyceride synthesis (not shown), LDLcholesterol concentrations also decrease modestly.

The mechanisms by which f brate-mediated PPAR activation raises plasma HDL depend at least in part on increased hepatic production o apolipoprotein AI. This would be expected to contribute directly to increased plasma HDL. Upregulation o ABCA1 in macrophages presumably promotes cholesterol e ux rom these cells in vivo. Hepatocytes also increase expression o SR-B1 in response to PPAR activation, providing a pathway or increased reverse cholesterol transport, with subsequent cholesterol excretion into bile. Fibrates also lower LDL levels modestly. The lower LDL levels result rom a PPAR -induced shi t in hepatocyte metabolism toward atty acid oxidation. PPAR increases the expression o numerous enzymes involved in atty acid transport and oxidation, thereby increasing atty acid catabolism and decreasing triglyceride synthesis and VLDL production. PPAR activation also results in LDL particles o larger size, which appear to be taken up more e f ciently by LDL receptors. Many o the e ects o PPAR on lipid metabolism remain the subject o basic and clinical investigation, which may lead to the development o more selective PPAR agonists that are capable o targeting selective aspects o lipid metabolism. Finally, f brates have a benef cial anti-in ammatory e ect, decreasing the vulnerability o atherosclerotic plaques to rupture. Gemf brozil and enof brate are the available f brates in the United States. Two other f brates, bezaf brate and ciprof brate , are available in Europe. Fibrates are indicated or treatment

354 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

o hypertriglyceridemia as well as hypertriglyceridemia with low HDL. They lower triglycerides by up to 50%, raise HDL by up to 20%, and lower LDL by up to 15%. In addition, f brates are the pre erred therapy or patients with dysbetalipoproteinemia. Because o their greater e f cacy, statins are pre erred over f brates or treatment o increased LDL levels. However, f brates (e.g., enof brate) can be used together with statins in cases o combined hyperlipidemia or when HDL cholesterol is decreased. Gastrointestinal discom ort is the most common adverse e ect o f brates. Rare adverse e ects include myopathy and arrhythmias. Increases in liver transaminases occur in about 5% o patients. Gastrointestinal disturbances and myopathy are less common with enof brate than with gemf brozil. Fibrates displace war arin rom albumin binding sites, resulting in increased ree war arin concentrations. Thereore, the response to war arin should be monitored when a f brate is co-administered. Gemf brozil-associated gallstone ormation is presumably a consequence o f brate-induced increases in biliary cholesterol excretion. However, screening or gallstones is not recommended. The e ect o a co-administered statin on f brate metabolism is described above.

Niacin Niacin (nicotinic acid, vitamin B3) is a water-soluble vita-

min. At physiologic concentrations, it is a substrate in the synthesis o nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are important co actors in intermediary metabolism. The pharmacologic use o niacin necessitates large doses (1,500–3,000 mg/day) and is independent o the conversion o nicotinic acid to NAD or NADP (Fig. 20-13). Niacin decreases plasma LDL-cholesterol and triglyceride concentrations and increases HDL cholesterol. Studies have identif ed a G protein-coupled receptor on adipocytes that appears to mediate the metabolic changes associated with niacin administration. Stimulation o this receptor by niacin decreases adipocyte hormone-sensitive lipase activity, leading to reduced peripheral tissue triglyceride catabolism and there ore decreased ux o ree atty acids to the liver. This decreases

Adipose tissue

Hormone -s e ns itive lipa s e

the rate o hepatic triglyceride synthesis and VLDL production, leading to decreases in triglycerides (by up to 45%) and LDL (by up to 20%). Niacin also increases the hal -li e o apoAI, the major apolipoprotein in HDL. The increase in plasma apoAI increases plasma HDL concentrations by up to 30% and presumably augments reverse cholesterol transport. Pharmacologic doses o niacin are available as oral agents or daily administration. The major adverse e ects o niacin are cutaneous ushing and pruritus (itching). The ushing is mediated by the G protein-coupled niacin receptor and involves the release o prostaglandins D2 and E2 within the skin. It can be mitigated by pretreatment with aspirin or another nonsteroidal anti-in ammatory drug (NSAID). These adverse e ects usually disappear a ter several weeks o niacin use. Timed-release ormulations o niacin are associated with less cutaneous ushing than the immediate-release dosage orm. In addition to ushing and pruritus, important adverse e ects o niacin include hyperuricemia, impaired insulin sensitivity, hepatotoxicity, and the potentiation o statin-induced myopathy. Hyperuricemia may precipitate gout. Impaired insulin sensitivity may precipitate diabetes in patients at risk, and niacin should be used with caution in diabetic patients. Rarely, niacin may cause myopathy. Concurrent administration o niacin with a statin slightly increases the risk o myopathy. Niacin is indicated or patients with elevations o both triglycerides and cholesterol, usually in combination with a statin. Because niacin is currently the most e ective agent available or raising HDL, it may also be the drug o choice or patients with modestly elevated LDL and decreased HDL. It is not clear whether both the LDL-lowering and HDL-raising e ects o niacin contribute to improved clinical outcomes.

Omega-3 Fatty Acids The omega-3 atty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), also re erred to as f sh oils, are e ective at reducing plasma triglycerides by up to 50% in patients with hypertriglyceridemia. The likely mechanism

Liver De cre a s e d pla s ma fre e fa tty a cids

De cre a s e d s ynthe s is of triglyce ride s

Peripheral cells De cre a s e d pla s ma VLDL

Chole s te rol de live ry Nia cin re ce ptor Niacin

Incre a s e d e xcre tion of chole s te rol in bile

De cre a s e d pla s ma LDL

Incre a s e d pla s ma HDL

Chole s te rol de live ry

Chole s te rol re mova l

De cre a s e d a poA-I cle a ra nce Niacin

FIGURE 20-13. Inf uence o niacin on lipid metabolism. Niacin lowers triglyceride and LDLlevels while increasing HDL. Activation by niacin o a G proteincoupled receptor on adipocytes results in decreased hormone-sensitive lipase activity in adipose tissue, which decreases the f ux o ree atty acids to the liver. The decreased ree atty acid f ux reduces hepatic triglyceride synthesis and limits VLDL synthesis. Because LDL is derived rom VLDL, the decreased VLDL synthesis decreases plasma concentrations o LDL cholesterol. Niacin also increases the hal -li e o apoAI, an important apolipoprotein in HDL. The increased apoAI levels directly increase levels o plasma HDL and may also augment reverse cholesterol transport, delivery o cholesterol rom HDL to the liver, and excretion o cholesterol in the bile.

C h a p t e r 2 0 Pharmacology of Cholesterol and Lipoprotein Metabolism 355

o triglyceride lowering involves regulation o nuclear transcription actors, including SREBP-1c and PPAR , to cause reduced triglyceride biosynthesis and increased atty acid oxidation in the liver. Omega-3 atty acids are available over the counter as nutritional supplements in the orm o atty acid ethyl esters. Lovaza ® , a prescription-strength orm o omega-3 atty acids, has also become available. Lovaza® is enriched (84%) in EPA and DHA, whereas most dietary supplements contain 13–63% f sh oils. The recommended dose o Lovaza® is 4 grams once a day. Omega-3 atty acids are generally added to therapy when plasma triglyceride concentrations exceed 500 mg/dL. The in uence o omega-3 atty acid use on clinical outcomes is uncertain.

PCSK9 Inhibitors When LDL-R degradation is reduced by genetic loss-o unction mutations in PCSK9, cardiovascular disease incidence is dramatically reduced by 88%. This observation has led to aggressive interest in PCSK9 as a therapeutic target. Monoclonal antibodies that target the LDL-R—interacting domain o PCSK9 are newly approved by the FDA or the treatment o patients with heterozygous FH or clinical atherosclerotic cardiovascular disease (ASCVD) who require additional therapy beyond diet and maximal statin therapy. These include evolocumab and alirocumab, which are administered subcutaneously and can lower LDL-C by 50–72%. These drugs have been well tolerated in short-term clinical trials and in post-marketing surveillance, with no major reported adverse e ects. The hal -li e o these antibodies is long, and the LDL-C lowering e ects persist or up to 2–4 weeks. Interestingly, PCSK9 degrades many receptor targets (LDL-R, apoE-R2, VLDL-R, and LRP1) that are also receptors or viruses, such as human rhinovirus and hepatitis C virus. This suggests that the incidence o viral in ection will need to be monitored closely in ongoing large-scale clinical trials o PCSK9 inhibitors.

CONCLUSION AND FUTURE DIRECTIONS LDL reduction by available lipid-lowering drugs—particularly statins—represents an important advance in reducing cardiovascular disease mortality. The recent FDA approval o inhibitors o VLDL secretion has provided valuable new therapies or the treatment o patients with homozygous and severe heterozygous FH. PCSK9 inhibitors will now o er options or patients who require additional LDL-C reduction despite maximal medical therapy. Future research will evaluate the long-term sa ety o these new medications, clari y the biology o sortilin, and examine the possible benef ts on cardiovascular disease o therapies that raise HDL-C (e.g., CETP inhibitors) and lower triglyceride levels.

Suggested Reading Ballantyne CM, ed. Clinical lipidology: a companion to Braunwald’s heart disease. 2nd ed. Philadelphia: Saunders/Elsevier; 2015;550 pp. (Concise chapters cover all aspects of lipoprotein metabolism and pharmacology.) Degoma EM, Rader DJ. Novel HDL-directed pharmacotherapeutic strategies. Nat Rev Cardiol 2011;8:266–277. (Review of treatments targeting HDL pathways.) Lukasova M, Malaval C, Gille A, Kero J, O ermanns S. Nicotinic acid inhibits progression o atherosclerosis in mice through its receptor GPR109A expressed by immune cells. J Clin Invest 2011;121:1163–1173. (Recent developments in biology of niacin and its therapeutic potential.) Rader D, Kastelein J. Lomitapide and mipomersen: two f rst-in-class drugs or reducing low-density lipoprotein cholesterol in patients with homozygous amilial hypercholesterolemia. Circulation 2014;129:1022–1032. (Summary of the role of VLDL synthesis inhibition therapies, including clinical trial data.) Shimada YJ, Cannon CP. PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the uture. Eur Heart J 2015;36:2415– 2424. (A comprehensive review of PCSK9 biology and pharmacology.) Strong A, Rader D. Sortilin as a regulator o lipoprotein metabolism. Curr Atheroscler Rep 2012;14:211–218. (Review of sortilin’s role in lipoprotein biology.)

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C h a p t e r 2 0 Pharmacology o Cholesterol and Lipoprotein Metabolism 357

Appiicaa l me mbra b ne bra ne

21

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NK NKC NKCC2

Ha ka n R. To ka a n d S e t h L. Alp eKr +

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358–359 3358 5 359 58–359 PHYSIOLOGY OF VOLU UME REG EGULAT ATIO ION N . . . . . . . . . .ROM . . . . 3588 R OM MK Determinantss of In Intravascular Volume . . . . . . . . . . . . . . . . . . . 3558 Volu olum me Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 Volume Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 60 2+ Renin-Angiotensin-Aldoster eron onee Sy SystemCa C . . . . . . . . . . . . . 36 360 60 Natr Na triu iure retiic Pe Pept ptid idess . . . . . . . . . . . . . . . . . . . .2.+. . . . . . . . . . . 363 633 Mg Mg Antid idiuretic Hormone nee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 + Renal Symppathetic Nee rves es. . . . . . . . . . . .Na . . . . . . . 10mV . . . . . . 3644 Renal Control of Na Excretion . . . . . . . . . . . . . . . . . . . . . . . . . 33644 Proximal Tubbul ulee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Thick Ascending Limb of the Loop of Henlee . . . . . . . . . . . . 366 3666 Distal Convoluted Tubulee . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 Collecting Duct. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 PATHOPHYSIOLOGY OF EDEMA FORMATION . . . . . . . . . . . . . . 370 Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370

INTRODUCTION Coordinated regulation o volume homeostasis and vascular tone maintains adequate tissue per usion in response to varying environmental stimuli. This chapter discusses the pharmacologically relevant physiology o volume regulation, with emphasis on the hormonal pathways and renal mechanisms that modulate systemic volume. (Control o vascular tone is discussed in Chapter 22, Pharmacology o Vascular Tone.) Dysregulation o volume homeostasis can result in edema, the pathologic accumulation o f uid in the extravascular space. Pharmacologic modulation o volume is targeted at reducing volume excess; this is an e ective treatment or hypertension and heart ailure (HF), as well as or cirrhosis and the nephrotic syndrome. The two broad classes o pharmacologic agents used to modi y volume status are modulators o neurohormonal regulators (such as angiotensin converting enzyme [ACE] inhibitors) and diuretics (agents that increase renal Na excretion). Drugs that modi y volume regulation also have many other clinically important e ects on the body, because these volume regulators act as diverse hormonal modulators in multiple physiologic pathways. Many o the clinical applications o these agents are discussed urther in Chapter 26, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure. 358

+ ClK Cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 Ci Nephroticc Sy Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372 CLC K2 CLC-K2 PHA PH ARMACO MACOLO L GIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 372 Agents That Modify Vollum A me Re Regu gula l tors . . . . . . . . . . . . . . . . . . 372 Inhibitors of the Renin-Angiotensinn Sy Systtem m . . . . . . . . . . . . 372 B-Type yp Natriuretic Peptide p . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 Vasopressin p i Receptor R Rece cept pptor Antagonis Anta A Antagonists ta ggonists is ttss and a nd dA Ag Agonis ggonists onists i s . . . . . . . 37 374 Agent A gents Th Thatt D Decrrease Decrea eaa se R Renal en nall N Naa Reabsorption Rea eabs bsor b orpt ptio tiion . . . . . . . . . . 374 Ca rbo Ca Carb rbbonic niic Anhydrase Anhydrase Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . 375 Osmo oti ticc Di Diur uret etic icss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Loop Diuretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 L 3755 Thiazidess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 Collecting Duct (Potassium-Sparing) Diureticss . . . . . . . . . 377 CONC CO NCLU LUSI SION ON AND FUT UTUR URE E DI DIRE RECT CTIO IONS NS . . . . . . . . . . . . . . . . . . 37 3788 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .379

PHYSIOLOGY OF VOLUME REGULATION An intricate set o mechanisms sense, signal, and modulate changes in plasma volume. Volume sensors are located throughout the vascular tree, including in the atria and in the kidneys. Many o the volume regulators activated by these sensors include systemic and autocrine hormones, while others involve neural circuits. The integrated result o these signaling mechanisms is to alter vascular tone and to regulate renal Na reabsorption and excretion. Vascular tone maintains end-organ tissue per usion; changes in renal Na excretion alter total volume status.

Determinants of Intravascular Volume Intravascular volume is a small proportion o total body water, but the amount o f uid in the vascular compartment critically determines the extent o tissue per usion. Approximately 2/3 o total body water is intracellular, while 1/3 is extracellular. O the extracellular f uid (ECF), approximately 3/4 resides in the interstitial space, while 1/4 o ECF is plasma. Fluid exchange between plasma and interstitial compartments occurs as a result o changes in capillary permeability, oncotic pressure, and hydrostatic pressure. Capillary permeability is determined largely by the junctions between

C h a p t e r 2 1 Pharmacology of Volume Regulation

Mr. R, a 70-ye a r-o ld m a le , is ta ke n b y a m b u la n ce to th e e m e rg e n cy d e p a rtm e n t a t 1:0 0 AM a te r wa kin g u p w ith s h o rtn e s s o b re a th o r th e o u rth n ig h t in a ro w. Ea ch tim e , h e “ e lt tig h t in th e ch e s t” a n d “ co u ld n’t g e t a b re a th ” ; th is d is co m o rt wa s re lie ve d s o m e w h a t b y s ittin g u p in b e d . He a ls o re ca lls p re vio u s e p is o d e s o s h o rtn e s s o b re a th w h ile clim b in g s ta irs . Phys ica l e xa m re ve a ls ta chyca rd ia (h e a rt ra te , 112/ m in ), m ild hyp e rte n s io n (b lo o d p re s s u re , 155/95 m m Hg ), d e cre a s e d o xyg e n s a tu ra tio n (90% o n ro o m a ir), in cre a s e d re s p ira to ry ra te (28/m in ), b ila te ra l p u lm o n a ry cra ckle s o n in s p ira tio n , a n d 1–2 e d e m a o th e e e t. Se ru m tro p o n in T le ve l (a m a rke r o ca rd io m yo cyte in ju ry) is n o rm a l, b u t s e ru m cre a tin in e (1.5 m g /d L) a n d b lo o d u re a n itro g e n (BUN, 30 m g / d L) a re m ild ly e le va te d . Urin a lys is is n o rm a l. Ele ctro ca rd io g ra m s h o w s e vid e n ce o a n o ld m yo ca rd ia l in a rctio n (Q wa ve in le a d s II, III, a n d V4–V6). Ech o ca rd io g ra p hy re ve a ls d im in is h e d le t ve n tricu la r e je ctio n ra ctio n (LVEF, 35%; th e ra ctio n o b lo o d in th e ve n tricle a t th e e n d o d ia s to le th a t is e je cte d w h e n th e ve n tricle co n tra cts ) w ith o u t ve n tricu la r d ila ta tio n .

individual endothelial cells lining a vascular space. The capillary beds o some organs are more permeable than those o others and, as a result, allow larger intercompartmental f uid shi ts. In the context o inf ammation and other pathologic conditions (see below), increased capillary permeability allows proteins to shi t, along with “oncotically obligated water,” between intravascular and perivascular compartments under the inf uence o the plasma oncotic pressure gradient. Oncotic pressure is determined by the molecular solute components o a f uid space that are di erentially partitioned between adjacent compartments (such constituents are said to be osmotically active). Because albumin, globulins, and other large plasma proteins are normally con ned to the plasma space, these oncotically active proteins serve to retain water in the vascular space. The hydrostatic pressure gradient across the capillary barrier between compartments is another orce or water movement. An elevated intracapillary pressure avors increased transudation o f uid rom plasma into the interstitial space. The relationship between f uid ltration and capillary permeability, oncotic pressure, and hydrostatic pressure is represented by the ollowing equation Equation 21-1 Fluid Filtration Kf (Pc Pif) ( c if) where Kf is the capillary permeability coe cient, Pc is capillary hydrostatic pressure, Pif is interstitial f uid hydrostatic pressure, c is capillary oncotic pressure, and if is interstitial f uid oncotic pressure. This equation emphasizes that transcapillary f uid movement is governed by intercompartmental gradients rather than by the absolute value o each

359

Ba s e d o n th e clin ica l f n d in g s o d e cre a s e d ca rd ia c o u tp u t, p u lm o n a ry co n g e s tio n , a n d p e rip h e ra l e d e m a , Mr. R is d ia g n o s e d w ith a cu te h e a rt a ilu re . His in cre a s e d cre a tin in e a n d BUN a ls o in d ica te a n e le m e n t o re n a l in s u f cie n cy. Ph a rm a co lo g ic th e ra p y is s ta rte d , in clu d in g a co ro n a ry va s o d ila to r, a n a n tih yp e rte n s ive ca lciu m ch a n n e l b lo cke r, a n d a lo o p d iu re tic. A te r Mr. R’s co n d itio n s ta b ilize s ove r th e co u rs e o 3 d a ys , th e d o s e o th e lo o p d iu re tic is d e cre a s e d a n d th e n d is co n tin u e d . Ele ctive co ro n a ry a n g io g ra p hy re ve a ls s ig n if ca n t s te n o s is o th e le t a n te rio r d e s ce n d in g co ro n a ry a rte ry. Mr. R u n d e rg o e s b a llo o n a n g io p la s ty a n d s te n t p la ce m e n t a n d re m a in s s ta b le . Mr. R is d is ch a rg e d o n a re g im e n th a t in clu d e s a n ACE in h ib ito r a n d s p iro n o la cto n e .

Questions 1 . What mechanisms led to Mr. R’s pulmonary congestion and pedal edema? 2 . Why was Mr. R given a loop diuretic? 3 . How do ACE inhibitors improve cardiovascular hemodynamics? 4 . Why was Mr. R prescribed spironolactone?

compartmental pressure. Note that the hydrostatic and oncotic gradient terms have opposing vectors and there ore avor f uid movement in opposite directions. Pc normally avors transudation rom the capillary lumen to the interstitium, whereas c normally avors f uid retention within the capillary lumen. The extent o f uid ltration that occurs along the length o the capillary di ers or each tissue’s capillary bed and is determined by cellular and junctional permeability properties o tissue-speci c capillary endothelial cells. In the example shown in Figure 21-1, liver capillaries lter f uid into the interstitium along their entire length. At the arterial end o the capillary bed, (Pc if) exceeds (Pif c), thus avoring plasma ltration rom the capillary into the interstitial space. Pc gradually decreases along the length o the capillary, and the rate o f uid ltration into the interstitium decreases. At the venous end o the capillary, hydrostatic f uid ltration and oncotic f uid absorption are almost balanced. Liver sinusoids, which trans er f uid into the interstitial space during per usion, return this f uid to the circulation via lymphatic f ow. In capillary beds o other tissues, the integrated oncotic pressure gradient avoring f uid f ow into the capillary balances the integrated hydrostatic pressure gradient, resulting in no net volume change between the vascular and interstitial spaces. Thus, the physiologic steady state o extracellular f uid represents a balance o driving orces between f uids o the intravascular and interstitial compartments. Pathologic alterations in the determinants o transcapillary f uid shi ts, coupled with changes in renal Na handling, can result in the ormation o edema, as discussed below.

360 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY A

Πc Arte ria l e nd

Πc

P if

Π if

Pc

Pc

P if

Π if

Ve nous e nd

Volume Regulators

Flow

Together, the low-pressure and high-pressure eedback systems integrate neurohumoral volume signals to maintain volume homeostasis in the ace o volume perturbations. The neurohormonal response to a change in volume status is controlled by our main systems: the renin-angiotensinaldosterone system (RAAS), natriuretic peptides, ADH, and renal sympathetic nerves. The RAAS, ADH, and renal sympathetic nerves are active in situations o intravascular volume depletion, while natriuretic peptides are released in response to intravascular volume overload.

B

o

ve

m

e

n

t

Out

0

l

u

i

d

m

The high-pressure system consists o specialized baroreceptors in the aortic arch, carotid sinus, and juxtaglomerular apparatus. These sensors modulate hypothalamic control o ADH secretion and sympathetic outf ow rom the brainstem. In addition, sympathetic input stimulates the juxtaglomerular apparatus to secrete renin, a proteolytic enzyme that activates the renin-angiotensin-aldosterone system (see below).

N

e

t

f

Renin-Angiotensin-Aldosterone System In Arte ria l e nd

P os ition a long ca pilla ry

Ve nous e nd

FIGURE 21-1. Capillary f uid ltration. The balance o hydrostatic pressure and oncotic pressure determines f uid ltration along the capillary. The example shown here is or a capillary in the liver in which f uid ltration exceeds f uid reabsorption. A. At the arterial end o the capillary, the capillary hydrostatic pressure (Pc ) is high (largest arrow), and the sum o Pc and interstitial oncotic pressure ( if) exceeds the sum o interstitial hydrostatic pressure (Pif) and capillary oncotic pressure ( c ). There ore, f uid moves out o the capillary into the interstitial space. As f uid continues to lter along the length o the capillary, the increased f uid ltration results in decreased Pc and increased c , thus decreasing the driving orce or f uid ltration rom the capillary to the interstitium. Throughout the length o the capillary, Pif and if remain relatively constant. B. A graphic representation o net f uid movement along the capillary length shows the decreasing driving orce or f uid ltration into the interstitium. In the capillary shown here, f uid is ltered into the interstitium along the entire capillary length; lymphatic vessels eventually return the excess interstitial f uid to the systemic circulation (not shown).

Volume Sensors Vascular volume sensors can be divided into low-pressure and high-pressure eedback systems. The low-pressure system consists o the atria and pulmonary vasculature. In response to decreased wall stress (e.g., caused by decreased intravascular volume), peripheral nervous system cells lining the atria and pulmonary vasculature transmit a signal to noradrenergic neurons in the medulla o the central nervous system (CNS). This signal is relayed to the hypothalamus, resulting in increased secretion by the posterior pituitary gland o antidiuretic hormone (ADH, also known as arginine vasopressin, AVP). ADH promotes vasoconstriction and antidiuresis (increased renal water reabsorption). Together with increased peripheral sympathetic tone, this maintains distal tissue per usion. In response to increased wall stress (e.g., caused by increased intravascular volume), cells o the atria produce and secrete natriuretic peptide, which promotes vasodilation and natriuresis (increased renal Na excretion).

Renin is an aspartyl protease that activates the RAAS by cleavage o the circulating prohormone angiotensinogen to generate angiotensin I. Renin is produced and secreted by the juxtaglomerular apparatus (J GA), a specialized set o granule-

containing smooth muscle cells derived rom the a erent arteriole that line the a erent and e erent arterioles o the renal glomerulus. The JGA is also adjacent to the macula densa, a nephron segment between the end o the thick ascending limb o the loop o Henle and the distal convoluted tubule, containing specialized tubular epithelial cells capable o sensing distal chloride (and/or sodium) delivery. The JGA in the renal cortex represents the major structural component o the RAAS and one o the most important regulatory sites o renal volume conservation and blood pressure maintenance. The ultimate result o renin secretion is vasoconstriction and Na retention, actions that maintain tissue per usion and increase extracellular f uid volume (Fig. 21-2). At least three mechanisms are thought to control juxtaglomerular cell renin release (Fig. 21-3). First, a direct pressure-sensing mechanism o the a erent arteriole, equivalent to an intrarenal baroreceptor, responds to changes in renal per usion pressure (arteriolar wall tension) to increase juxtaglomerular cell release o renin. The detailed molecular mechanism o this sensory transduction is unknown in humans; in rodents, it involves autocrine prostaglandin and purinergic signaling. Second, sympathetic innervation o juxtaglomerular cells promotes renin release via 1adrenoceptor stimulation. Third, the autoregulatory mechanism known as tubuloglomerular eedback senses distal nephron delivery o chloride (and/or sodium) to modulate renin release. Nephron anatomy is organized such that the distal end o the cortical thick ascending limb (TAL) o each nephron is closely apposed to the juxtaglomerular mesangium o the same nephron. This spatial proximity allows rapid integrative regulation o a erent arteriolar diameter and glomerular mesangial contractility by distal nephron electrolyte concentration and/or salt load. Macula densa cells o the cortical thick ascending limb respond to increased luminal NaCl delivery by increasing extracellular adenosine in the juxtaglomerular interstitium, thereby activating A1 receptors on the juxtaglomerular mesangial cells to decrease renin release. Conversely, decreased luminal NaCl delivery

C h a p t e r 2 1 Pharmacology o Volume Regulation Proteolytic cleavage Angiote ns inoge n

P rore nin

361

Non-e nzyma tic conforma tiona l cha nge

(s e cre te d by live r)

Renin

Activa te d prore nin

Angiotensin converting enzyme

P rore nin re ce ptor

(s e cre te d by kidne y)

Angiote ns in I

(e xpre s s e d in lung e ndothe lium)

Angiote ns in II

Adre na l corte x (zona glome rulos a )

Re na l proxima l tubule

(incre a s e d Na Cl a bs orption)

Re na l e ffe re nt a rte riole s

Hypotha la mus

He a rt

(thirs t; incre a s e d ADH s e cre tion)

(ca rdiova s cula r re mode ling)

(va s ocons triction; ma inta ins GFR)

Aldos te rone (incre a s e d Na Cl a bs orption) a cting a t 1. Me dulla ry thick a s ce nding limb of He nle 2. Dis ta l tubule 3. Colle cting duct

FIGURE 21-2. The renin-angiotensin-aldosterone axis. Angiotensinogen is a prohormone secreted into the circulation by hepatocytes. Renin, an aspartyl protease secreted by juxtaglomerular cells o the kidney, cleaves angiotensinogen to the decapeptide angiotensin I, which has no known intrinsic biological activity. Prorenin, the proenzyme o renin, may have additional biologic e ects by signaling through the prorenin receptor. Angiotensin converting enzyme (ACE), a protease expressed on pulmonary capillary endothelium (and elsewhere in tissues), cleaves angiotensin I to the octapeptide angiotensin II. Angiotensin II has at least our actions via the angiotensin II type 1 receptor (AT1R) that increase intravascular volume and maintain tissue per usion. First, angiotensin II stimulates zona glomerulosa cells o the adrenal cortex to secrete aldosterone, a hormone that increases renal NaCl reabsorption at multiple segments along the nephron. Second, angiotensin II directly stimulates renal proximal tubule reabsorption o NaCl. Third, angiotensin II causes e erent arteriolar vasoconstriction, an action that increases intraglomerular pressure and thereby increases GFR. Fourth, angiotensin II stimulates hypothalamic thirst centers and promotes ADH secretion. Angiotensin II has additional tissue-specif c and cellular e ects that can activate the sympathetic nervous system and promote cardiovascular remodeling. Aldosterone (via the mineralocorticoid receptor; not shown) and prorenin (via the prorenin receptor) can have similar e ects on the cardiovascular system.

activates a mesangial prostaglandin signaling cascade that culminates in increased renin release. Macula densa cells sense luminal NaCl delivery by monitoring both luminal NaCl concentration and luminal f uid f ow rate as sensed by shear stress. NaCl delivery may be sensed directly by receptors in the apical sensory monocilia o macula densa cells; f uid f ow may be sensed by direct bending o the monocilia. Molecular components in the extraciliary apical membrane likely also contribute to these signal transduction processes. Prorenin, the renin proenzyme, was or many years considered an inactive precursor o renin lacking any intrinsic unction. It now seems, however, that prorenin can be activated either by proteolytic cleavage to renin or by nonenzymatic con ormational change induced by acid pH or elevated temperature. Renin activation typically occurs be ore its release rom juxtaglomerular cells. Proconvertase I and cathepsin B are among the several enzymes that have been proposed to mediate proteolytic activation o renin; the potential roles o plasma and tissue kallikrein in renin cleavage have been disputed. Recent studies have suggested that prorenin has cleavage-independent unctions mediated through its binding to the (pro)renin receptor (PRR). Reversible binding o prorenin to PRR in tissues promotes a nonenzymatic conormational change that allows the uncleaved prorenin to act as an angiotensinogen convertase, thereby converting

angiotensinogen to angiotensin I (Ang I; see below). In addition, binding o prorenin to PRR triggers downstream activation o the MAP kinase ERK1/2 signaling pathway, leading to up-regulation o pro brotic and cyclooxygenase-2 genes and to regulation o the vacuolar H ATPase in the collecting duct (see below). Interestingly, circulating plasma levels o prorenin are higher than those o renin, particularly in the setting o diabetic kidney disease. Transgenic mice overexpressing PRR develop hypertension and glomerulosclerosis, whereas genetic engineering o PRR loss-o - unction leads to embryonic lethality, suggesting that essential cellular unctions o PRR are not yet understood. Secreted renin (produced by cleavage o prorenin) acts as a protease to cleave the rst 10 amino acids o the circulating prohormone angiotensinogen, generating angiotensin I (Ang I). Ang I (Ang 1-10) is then cleaved to the active octapeptide angiotensin II (Ang II, AT II, or Ang 1-8) by the carboxypeptidase angiotensin converting enzyme (ACE) located on the endothelial cell sur ace. Although ACE is expressed primarily in the pulmonary vascular endothelium and coronary circulation, ACE activity regulates local production o AT II in all vascular beds. Indeed, an incompletely understood “local” renin-angiotensin system is also expressed in the vasculature, producing these substances as autocrine actors independently o the kidney and liver. ACE has a broad proteolytic substrate speci city that includes

362 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

FIGURE 21-3. Modulation of renin release. Renin is reMa cula de ns a ce ll

J uxta glome rula r ce ll Ade nos ine (A) β1 -a gonis t

Tubula r lume n Na + ClK+

β1 -AR A1 re ce ptor ? Gs

Na + 2Cl[Na +]

NKCC2

K+

[Cl-]

Gi

S UCNR1 COX-2 G q/11

cAMP P ros ta gla ndins (P G) Renin

Gs

P G re ce ptor

many other substrates such as neuropeptides and kinins (e.g., bradykinin) that are venodilatory autacoids released in response to inf ammation. For this reason, ACE is also known as kininase II. Kininase activity has important pharmacologic consequences, as discussed below. The recently identi ed ACE homolog ACE2 is highly expressed in the kidney, where it is localized predominantly in tubular epithelial cells and less prominently in glomeruli and the renal vasculature. ACE2 degrades both Ang I and Ang II to the heptapeptide Ang 1-7. In contrast to the vasoconstrictor and pro-proli erative peptide Ang II, Ang 1-7 is considered an antiproli erative vasodilator that counteracts the cardiovascular and baroref ex actions o Ang II. Ang 1-7 binds to the G protein-coupled receptor MAS rather than to the angiotensin II (AT II) receptor (see below). ACE2 activity is altered in diabetic kidney disease and hypertensive renal disease and in experimental models o kidney injury. Dissociation between tubular and glomerular ACE2 expression can occur in diabetic kidney disease, in which ACE2 expression is increased in tubular epithelial cells but decreased in glomeruli. In addition to Ang I (Ang 1-10), Ang II (Ang 1-8), and Ang 1-7, angiotensin III (Ang 2-8) and angiotensin IV (Ang 3-8) have been identi ed. Ang 2-8 is a less potent vasopressor than Ang II but retains 100% o its aldosteronestimulating activity. The vasopressor activity o Ang 3-8 resembles that o Ang 2-8. Ang II (AT II) binding to the AT II receptor subtype 1 (G protein-coupled AT1 receptor, AT1R) produces at least our stimulatory physiologic responses: (1) stimulation o

S uccina te

leased by juxtaglomerular cells in response to diverse stimuli that signal intravascular volume depletion. First, decreased pressure in the a erent arteriole (not shown) stimulates increased renin release promoted by intrarenal baroreceptors, possibly by releasing prostaglandins. Second, juxtaglomerular cells express 1-adrenergic receptors ( 1-AR) coupled to Gs , which stimulates adenylyl cyclase to increase the intracellular level o cAMP and thereby stimulates renin release. Third, cells lining the diluting segments o the nephron modulate renin release according to luminal NaCl f ux and local accumulation o the tricarboxylic acid (TCA) cycle intermediate succinate, ref ecting tissue energy balance. Succinate activates the G protein-coupled succinate receptor SUCNR1, leading via MAP kinase activation to stimulation o cyclooxygenase (COX-2)-mediated prostaglandin production, ultimately increasing renin release. Succinate accumulation also stimulates eNOS and nitric oxide (NO) production, leading to vasodilation o the a erent arteriole (not shown). Decreased NaCl f ux leads to decreased Cl entry through the Na /2Cl /K transporter (NKCC2) on the apical membrane o macula densa cells in the distal convoluted tubule, increasing COX-2-dependent production o prostaglandins PGE2 and PGI2. These prostaglandins activate juxtaglomerular cell prostaglandin receptors to stimulate renin release by increasing cAMP production. In contrast, increased NaCl delivery to the cortical thick ascending limb (TAL) leads, through still-debated mechanisms, to increased generation o adenosine in the juxtaglomerular mesangial interstitium. Activation o Gi-coupled A1 adenosine receptors o the juxtaglomerular cell decreases intracellular cAMP, which leads to decreased renin release.

aldosterone secretion by zona glomerulosa cells o the adrenal glands, (2) increased reabsorption o NaCl rom the proximal tubule and other nephron segments, (3) arteriolar vasoconstriction, and (4) central stimulation o thirst and ADH secretion. All our o these actions increase intravascular volume and there ore help to maintain per usion pressure: aldosterone secretion increases distal tubule Na reabsorption; proximal tubule NaCl reabsorption increases the raction o ltered Na that is reabsorbed; arteriolar vasoconstriction maintains blood pressure; stimulation o thirst increases ree water absorbed into the vasculature; and secretion o ADH increases collecting duct ree water absorption. Ang II also negatively regulates renin secretion through binding to AT1R on juxtaglomerular cells. Ang II has many other AT1R-mediated tissue- and cell type-speci c e ects, leading to sympathetic nervous system activation, generation o reactive oxygen species (ROS), and cell growth. Conversion o Ang I to Ang II by tissue-speci c ACE (chymase) o cardiac myocytes can promote cardiac remodeling, hypertrophy, and brosis. Similar cardiovascular e ects are also potentiated through aldosterone activation o extrarenal mineralocorticoid receptors. The actions o Ang II are best understood in vascular smooth muscle cells, where AT1R activates phospholipase C, leading to release o Ca2 rom intracellular stores, activation o protein kinase C, and vasoconstriction. Inhibition o AT1R decreases vascular smooth muscle cell contractility and thereby decreases systemic vascular resistance and blood pressure (see the ollowing discussion). The related

C h a p t e r 2 1 Pharmacology o Volume Regulation

G protein-coupled AT II receptor AT2R has a vasodilator role, in part by increasing nitric oxide production. AT2R is highly expressed in etal kidney and intestine, whereas high expression in adults is restricted to myometrium, with lower levels in adrenals and oviducts. AT2R action has recently been implicated in some types o re ractory pain syndromes.

Natriuretic Peptides

Natriuretic peptides are hormones released by atria, ventricles, and vascular endothelium in response to volume overload. The classical natriuretic peptides are A-type, B-type, and C-type natriuretic peptides. A-type natriuretic peptide (ANP) is released primarily by the atria, while B-type natriuretic peptide (BNP) is released mainly by the ventricles. C-type natriuretic peptide (CNP) is released by vascular endothelial cells. The natriuretic peptide uroguanylin (UGN) is released by enterocytes in response to dietary ingestion o salt. Vascular natriuretic peptides are released in response to increased intravascular volume, an e ect that may be signaled by increased stretch o natriuretic peptide-secreting cells. Circulating natriuretic peptides bind to one o three receptors, termed NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are transmembrane proteins with cytoplasmic guanylyl cyclase domains (see Chapter 1, Drug–Receptor Interactions); activation o these receptors increases intracellular

A

cGMP levels. NPR-C lacks an intracellular guanylyl cyclase domain and may serve as a “decoy” or “bu er” receptor to reduce the level o circulating natriuretic peptides available to bind to the two signaling receptors. Both ANP and BNP bind with high a nity to NPR-A, while only CNP binds to NPRB. All three natriuretic peptides bind to NPR-C (Fig. 21-4A). Deletion o the ANP gene (Nppa) in mice causes salt-sensitive hypertension. Common human allelic gene variants in the ANP (NPPA) and BNP (NPPB) genes have been associated with higher levels o ANP and BNP, higher blood pressure, and risk o hypertension. In mice, UGN binds and activates transmembrane guanylyl cyclase C in both enterocytes (promoting enteric Cl secretion) and renal proximal tubule cells (reducing renal Na and Cl reabsorption). UGN also promotes natriuresis in the renal collecting duct by additional, less well characterized mechanisms. Natriuretic peptides a ect the cardiovascular system, the kidney, and the central nervous system. Integration o natriuretic peptide-derived signals serves to decrease volume overload and its sequelae. ANP relaxes vascular smooth muscle by increasing intracellular cGMP, which causes dephosphorylation o myosin light chain and subsequent vasorelaxation (see Chapter 22). ANP also increases capillary endothelial permeability, which reduces blood pressure by avoring f uid ltration rom the plasma into the interstitium (see Equation 21-1).

B

GTP

Biologica l e ffe cts, including incre a s e d na triure s is

Apica l me mbra ne

Ba s ola te ra l me mbra ne

NP R-A

Va s opre s s in/ ADH Lume n of colle cting duct

cGMP

363

Ve s icle conta ining AQP 2

ANP /BNP

GDP

s

GTP

s

V2 -re ce ptor

AQP 2 NP R-C De gra da tion

Inte rna liza tion

Wa te r

cAMP Tra ns loca tion/ins e rtion

Wa te r

ATP

Ade nylyl cycla s e

FIGURE 21-4. Natriuretic peptide and antidiuretic hormone signaling pathways. A. A-type and B-type natriuretic peptides (ANP and BNP) are hormones secreted in response to volume overload. These peptides bind to natriuretic peptide receptor-A (NPR-A) and natriuretic peptide receptor-C (NPR-C). NPR-A is a transmembrane receptor with intrinsic guanylyl cyclase activity associated with its cytoplasmic domain. Increased intracellular cGMP levels mediate the e ects o natriuretic peptides, including increased natriuresis. NPR-C is believed to be a “decoy receptor” because the protein lacks the intracellular catalytic domain. Binding o natriuretic peptide to NPR-C may result in internalization and degradation o the receptor together with its bound natriuretic peptide. A third natriuretic peptide, CNP, is expressed by vascular endothelial cells and binds exclusively to NPR-B (not shown). B. Antidiuretic hormone (ADH), also known as vasopressin, is secreted by the hypothalamus in response to increased osmolality and volume depletion. ADH mediates renal collecting duct water reabsorption by activating the Gs -coupled V2 vasopressin receptor. Activation o Gs leads to increased adenylyl cyclase activity and increased cAMP levels. cAMP increases collecting duct water reabsorption by promoting the translocation and insertion o aquaporin 2 water channel (AQP2)-containing vesicles into the collecting duct apical membrane. The increased expression o apical-membrane AQP2 results in increased water f ux across the collecting duct and there ore increased reabsorption o ltered water. Hydrolysis o cAMP by phosphodiesterase leads to removal o AQP2 rom the luminal membrane by endocytosis o AQP2-containing vesicles (not shown).

364 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

In the kidney, natriuretic peptides promote both increased glomerular ltration rate (GFR) and natriuresis. GFR is increased because o constriction o the e erent arteriole and dilation o the a erent arteriole, resulting in higher intraglomerular pressure and there ore increased plasma ltration. The natriuretic e ects on the kidney result rom antagonism o ADH action in the collecting ducts and antagonism o Na reabsorption in multiple nephron segments. The central e ects o natriuretic peptides are less well understood, but they include decreased perception o thirst (and there ore decreased f uid intake), decreased release o antidiuretic hormone, and decreased sympathetic tone. The signaling mechanisms mediating these actions are uncertain, but may be via CNP, as this natriuretic peptide is expressed at high levels in the brain. Although many o the e ects o natriuretic peptides remain incompletely understood, these hormones appear to play an important role in regulating the pathophysiology o volume excess. Much interest has recently ocused on the relationship between natriuretic peptides and heart ailure. In particular, BNP and N-terminal proBNP (NT-proBNP) have emerged as promising markers or heart ailure diagnosis, prognosis, and treatment. The physiology and pharmacology o natriuretic peptides and their receptors remain subjects or active investigation.

Renal Control of Na Excretion Over the course o 24 hours, the kidneys lter approximately 180 L o f uid. To increase or decrease body f uid volume, the kidneys must increase or decrease renal Na reabsorption rom the large daily volume o glomerular ltrate. For this reason, the neurohormonal mechanisms controlling extracellular volume status have important actions on the kidney. An understanding o the renal control o Na excretion is crucial to understanding the role o the kidney in regulation o body f uid volume. The renal glomerulus produces an ultra ltrate o plasma that f ows through and is processed by renal tubules o the nephron, the unctional unit o the kidney (Fig. 21-5). The postglomerular nephron is responsible or solute and water reabsorption rom the ltrate, as well as or excretion o

Carbonic anhydrase inhibitors

JG

Glome rulus

1 PT

Affe re nt a rte riole Effe re nt a rte riole

Antidiuretic Hormone

Antidiuretic hormone (ADH, arginine vasopressin, or vasopressin) is a nonapeptide hormone secreted by the posterior

pituitary gland in response to increased plasma osmolality or severe hypovolemia. ADH constricts the peripheral vasculature and promotes water reabsorption in the renal collecting duct. Its actions are mediated by two distinct G protein-coupled receptors. The V1 receptor, present predominantly in vascular smooth muscle cells, stimulates vasoconstriction through a Gq-mediated mechanism. The V2 receptor, expressed in collecting duct principal cells, stimulates water reabsorption by a Gs-mediated mechanism (Fig. 21-4B). This Gs signal increases cytosolic cAMP, which leads to activation o protein kinase A (PKA). PKA phosphorylates the water channel aquaporin 2 and activates transport and usion o aquaporin 2-containing vesicles into the apical membrane o the principal cell. Increased aquaporin 2 expression at the apical membrane promotes increased water reabsorption. Regulation of renal water reabsorption in the collecting duct modulates urine and plasma osmolality and serves as a reserve mechanism or increasing intravascular volume in situations o severe dehydration.

Renal Sympathetic Nerves

Renal sympathetic nerves innervate both a erent and e erent arterioles. In response to a decrease in intravascular volume, the renal sympathetic nerves decrease GFR by stimulating constriction o the a erent arteriole to a greater degree than the e erent arteriole. The decreased GFR resulting rom pre erential constriction o the a erent arteriole ultimately leads to decreased natriuresis. Renal sympathetic nerves also increase renin production by stimulation o 1adrenergic receptors on juxtaglomerular mesangial cells and increase proximal tubule NaCl reabsorption. Since transplanted kidneys unction normally in the initial absence o sympathetic nerve input, renal innervation is not required or clinically normal kidney unction.

CCD DCT

3 4 Thiazide diuretics CTAL Loop diuretics

2

Potassiumsparing diuretics

OMCD

MTAL

IMCD ATL

TDL

FIGURE 21-5. Nephron anatomy and sites of action of diuretics. Nephron f uid ltration begins at the glomerulus, where an ultra ltrate o the plasma enters the renal epithelial (urinary) space. This ultra ltrate then f ows sequentially through our axially distinct nephron segments (1–4). From the glomerulus, ultra ltrate travels to the proximal tubule (PT) (1), then to the loop o Henle (2), which includes the thin descending limb (TDL), ascending thin limb (ATL), medullary thick ascending limb (MTAL), and cortical thick ascending limb (CTAL) o Henle. The distal convoluted tubule (DCT) (3) includes the macula densa and juxtaglomerular (J G) apparatus. The collecting duct (4) consists o the cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). Pharmacologic agents inhibit speci c solute transporters within each segment o the nephron. Carbonic anhydrase inhibitors act primarily at the proximal tubule; loop diuretics act at the medullary and cortical thick ascending limbs; thiazide diuretics inhibit solute transport in the distal convoluted tubule; and potassium-sparing diuretics inhibit collecting-duct Na reabsorption.

C h a p t e r 2 1 Pharmacology o Volume Regulation

metabolic waste products and xenobiotics, including drugs. The renal tubular epithelial cells o the postglomerular nephron enclose a lengthy tubular lumen, the “urinary space,” which leads to the ureters, urinary bladder, and urethra. The initial glomerular ultra ltrate contains solutes o low molecular weight at concentrations similar to those in the plasma. As the ultra ltrate passes through the nephron, substratespeci c transporters and channels in the luminal (apical) membrane o polarized renal tubular epithelial cells sequentially alter the solute concentrations o the tubular f uid. The unction o these transporters and channels is, in turn, inf uenced by changes in solute concentrations in the cells themselves, as regulated in part by channels and transporters on the contraluminal (basolateral) side o the cells. Systemic volume regulation by the kidney is accomplished by tubular solute reabsorption through integrated action o ion channels and ion transporters in the apical and basolateral membranes o tubular epithelial cells and by the accompanying reabsorption o water. The postglomerular nephron exhibits remarkable heterogeneity along its length. Four segments o the nephron are especially relevant to the pharmacology o systemic volume regulation (Fig. 21-5). These are the proximal tubule , the thick ascending limb (TAL) o the loop o Henle, the distal convoluted tubule (DCT), and the cortical collecting duct (CCD). In each tubular segment, a complex but tightly choreographed group o segment-speci c ion transporters and channels collaborate in the reabsorption o NaCl rom the lumen across the cellular monolayer o tubular epithelium into the interstitial space. NaCl reabsorption is key or systemic water retention. Solute and water transport across each segment requires coordination o transporter unction in the luminal and basolateral membranes. In addition, paracellular transport o ions across the tight junctions between cells requires regulated communication between adjacent cells o the tubular epithelium. Integration o the transcellular and paracellular components o transepithelial transport requires integration o signals transmitted by sensors o extracellular and intracellular ion concentrations and o intracellular, local extracellular, and systemic volume. Alteration o ion transport by drugs in any nephron segment can induce compensatory regulation locally and in more distally located nephron segments.

Proximal Tubule

The proximal tubule (PT) is the rst reabsorptive site in the nephron. It is responsible or approximately two-thirds o sodium reabsorption, 85–90% o bicarbonate reabsorption, and approximately 60% o chloride reabsorption (Fig. 21-6). Speci c sodium-coupled symporters in the proximal tubule apical membrane drive renal reabsorption o all glucose, amino acids, phosphate, and sul ate rom the glomerular ltrate. The proximal tubule also mediates secretion and reabsorption o weak organic acids and weak organic bases; these are coupled to processes o sodium or proton symport or antiport or to anion exchange mechanisms. Among these weak acids and bases are many o the drugs used to regulate systemic volume (see below). Bicarbonate reabsorption requires the coordinated action o apical and basolateral ion transporters together with apical and intracellular enzymatic activities (Fig. 21-6). At the luminal sur ace o the proximal tubule, ltered bicarbonate encounters active proton secretion across the proximal tubule

Apica l me mbra ne

365

Ba s ola te ra l me mbra ne

Lume n of proxima l tubule

3HCO 3 -

Na +

NBCe 1

NHE3

Na +

H+

vH+ ATP ase

H+ + HCO 3 H2 CO 3

H2 CO 3 3Na +

H+

Na +/K+

CAII CAIV

Acetazolamide

ATPase

2K+ Acetazolamide

CO 2 + H2 O

CO 2 + H2 O

FIGURE 21-6. Proximal tubule cell. A signif cant percentage o f ltered Na in the proximal tubular lumen is reabsorbed via the NHE3 Na /H exchanger. The activity o NHE3, together with that o an apical membrane vacuolar ATPase (vH ATPase), results in signif cant H extrusion into the proximal tubule urinary space. H extrusion is coupled to HCO3 reabsorption by the action o apical membrane carbonic anhydrase IV (CAIV), which catalyzes the cleavage o HCO3 into OH and CO2. OH combines with H to orm water, while CO2 di uses into the cytoplasm o the epithelial cell. The cytoplasmic enzyme carbonic anhydrase II (CAII) catalyzes the ormation o HCO3 rom CO2 and OH ; the HCO3 is then transported into the interstitium together with Na . The net result o this process is reabsorption o HCO3 and Na by the basolateral co-transporter NBCe1. Acetazolamide inhibits both iso orms o carbonic anhydrase; the decreased carbonic anhydrase activity results in decreased Na and HCO3 absorption.

brush-border microvilli. Two-thirds o the proton e f ux is in exchange or inf ux o Na , largely via the NHE3 Na /H exchanger. The remaining third o proton e f ux is mediated by the vacuolar H ATPase (vH ATPase). The HCO3 permeability o the luminal membrane o the proximal tubular cell is low. However, the outer leaf et o the luminal membrane harbors the glycosylphosphatidylinositol-linked exoenzyme carbonic anhydrase IV (CAIV). CAIV converts luminal HCO3 to CO2 and OH . The OH is rapidly hydrated to water by the abundance o local protons, and the CO2 reely di uses into the cytoplasm o the proximal tubular epithelial cell. The intracellular CO2 is rapidly rehydrated to HCO3 by cytoplasmic carbonic anhydrase II (CAII); this reaction consumes the intracellular OH accumulated as a result o the H -extruding activities o apical NHE3 and vH ATPase. The HCO3 produced by the CAII reaction is then co-transported with Na across the basolateral membrane o the epithelial cell, accounting or the net reabsorption o sodium and bicarbonate. The Na /HCO3 co-transporter NBCe1 mediates electrogenic basolateral e f ux o three HCO3 ions with each co-transported Na ion. Basolateral K channels maintain an inside-negative membrane potential to enhance the driving orce or net e f ux o two negative charges per NBCe1 transport cycle. Emerging

366 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

evidence also suggests the presence o several types o transmembrane ecto-carbonic anhydrases in the basolateral membrane that help dissipate the local accumulation o bicarbonate within the interstitial space between the epithelial cells and peritubular capillaries. Solute absorption in the proximal tubule is iso-osmotic— water accompanies reabsorbed ions to maintain osmotic balance. In the past, water f ow was assumed to be largely paracellular. However, data rom mice genetically modied to lack the aquaporin water channel AQP1 (and rom rare cases o humans lacking AQP1) demonstrate that most water reabsorption across the proximal tubule—and, beyond that, across the thin descending limb o Henle—is transcellular. Aquaporins are central to transepithelial water permeability in all water-permeable nephron segments. Thus, the transition rom the water-permeable thin descending limb o Henle to the water-impermeable ascending thin limb is paralleled by decreased AQP1 expression. Proximal tubule Na reabsorption is a key component o the intrarenal RAAS. Ang II is ltered into the tubular f uid and activates AT1 receptors expressed on the apical membrane o renal tubular epithelial cells. Proximal tubular lumen concentrations o Ang I and Ang II have been ound to be higher than their corresponding plasma concentrations, leading to the discovery that proximal tubular epithelial cells express angiotensinogen that is converted to Ang I and Ang II through intrarenal (pro)renin and ACE activity. Elevated Ang II levels sustain or up-regulate tubular epithelial cell AT1R but downregulate vascular AT1R. The critical importance o kidney AT1R has been demonstrated by using proximal tubule-speci c AT1R knockout mice (with intact expression o AT1R in all other tissues). In usion o Ang II into these mice ailed to increase blood pressure to the same degree as in control mice. The reduced positive Na balance observed in these mice was consistent with acilitated natriuresis as a mechanism or resistance to hypertension.

Thick Ascending Limb of the Loop of Henle

The tubular f uid emerging rom the ascending thin limb is hypertonic and has an elevated NaCl concentration. The three nephron segments into which this f uid f ows, the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the connecting tubule or segment (CNT), together constitute “the diluting segment.” The apical membrane o the thick ascending limb o Henle is devoid o aquaporins, as is the apical membrane o the rest o the diluting segment; there ore, these nephron segments reabsorb NaCl and urea without accompanying water (Fig. 21-7), thus diluting the solutes o the tubular f uid. Reabsorption o NaCl and urea across the TAL provides the interstitial solute that generates and maintains the corticomedullary osmotic gradient o the kidney, allowing operation o the “countercurrent multiplier” that can concentrate the urine o humans to 1,200 mOsM and that o desert rodents to 4,000 mOsM. The TAL reabsorbs between 25% and 35% o the ltered Na load by means o the luminal membrane Na -K -2Cl co-transporter, NKCC2. The Cl imported by NKCC2 exits the basolateral side o the cell via CLC-K2 chloride channels. CLC-K2’s -subunit, Barttin, which colocalizes with CLC-K2 at the basolateral membrane, is essential or intracellular tra cking and unction o CLC-K2. (Barttin also serves as the -subunit or a related Cl channel, CLC-K1, in potassium-secreting stria vascularis epithelial cells o the

Apica l me mbra ne

Ba s ola te ra l me mbra ne

Lume n of me dulla ry thick a s ce nding limb

Loop diuretics

3Na + Na +/K+ ATP a s e

Na + 2Cl-

2K+

NKCC2

K+

K+ ROMK

ClCLC-K2

Ca 2 + Mg 2 + Na + 10mV

FIGURE 21-7. Medullary thick ascending limb cell. The medullary thick ascending limb o the loop o Henle absorbs Na through an apical membrane Na /K /2Cl (NKCC2) transporter. The Na /K ATPase pumps sodium rom the cytoplasm into the interstitium, and a basolateral Cl channel (CLC-K2) transports Cl into the interstitium. K is primarily recycled back into the urinary space via a luminal K channel (ROMK). The combined activities o apical ROMK and basolateral CLC-K2 result in a lumen-positive transepithelial potential di erence (approximately 10 mV) that drives paracellular absorption o cations, including Ca 2 and Mg2 . Loop diuretics inhibit NKCC2, resulting in signif cantly increased renal sodium excretion. Attenuation o the positive transepithelial potential by loop diuretics also increases the excretion o Ca 2 and Mg2 .

inner ear.) The Na imported rom the lumen via NKCC2 leaves the basolateral side o the cell via the Na /K ATPase. Because Cl carries a negative charge, exit o unaccompanied Cl through basolateral CLC-K2 depolarizes the cell. The stoichiometry o the Na /K ATPase, 3Na outward per 2 K inward, partly counters this depolarization; additional repolarization o the cell is accomplished by the apical K channel ROMK, which recycles back into the lumen the K imported into the cell via NKCC2. Reduced unction o any one o these transporters or channels, secondary either to pharmacologic inhibition (e.g., with loop diuretics, inhibiting NKCC2 unction) or to losso - unction mutation, is associated with renal salt wasting. Several TAL transporter gene de ects cause Bartter syndrome, which is characterized by renal salt wasting, possible hypotension, hypokalemia, metabolic alkalosis, and, in some patients, hypercalciuria (Table 21-1). The coordinated operation o these apical and basolateral transporters and channels generates a lumen-positive electrical potential across the TAL. The transepithelial potential di erence in the TAL constitutes the electrical driving orce avoring paracellular reabsorption o additional Na rom lumen to interstitium. The paracellular component o Na reabsorption reduces the energetic cost to TAL epithelial cells (measured as ATP consumption), because Na /K transport consumes most o the ATP in the TAL cell. Even with the energy conserved by the paracellular Na absorptive pathway, the TAL working at maximal capacity can consume up to 25% o the body’s total ATP production, or approximately

C h a p t e r 2 1 Pharmacology o Volume Regulation

367

TABLE 21-1 Bartter Syndrome: Renal Salt Wasting in the Thick Ascending Limb (TAL) Associated with Hypercalciuria SYNDROME Bartter

RENIN

ALDOSTERONE

TREATMENT

GENE LOCUS

GENE

15q21

SLC12A1 (type 1, neonatal)

INHERITANCE

K

pH

AR

↓

↑

↑

↑

AR

↓

↑

↑

↑

11q24

KCNJ 1 (type 2, neonatal)

AR

↓

↑

↑

↑

1p36

CLCNKB (type 3, classic)

AR

↓

↑

↑

↑

1p32

BSND (type 4, associated with dea ness)

AD

↓

↑

↑

↑

3q21

CASR (type 5 or autosomal dominant hypocalcemia)

Increase salt intake ( or all types)

SLC12A1 (NKCC2) and KCNJ 1 (ROMK) are located at the apical membrane o the TAL epithelial cells, whereas CLCNKB (CLC-K2), BSND (Barttin), and CASR (calciumsensing receptor) are located at the basolateral membrane. AR, autosomal recessive; AD, autosomal dominant.

65 moles per day at rest. The lumen-positive transepithelial potential o the TAL also drives paracellular reabsorption o luminal calcium and magnesium ions. This paracellular transport is now known to be mediated by claudins 16 and 19, which orm tight junctional, heteromeric, cation-selective channels comprised o the ectodomains o claudin polypeptides rom adjacent TAL epithelial cells. Recent studies have demonstrated that claudins, in addition to their barrier unction, regulate paracellular transport across epithelia by interacting with multiple intra- and extracellular signaling pathways. For example, the extracellular calcium-sensing receptor (CaSR), a widely expressed G protein-coupled receptor, responds to changes in basolateral (serum) [Ca2 ] by modi ying claudin expression in TAL cells, thereby modulating TAL permeability to paracellular calcium.

Distal Convoluted Tubule

This continuation o the diluting segment actively reabsorbs between 2% and 10% o the ltered NaCl load, while remaining impermeable to luminal water (Fig. 21-8). Luminal Na enters the epithelial cells o the distal convoluted tubule (DCT) via the electroneutral, K -independent NCC Na -Cl co-transporter. Basolateral exit o Na is mediated by Na / K ATPase in coordination with basolateral K recycling by the heterotetrameric K channel Kir4.1/5.1 (KCNJ10/ KCNJ16). The Cl imported across the apical membrane exits via basolateral anion pathways that include both electrogenic Cl channels and (at least in the mouse) electroneutral K -Cl co-transport. Pharmacologic inhibition o the NCC sodium channel with thiazide diuretics leads to increased Na excretion accompanied by hypokalemia and other electrolyte and acid–base changes. Genetic loss o unction o NCC underlies Gitelman syndrome, which is characterized by salt wasting much milder than that with Bartter syndrome. Genetic loss o unction o Kir4.1 underlies SeSAME syndrome (also known as EAST syndrome), in which hypokalemia, hypomagnesemia, and metabolic alkalosis is accompanied by epilepsy, ataxia, sensorineural dea ness, and intellectual dys unction; the latter mani estations ref ect loss o Kir4.1 expression in the brain (Table 21-2). Mutations in WNK protein kinases o the distal convoluted tubule and collecting duct (WNK4 and WNK1), as well as in

Apica l me mbra ne

Ba s ola te ra l me mbra ne

Lume n of dis ta l convolute d tubule Ca 2 +

Thiazides

NCX1

Na +

3Na + NCC

3Na +

Cl-

Na +/K+ ATPa s e

2K+ Ca 2 +

K+ Kir 4.1/5.1

TRP V5

ClMg 2 + TRP M6/ TRP M7

g Cl-

Mg 2+ g Mg 2+

FIGURE 21-8. Distal convoluted tubule cell. Distal convoluted tubule (DCT) cells absorb Na via the NaCl co-transporter (NCC) at the apical membrane. NCC abundance is up-regulated by activation o the mineralocorticoid receptor through aldosterone (not shown). NCC activation by phosphorylation is mediated by SPAK, under the inf uence o “upstream” WNKand other kinases (not shown). Positive and negative regulation o NCC may arise rom antagonistic actions o the di erent WNK kinases and their interactions with the Cullin3-KLHL3 E3 ligase ubiquitination system (not shown). Na is transported across the basolateral membrane into the interstitium via the Na /K ATPase, aided by basolateral K recycling via the heterotetrameric K channel Kir4.1/5.1. Cl exits the cell across the basolateral membrane via Cl channels (gCl ) and likely also via K -Cl co-transporters (not shown). DCT epithelial cells absorb Ca 2 via apical membrane Ca 2 channels (TRPV5), and Ca 2 is transported across the basolateral membrane into the interstitium by the Na / Ca 2 exchanger NCX1 and by the Ca 2 ATPase PMCA (not shown). Mg2 is absorbed by magnesium-selective heteromeric TRPM6/TRPM7 channels at the apical membrane and transported across the basolateral membrane via pathways that are incompletely characterized (gMg2 ). Thiazides inhibit NCC, resulting in increased Na excretion. Thiazides also increase epithelial cell absorption o Ca 2 and Mg2 by an unknown mechanism (not shown).

368 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

TABLE 21-2 Syndromes of Renal Salt Wasting Associated with Low Blood Pressure Caused by Defects in the Distal Nephron SYNDROME

TREATMENT

GENE LOCUS

GENE

INHERITANCE

K

pH

RENIN

ALDOSTERONE

Gitelman

AR

↓

↑

↑

↑

Increase salt intake

16q13

SLC12A3 (NCC)

EAST (also known as SeSAME)

AR

↓

↑

↑

↑

Increase salt intake

1q23

KCNJ 10 (Kir4.1)

Pseudohypoaldosteronism type 1 (PHA type I)

AD AR AR AR

↑

↓

↑

↑

Increase salt intake

4q31 12p13 16p13 16p13

NR3C2 (type 1A) SCNN1A (type 1B) SCNN1B (type 1B) SCNN1G (type 1B)

Renal tubular dysgenesis (RTD)

AR

↑

↓

↑ or ↓

↓

Vasopressors

1q32 1q42 3q24

REN (renin) AGT (angiotensinogen) ACE (angiotensin converting enzyme) AGT1R (angiotensin II type 1 receptor)

17q23

AR, autosomal recessive; AD, autosomal dominant; EAST, epilepsy, ataxia, sensorineural dea ness, tubulopathy; Kir 4.1, inward rectif er-type K -channel, member 4.1; NR3C2, nuclear receptor sub amily 3, group C, member 2 (gene encoding or the mineralocorticoid receptor); SCNN1A, 1B, or 1G, Na channel, non-voltage-gated 1, -subunit, -subunit, or -subunit (genes encoding ENaC subunits).

the E3 ubiquitin ligase components Kelch-like 3/KLHL3 and Cullin 3/CUL3, each lead to increased stability and unction o NCC in the apical membrane, resulting in hyperkalemic hypertension associated with metabolic acidosis (pseudohypoaldosteronism type 2 or PHA2; Fig. 21-8 and Table 21-3).

The DCT also mediates transepithelial reabsorption o luminal calcium and magnesium ions via calcium-specif c TRPV5 channels and magnesium-specif c TRPM6/TRPM7 channels in the apical membrane. The reabsorbed calcium is transported across the DCT cell basolateral membrane

TABLE 21-3 Rare Inherited Forms of High Blood Pressure due to Increased Salt Reabsorption in the Distal Nephron (Distal Convoluted Tubule and/or Collecting Duct) ALDO

TREATMENT

GENE LOCUS

↓

↓

ENaC inhibitors

16p12

ENaC (epithelial Na channel)

↑

↓

N (↑ )

Corticosteroid therapy

8q24

Chimeric gene: 11- -hydroxylase / aldosterone synthase

↓

↑

↓

↓

Spironolactone (ENaC inhibitors)

16q22

11- -hydroxysteroid dehydrogenase

de novo* de novo de novo de novo

↓

↑

↓

↑

Spironolactone (adrenal adenomectomy)

11q24 3p21 1p13 Xq28

KCNJ 5 CACNA1D ATP1A1 ATP2B3

Congenital adrenal hyperplasia

AR

↓

↑

↓

↓

Corticosteroid therapy

10q24 8q24

17- -hydroxylase 11- -hydroxylase

Pseudohypoaldosteronism type 2 (Gordon syndrome)

AD

↑

↓

↓

N (↑ )

Thiazide diuretics

12p13 17q21 5q31 2q36

WNK1 WNK4 Kelch-like3 Cullin3

SYNDROME

INHERITANCE

K

pH

RENIN

Liddle

AD

↓

↑

Glucocorticoid-remediable aldosteronism

AD

↓

Apparent mineralocorticoid excess

AR

Aldosterone-producing adrenal adenomas

DISEASE GENE(S)

These syndromes eature intravascular volume expansion associated with acid–base and electrolyte abnormalities. Aldo, aldosterone; AD, autosomal dominant; AR, autosomal recessive; N, normal; KCNJ 5, K inwardly-recti ying channel, sub amily J , member 5; CACNA1D, calcium channel, voltage-dependent, L type, -1D subunit; ATP1A1, Na /K ATPase -1 subunit; ATP2B3, PMCA3 (plasmalemmal Ca 2 ATPase 3). *Rarely, can also be inherited in Mendelian (autosomal dominant) ashion.

C h a p t e r 2 1 Pharmacology o Volume Regulation

via specif c NCX Na /Ca2 exchangers and Ca2 ATPases. The still undef ned basolateral exit pathway(s) or DCT cell Mg2 may include the magnesium transporter polypeptides SLC41A3 and CNNM2, with additional required basolateral unction o FXYD2 (the 1 subunit o the basolateral Na / K ATPase) and the heterotetrameric K channel Kir4.1/5.1.

Collecting Duct

This terminal portion o the nephron is divided into cortical, outer medullary, and inner medullary collecting duct (CD) segments (Fig. 21-9). The cortical and outer medullary CD segments consist o two cell types: principal cells and intercalated cells . Principal cells reabsorb between 1% and 5% o the f ltered sodium load, depending on plasma aldosterone levels (aldosterone increases sodium reabsorption and water retention, see below). Luminal Na enters the principal cells at the apical membrane o the cortical collecting duct via heterotrimeric epithelial Na channels, ENaC, consisting o -, -, and -subunits. Loss-o - unction mutations in each o the three ENaC subunits cause a syndrome o low blood pressure associated with hyperkalemia and metabolic acidosis (pseudohypoaldosteronism type 1B; Table 21-2). In contrast, ENaC gain-o - unction mutations in - or -subunits cause severe hypertension associated with hypokalemia and metabolic alkalosis (Liddle syndrome; Table 21-3). Several other gene de ects can increase ENaC activity by enhancing activation o the mineralocorticoid receptor. WNK kinase regulation o NCC and (likely also) ENaC contributes to the ability o aldosterone to increase Na reabsorption by DCT and CCD cells in response to hypovolemia. WNK kinase regulation o ROMK contributes to the ability o aldosterone to increase K secretion by CCD cells in response to hyperkalemia. ENaC activity at the apical cell sur ace is regulated by the serum/glucocorticoid regulated kinase 1 (SGK1). SGK1

phosphorylates and sequesters ENaC’s ubiquitin ligase Nedd4-2, thereby preventing endocytosis o ENaC rom the apical cell sur ace and increasing ENaC activity. Serine proteases also activate ENaC under physiologic and pathophysiologic conditions by excision o amino acids rom extracellular loops o its - and -subunits, thereby increasing the probability o ion channel opening. The protease urin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1), anchored to the apical cell sur ace, mediates important extracellular cleavage. Soluble protease activity is very low in urine under physiologic conditions but can rise in proteinuric conditions. In nephrotic syndrome (proteinuria 3.5 g/day), the dominant soluble protease activity is plasmin, which is generated rom f ltered plasminogen by the action o urokinase-type plasminogen activator. Plasmin activates ENaC directly at high concentrations and through CAP-1 activation at lower concentrations. This f ltered protease activity may thus contribute to the edema that accompanies nephrotic-range proteinuria. Intracellular Na exits the basolateral side o the cell via the Na /K ATPase. Principal cells also secrete K into the

Apica l me mbra ne

Ba s ola te ra l me mbra ne

Inte rca la te d ce ll Type B, Non-A Non-B ClNa +

cells (lower cell) absorb Na via an apical membrane Na channel (ENaC). Cytoplasmic Na is transported across the basolateral membrane via the Na /K ATPase. In addition, collecting duct cells express apical membrane K channels (especially ROMK) that allow K to exit into the urinary space. ENaC expression and apical sur ace localization is modulated by aldosterone. The kinase SGK1 increases membrane expression o ENaC via phosphorylation and sequestration o the E3 ubiquitin ligase Nedd4-2, which otherwise promotes ENaC endocytosis (not shown). The WNK/SPAK-OSR1 kinase cascade likely regulates SGK1 and Nedd4-2 in the cortical collecting duct, as well as ENaC and ROMK, by mechanisms that are still being elucidated. Aldosterone binds to the mineralocorticoid receptor, which then increases transcription o the gene encoding ENaC as well as genes encoding other proteins involved in Na reabsorption (such as Na /K ATPase). The collecting duct principal cell is the site o action o the two classes o potassium-sparing diuretics. Mineralocorticoid receptor antagonists such as spironolactone and eplerenone (not shown) competitively inhibit the interaction o aldosterone with the mineralocorticoid receptor and thereby decrease expression o ENaC. Direct inhibitors o ENaC, such as amiloride and triamterene, inhibit Na inf ux through ENaC channels at the apical plasma membrane. Cortical collecting duct intercalated cells o type B or o “non-A, non-B” type (upper cell, within dashed box) have been proposed to mediate electroneutral reabsorption o NaCl. Based on studies with knockout mice, apical NaCl uptake via parallel operation o pendrin and SLC4A8 is coupled to basolateral NaCl e f ux via SLC4A9 in parallel with KCC4 and CLCKb. Transepithelial NaCl transport by this cell type appears to be energized by basolateral vH ATPase rather than by Na /K ATPase. Note that this proposed mechanism o distal nephron electroneutral NaCl reabsorption is still under investigation.

vH+

H+

ATPas e

Na +

S LC4A8

S LC4A9

HCO 3 -

(HCO 3 -)n K+

HCO 3 -

KCC4

Pe ndrin

FIGURE 21-9. Cortical collecting duct. Cortical collecting duct principal

369

Cl-

Cl-

ClCLC-Kb

P rincipa l ce ll

Amiloride, Triamterene

+ ENa C e xpre s s ion 3Na Na +/K+ ATP a s e e xpre s s ion

Na +

Na +/K+ ATPa s e

2K+

ENa C

Nucle us

K+

Mine ra locorticoid re ce ptor

ROMK

Lume n of cortica l colle cting duct Spironolactone

Aldos te rone

O

O

O

HO

H H O

O

H

H

H

S

O

O

H

OH

370 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

lumen to maintain tight control o plasma [K ], as well as to minimize the transepithelial potential di erence resulting rom Na reabsorption. In addition, cortical and outer medullary principal cells, as well as cells o the inner medullary collecting duct, express vasopressin (ADH)-responsive water channels. ADH activates water reabsorption by stimulating a Gs protein-coupled V2 receptor in the basolateral membrane; in turn, Gs protein signaling promotes the reversible insertion into the apical membrane o intracellular vesicles containing aquaporin 2 (AQP2) water channels (Fig. 21-4B). At least two subtypes o intercalated cells (IC) contribute to systemic acid–base balance through cell type-speci c polarized expression o the vacuolar H ATPase (vH ATPase). Type A IC secrete protons via the apical H ATPase and reabsorb bicarbonate through the basolateral Cl /HCO3 exchanger (also known as kidney AE1). Type B IC secrete HCO3 through the apical Cl /HCO3 exchanger pendrin and reabsorb protons via the basolateral H ATPase. Pendrin likely also mediates apical Cl reabsorption via a third IC type, the “non-A, non-B” IC. In rodents, pendrin-mediated electroneutral NaCl reabsorption may be equivalent in magnitude to ENaC-mediated electrogenic Na absorption accompanied by paracellular Cl absorption across the CCD. Pendrin-mediated Cl absorption and HCO3 secretion are positively regulated by luminal -ketoglutarate ( -KG) secreted by the proximal tubule, acting through the G protein-coupled OXGR1 -KG receptor o non-A, non-B intercalated cells. Intercalated cells also mediate K absorption by electroneutral luminal H /K ATPases, f ow-sensitive K secretion by Ca2 -activated maxi-K channels, and NH4 secretion by NH3/NH4+ transporter proteins related to the RHAG component o the erythroid Rhesus (Rh) antigens.

PATHOPHYSIOLOGY OF EDEMA FORMATION Edema is de ned as the accumulation o f uid in the interstitial space. Edema can be either exudative (having a high protein content) or transudative (having a low protein content, essentially a plasma ultra ltrate). Exudative edema occurs as part o the acute inf ammatory response (see Chapter 42, Principles o Inf ammation and the Immune System). The type o edema considered here is transudative edema , which can result rom pathologic renal retention o Na . Under physiologic conditions, any increased f uid ltration across the capillary membrane is quickly counterbalanced by homeostatic mechanisms. This return to a physiologic set-point is mediated by three actors: oncotic orces, lymphatic drainage, and long-term modulation o volume by physiologic sensors and signals. Oncotic orces play an immediate role in f uid shi ts between compartments. For example, increased f uid shi t to the interstitial space will result in increased interstitial hydrostatic pressure and increased plasma oncotic pressure. Both o these variables avor f uid shi t back into the intravascular space (Fig. 21-1). The lymphatic system can also increase return o ltered f uid dramatically, thereby decreasing the amount o ltered f uid that remains in the interstitial space. Over a period o days to weeks, volume sensors and signals respond to changes in volume by altering the extent o natriuresis or sodium reabsorption necessary to maintain a constant intravascular volume. These combined systems closely monitor and regulate intravascular volume. There ore, the

pathophysiology o transudative edema ormation almost always requires an element o pathologic renal Na retention. The three most common clinical situations resulting in edema ormation are heart ailure, cirrhosis, and nephrotic syndrome. All o these diseases mani est deranged Na reabsorption caused by pathologic alterations in volume regulation. Understanding the pathophysiology o edema ormation in these diseases provides a rationale or the therapeutic use o natriuretic agents.

Heart Failure Heart ailure (HF) is de ned by the inability o the heart to per use tissues and organs adequately. Insu cient cardiac output and subsequent decreased blood f ow through the arterial vascular bed leads to congestion in the venous “capacitance” vessels. The resulting increase in capillary hydrostatic pressure avors f uid transudation into tissue interstitial spaces. Right heart ailure leads initially to peripheral edema, whereas le t heart ailure can lead rst to pulmonary edema. In the introductory case, Mr. R’s compromised cardiac unction caused pulmonary venous congestion and peripheral edema; the pulmonary congestion was responsible or his sensation o dyspnea. The pathophysiology o heart ailure is discussed in urther detail in Chapter 26; the current discussion is restricted to the pathophysiology o edema ormation. The undamental cause o Na retention in HF is perceived volume depletion (Fig. 21-10). The inadequate arterial blood f ow is perceived by high-pressure volume receptors, including the juxtaglomerular apparatus, as a decrease in intravascular volume. The kidney there ore increases renin production, leading to increased angiotensin II (AT II) production and secretion o aldosterone by the adrenal cortex. AT II and aldosterone both increase renal Na absorption. Other important mediators o increased renal Na reabsorption may include renal sympathetic innervation and autacoids such as endothelin and prostaglandins; these pathways act to maintain renal per usion pressure and glomerular ltration raction in the presence o (renally) perceived volume depletion. Under physiologic conditions, low-pressure systems such as neural responses and natriuretic peptides sense the increased pressure resulting rom venous congestion and there ore promote natriuresis. This response limits the extent o renal Na reabsorption and prevents pathologic extracellular f uid volume expansion. However, both neural and natriuretic peptide signaling pathways are disrupted in heart ailure. Heart ailure activates excessive sympathetic responses, in part to increase ventricular inotropy through the action o norepinephrine, thus augmenting ejection raction and maintaining cardiac output. Plasma natriuretic peptide is signi cantly increased in heart ailure, but coexisting end-organ resistance may blunt the natriuretic response to the increased concentration o circulating hormone. Diuretics and ACE inhibitors have ound signi cant application in the interruption o heart ailure pathophysiology. As discussed below, diuretics decrease renal Na reabsorption and thereby reduce the extracellular volume expansion that avors edema ormation. As demonstrated in the introductory case, diuretics can be used in an acute setting to reduce pulmonary edema. Over the longer term, decreased Na retention also a ects a terload by reducing intravascular volume, which can lower ventricular systolic pressure and systemic blood pressure. ACE inhibitors may interrupt

C h a p t e r 2 1 Pharmacology o Volume Regulation Compromis e d ca rdia c function

A "Underfill model"

De cre a s e d a rte ria l blood pre s s ure

371

B "Overflow model"

He pa tic ve nous outflow obs truction

Live r injury le a ding to pos t-s inus oida l obs truction

As cite s forma tion

He pa to-re na l re fle x

De cre a s e d intra va s cula r volume

P rima ry re na l Na + re te ntion

Low ve nous filling pre s s ure

Incre a s e d pla s ma volume

Low ca rdia c output

As cite s forma tion

Fluid tra ns uda tion

S ys te mic a nd pulmona ry conge s tion, de cre a s e d lympha tic dra ina ge

Extra ce llula r fluid volume e xpa ns ion

Ele va te d ca rdia c a tria l pre s s ure

Re na l s e ns ors pe rce ive de cre a s e d volume

Chronic dila tion of ca rdia c cha mbe rs

Atte nua tion of na triure tic re s pons e

Re na l s odium re te ntion

FIGURE 21-10. Mechanisms o Na retention in heart ailure. In heart ailure, compromised cardiac unction leads to decreased arterial blood pressure and subsequent activation o renal volume sensors. These sensors activate renal sodium retention to expand extracellular volume and thereby correct the decreased arterial blood pressure. The expansion o extracellular volume increases cardiac atrial pressure. In the ailing heart, the increased atrial pressure leads to increased hydrostatic pressure in the pulmonary and systemic circuits, leading to f uid transudation and edema. In addition, evidence suggests that chronic dilation o the cardiac chambers leads to local resistance to stimulation by natriuretic peptide; in the absence o an appropriate natriuretic response, the kidney continues reabsorbing Na despite the increased extracellular volume.

pathologic paracrine signaling pathways that otherwise lead to deterioration o cardiac tissue and worsening o HF (see below).

Cirrhosis Cirrhosis is caused by hepatic parenchymal brosis resulting

rom chronic inf ammation or hepatotoxic insult. The brotic changes alter hepatic hemodynamics by obstructing venous outf ow rom the liver and increasing hydrostatic pressure in the portal vein. The obstruction to f ow causes portosystemic shunting o blood away rom the liver and into the systemic circulation. Hepatocellular injury disrupts the synthetic and metabolic unctions o the liver, leading to decreased production o albumin and o other important macromolecular contributors to plasma oncotic pressure. Liver dys unction decreases biosynthesis and secretion o peptide hormones, serum hormone-binding proteins, and coagulation actors (thereby increasing the risk or bruising and bleeding). The mechanism o renal Na retention in cirrhosis remains controversial, as ref ected in two proposed models (Fig. 21-11). The under ll model (Fig. 21-11A) suggests that obstruction o hepatic venous outf ow leads to an increase in intrahepatic hydrostatic pressure. The increased hydrostatic pressure causes increased f uid transudation across the hepatic sinusoids, increasing lymphatic f ow through the thoracic duct. Under physiologic conditions, the lymphatic

Activa tion of ba rore ce ptors Re na l Na + re te ntion

FIGURE 21-11. Proposed mechanisms o Na retention in cirrhosis. The postsinusoidal obstruction in cirrhosis is associated with renal Na retention as well as the accumulation o ascites f uid. Two models have been proposed to explain the mechanisms o these e ects. A. Hepatic venous outf ow obstruction causes increased hydrostatic pressure, which initiates ascites ormation. The accumulation o ascites f uid decreases intravascular volume, leading to low venous lling pressure, decreased cardiac output, and subsequent activation o arterial baroreceptors that initiate renal Na retention. B. Postsinusoidal obstruction activates the hepatorenal ref ex, an autonomic response involving the liver and kidney that initiates renal Na reabsorption by a poorly understood mechanism. The renal Na retention leads to an expansion o plasma volume, increased hydrostatic pressure in the portal circuit, and the ormation o ascites.

system is able to increase its f ow dramatically and thereby limit the extent o interstitial f uid accumulation. In cirrhosis, however, lymphatic f ow can exceed 20 L/day, overwhelming the ability o the lymphatic system to return transudate to the systemic circulation and leading to the ormation o ascites (an accumulation o serous f uid in the abdominal cavity). Ascites ormation decreases intravascular volume, because f uid is shunted rom the plasma into the abdominal cavity. The decreased intravascular volume leads to a decrease in cardiac output, with subsequent activation o baroreceptors that increase renal Na retention. Thus, the under ll model is conceptually similar to the mechanism o edema ormation in heart ailure, in that the kidney initiates Na reabsorption in response to a perceived decrease in intravascular volume. The overf ow model postulates that ascites ormation involves an element o primary renal Na retention (Fig. 21-11B). In this model, postsinusoidal obstruction activates the hepatorenal ref ex, an incompletely characterized autonomic response that increases renal Na retention. This pathologic Na retention leads to intravascular volume expansion, increased portal hydrostatic pressure, and ormation o ascites. Although not well understood, this mechanism is consistent with a number o experimental model systems demonstrating that renal Na retention in cirrhosis occurs be ore the development o ascites.

372 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Ascites ormation may well involve elements o both the under ll and overf ow models. Both models begin with the observation that cirrhosis leads to signi cant hepatic outf ow obstruction, and both must consider compromised portal hemodynamics, decreased hepatic synthetic and secretory unctions leading to decreased plasma oncotic pressure, and poorly characterized neural or hormonal interactions between the liver and the kidney. Elucidation o the mechanism o the hepatorenal ref ex may lead in the uture to more e ective pharmacologic interventions to manage the development o ascites in cirrhosis.

Nephrotic Syndrome Nephrotic syndrome is characterized by massive proteinuria ( 3.5 g/day), edema, hypoalbuminemia, and o ten hypercholesterolemia. The primary cause o nephrotic syndrome is glomerular dysfunction, which may be due to immune complex disease, diabetes, lupus, amyloidosis, or genetic or other conditions a ecting glomerular unction. A classical explanation o edema ormation in nephrotic syndrome ollows this sequence. First, massive proteinuria leads to decreased plasma oncotic pressure, reducing the orces avoring f uid retention in the capillary and leading to f uid transudation into the interstitium. The increased net f uid transudation decreases intravascular volume, activating volume sensors to enhance renal Na retention. The resulting expansion in f uid volume, in the absence o adequate compensatory albumin synthesis, maintains low plasma oncotic pressure and continued edema ormation. In this view, renal Na retention is secondary to decreased renal arterial per usion. However, the edema o nephrotic syndrome may also be caused by intrinsic changes in capillary junctional permeability and/or by primary renal Na retention. The postulated primary Na retention o nephrotic syndrome may be localized to the distal nephron, arising rom resistance to natriuretic peptides, increased sympathetic nervous system activity, or increased ENaC activation by ltered luminal proteases, as described above. Although treatment o nephrotic syndrome can include diuretics to counter renal Na retention, correction o edema typically requires correction o the underlying glomerular disorder, eventually leading to decreased proteinuria and correction o the edema. The glucocorticoids and immunosuppressants used to treat some orms o nephrotic syndrome can themselves promote urther sodium retention. Diuretics are used in the short term to minimize edema ormation.

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic modulators o extracellular f uid volume can be divided into agents that modi y neurohormonal volume regulators and agents that act directly on the nephron segments to alter renal Na handling. The ormer category includes agents that interrupt the renin-angiotensin axis, alter circulating levels o natriuretic peptides, or interrupt ADH signaling. The latter category includes the various classes o diuretics, which directly target renal ion transporter or channel unction or expression to increase renal Na excretion. Neurohormonal volume regulators may also act directly on Na reabsorption through mechanisms less well understood than those o the diuretics.

Agents That Modify Volume Regulators Inhibitors of the Renin-Angiotensin System

Four clinically available pharmacologic strategies are used to interrupt the renin-angiotensin-aldosterone system (RAAS). First, inhibition o the enzymatic activity o renin prevents generation o angiotensin I. Second, ACE inhibitors interrupt the conversion o angiotensin I to angiotensin II. Third, angiotensin receptor antagonists are competitive antagonists at the AT1 receptor and thus inhibit the target-organ e ects o angiotensin II. Fourth, antagonists o the mineralocorticoid receptor block aldosterone action at the nephron collecting duct. The rst three classes o agents are discussed here; antagonists o aldosterone action are considered diuretics and are addressed below (see “Collecting Duct [PotassiumSparing] Diuretics”). Renin Inhibitors Aliskiren is the rst and currently only approved inhibitor o

the enzymatic activity o renin. It binds to the active site o renin, inhibiting the binding o renin to angiotensinogen and blocking the conversion o angiotensinogen to angiotensin I, the rate-determining step o the RAAS cascade. Aliskiren is an e ective antihypertensive and has been approved or use in hypertensive patients with renal insu ciency. Aliskiren is approved or use with thiazide diuretics, which improve the e cacy o drugs targeting the RAAS. Aliskiren may also be use ul in slowing the progression o heart ailure and chronic kidney disease (Fig. 21-12). Aliskiren should not be combined with ACE inhibitors or angiotensin receptor blockers (ARBs) in patients with diabetes or chronic kidney disease o stage 3 (GFR 60 mL/min) or higher. The risks include worsening o kidney unction, hypotension, and hyperkalemia. Aliskiren is contraindicated in pregnancy, since drugs that act directly on the renin-angiotensin system are teratogenic.

Angiotensin Converting Enzyme Inhibitors

Pharmacologic interruption o the renin-angiotensin axis is achieved most commonly via inhibition o angiotensin converting enzyme (ACE). Because angiotensin II is the primary mediator o the activity o the renin-angiotensinaldosterone system, decreased conversion o angiotensin I to angiotensin II inhibits arteriolar vasoconstriction, decreases aldosterone synthesis, inhibits renal proximal tubule NaCl reabsorption, and decreases ADH release. All o these actions result in decreased blood pressure and increased natriuresis. In addition, because ACE proteolytically cleaves bradykinin (among other substrates), ACE inhibitors also increase levels o bradykinin and other kinins. Bradykinin causes vascular smooth muscle relaxation by binding to bradykinin receptors on endothelial cell sur aces, leading to intracellular Ca2 mobilization, eNOS activation, and increased NO production (see Chapter 22). Thus, ACE inhibitors decrease blood pressure both by decreasing angiotensin II levels and by increasing bradykinin levels (Fig. 21-12). The contribution o reduced plasma aldosterone levels to the antihypertensive e ects o ACE inhibitors remains unclear. This uncertainty is related to the observation that the renal vasoconstrictive e ects o angiotensin II occur primarily at the e erent arteriole o the glomerulus. A pre erential decrease in e erent relative to a erent arteriolar tone reduces intraglomerular pressure, resulting in decreased GFR. This reduction in GFR may counterbalance the anticipated reduction

C h a p t e r 2 1 Pharmacology of Volume Regulation Angiote ns inoge n

Kininoge n

Re nin inhibitor

Renin Angiote ns in I

Bra dykinin ACE inhibitor

ACE

Kallikrein

Angiote ns in II

Kininase II

Ina ctive

AT1 re ce ptor a nta gonis ts

Aldos te rone s e cre tion (me dia te d by AT1 re ce ptors )

Incre a s e d Na + a nd H2 O re a bs orption

Va s ocons triction

Va s odila tion

(me dia te d by AT1 re ce ptors )

Incre a s e d pe riphe ra l va s cula r re s is ta nce

Incre a s e d blood pre s s ure

De cre a s e d pe riphe ra l va s cula r re s is ta nce

De cre a s e d blood pre s s ure

FIGURE 21-12. Effects of renin-angiotensin system inhibitors on blood pressure. Renin inhibitors prevent the conversion of angiotensinogen to angiotensin I. ACE inhibitors prevent the conversion of angiotensin I to angiotensin II (both in the lung and locally in blood vessels and tissues) and inhibit the inactivation of bradykinin. Both actions of ACE inhibitors lead to vasodilation. The inhibition of angiotensin I conversion decreases AT1-mediated vasoconstriction and decreases aldosterone secretion; both of these effects act to decrease blood pressure. The inhibition of kininase II activity results in higher bradykinin levels, which promote vasodilation. The increased vasodilation decreases peripheral vascular resistance, which decreases blood pressure. AT1 antagonists (also known as angiotensin receptor blockers, or ARBs) similarly decrease aldosterone synthesis and interrupt AT1-mediated vasoconstriction, but do not alter bradykinin levels. Note that bradykinin-induced cough is a major adverse effect of ACE inhibitors but not of AT1 antagonists.

in Na and H2O retention that should occur as a result o the reduced aldosterone levels. ACE inhibitors exhibit three patterns o metabolism. The prototypical ACE inhibitor, captopril, represents the f rst pattern: it is active as administered but is also biotrans ormed to an active metabolite. The second and most common pattern, exemplif ed by enalapril and ramipril, is that o an ester prodrug converted in the plasma to an active metabolite. The active orms o each o these drugs are denoted by the letters “-at” added to the drug name; thus, enalaprilat and ramiprilat are the active orms o enalapril and ramipril, respectively. Lisinopril exemplif es the third pattern, in which the drug is administered in active orm and excreted unchanged by the kidneys. Captopril, enalapril, ramipril, and lisinopril have all been studied in large-scale clinical trials and are among the ACE inhibitors in clinical use (see Drug Summary Table). Fosinopril is unique among approved ACE inhibitors in its excretion by both renal and hepatic pathways rather than only via the kidneys. This characteristic makes osinopril a sa er drug choice than other ACE inhibitors or heart ailure

373

patients with decreased renal unction resulting rom poor per usion. Although ACE inhibitors are generally well tolerated, important adverse e ects o these agents include cough and angioedema caused by potentiation o bradykinin action. The cough, occurring in up to 20% o patients taking captopril, is usually dry and nonproductive. While not causing serious physiologic e ects, the cough may cause discom ort, impair voice quality, and limit patient adherence. Angioedema (rapid swelling/edema o the dermis, subcutaneous tissue, mucosa, and submucosal tissues), which can occur in 0.1–0.2% o patients, is a potentially li e-threatening cause o airway obstruction. Lisinopril-induced angioedema that is isolated to the small intestine can present with abdominal pain. These adverse e ects usually occur during the f rst week o therapy and may require emergent intervention. ACE inhibitors can precipitate f rst-dose hypotension and/ or acute renal failure and thus are administered at a low initial dose. These adverse e ects are more common in patients with bilateral renal artery stenosis. In such patients, renal unction can depend on increased angiotensin II activity, because elevated angiotensin II maintains GFR by pre erential constriction o the e erent arteriole. For this reason, bilateral renal artery stenosis is a relative contraindication to ACE inhibitor therapy. ACE inhibitors reduce aldosterone synthesis and so can produce hyperkalemia . Hyperkalemia is more commonly observed when ACE inhibitors are used in conjunction with potassium-sparing diuretics such as spironolactone, amiloride, and triamterene (see below). ACE inhibitors are widely used to treat hypertension, heart ailure, acute myocardial in arction, and chronic kidney disease. In many cases, ACE inhibitors are increasingly considered f rst-line agents or hypertension, especially when a patient has concomitant le t ventricular wall dysunction or diabetes (see Chapter 26). ACE inhibitors have broad applicability to all orms o hypertension, including hypertension in which there is no clear increase in plasma renin levels. By incompletely understood mechanisms that may involve inhibition o paracrine growth actors and hormones that stimulate pathologic tissue hypertrophy and f brosis, long-term use o ACE inhibitors retards progression o the cardiac contractile dys unction observed in heart ailure and a ter myocardial in arction. ACE inhibitors can also delay progression o diabetic nephropathy, likely through attenuation o renal paracrine signaling pathways, with consequent improvement in renal hemodynamics. As noted above, worsened clinical outcomes have led to discontinuation o the combined use o ACE inhibitors and aliskiren. Combined administration o ACE inhibitors and angiotensin receptor blockers (ARBs) is similarly no longer recommended, as it increases the risks o hyperkalemia and acute kidney injury compared to monotherapy. Like aliskiren, ACE inhibitors are teratogenic and are there ore contraindicated in pregnancy. Angiotensin Receptor Antagonists

AT1 receptor antagonists, such as losartan and valsartan, inhibit the action o angiotensin II at its receptor (Fig. 21-12). Compared to ACE inhibitors, AT1 receptor antagonists may allow more complete inhibition o the actions o angiotensin II, because ACE is not the only enzyme that can generate angiotensin II. In addition, because AT1 receptor antagonists have no e ect on bradykinin metabolism, their use may

374 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

minimize the incidence o drug-induced cough and angioedema. However, the inability o AT1 receptor antagonists to potentiate the vasodilatory e ects o bradykinin may result in less e ective vasodilation. Unlike ACE inhibitors, AT1 receptor antagonists may indirectly increase vasorelaxant AT2 receptor activity. Both ACE inhibitors and AT1 antagonists induce increased renin release as a compensatory mechanism; in the case o AT1 blockade, the increased angiotensin II that results could lead to increased interaction o angiotensin II with AT2 receptors. AT1 receptor antagonists are approved or the treatment o hypertension. Although these agents were initially prescribed only or patients with intolerable adverse reactions to ACE inhibitors, they are now considered rst-line treatments or hypertension. AT1 receptor antagonists are also under study or the treatment o heart ailure. Recent trials have suggested that the combination o an AT1 receptor antagonist and an ACE inhibitor may have some clinical bene t in severe heart ailure, and studies testing such combinations in the treatment o chronic kidney disease and cardiac disease progression are currently underway. Combined therapy using AT1 receptor antagonists and aliskiren is also under investigation or treatment o hypertension, heart ailure, and renal ailure. AT1 receptor antagonists may protect against stroke, not only by controlling hypertension but also through bene cial secondary e ects. These include reduced platelet aggregation, decreased serum uric acid levels, decreased incidence o atrial brillation, and antidiabetic e ects. The mechanisms o these secondary e ects remain to be elucidated.

B-Type Natriuretic Peptide

Nesiritide , a recombinant human-sequence B-type natri-

uretic peptide (BNP), can be used or short-term management o decompensated heart ailure. Because nesiritide is a peptide, it is ine ective when given orally. In clinical trials o nesiritide in acute heart ailure, the drug decreased pulmonary capillary wedge pressure (a measure o hydrostatic pressure in the pulmonary system), decreased systemic vascular resistance, and improved cardiac hemodynamic parameters such as stroke volume. Although nesiritide was not more e cacious in these trials than the more commonly used dobutamine (see Chapter 26), nesiritide may be associated with a lower incidence o arrhythmias than dobutamine. At low doses, nesiritide appears to promote water excretion to a greater degree than sodium excretion. Hypotension is a major adverse e ect o nesiritide, ref ecting the vasorelaxant properties o the natriuretic peptides. The risk o hypotension is increased by co-administration o nesiritide with an ACE inhibitor. Nesiritide treatment is also associated with an increased risk o renal dys unction. These adverse e ects have not been reported in preliminary clinical trials o an investigational peptide related to ANP, which exhibits power ul natriuretic as well as diuretic properties.

Vasopressin Receptor Antagonists and Agonists

The tetracycline analogue demeclocycline has long been used in the treatment o syndromes o inappropriate ADH secretion (SIADH), when dietary water restriction is not easible or su cient. Its mechanism o action is uncertain, although vasopressin receptor type 2 (V2R) blockade has been suggested. Demeclocycline use in the setting o chronic kidney disease has been associated with precipitation o

acute kidney injury. Demeclocycline shares with other tetracyclines an increased risk o photosensitivity, leading to the recommendation to minimize sun or UV exposure during treatment. Conivaptan is the rst speci c nonpeptide vasopressin receptor antagonist approved or treatment o euvolemic hyponatremias (SIADH). Its disadvantages include a requirement or intravenous administration and some V1 receptor antagonist activity. However, the V2-selective receptor antagonist tolvaptan is orally bioavailable. In clinical trials, V2 receptor antagonists have also shown bene t in the treatment o other conditions associated with inappropriate ADH-induced water retention, including heart ailure and cirrhotic ascites. V2 receptor antagonists are also showing promise as agents to retard vasopressin-driven renal cyst growth in autosomal dominant polycystic kidney disease. V2R antagonists are sometimes used o -label (primarily by cardiologists) or management o volume overload in patients with severe heart ailure. Congenital nephrogenic diabetes insipidus may result rom mutations in either the V2 receptor or the aquaporin AQP2 in the principal cells o the collecting duct (see Fig. 21-4B). Some V2 receptor mutations are associated with trapping o newly synthesized receptor polypeptides inside the principal cell. Vasopressin receptor antagonists may act as molecular chaperones or a subset o these mutant receptors; in these cases, antagonist binding presumably promotes a receptor con ormation that allows insertion o the mutant protein into the apical membrane o the cell. Cell-permeant, vasopressin-mimetic small molecules have also been shown to activate mutant V2 receptors inside cells, generating su cient cAMP to mobilize aquaporin 2 water channels to the apical sur ace. This strategy is thus ar the most promising approach to the treatment o V2 receptor-linked nephrogenic diabetes insipidus. Similar strategies are being adopted or many hereditary diseases o G protein-coupled receptors. Terlipressin is an investigational vasopressin analog with moderate V1 receptor agonist activity and speci city. It may have potential clinical application in reducing portal hypertension and improving renal hemodynamics in liver ailure and ascites.

Agents That Decrease Renal Na Reabsorption As discussed above, the kidney modi es the ionic composition o the glomerular ltrate by the concerted action o ion transporters and channels in both apical and basolateral membranes o renal tubular epithelial cells. This transepithelial ion transport can be modulated pharmacologically by the actions o diuretic drugs to regulate urinary volume and composition. Pharmacologic inhibition o ion reabsorption leads to reduction o the osmotic driving orce that avors water reabsorption in the water-permeable segments o the nephron. Diuretics target sodium reabsorption along our segments o the nephron: the proximal tubule, medullary thick ascending limb, distal convoluted tubule, and collecting duct. The kidney concentrates and secretes these drugs into the tubule lumen, allowing diuretics to reach higher concentrations in the tubule than in the blood. Because o this concentrating e ect, therapeutic diuretic doses are o ten accompanied by low blood levels o diuretics and by mild extrarenal adverse e ects.

C h a p t e r 2 1 Pharmacology of Volume Regulation

Carbonic Anhydrase Inhibitors

Carbonic anhydrase inhibitors, exempli ed by acetazolamide , inhibit sodium reabsorption by noncompetitively and reversibly inhibiting cytoplasmic carbonic anhydrase II and luminal carbonic anhydrase IV in proximal tubule cells (Fig. 21-6). Inhibition o carbonic anhydrase leads to increased delivery o sodium bicarbonate to more distal segments o the nephron. Much o this sodium bicarbonate is initially excreted, resulting in an acute decrease in plasma volume (diuresis). However, over the course o several days o therapy, the diuretic e ect o the drug is diminished by compensatory up-regulation o NaHCO3 reabsorption and compensatory increased NaCl reabsorption across more distal nephron segments (by incompletely understood mechanisms). Use o carbonic anhydrase inhibitors is o ten associated with mild to moderate metabolic acidosis, arising not only rom inhibition o proximal tubular H secretion but also rom inhibition o carbonic anhydrase in acid-secreting intercalated cells o the collecting duct. The alkalinized urine resulting rom carbonic anhydrase inhibition increases the urinary excretion o organic acid anions, including aspirin. The clinical use o carbonic anhydrase inhibitors is primarily restricted to several carbonic anhydrase-dependent conditions (see below). In addition, carbonic anhydrase inhibitors are occasionally used to restore acid–base balance in heart ailure patients with metabolic alkalosis due to treatment with loop diuretics. Carbonic anhydrase inhibitors have ophthalmologic applications. The ciliary process epithelium o the anterior chamber o the eye secretes sodium chloride into the aqueous humor. This NaCl secretion requires carbonic anhydrase activity, because a portion o the basolateral Cl uptake by the ciliary epithelium requires coupled Cl -HCO3 and Na -H exchange as well as Na -HCO3 symport. The basolateral membrane Na -K -2Cl co-transporter NKCC1 mediates most o the remaining Cl uptake by ciliary epithelial cells. Glaucoma is characterized by increased pressure in the anterior chamber o the eye. This is usually attributed to partially obstructed outf ow o aqueous humor, but in some cases, overproduction o aqueous humor may also contribute. Inhibition o carbonic anhydrase in the ciliary process epithelium reduces secretion o aqueous humor and may thereby reduce elevated intraocular pressure. Topical lipophilic carbonic anhydrase inhibitors such as brinzolamide are o ten used in concert with topical -adrenergic antagonists in the treatment o glaucoma (see Chapter 11, Adrenergic Pharmacology). Ascent to altitudes higher than 3,000 m above sea level predisposes several body organs, including the brain, to edema and ionic disequilibria. Symptoms o acute mountain sickness can include nausea, headache, dizziness, insomnia, pulmonary edema, and con usion. Carbonic anhydrase is involved in the secretion o chloride and bicarbonate into the cerebrospinal f uid by the choroid plexus o the cerebral ventricles, and inhibition o carbonic anhydrase can be used prophylactically against acute mountain sickness. The stillcontroversial mechanism(s) o action include e ects on the choroid plexus and ependyma, on the respiratory control centers o the brain, and on the blood–brain barrier. Carbonic anhydrase inhibitors are also used in the treatment o epilepsy, although the antiepileptic mechanism o some o these drugs may not require inhibition o carbonic anhydrase. One such antiepileptic drug, topiramate , can produce

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mild to moderate acidosis due to impaired renal acidi cation o the urine. The treatment o hyperuricemia or gout (see Chapter 49, Integrative Inf ammation Pharmacology: Gout) may involve alkalinization o the urine to increase the urinary solubility o uric acid. Increased uric acid solubility prevents uric acid precipitation in the urine and consequent uric acid nephropathy and nephrolithiasis (kidney stones). Urinary alkalinization can be achieved by oral bicarbonate, supplemented as needed by a carbonic anhydrase inhibitor to reduce renal reabsorption o the ltered bicarbonate.

Osmotic Diuretics

Osmotic diuretics, such as mannitol, are small molecules that are ltered at the glomerulus but not subsequently reabsorbed in the nephron. Thus, they constitute an intraluminal osmotic orce that limits reabsorption o water across water-permeable nephron segments. The e ect o osmotic agents is greatest in the proximal tubule, where most isoosmotic reabsorption o water takes place. By causing water loss in excess o sodium excretion, osmotic diuresis can sometimes lead to unintended hypernatremia. Alternatively, the increased urine volume associated with osmotic diuresis can also promote vigorous natriuresis. There ore, care ul monitoring o clinical volume status and serum electrolytes is warranted. Mannitol is used primarily or rapid (emergent) treatment o increased intracranial pressure . In the setting o head trauma, brain hemorrhage, or a symptomatic cerebral mass, the increased intracranial pressure can be relieved, at least transiently, by the acute reduction in cerebral intravascular volume that ollows the mannitol-induced reduction in systemic vascular volume. Osmotic diuresis can also occur as a result o pathologic states. Two common examples o this phenomenon are hyperglycemia and the use o radiocontrast dyes. In diabetic hyperglycemia, the ltered glucose load exceeds the reabsorptive capacity o the proximal tubule or glucose. As a result, signi cant quantities o glucose remain in the lumen o the nephron and act as an osmotic agent to increase f uid retention in the tubular lumen, thereby decreasing f uid reabsorption. Radiocontrast agents used or radiologic imaging studies are ltered at the glomerulus but not reabsorbed by the tubular epithelium. Thus, these dyes constitute an osmotic load and can produce osmotic diuresis. In patients with borderline cardiovascular status, the consequent reduction in intravascular volume can lead to hypotension or to renal and/or cardiac insu ciency secondary to reduced organ per usion.

Loop Diuretics

The so-called loop diuretics act at the TAL o the loop o Henle. These agents reversibly and competitively inhibit the Na -K -2Cl co-transporter NKCC2 in the apical (luminal) membrane o TAL epithelial cells (Fig. 21-7). In addition to the primary e ect o inhibiting Na reabsorption across the TAL, inhibition o transcellular NaCl transport secondarily reduces or abolishes the lumen-positive transepithelial potential di erence across the TAL. Consequently, paracellular reabsorption o divalent cations, particularly calcium and magnesium, is also inhibited. The increased delivery o luminal calcium and magnesium to downstream reabsorptive sites in the distal convoluted tubule can lead to increased urinary excretion o calcium and magnesium. The resultant

376 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

hypocalcemia and/or hypomagnesemia can be clinically signi cant in some patients who require prolonged administration o loop agents. Furthermore, increased downstream delivery o sodium increases the Na load presented to principal cells o the collecting duct. The increased Na load stimulates increased secretion o K and protons, predisposing to hypokalemia and metabolic alkalosis. Together, the clinical consequences o loop diuretic treatment are o ten described as volume-contraction alkalosis . Diuretic-associated hypokalemia can predispose to cardiac arrhythmias in the setting o coronary or cardiac insu ciency. The prototypical loop diuretic is furosemide . Other drugs in this class include bumetanide , torsemide , and ethacrynic acid. All o these agents are generally well tolerated. Apart rom their e ects on renal electrolyte handling, loop diuretics are associated with dose-related ototoxicity, presumably because o altered electrolyte handling in the endolymph. For this reason, co-administration o loop diuretics with aminoglycosides (which are also ototoxic; see Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation) should be avoided. The major di erences among the loop diuretics are in potency and incidence o allergies. Bumetanide is approximately 40 times more potent than the other loop diuretics. Furosemide, bumetanide, and torsemide are all sulfonamide derivatives , while ethacrynic acid is not o this structural class. Thereore, ethacrynic acid is a therapeutic option or patients who are allergic to “sul a” drugs. The high sodium reabsorption capacity o the TAL makes loop diuretics a rst-line therapy or acute relie o pulmonary and peripheral edema in the context o heart ailure. Loop diuretics are capable o reducing intravascular volume to the extent that lling pressures are decreased below the threshold or pulmonary and peripheral edema. This was the rationale or the intravenous urosemide used to treat Mr. R’s pulmonary edema and peripheral edema in the introductory case. Hypoalbuminemia, resulting rom decreased synthesis o albumin (liver disease) or increased clearance o the protein (nephrotic proteinuria), can diminish intravascular oncotic pressure and cause edema. These edematous states can also be treated with low-dose loop diuretics. Loop agents can be used therapeutically to increase calcium diuresis, and thereby provide acute relie o hypercalcemia , in states such as hyperparathyroidism or malignancy-associated hypercalcemia caused by tumor secretion o parathyroid hormone-related protein or other calciotropic hormones (see Chapter 32, Pharmacology o Bone Mineral Homeostasis). Loop agents are also used to counteract hyperkalemia caused by potassium-retaining adverse e ects o other drugs or by renal insu ciency with impaired urinary K excretion in the context o normal or elevated dietary K intake. In acute renal failure , the increased urine f ow elicited by loop diuretics can acilitate clinical management o f uid balance in the ace o decreased glomerular ltration. However, there is no evidence to support the o t-repeated claim that increased urine output itsel intrinsically enhances renal tubular epithelial cell recovery rom the ischemic or toxic event that precipitated the acute renal ailure.

Thiazides

Thiazide diuretics inhibit sodium chloride reabsorption in the distal convoluted tubule (Fig. 21-8). These agents act rom the apical (luminal) side as competitive antagonists o

the NCC Na -Cl co-transporter in the luminal membrane o distal convoluted tubule cells. The modest natriuresis produced by thiazides stems rom the act that 90% o sodium reabsorption occurs upstream o their site o action in the nephron; nonetheless, thiazides do cause a modest reduction in intravascular volume. The decrease in intravascular volume, possibly combined with a poorly understood direct vasodilatory e ect, decreases systemic blood pressure. The distal tubule is also a site o parathyroid hormoneregulated reabsorption o calcium via voltage-independent TRPV5 Ca2 channels. Thiazides promote increased transcellular calcium reabsorption in the distal convoluted tubule. Thiazides have been used to decrease urinary Ca2 wasting in osteoporosis (although this is no longer common practice in the absence o hypercalciuria) and to diminish hypercalciuria in patients at risk or nephrolithiasis . The mechanism by which inhibition o NaCl uptake enhances apical Ca2 entry remains incompletely understood, but part o the response is mediated by increased expression o the apical membrane TRPV5 Ca2 channel and the basolateral membrane Na /Ca2 exchanger. Additionally (and more speculatively), the decreased intracellular Cl concentration that results rom thiazide inhibition o apical Na -Cl co-transport may avor Cl entry via basolateral Cl channels, and the consequent membrane hyperpolarization may avor apical Ca2 entry. In mice, the inhibitory action o thiazide diuretics on distal tubular Na reabsorption and the stimulatory inf uence o thiazides on Ca2 reabsorption both require expression o the small intracellular Ca2 binding protein parvalbumin, but the mechanism connecting these processes remains unde ned. Hydrochlorothiazide is the prototypical thiazide diuretic. In addition to its e ects on renal electrolyte handling, hydrochlorothiazide decreases glucose tolerance and may unmask diabetes in patients at risk or impaired glucose metabolism. The mechanism o this e ect is unknown but may be attributable to drug-induced impairment o insulin secretion and/ or decreased peripheral insulin sensitivity. Thiazide diuretics should not be administered concurrently with antiarrhythmic agents that prolong the QT interval (e.g., quinidine, sotalol), since co-administration o these drugs predisposes patients to torsades de pointes (polymorphic ventricular tachycardia; see Chapter 24, Pharmacology o Cardiac Rhythm). The mechanism o this adverse e ect may be related to thiazideinduced hypokalemia, which increases the potential or cardiac arrhythmias (see Chapter 24). Thiazide diuretics are rst-line agents or treatment o hypertension (see Chapter 26). In numerous randomized clinical trials, these drugs have been shown to reduce both cardiovascular-related and total mortality. In addition, thiazide diuretics are o ten used together with loop agents or their synergistic diuretic e ects in heart ailure. This synergism arises because the increased Na load, delivered rom the loop diuretic-blocked TAL to the thiazide diureticblocked DCT, proceeds urther downstream to the collecting duct, which has only a limited ability to up-regulate compensatory Na reabsorption. The dose o thiazide must be care ully considered in this setting, or as with loop diuretics, thiazides can increase K and H secretion by increasing Na presentation to the collecting duct, thus leading to hypokalemic metabolic alkalosis. Hydrochlorothiazide should be taken several times a day. A longer acting thiazide, chlorthalidone , can be taken once a day and may better prevent the nocturnal blood pressure

C h a p t e r 2 1 Pharmacology of Volume Regulation

elevation that correlates, over time, with end-organ damage. The majority o the clinical trials that documented bene cial e ects o thiazide diuretics or treatment o hypertension were based on chlorthalidone. Patients with impaired secretion o vasopressin by the posterior pituitary gland, or with impaired signaling by the V2 vasopressin receptor in collecting duct principal cells, ail to reabsorb water in the terminal nephron. These patients generate large volumes o hypotonic urine. Central diabetes insipidus (de ective pituitary secretion o vasopressin) can be treated with the exogenous vasopressin agonist desmopressin (see Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland). Patients with nephrogenic diabetes insipidus do not respond to desmopressin; paradoxically, however, thiazide diuretics can produce a modest decrease in urine f ow in this setting. It is thought that, by reducing intravascular volume and decreasing glomerular ltration rate, thiazides reduce the volume o tubular f uid delivered to the collecting duct and thereby decrease urine volume. For nephrogenic diabetes insipidus associated with chronic lithium therapy, traditional treatment with thiazides will likely be supplanted by treatment with amiloride (see below) and, possibly, with acetazolamide.

Collecting Duct (Potassium-Sparing) Diuretics

In contrast to all other diuretic classes, potassium-sparing diuretics increase nephron reabsorption o potassium. Agents in this class interrupt Na reabsorption by principal cells o the collecting duct by one o two mechanisms. Spironolactone and eplerenone inhibit biosynthesis o new Na channels in principal cells, while amiloride and triamterene block the activity o Na channels in the luminal membranes o these cells (Fig. 21-9). The epithelial sodium channel (ENaC) o collecting duct principal cells comprises a complex o partly homologous , , and subunits. Control o sodium channel expression is regulated primarily by aldosterone, which is secreted by the adrenal cortical zona glomerulosa under the regulation o angiotensin II and plasma potassium. Circulating aldosterone di uses into collecting duct principal cells and binds to an intracellular mineralocorticoid receptor. Activation o the mineralocorticoid receptor increases transcription o mRNAs that encode proteins involved in Na handling, including ENaC expressed in the apical membrane and Na / K ATPase expressed in the basolateral membrane. Increased ENaC expression allows increased Na inf ux across the luminal membrane, while increased Na /K ATPase activity allows increased Na e f ux rom the cytoplasm across the basolateral membrane into the interstitium. These two actions o aldosterone, mediated by complex, multistep signaling pathways, increase transepithelial Na reabsorption and hence increase both the Na content o the extracellular space and the intravascular volume. Spironolactone and eplerenone inhibit aldosterone action by binding to and preventing nuclear translocation o the mineralocorticoid receptor. Recent studies suggest that up to 20% o patients with essential hypertension have elevated aldosterone levels. Mineralocorticoid receptor antagonists are used to treat hypertension, and they seem to have greater e cacy in obesity-associated hypertension. This increased sensitivity o obese individuals has been attributed to increased adrenal aldosterone synthesis secondary to actors released by the increased mass o adipocytes. Unlike most diuretics, which must reach their luminal sites o action

377

through glomerular ltration o the albumin-unbound raction, spironolactone requires neither albumin-binding nor glomerular ltration to reach its target receptor and thus can exhibit greater e cacy in the settings o liver ailure and nephrotic syndrome. The ability o spironolactone to crossreact with and inhibit the androgen receptor can cause male impotence and gynecomastia, but the more selective eplerenone, primarily used or the treatment o chronic heart ailure, has a lower incidence o these adverse e ects. Amiloride and triamterene are competitive inhibitors o the ENaC Na channel in the apical membrane o collecting duct principal cells. These agents are also used to treat hypertension. Both types o potassium-sparing diuretics can cause hyperkalemia , because inhibition o electrogenic Na uptake by either mechanism decreases the normal transepithelial lumen-negative potential and thus decreases the driving orce or potassium secretion rom collecting duct cells. Decreased Na uptake through ENaC may also diminish H secretion, leading to metabolic acidosis . The longer hal li e o amiloride may be pre erable in the treatment o some patients. Triamterene occasionally induces crystalluria and, more rarely, triamterene stone ormation, sometimes with reversible acute kidney injury. Used in isolation, potassium-sparing diuretics are mild diuretics because the collecting duct reabsorbs only 1–5% o ltered sodium. However, they can be strong potentiators o more proximally acting diuretics, including loop diuretics. Potassium-sparing diuretics are occasionally used to counteract the potassium-wasting e ects o the thiazides. Amiloride and triamterene are drugs o choice to treat Liddle’s syndrome, a rare, Mendelian orm o hypertension resulting rom gain-o - unction mutations in - or -subunits o the ENaC Na channel o the principal cell (Table 21-3). The antidepressant lithium (Li ) is reabsorbed by ENaC in an amiloride-sensitive manner. In experimental animals, amiloride attenuates or prevents both acute and chronic impairment o urinary concentrating ability by Li . Amiloride may thus also serve to reduce the elevated risk o renal cancer potentially associated with long-term Li use. Potassium-sparing diuretics are used clinically to treat hypokalemic alkalosis secondary to the mineralocorticoid excess that can accompany heart ailure, liver ailure, and other disease processes associated with diminished aldosterone metabolism. The mild diuretic action o spironolactone or eplerenone minimizes the risk o cardiovascular compromise rom excessively rapid or extensive diuresis when diminished oncotic pressure impairs the mobilization o extravascular f uid into the vasculature. There ore, mineralocorticoid receptor antagonists are the diuretics o choice or treatment o ascites and edema associated with impaired plasma protein biosynthesis secondary to liver ailure. Studies have suggested that mineralocorticoid receptor antagonists preserve cardiac unction in the setting o coronary ischemia and that these agents retard the development o heart ailure. Both spironolactone and eplerenone reduce mortality in patients with heart ailure and in patients with signi cant cardiac dys unction (ejection raction 40%) a ter myocardial in arction. This is why Mr. R was prescribed spironolactone in the introductory case. In addition, as in Mr. R’s case, patients with heart ailure are o ten prescribed ACE inhibitors in combination with spironolactone or eplerenone. Because both o these drug classes also decrease K excretion, plasma K levels should be monitored care ully.

378 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

The mechanism by which mineralocorticoid receptor antagonists preserve cardiac unction may be related to inhibition o cardiac brosis resulting, in part, rom a paracrine aldosterone signaling pathway involving mineralocorticoid receptor action in macrophages. Mineralocorticoid antagonists also minimize the aldosterone-dependent decrease in the activity o glucose-6-phosphate dehydrogenase, an important cellular de ense against oxidant stress in endothelial and epithelial cells. Mineralocorticoid antagonists have similar e ects in slowing the progression o chronic kidney disease and the development o renal brosis.

CONCLUSION AND FUTURE DIRECTIONS This chapter has reviewed the physiology and pathophysiology o extracellular volume regulation. Control o intravascular volume maintains adequate per usion pressure to organs and ensures that the kidney is able to lter waste products rom the plasma. Regulation o extracellular volume is accomplished by integrated neurohormonal mechanisms that respond to changes in arterial and atrial wall stress. These hormones modulate numerous steps in renal Na handling and thereby maintain a homeostatic balance between dietary Na intake and Na excretion. Edema can develop when the capillary hydrostatic pressure gradient avoring f uid ltration exceeds the opposing oncotic orces avoring f uid entry into the intravascular space. Pharmacologic treatment o dysregulated extracellular volume involves modi cation o neurohormonal signaling and direct inhibition o renal Na reabsorption. ACE inhibitors prevent the conversion o angiotensin I to angiotensin II; drugs in this class have important vasodilatory actions. Angiotensin receptor antagonists and renin inhibitors are also use ul in interrupting the angiotensin-aldosterone axis. Both ACE inhibitors and angiotensin receptor antagonists have bene cial e ects in slowing the progression o hypertrophy and brosis in the heart, the kidney, and the vasculature. B-type natriuretic peptide (nesiritide) is used in the treatment o decompensated heart ailure, and terlipressin is under investigation or the treatment o portal hypertension. Diuretics are agents that alter nephron Na reabsorption and secondarily alter the reabsorption and secretion o other ions. Essential to understanding diuretic mechanisms is an appreciation o the unctional organization o the nephron. With the exception o osmotic diuretics, which increase urinary f ow by osmotic retention o water throughout the nephron, speci c classes o diuretic drugs target each o the our segments o the nephron. Carbonic anhydrase inhibitors such as acetazolamide decrease sodium and bicarbonate reabsorption in the proximal tubule; loop agents such as urosemide decrease sodium and chloride reabsorption by the apical Na -K -2Cl (NKCC2) pump in the thick ascending limb o the loop o Henle; thiazides such as hydrochlorothiazide inhibit the apical Na -Cl co-transporter (NCC) in the distal convoluted tubule; and potassium-sparing diuretics such as spironolactone and amiloride inhibit, respectively, the aldosterone receptor and the ENaC apical Na channel in the collecting duct. The most important use o diuretics is in the treatment o hypertension; the second most important use is to treat edema o any cause.

Future developments in the pharmacology o extracellular volume regulation will likely ocus on interrupting or enhancing the hormonal pathways implicated in the disruption o volume homeostasis, as well as on the solute and water transporters themselves. New drugs to interrupt the renin-angiotensin-aldosterone axis may include neutral endopeptidase inhibitors, (pro)renin receptor antagonists, AT2 receptor agonists, selective endothelin receptor antagonists, and natriuretic peptides o increased potency and selectivity. The latter will likely play an increasingly important role in the management o decompensated heart ailure and possibly the ascites o liver ailure. Drugs acting on the renin-angiotensin-aldosterone axis will also likely be use ul in slowing the rate o renal and cardiac brosis, rein orcing or improving on the actions o ACE inhibitors, AT1 receptor antagonists, and mineralocorticoid receptor blockers. These drugs also have general and cell type-speci c trophic actions. One example is provided by the role o the AT1 receptor in promoting proli eration o epidermal growth actor receptor ERBB2-negative mammary tumor cells in culture and in xenogra ts. AT1 receptor blockers have slowed mammary cell tumor growth in xenogra t models. Thus, AT1 blockade is a reasonable candidate adjunct therapy or breast tumors that may not respond to more conventional therapy. Promising inhibitors o the renal outer medullary potassium channel ROMK are in late-stage development. Inhibitors o the intercalated cell NaCl reabsorption pathway, targeting the apical Cl /HCO3 exchanger pendrin or the Na -dependent exchanger SLC4A8, are in early-stage development. These will most likely take the orm o inhibitors o the transport proteins themselves, but inhibitors o OXGR1 or the proximal tubular -KG secretory pathway are also potential drug candidates. Drug therapies targeting the WNK and SPAK kinases are in early stages o development. The extrarenal WNK and SPAK kinases are also potential targets or treatment o cystic brosis and central nervous system diseases, including autism, epilepsy, and stroke. “Glif ozin” inhibitors o proximal tubular Na -glucose cotransporter SGLT2, although not approved or treatment o hypertension, have some antihypertensive e ects and may be use ul additions to drug regimens targeting more distal tubular segments, especially i current sa ety concerns are addressed. Speci c V2 vasopressin receptor antagonists such as tolvaptan will be used increasingly in hypervolemic conditions accompanied by elevated ADH levels or action. V2 receptor antagonists have also shown promise in retarding progression o cyst growth in autosomal dominant polycystic kidney disease. The synthetic A-type natriuretic peptide carperitide, currently approved in Japan to treat heart ailure through its diuretic e ects and direct vasodilation, could be used in combination with loop diuretics and tolvaptan. Aquaporin blockers (aquaretics) and urea transporter inhibitors (urearetics) are under development or regulation o f uid homeostasis, and aquaglyceroporin blockers are under investigation as treatments or skin conditions and as modulators o lipid metabolism. Chloride channel blockers and potassium channel blockers are under development to treat the volume depletion o severe toxigenic and in ectious diarrhea as well as rare congenital diarrheas. Chloride channel activators and potassium channel activators are being developed to treat the pulmonary, gastrointestinal, and genitourinary hyposecretion disorders o cystic brosis, sicca

C h a p t e r 2 1 Pharmacology of Volume Regulation

syndromes, and inf ammatory biliary cirrhosis. Carbonic anhydrase II has recently been shown to act as a nitrite reductase and thereby to generate nitric oxide at the acidic pH o ischemic or hypoxic tissue. Surprisingly, this nitrite reductase activity is activated by sul onamide carbonic anhydrase inhibitors even as they inhibit carbonic anhydrase activity. This property may explain the vasodilation associated with use o carbonic anhydrase inhibitors and encourages consideration o new uses or this old drug class.

Suggested Reading Christova M, Alper SL. Core curriculum in nephrology. Tubular transport: core curriculum 2010. Am J Kidney Dis 2010;56:1202–1217. (Annotated review of transport by renal tubular epithelial cells.) Danziger J, Zeidel M. Osmotic homeostasis. Clin J Am Soc Nephrol 2015;10:852–862. (Reviews mechanisms of water homeostasis and disorders of water balance.) Ellison EH. Physiology and pathophysiology o diuretic action. In: Alpern RJ, Hebert SC, eds. The kidney: physiology and pathophysiology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2013:1353–1404. (Full discussion of the physiology and pathophysiology of diuretics.)

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Ernst ME, Moser M. Drug therapy: use o diuretics in patients with hypertension. N Engl J Med 2009;361:2153–2164. (Clinical pharmacology of diuretics.) Palmer L, Schnermann J. Integrated control o sodium transport along the nephron. Clin J Am Soc Nephrol 2015;10:676–687. (Reviews renal mechanisms that integrate control of Na reabsorption, Na excretion, and K excretion.) Seva Pessoa B, van der Lubbe N, Verdonk K, Roks AJ, Hoorn EJ, Danser AH. Key developments in renin-angiotensin-aldosterone system inhibition. Nat Rev Nephrol 2013;9:26–36. (Recent advances in renin-angiotensin physiology.) Townsend RR, Peixoto AJ. Hypertension. NephSAP (Am Soc Nephrol) 2014;13:57–131. (Updated nephrology board review summary and questions about hypertension and antihypertensive therapy.) Verbalis JG, Goldsmith SR, Greenberg A, et al. Diagnosis, evaluation, and treatment o hyponatremia: expert panel recommendations. Am J Med 2013;126(suppl 1):S1–S42. (Includes update on clinical physiology of and indications for use of vasopressin receptor antagonists.) Vongpatanasin W. Resistant hypertension: a review o diagnosis and management. JAMA 2014;311:2216–2224. (Clinical review of resistant hypertension.) Zois NE, Bartels ED, Hunter I, Kousholt BS, Olsen LH, Goetze JP. Natriuretic peptides in cardiometabolic regulation and disease. Nat Rev Cardiol 2014;11:403–412. (Overview of natriuretic peptide physiology in volume regulation.)

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384 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Ade nylyl yl cycla s e NO O

P LC LC DAG DA D AG A G

Gs

Gi

G 12/13

Rh RhoG h oG G EF Rhoo A Rh

Gq

22

Pharmacology of Vascular Tone s GC

ROCK CK K

IP 3

P KC

Va s culaa r s mo moot othh ot muu s ccle ce ll pla s maa m e m br braa n

c GMP G

PKA

GTP GT

MHC-MLC W illia m M . O ld h a m a n d JPoK sGe p h Lo s c a lzo KG MLCK Ca 2+

Ca 2+

ML LC p h o s p h a ta s e

Ca 2 +/C a M C

INTROD ODUC DUC DU UCTION U C & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385–38M MHC MH C -ML MLC ML Cc OPxiddde Donor 3922 orss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Mechanism of Actiionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 PHYSIOLOGY PHY O Y OF VASCULAR TONE. . . . . . . . . . . . . . . . . . . . . . . . 38 3855 e ptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Chemical Classes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3933 3 rre Vess eel cce Physiology 3855 S a rcopla s m mic Blood IP Pharmacokineticss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 Vascular Smoothh Mu M scle Con ontr trac acti tioon re ti ticu icullum lu m Co ntraccPharma C ttio io o n a co colo logi g c Tolerancee . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 a nd Relax elaxati tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 Toxicities and Contr trai aind ndic ications . . . . . . . . . . . . . . . . . . . . . . 395 Regulation of Vascular Tone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 cGMP Potentiators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 395 Siignall Tra rans nsdu duct ction Pathwayss . . . . . . . . . . . . . . . . . . . . . . . 387 Phosphodiesterase Type V Inhibit itor orss . . . . . . . . . . . . . . . . . . 39 3955 Environmental Factorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 Rioc Ri ocig iggua uatt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 Endothelial Factorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3888 Nesi Ne siri riti tide d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3966 Autonomic Nervous System m . . . . . . . . . . . . . . . . . . . . . . . . . 389 Prostacyclin Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 Humorall Regulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 Hu Endothelin Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . 39 3966 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 390 Sympathetic Nervous System Antagonists . . . . . . . . . . . . . . . 397 Ca 2 Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 s . . . . . . . . . . . . . . . . . . . . . . . . . . 397 1-Adrenergic Antagonists Mechanism of Actionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 -Adrenergic Antagonistss . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Chemical Ch i l Classes Cl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 Renin-Angiotensin-Aldosterone System Blockers . . . . . . . . . 397 Pharmacokineticss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Toxicities and Contraindications . . . . . . . . . . . . . . . . . . . . . . 391 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 397 K Channel Openers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .397 Hydralazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

INTRODUCTION While the heart provides cardiac output, the blood vessels play a critical role in the distribution o oxygen and nutrients to metabolically active tissues. Blood f ow to these tissues is exquisitely controlled by a variety o stimuli that act on vascular smooth muscle cells to regulate vascular tone (i.e., the degree o contraction o vascular smooth muscle). Dysregulation o vascular tone contributes to the pathogenesis o a variety o diseases, including hypertension, coronary artery disease, Raynaud’s phenomenon, and migraine headache. Multiple signal transduction pathways converge on the vascular smooth muscle contractile apparatus, o ering numerous targets or pharmacologic intervention. Many success ul therapies have already been developed based on a molecular understanding o the regulation o vascular tone. New targets continue to be identi ed, o ering hope that, in the uture, even better therapies will be available to treat patients with vascular disease.

PHYSIOLOGY OF VASCULAR TONE Vascular tone is a key regulator o tissue per usion, which determines whether tissues receive su cient oxygen and nutrients to

meet their metabolic demands. Blood f ow distribution and circulating blood volume are tightly controlled by the tone o resistance arterioles and capacitance veins, respectively. Vascular smooth muscle cells are the unctional regulatory unit o vessel tone in these regions, integrating a variety o signals to optimize their contractile state. In general, these regulatory units act through the signal transduction pathways discussed in this chapter, many o which are targets or therapeutic intervention.

Blood Vessel Physiology Poiseuille’s law approximates f ow through blood vessels: 4 P r Equation 22-1 Flow L where P is the pressure drop across a length (L) o vessel, r is the vessel radius, and is blood viscosity. This relationship demonstrates that small changes in the tone o circumerential layers o vascular smooth muscle cells, and thus vessel diameter, can have a signi cant impact on blood f ow. The tone o the arterial portion o the circulation and the tone o the venous portion o the circulation have important yet distinctive e ects on the cardiovascular system. Arterial tone directly controls systemic vascular resistance (SVR) 385

386 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

GF, a 63-ye a r-o ld m a n w ith a h is to ry o h yp e rte n s io n , d ia b e te s , a n d h yp e rch o le s te ro le m ia , b e g in s to d e ve lo p e p is o d e s o ch e s t p a in w ith e xe rtio n . On e w e e k a te r h is f rs t e p is o d e , a b o u t o ch e s t p a in i o ccu rs w h ile h e is m o w in g th e la w n . Tw e n ty m in u te s a te r th e o n s e t o h is p a in , GF ta ke s tw o o h is w i e ’s s u b lin g u a l n itro g lyce rin ta b le ts . With in a e w m in u te s , h e e e ls m u ch b e tte r. GF e e ls s o w e ll th a t h e d e cid e s to ta ke o n e o th e s ild e n a f l (Via g ra ®) p ills th a t a rie n d h a d p re vio u s ly o e re d to h im . A e w m in u te s a te r ta kin g s ild e n a f l, h e e e ls u s h e d , d e ve lo p s a th ro b b in g h e a d a ch e , a n d s e n s e s h is h e a rt ra cin g . Up o n s ta n d in g , GF b e co m e s lig h th e a d e d a n d a in ts . He is ta ke n im m e d ia te ly to th e e m e rg e n cy d e p a rtm e n t, w h e re h e is o u n d to h a ve s e ve re hyp o te n s io n . He is q u ickly p la ce d in a s u p in e p o s itio n w ith h is le g s ra is e d a n d m o n ito re d u n til h e re ga in s co n s cio u s n e s s . Th e p hys icia n co n s id e rs a d m in is te rin g a n -a d re n e rg ic a g o n is t, s u ch a s

and, with cardiac output (CO), is an important determinant o mean arterial blood pressure (MAP): MAP

SVR

CO

Equation 22-2

Perhaps more importantly, these changes in arteriolar resistance regulate blood f ow into tissue capillary beds, where increased smooth muscle cell contraction increases vascular resistance and decreases distal per usion. At the organismal level, a coordinated response o resistance vessels is absolutely required to redirect oxygen and nutrients to the tissues most in need. Venous tone, by contrast, plays an important role in determining circulating blood volume. Veins are highly compliant (i.e., can accommodate large changes in volume with little change in pressure) and contain approximately 70% o blood volume during rest. Venoconstriction mobilizes these stores to increase the e ective circulating blood volume, allowing per usion o additional vascular beds (i.e., venoconstriction can be thought o as an “autotrans usion”). The heart and blood vessels orm an integrated and interdependent system, and physiologic or pathophysiologic changes in vascular tone can have a signi cant impact on tissue per usion as well as cardiac output. Vasoconstriction and the resulting increase in vascular resistance increases ventricular a terload, or the systolic ventricular wall stress. The volume and thickness o the le t ventricle also contribute to the net stress experienced by the contracting ventricle. Venoconstriction and the resulting increase in blood return to the heart increases ventricular preload, de ned as end-diastolic ventricular wall stress. These changes directly a ect cardiac stroke volume through the Frank-Starling mechanism and indicate the close coupling between vascular and cardiac physiology (Fig. 22-1).

Vascular Smooth Muscle Contraction and Relaxation As in cardiac and skeletal muscle cells, vascular smooth muscle cells use cyclic interactions between actin and myosin to

p h e nyle p h rin e , b u t th e ra p id im p rove m e n t in GF’s b lo o d p re s s u re a te r h e is p la ce d in a s u p in e p o s itio n s u g g e s ts th a t p h a rm a co lo g ic in te rve n tio n is u n n e ce s s a ry. A te r GF re co ve rs , h is p h ys icia n d is cu s s e s w ith h im th e d a n g e rs o ta kin g m e d ica tio n s w ith o u t a p re s crip tio n a n d , s p e cif ca lly, th e ris k o co n cu rre n t a d m in is tra tio n o o rga n ic n itra te s a n d s ild e n a f l.

Questions 1 . What is the mechanism by which sublingual nitroglycerin acts so quickly to relieve chest pain? 2 . What are the common adverse e ects o nitroglycerin? 3 . How can sildenaf l and organic nitrates interact to precipitate severe hypotension? 4 . Are non-nitrate antihypertensives, such as calcium channel blockers, also contraindicated or men taking sildenaf l? 5 . How can the mechanisms o action o drugs be used to predict possible drug–drug interactions or lack o interactions?

generate orce. This process is regulated by the intracellular calcium (Ca 2 ) concentration, which is normally 10,000 times lower than the extracellular concentration (2 mM). The steep transmembrane Ca2 gradient is maintained by the relative impermeability o the plasma membrane to Ca2 and by the actions o membrane pumps that actively export Ca2 S troke volume a nd myoca rdia l O 2 de ma nd Ve ntricula r wa ll s tre s s

P re loa d

Afte rloa d He a rt (pump)

Ve nous tone Ve ins

Arte riola r tone Arte rie s

Ca pilla rie s

Ve ins (ca pa cita nce ve s s e ls )

Arte riole s (re s is ta nce ve s s e ls )

FIGURE22-1. Coupling of vascular tone and cardiac output. Cardiac stroke volume and myocardial oxygen demand are determined, in part, by ventricular wall stress, which is a unction o ventricular preload, a terload, volume, and thickness. Changes in vascular tone are coupled to cardiac output through their e ects on preload and a terload. Contraction o resistance arterioles increases ventricular a terload while contraction o capacitance veins increases ventricular preload.

C h a p t e r 2 2 Pharmacology o Vascular Tone

L-type Ca 2+ cha nne l

Ca 2+

Extra ce llula r s pa ce

Ca 2+

Ca 2+ ATP a s e

Ca 2+

Na +

Cytos ol

Va s cula r s mooth mus cle ce ll

Na +/Ca 2+ e xcha nge r Ca 2+

Ca 2+

S a rcopla s mic re ticulum

Ca 2+

387

Ca 2+

Actin-myos in cros s -bridge s Ca 2+

Ca 2+ Contraction

Na +

Ca 2+ ATP

IP 3 re ce ptors a nd rya nodine re ce ptors

FIGURE 22-2. Regulation of intracellular Ca 2 in vascular smooth muscle cells. Cytosolic Ca 2 concentration is 100 nM, while the extracellular and sarcoplasmic reticulum Ca 2 concentration is 2 mM. Upon activation by contractile stimuli, Ca 2 di uses into the cytoplasm down its concentration gradient through L-type Ca 2 channels in the plasma membrane and IP3 or ryanodine receptors in the sarcoplasmic reticulum. Increased cytoplasmic Ca 2 triggers actin–myosin cross-bridge ormation and cell contraction. Ca 2 is cleared rom the cytoplasm by Ca 2 ATPases in the plasma membrane and sarcoplasmic reticulum and Na /Ca 2 exchangers in the plasma membrane.

rom the cell. Contractile stimuli serve to increase intracellular Ca2 through two mechanisms. First, Ca2 can di use down its concentration gradient into the cell through Ca2 selective channels in the plasma membrane that can be opened by the activation o cell sur ace receptors (receptor-operated Ca2 channels), by mechanical stretch, or by membrane depolarization (voltage-dependent or L-type Ca2 channels). Second, Ca2 can be released rom intracellular stores by activation o inositol 1,4,5-trisphosphate (IP3) receptors and Ca2 -induced Ca2 release through ryanodine receptors located in the sarcoplasmic reticulum. Upon termination o the contractile stimulus, Ca2 is removed rom the cytoplasm through active transport by Ca2 ATPases in the plasma membrane and sarcoplasmic reticulum and Na /Ca2 exchangers in the plasma membrane (Fig. 22-2). Increases in intracellular Ca2 are tightly coupled to vasoconstriction. Ca2 binds to calmodulin (CaM), and the Ca2 /CaM complex binds to and activates myosin light chain kinase (MLCK). Active MLCK phosphorylates myosin light chain (MLC), permitting myosin interactions with actin f laments, leading to cross-bridge cycling and smooth muscle contraction. Vasodilation occurs as a consequence o decreasing intracellular Ca2 and dephosphorylation o MLC by MLC phosphatase (Fig. 22-3).

signals that activate them will be discussed in subsequent sections (Fig. 22-4). Vasoconstriction is potentiated by three signaling pathways. First, activation o G protein-coupled receptors (GPCR) associated with heterotrimeric Gq proteins activates phospholipase C (PLC) to produce IP3 and diacylglycerol (DAG). IP3 stimulates Ca2 release rom intracellular stores by activating IP3 receptors in the sarcoplasmic reticulum. DAG activates protein kinase C, which also promotes contraction through a variety o phosphorylation events. Second, GPCRs coupled to heterotrimeric G12/13 proteins stimulate nucleotide exchange on the small G protein RhoA. RhoA activates Rho-kinase to phosphorylate and inactivate MLC phosphatase, thereby maintaining MLC phosphorylation. This pathway provides a mechanism to sustain smooth muscle contraction beyond transient increases in intracellular Ca2 . Third, activation o Gi-coupled receptors inhibits adenylyl cyclase and thereby Va s cula r s mooth mus cle ce ll Ca 2+ Ca 2+/Ca M MLCK

Regulation of Vascular Tone Vascular smooth muscle cells integrate a variety o signals to regulate vascular tone, including local environmental actors, endothelium-derived signaling molecules, neurotransmitters, and hormones. Extracellular stimuli generally converge on shared intracellular signal transduction pathways that regulate the smooth muscle contractile apparatus. These signaling cascades are targeted by the drugs discussed in this chapter and, thus, provide the ramework or understanding the mechanisms o drug action.

Signal Transduction Pathways

Intracellular signaling pathways are o ten shared among a variety o extracellular stimuli. These intracellular pathways will be reviewed in this section, and the extracellular

MHC-MLC

MHC-MLC P MLC p h o s p h a ta s e

Relaxation

Contraction

FIGURE 22-3. Vascular smooth muscle cell contractile apparatus. Increased cytoplasmic Ca 2 binds and activates calmodulin (CaM). The Ca 2 / CaM complex stimulates myosin light chain kinase (MLCK) to phosphorylate myosin light chain (MLC), which permits myosin heavy chain (MHC) binding to actin, orming actin–myosin cross-bridges. Actin and myosin f laments slide past one another as a consequence o ATP hydrolysis by myosin. Contraction ceases with the dephosphorylation o MLC by MLC phosphatase.

388 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Ade nylyl cycla s e NO

P LC DAG

Gs

Gi

G 12/13

RhoGEF RhoA

Gq

s GC

ROCK IP3

Va s cula r s mooth mus cle ce ll pla s ma me mbra ne

P KC

cGMP

PKA

GTP MHC-MLC MLCK Ca 2+

Ca 2+

P KG

MLC p h o s p h a ta s e

Ca 2+/Ca M MHC-MLC P

S a rcopla s mic re ticulum

IP 3 re ce ptor Contraction

FIGURE 22-4. Intracellular signaling in vascular smooth muscle cells. Activation of Gq-coupled receptors stimulates phospholipase C (PLC) to hydrolyze membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 activates receptors in the sarcoplasmic reticulum to release stored Ca 2 , while DAG activates protein kinase C (PKC). PKC phosphorylates Ca 2 channels and components of the contractile apparatus to promote smooth muscle contraction (not shown). Production of cAMP by adenylyl cyclase is regulated by Gi (inhibitory)- and Gs (stimulatory)coupled receptors. Cytosolic cAMP activates protein kinase A (PKA), which phosphorylates and inactivates MLCK. Thus, Gs -coupled receptors that increase cAMP cause smooth muscle relaxation, while Gi-coupled receptors that decrease cAMP cause contraction. G12/13-coupled receptors activate Rho kinase (ROCK) by stimulating nucleotide exchange (mediated by Rho guanine nucleotide exhange factor [RhoGEF]) on the small G protein RhoA. ROCKinhibits myosin light chain (MLC) phosphatase, thereby potentiating smooth muscle contraction. Nitric oxide (NO) stimulates cGMP production by soluble guanylyl cyclase (sGC), leading to protein kinase G (PKG) activation. PKG activates MLC phosphatase to induce smooth muscle relaxation. MHC, myosin heavy chain.

decreases production o cyclic adenosine monophosphate (cAMP). Reduced cAMP decreases protein kinase A (PKA) activity, thereby relieving inhibition o MLCK. Vasodilation is potentiated by two signaling pathways. First, Gs-coupled receptors stimulate cAMP ormation, PKA activation, MLCK inhibition, and ATP-regulated K channel (K ATP) opening. Second, nitric oxide (NO) activates soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). This second messenger activates protein kinase G (i.e., cGMP-dependent protein kinase), which phosphorylates a variety o downstream targets that activate MLC phosphatase to inhibit myosin-actin cycling, inhibit Ca2 mobilization and increase sequestration, and activate Ca2 -dependent K channels. These concerted actions lead to smooth muscle relaxation. One common eature o these vasodilatory pathways is the opening o plasma membrane K channels, leading to membrane hyperpolarization. When K channels open, K exits the cell down its concentration gradient, moving the Nernst equilibrium potential o the plasma membrane down toward 90 mV (the Nernst potential or K ) rom some higher resting value (thereby hyperpolarizing the membrane). This change in potential makes it more di cult or the plasma membrane to depolarize su ciently or the voltage-gated L-type Ca2 channels to open, thereby inhibiting smooth muscle cell contraction.

circulation is particularly sensitive to f uctuations in pH and CO2, which is why acute hyperventilation (which decreases pH and CO2, leading to vasoconstriction and decreased cerebral blood f ow) is one therapy or intracranial hypertension. Increases in extracellular K activate inward recti er K channels, thereby causing hyperpolarization o the plasma membrane and inhibiting voltage-gated Ca2 channel opening. Adenosine activates A2 Gs-coupled receptors. Systemic vessels also respond to decreased O2 by vasodilation, in contrast to pulmonary vessels that vasoconstrict (i.e., hypoxic vasoconstriction) to preserve ventilation–per usion matching in the lung. The molecular mechanisms mediating these disparate responses remain active areas o research. In addition to metabolic actors, vascular smooth muscle cells contract in response to stretch through the opening o stretch-activated Ca2 channels in the cell membrane. This myogenic ref ex protects distal capillary beds rom high pressures by increasing vascular resistance. In combination with local control by metabolic actors, the myogenic ref ex is an important mechanism o vascular autoregulation where the vessels adjust resistance in an attempt to maintain steady blood f ow over a range o per usion pressures (recall that Flow Pressure/Resistance). Autoregulation is particularly evident in vascular beds that are sensitive to ischemia, such as the brain, heart, and kidneys.

Environmental Factors

Vascular endothelial cells play a critical role in regulating vascular smooth muscle tone through direct cellular contacts and the elaboration o signaling molecules. Among these signaling molecules, NO, endothelium-derived hyperpolarizing actors, prostacyclin, and endothelin are the most relevant pharmacologically.

Arteriolar smooth muscle cells coordinate blood f ow into the capillary beds o metabolically active tissues. In regions where tissue metabolic demand exceeds supply, increases in H (as lactic acid), CO2, K , and adenosine ( rom ATP utilization) all lead to vasodilation and increased blood f ow. The cerebral

Endothelial Factors

C h a p t e r 2 2 Pharmacology of Vascular Tone

The obligatory role o endothelial cells in regulating vascular tone was rst recognized with the observation that acetylcholine causes vasoconstriction when applied directly to de-endothelialized blood vessels but causes vasodilation when applied to normally endothelialized vessels. This nding suggested that endothelial cells produce a vasodilatory compound, initially termed endothelium-derived relaxing actor (EDRF) and subsequently proven to be NO. Nitric oxide is a membrane-permeable gas that reacts with a variety o biomolecules to elicit cellular responses, particularly activation o sGC in vascular smooth muscle cells, leading to cGMP production. Vascular endothelial cells synthesize NO in response to a variety o stimuli, including shear stress, acetylcholine, histamine, bradykinin, sphingosine-1-phosphate, serotonin, substance P, and ATP. These actors increase intracellular Ca2 , thereby stimulating the Ca2 /CaM-activated endothelial NO synthase (eNOS). Endothelial eNOS produces NO rom arginine, and NO subsequently di uses to the vascular smooth muscle cell to initiate downstream signaling events and vasorelaxation (Figs. 22-4 and 22-5). Agonis t (e.g., a ce tylcholine , bra dykinin)

Ca 2+

Endothe lia l ce ll

Ca 2+

Ca 2+ Ca 2+/Ca M e NOS

e NOS

L-Arg

Ca 2+-de pe nde nt K+ cha nne l

NO

NO

K+

S a rcopla s mic re ticulum

Guanylyl cycla s e

NO

Va s cula r s mooth mus cle ce ll

Gu a n ylyl c yc la s e

Hype rpola riza tion Relaxation

FIGURE 22-5. Endothelial regulation of nitric oxide-mediated vascular smooth muscle relaxation. Endothelial cell production of nitric oxide (NO) controls the extent of vascular smooth muscle cell relaxation. Production of NO is stimulated by agonists such as acetylcholine or bradykinin. Stimulation of receptors by these agonists activates Ca 2 second messenger systems and promotes direct entry of Ca 2 into the cytosol. The increased cytosolic Ca 2 activates a Ca 2 /calmodulin (Ca 2 /CaM) complex that stimulates endothelial nitric oxide synthase (eNOS), an enzyme that catalyzes the formation of NO from L-arginine (L-Arg, an amino acid). Nitric oxide diffuses from the endothelial cell into subjacent vascular smooth muscle cells, where it activates guanylyl cyclase, promoting smooth muscle cell relaxation. NO can also directly activate Ca 2 -dependent K channels. This parallel signaling pathway contributes to relaxation by hyperpolarizing the smooth muscle cell. The active form of each enzyme is shown in italicized blue.

389

Interestingly, measured levels o NO cannot explain all o the endothelium-dependent responses o vascular smooth muscle cells. Indeed, endothelium-dependent vasodilation can be caused by smooth muscle cell hyperpolarization that is not due to NO. Several molecules have been implicated as endothelium-derived hyperpolarizing factors (EDHFs), including epoxyeicosatrienoic acids (arachidonic acid metabolites), hydrogen peroxide, carbon monoxide, hydrogen sul de, C-natriuretic peptide, and K itsel . These mediators open a variety o K channels on smooth muscle cells, leading to membrane hyperpolarization and smooth muscle cell relaxation. Hydrogen sul de has a variety o additional e ects that lead to smooth muscle relaxation, largely mediated by covalent S-sul hydration o target proteins, including not only K channels but also Ca2 channels, and by stimulating release o other EDHFs. Prostacyclin is also a vasodilatory molecule produced in endothelial cells rom arachidonic acid in reactions that involve the cyclooxygenase (COX) enzymes. Prostacyclin activates Gs-coupled receptors on the vascular smooth muscle cells, leading to vasodilation. Since the COX enzymes are inhibited by nonsteroidal anti-inf ammatory drugs, such drugs should be used with caution in patients with hypertension because they decrease prostacyclin production. In contrast to the potent vasodilating e ects o the molecules described above, endothelial cells also produce the most potent endogenous vasoconstrictor known, endothelin-1 (ET-1). ET-1 is a 21-amino acid peptide synthesized as preproendothelin, which is cleaved to big endothelin and subsequently to ET-1 by endothelin converting enzyme. ET-1 is released by endothelial cells in response to mechanical stress and vasoactive agents (e.g., vasopressin, angiotensin II), while its release is inhibited by prostacyclin, NO, and atrial natriuretic peptide. ET-1 binds to two receptor subtypes, ETA and ETB, and both are Gq-coupled receptors. Both subtypes are located on vascular smooth muscle cells and mediate vasoconstriction. Interestingly, endothelial cells express ETB receptors, which, when occupied by ET-1, activate eNOS and COX, leading to NO and prostacyclin release. This negative eedback pathway is one mechanism by which endothelial cells assist in modulating vascular tone (Fig. 22-6).

Autonomic Nervous System

Vascular smooth muscle cells receive input rom the sympathetic nervous system, which is an important determinant o vascular tone. Sympathetic nerves innervate vascular smooth muscle cells in both arteries and veins, and sympathetic activation leads to both vasoconstriction and venoconstriction. Sympathetic postganglionic neurons release norepinephrine, which binds to postsynaptic 1- and 2-adrenergic receptors coupled to Gq and Gi, respectively, leading to contraction o the smooth muscle cell (Fig. 22-4). The presynaptic nerve terminal also expresses 2-adrenergic autoreceptors, which inhibit urther release o norepinephrine in a negative eedback loop. Sympathetic activation also induces epinephrine release rom the adrenal medulla. In contrast to norepinephrine, epinephrine activates both - and 2-adrenergic receptors on vascular smooth muscle cells. The 2 receptors activate the Gs signaling pathway, leading to smooth muscle cell relaxation (Fig. 22-4). Thus, the e ects o epinephrine on a given vascular bed depend on the dose ( 2 receptors have a higher a nity or epinephrine and, thus, are activated at lower epinephrine

390 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

production o cGMP. Histamine and bradykinin also act as vasodilators. Histamine activates Gs-coupled H2 receptors on vascular smooth muscle cells to cause vasodilation (Fig. 22-4) and Gq-coupled H1 receptors on endothelial cells to cause NO generation (Fig. 22-5). Bradykinin also stimulates NO production through the 2 receptor on endothelial cells (Fig. 22-5).

Lume n Endothe lia l ce lls

Ara chidonic a cid COX

L-Arg

P ros ta cyclin

Endothe lin pre curs ors

eNOS NO

PHARMACOLOGIC CLASSES AND AGENTS

Endothe lin-1

The pharmacologic agents considered in this chapter are all vasodilators, that is, drugs that act on vascular smooth muscle cells or endothelial cells to decrease vascular tone. This e ect can be accomplished either by inhibiting components o contractile signal transduction pathways or by potentiating e ects o relaxing signal transduction pathways.

ETB

Endothelin-1 P ros ta cyclin

Endothelin-1 NO

IP

NO

Relaxation

ETA

ETB

Contraction

Va s cula r s mooth mus cle ce lls

FIGURE 22-6. Effects of endothelin on the blood vessel wall. Endothelin mediates both contraction and relaxation of vascular smooth muscle cells. Endothelin precursors in endothelial cells are processed to ET-1. ET-1 is secreted on the basal side of the endothelial cell, where it interacts with Gq-coupled ETA and ETB receptors on vascular smooth muscle cells to stimulate vasoconstriction. ETB receptors are also expressed on endothelial cells. Endothelial cell ETB activation stimulates cyclooxygenase (COX), which catalyzes the formation of prostacyclin from arachidonic acid. Prostacyclin diffuses from the endothelial cell to the vascular smooth muscle cell membrane, where it binds to and activates the prostacyclin (IP) receptor. ETB activation also stimulates endothelial nitric oxide synthase (eNOS), which catalyzes the formation of NO from arginine (L-Arg). Both prostacyclin and NO stimulate vascular smooth muscle cell relaxation.

concentrations than receptors) and the relative composition o receptors expressed on the target cells. For example, during a “ ght or f ight” response, blood f ow is diverted away rom skin and viscera, where 2, and toward skeletal muscle, where 2 . While most blood vessels lack parasympathetic innervation, acetylcholine does cause vasodilation through M3muscarinic receptor-mediated NO release rom vascular endothelial cells (Fig. 22-5).

Humoral Regulators

In addition to the autonomic nervous system, several humoral mediators contribute to the regulation o vascular tone and integrate renal and cardiovascular unction. Among these, angiotensin II and vasopressin are potent vasoconstrictors through their activation o Gq-coupled AT1 and V1 receptors, respectively (Fig. 22-4). These mediators act to increase both vascular resistance and intravascular volume in response to hypovolemia (i.e., hemorrhagic shock) through coordinated actions in the blood vessels and kidney. Atrial and brain natriuretic peptides are released in response to hypervolemia and induce vasodilation through the activation o membrane-bound guanylyl cyclase receptors and

Ca 2 Channel Blockers Ca2 channel blockers are among the most prescribed agents or the management o hypertension and angina owing to their e ectiveness and ease o use. These agents are primarily arterial vasodilators, having little impact on the venous system. Importantly, Ca2 channel blockers bind to both vascular smooth muscle cells and cardiac myocytes, which accounts or their e ectiveness as positive lusitropic agents (i.e., agents that relax the myocardium) and in the management o certain cardiac arrhythmias.

Mechanism of Action

Ca2 inf ux through L-type Ca2 channels is an important determinant o vascular tone and cardiac contractility. Ca2 channel blockers inhibit Ca2 inf ux through these channels. In smooth muscle cells, decreased Ca2 entry keeps intracellular Ca2 concentrations low, thereby reducing Ca2 /CaMmediated activation o MLCK, actin–myosin cross-bridge ormation, and smooth muscle cell contraction. Vasodilation o resistance arterioles decreases systemic vascular resistance and blood pressure and, consequently, ventricular a terload. Additionally, drug-induced dilation o the coronary arteries augments myocardial oxygen supply, helping to alleviate the symptoms o angina in a ected patients (Fig. 22-7). Although Ca2 channel blockers can relax many di erent types o smooth muscle (e.g., bronchiolar and gastrointestinal), they appear to have the greatest e ect on vascular smooth muscle. In cardiac myocytes, reduced Ca2 inf ux decreases myocardial contractility, increases lusitropy, and decreases sinoatrial (SA) node pacemaker rate and atrioventricular (AV) node conduction velocity. Ca2 channel blockers do not signi cantly a ect skeletal muscle, since these cells depend mainly on intracellular Ca2 stores in the sarcoplasmic reticulum to support excitation–contraction coupling and do not rely on transmembrane Ca2 inf ux.

Chemical Classes

Three chemical classes o Ca2 channel blockers are currently in clinical use: dihydropyridines (e.g., nifedipine , amlodipine , and felodipine ), benzothiazepines (e.g., diltiazem), and phenylalkylamines (e.g., verapamil). All three classes inhibit L-type Ca2 channels, but each class has distinctive pharmacologic e ects owing to di erences in drug binding sites on the channels, di erent a nities or particular channel con ormations (e.g., closed, open, or inactivated), and di erent a nities or the subtypes o L-type Ca2 channels.

C h a p t e r 2 2 Pharmacology o Vascular Tone S A node

AV node

Automa ticity

Conduction

Ca rdia c myocyte s Afte rloa d Myoca rdia l O 2 de ma nd

Corona ry a rte rie s Va s odila tion Myoca rdia l O 2 s upply

Ve ins

P e riphe ra l ve ins

Arte rie s He a rt (pump)

Minima l ve nodila tion

P e riphe ra l a rte riole s Va s odila tion Afte rloa d Myoca rdia l O 2 de ma nd

FIGURE 22-7. Sites of action of Ca 2 channel blockers. Ca 2 channel blockers dilate coronary arteries and peripheral arterioles, but not veins. They also decrease cardiac contractility, automaticity at the SA node, and conduction at the AV node. Dilation o the coronary arteries increases myocardial O2 supply. Dilation o systemic (peripheral) arterioles decreases a terload and thereby decreases myocardial O2 demand. However, some Ca 2 channel blockers (especially dihydropyridines) cause ref ex tachycardia, which can paradoxically increase myocardial O2 demand. Decreased cardiac contractility and decreased SA node automaticity also decrease myocardial O2 demand. The inhibition o AV node conduction by some Ca 2 channel blockers makes them use ul as antiarrhythmic agents. Note that the e ects diagrammed here are representative e ects o the class o drugs; individual agents are more or less selective or each o these e ects.

All three classes o Ca2 channel blockers bind to separate but allosterically connected binding sites on the 1 ion poreorming subunit o the channel. Dihydropyridines inhibit the Ca2 channel rom the extracellular space through a binding site buried in the lipid bilayer. Diltiazem binds the ion pore rom the extracellular side o the channel, while verapamil is thought to enter the cell and block the cytoplasmic opening o the pore. Dihydropyridines exhibit much greater arterial vasodilation than non-dihydropyridines, while having relatively little impact on cardiac tissue (i.e., there is less depression o myocardial contractility, less impairment o SA node automaticity, and less slowing o AV node conduction velocity). These di erential e ects are due, in part, to the pre erence o dihydropyridines to bind to inactivated channels. Since smooth muscle cells have a relatively depolarized resting membrane potential ( 70 mV) compared to cardiac myocytes ( 100 mV), their calcium channels are more likely to be in the inactive con ormation and are, there ore, inhibited by lower concentrations o dihydropyridine agents. By contrast, non-dihydropyridines bind to the open con ormation and prolong the channel re ractory period (i.e., increase the channel recovery time), thereby decreasing the number o conducting channels as depolarizations occur more requently.

391

Thus, non-dihydropyridines are more e ective in tissue with requent channel openings (i.e., SA node, AV node, and cardiac myocytes), and channel inhibition increases in proportion to heart rate. The negative chronotropic and inotropic e ects o non-dihydropyridine agents appear greater or verapamil than diltiazem. L-type Ca2 channels are a amily o our di erent multimeric protein complexes de ned by the gene encoding the pore- orming 1 subunit (CaV1.1–1.4). CaV1.2 and CaV1.3 are the iso orms ound on cardiac myocytes and vascular smooth muscle cells. Alternate splicing o the CaV1.2 gene in heart and vascular smooth muscle cells also contributes to pre erential inhibition o smooth muscle by dihydropyridines.

Pharmacokinetics

Ca2 channel blockers are typically administered in oral dosage orms, although intravenous ormulations o diltiazem and verapamil are available. Clevidipine is a dihydropyridine agent that is available only as an intravenous ormulation. Three pharmacokinetic properties o most Ca2 channel blockers are suboptimal. First, orally administered Ca2 channel blockers undergo signi cant rst-pass metabolism in the gut and liver, signi cantly reducing bioavailability to 10–30%. Second, most o these drugs have a rapid onset o action, between 20 minutes and 2 hours. In the case o ni edipine, oral administration can lead to a rapid and precipitous all in blood pressure, resulting in severe ref ex tachycardia. This worsens myocardial ischemia by increasing myocardial O2 demand and it also decreases O2 supply as a result o shortened diastole. Third, these agents typically have short elimination hal -lives (2–10 hours), necessitating short dosing intervals or extended-release preparations (available or all commonly administered Ca2 channel blockers). Amlodipine was developed in an attempt to overcome the pharmacokinetic limitations o ni edipine. This drug has an increased oral bioavailability o 60%, a long time to onset o 6 hours, and a long elimination hal -li e o 40 hours. These kinetic properties are likely due, in part, to its lipophilic character and its positive charge at physiologic pH, which lead to increased association with negatively charged plasma membranes. All Ca2 channel blockers are metabolized by the liver. Diltiazem is primarily excreted by the liver, while dihydropyridines and verapamil are primarily excreted in the urine.

Toxicities and Contraindications

The toxicities o Ca2 channel blockers are mainly a consequence o the mechanism o action. Like all vasodilators, Ca2 channel blockers can cause headache, dizziness, lightheadedness, and f ushing. Constipation is a common adverse e ect o verapamil that is likely caused by excessive smooth muscle relaxation in the gastrointestinal tract. These agents also cause peripheral edema by increasing the transcapillary hydrostatic pressure. Venodilating agents can mitigate this e ect, while diuretics are less e cacious with this class o agents. When taken at doses that lead to drug concentrations higher than the therapeutic window, the negative chronotropic and inotropic e ects o verapamil and diltiazem can lead to bradycardia, AV block, and heart ailure. Patients taking -adrenergic blockers (which are also negative inotropes) are o ten advised not to use diltiazem or verapamil concomitantly because o the increased likelihood o excessive cardiac

392 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

depression. Some studies have suggested that Ca2 channel blockers increase the risk o mortality in patients with heart ailure, and Ca2 channel blockers are contraindicated in the management o heart ailure. Some reports also suggest that the short-acting agents, such as ni edipine, are associated with an increased risk o myocardial ischemia and in arction due to impairment o myocardial O2 supply (see above).

K Channel Openers K channel openers cause direct arterial vasodilation by opening K ATP channels in the plasma membrane o vascular smooth muscle cells, leading to membrane hyperpolarization and preventing Ca2 channel opening (see above). While not commonly employed as rst-line agents owing to the multiple adverse e ects described below, K channel openers represent a use ul amily o drugs or re ractory hypertension because o this unique mechanism o action. The K ATP channel openers include minoxidil and nicorandil. These drugs act primarily on arterial smooth muscle cells and there ore decrease arterial blood pressure. Adverse e ects o K channel openers include headache and f ushing, caused by excessive dilation o cerebral and cutaneous arteries, respectively. Similar to Ca2 channel blockers, these agents can cause peripheral edema, necessitating the use o diuretics. When arterial vasodilators are used as monotherapy, the decrease in arterial pressure o ten elicits sympathetic activation, ref ex tachycardia, and increased myocardial O2 demand. Use o -adrenergic blockers can mitigate these adverse e ects and preserve the therapeutic utility o arterial vasodilators. Minoxidil can also cause hypertrichosis; as a result, it is more commonly prescribed as a therapy or male pattern baldness.

Hydralazine Hydralazine is an arteriolar vasodilator that is sometimes used in the treatment o hypertension and, in combination with isosorbide dinitrate, in the treatment o heart ailure. The mechanism o action o hydralazine remains unclear; current evidence suggests that hydralazine may cause membrane hyperpolarization by opening K ATP channels and inhibiting IP3-induced Ca2 release rom the sarcoplasmic reticulum in vascular smooth muscle cells. More recently, hydralazine has been shown to decrease the promoter methylation and, thereby, increase the gene expression o SERCA2a, which is the Ca2 pump that transports cytoplasmic Ca2 back into the sarcoplasmic reticulum. While this mechanism would not account or the acute e ects o hydralazine, it may play an important role in patients on chronic therapy. Hydralazine appears to prevent the development o nitrate tolerance, perhaps by inhibiting vascular superoxide production and by scavenging peroxynitrite ormed rom superoxide and NO. The combination o hydralazine and nitrates has a demonstrated mortality bene t in patients with persistent symptoms o advanced systolic heart ailure despite optimal medical therapy. The data suggest that this e ect is stronger in black Americans, possibly because o decreased NO bioavailability or NO signaling in this population. Hydralazine is not rst-line therapy or hypertension owing to the requirement or requent dosing and the rapid development o tachyphylaxis to its antihypertensive e ects. As the bene ts o combination therapy or hypertension and heart ailure are becoming better appreciated, it may be

possible or hydralazine to be used more e ectively, especially in patients or whom other vasodilators (e.g., angiotensin converting enzyme [ACE] inhibitors) are contraindicated. An intravenous ormulation o hydralazine is available or acute blood pressure control in hospitalized patients. Hydralazine typically has low bioavailability because o extensive rst-pass metabolism. The rate o its metabolism depends on whether the patient is a slow or ast acetylator. In slow acetylators (see Chapter 4, Drug Metabolism), hydralazine has a slower rate o hepatic degradation and, thus, higher bioavailability and higher plasma concentrations. A rare adverse e ect o hydralazine is the development o a reversible lupus erythematosus-like syndrome; this e ect occurs primarily in slow acetylators.

Nitric Oxide Donors Nitric oxide donors include organic nitrates, sodium nitroprusside, and inhaled nitric oxide itsel . The organic nitrates represent one o the oldest cardiac therapies still in use. Indeed, glyceryl trinitrate, more commonly denoted nitroglycerin (NTG), was rst employed or relie o angina symptoms over 100 years ago. Indications or the use o organic nitrates now include the classic indication o stable angina pectoris as well as unstable angina, acute myocardial in arction, hypertension, and heart ailure.

Mechanism of Action

Within the body, nitric oxide donors are chemically reduced to release NO, the rst described gasotransmitter, or gaseous signaling molecule. Nitric oxide reacts with a variety o biomolecules; however, soluble guanylyl cyclase is recognized as its primary physiologic receptor. Activation o guanylyl cyclase by NO induces smooth muscle relaxation as described above (Fig. 22-4). Although NO can dilate both arteries and veins, venous dilation predominates at therapeutic doses. This e ect contrasts with Ca2 channel blockers and K channel openers, which cause primarily arteriolar vasodilation. Venodilation increases venous capacitance, leading to a decrease in blood return to the right ventricle and, consequently, to decreased right and le t ventricular end-diastolic pressure and volume. Decreased preload decreases myocardial O2 demand while acilitating subendocardial per usion owing to decreased diastolic wall stress. Mild e ects on arterial vasodilation decrease a terload and improve blood f ow through coronary arteries, all contributing to improved myocardial O2 supply/ demand matching. Moreover, this hemodynamic pro le is bene cial in patients with congestive heart ailure, since decreases in ventricular preload minimize the development o pulmonary edema while decreases in a terload improve ventricular stroke volume (Fig. 22-8). In the coronary circulation, NTG predominantly dilates large epicardial coronary arteries with only minimal e ects on the coronary resistance vessels. This pre erential action minimizes the development o the coronary steal phenomenon, where vasodilators, by decreasing the resistance o normal arteries, can divert (“steal”) blood f ow away rom regions o the heart with stenotic coronary arteries that are already maximally dilated. The generation o NO rom organic nitrates can also cause relaxation o other types o smooth muscle, including esophageal, bronchial, biliary, intestinal, and genitourinary. Indeed, the ability o NTG to relieve the angina-like chest

C h a p t e r 2 2 Pharmacology o Vascular Tone He a rt (pump) Ve ins

Ca pa cita nce ve s s e ls

Organic nitrates Arte rie s

Myoca rdia l O 2 s upply by dila ting la rge e pica rdia l a rte rie s

Re s is ta nce ve s s e ls

Sodium nitroprusside (SNP)

393

Organic nitrates (RNO2) Enzymes and extracellular reductants

Spontaneous

Nitric oxide (NO)

S -Nitros othiol (RS NO)

RNO 2 Enzymes and intracellular reductants RS NO

Organic nitrates

Organic nitrates

P re loa d Myoca rdia l O 2 de ma nd

Afte rloa d Myoca rdia l O 2 de ma nd

FIGURE 22-8. Sites of action of organic nitrates. Organic nitrates exert the majority o their vasodilator action on venous capacitance vessels. This selectivity results in greatly decreased preload, with resulting decreased myocardial O2 demand. Organic nitrates also mildly dilate arteriolar resistance vessels, with resulting decreased a terload and decreased myocardial O2 demand. Myocardial O2 supply is mildly increased by dilation o large epicardial arteries.

Endothe lia l ce ll

S NP

Enzymes and intracellular reductants RNO 2 RS NO

NO

NO

Guanylyl cyclase

Relaxation

pain o esophageal spasm can occasionally result in a misdiagnosis o coronary artery disease. These actions o nitrates on nonvascular smooth muscle are usually o limited clinical signif cance. Nitric oxide generated rom organic nitrates also unctions as an antiplatelet agent. Nitric oxide-mediated increases in platelet cGMP inhibit platelet aggregation. Together with the vasodilatory e ect o nitrates, this antiplatelet e ect may decrease the likelihood o coronary artery thrombosis. Nitrate-induced inhibition o platelet aggregation may be especially important in the treatment o rest angina (i.e., chest pain that occurs spontaneously at rest) because rest angina requently results rom the ormation o occlusive platelet aggregates at the site o atherosclerotic coronary artery lesions. Rest angina is also known as unstable angina because the thrombotic occlusions that cause rest angina can evolve into complete occlusion, resulting in myocardial in arction.

Chemical Classes

Organic nitrates do not release NO directly but are chemically or enzymatically reduced to orm S-nitrosothiols with available sul hydryl groups on proteins or glutathione (Fig. 22-9). The S-nitrosothiols are subsequently reduced to generate ree NO, which then activates sGC. The reduction o organic nitrates can be catalyzed in tissues expressing specif c enzymes, such as mitochondrial aldehyde dehydrogenase, suggesting one mechanism by which their e ects may be “targeted” to specif c vascular tissues. (In contrast, the inorganic nitrate sodium nitroprusside does not mani est tissue-specif c e ects; see below). Interestingly, S-nitrosylation itsel is becoming increasingly recognized as an important post-translational modif cation o proteins, with a variety o e ects on protein structure and unction, and has been implicated in the pathogenesis o a variety o diseases (e.g., Alzheimer’s disease). Several di erent preparations o organic nitrates are available. The most commonly used organic nitrates include NTG, isosorbide dinitrate , and isosorbide 5-mononitrate . Although these drugs share a common mechanism o action, they di er

Va s cula r s mooth mus cle ce ll

FIGURE 22-9. Biotransformation of organic nitrates and sodium nitroprusside. Organic nitrates and sodium nitroprusside increase local levels o nitric oxide (NO) by di erent mechanisms. Organic nitrates have the chemical structure RNO2. The nitro group is reduced to orm NO in the presence o specif c enzymes and extracellular or intracellular reductants (e.g., thiols). In comparison, sodium nitroprusside releases NO spontaneously without enzymatic aid. Both agents e ect relaxation via the ormation o NO. However, the requirement o organic nitrates or specif c cellular enzymes or reductants may result in tissue selectivity. Because sodium nitroprusside spontaneously converts to NO, it does not dilate vascular beds selectively.

in their routes o administration and pharmacokinetics, leading to important di erences in therapeutic utility. Sodium nitroprusside is an inorganic compound in which one nitrosyl group and f ve cyanide groups are coordinated to an iron atom (Fig. 22-10). Upon in usion, sodium nitroprusside is reduced by oxyhemoglobin and subsequently releases NO and f ve cyanide molecules. As a result o the nonenzymatic release o NO, sodium nitroprusside’s action does not appear to be targeted to specif c vessels, and, consequently, the drug dilates both arteries and veins. This agent is used intravenously or e ective hemodynamic control in hypertensive emergencies and severe heart ailure. Owing to its rapid onset o action, short duration o action, and high e f cacy, sodium nitroprusside must be in used with continuous blood pressure monitoring and care ul titration o dose to e ect. In addition to NO release, decomposition o sodium nitroprusside releases cyanide, which is metabolized to thiocyanate by thiosul ate sul otrans erase (rhodanese) in the liver. Thiocyanate is subsequently excreted by the kidneys (Fig. 22-10). Excessive cyanide accumulation can lead to acid–base disturbances, cardiac arrhythmias, and death. Thiocyanate toxicity can occur in patients with impaired renal unction, causing disorientation, psychosis, muscle spasms, and seizures. The accumulation o methemoglobin is usually not clinically

394 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

NO

A

NC NC

Fe

+2

CN CN

CN Nitroprusside B Sodium nitroprusside

NO

Cya nide

Va s odila tion

Live r

Thiocya na te

Re na l e xcre tion

S ulfhydryl donor

FIGURE 22-10. Chemical structure and metabolism of sodium nitroprusside. A. Sodium nitroprusside is a complex o iron, cyanide (CN), and a nitrosyl (NO) group. B. Sodium nitroprusside spontaneously decomposes to release NO and cyanide. NO e ects vasodilation; cyanide is metabolized in the liver to thiocyanate, which undergoes renal excretion. Cyanide toxicity can result rom prolonged administration o the drug or rom the presence o renal insu f ciency.

signif cant given the high dose o sodium nitroprusside required to generate signif cant amounts o methemoglobin. Inhaled NO gas can be used to dilate the pulmonary vasculature selectively. Because NO is rapidly inactivated by binding to blood hemoglobin, NO gas has little e ect on systemic blood pressure when administered by inhalation. Therapy with inhaled NO has established e f cacy in the treatment o primary pulmonary hypertension o the newborn. In adults with severe acute respiratory distress syndrome, inhaled NO improves ventilation–per usion matching and oxygenation by vasodilating vessels adjacent to ventilated alveoli but has not been shown to improve mortality.

and produces vasodilation. Indeed, oral nitrate administration lowers blood pressure and its e ects can be abrogated by antibacterial mouthwash. This may account or some o the salutary e ects o a diet rich in ruits and vegetables, which are good sources o nitrate. The potential therapeutic applications o this pathway are now under study. Nitroglycerin has a short hal -li e (approximately 5 minutes). NTG is denitrated into biologically active glyceryl dinitrate metabolites that have longer hal -lives (approximately 40 minutes) (Fig. 22-11, top). Equivalent doses o isosorbide dinitrate can be more e ective than NTG because isosorbide dinitrate has a longer hal -li e (about 1 hour). The partially denitrated metabolites o isosorbide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate, have even longer hal -lives (up to 2 and 4 hours, respectively) (Fig. 22-11, bottom). Isosorbide 5-mononitrate has become a popular therapeutic agent, not only because it has prolonged therapeutic e ects but also because it is well absorbed rom the gastrointestinal tract and is not susceptible to extensive f rst-pass metabolism in the liver. The bioavailability o orally administered isosorbide 5-mononitrate ONO 2 O 2 NO

Nitroglycerin (Glyceryl trinitrate)

OH

ONO 2 O 2 NO

OH

O 2 NO

Glyce ryl 1,2-dinitra te

ONO 2

Glyce ryl 1,3-dinitra te

O 2 NO

H O

Pharmacokinetics

The pharmacokinetics o the di erent nitrate ormulations provide a basis or the pre erential use o specif c agents and dosage orms in certain settings. For example, the rapid onset o action o sublingual nitrate preparations is desirable or rapid relie o acute angina attacks, while longer acting nitrates are more valuable or angina prophylaxis in the long-term management o coronary artery disease. Orally administered NTG and isosorbide dinitrate have low bioavailability because organic nitrate reductases in the liver rapidly metabolize these drugs. NTG or isosorbide dinitrate can be administered sublingually to circumvent the f rst-pass e ect and to attain therapeutic blood levels within minutes. Intravenous administration o NTG is indicated when continuous titration o drug e ect is necessary, or example, in the treatment o unstable angina or acute heart ailure. Slow-release transdermal and buccal preparations o NTG provide therapeutic steady-state levels o NTG that can be use ul or angina prevention in patients with stable coronary artery disease. Interestingly, nitrate rom dietary sources also plays an important role in blood pressure regulation. Facultative anaerobes on the tongue utilize nitrate as an electron acceptor, releasing nitrite into the saliva. In the acidic environment o the stomach, some nitrite is reduced to NO, which is absorbed

ONO 2

O H

ONO 2

Isosorbide dinitrate

HO

O 2 NO

H

H

O O

O O

H

ONO 2

Is os orbide 2-mononitra te

H

OH

Isosorbide 5-mononitrate

FIGURE 22-11. Chemical structures and metabolism of nitroglycerin and isosorbide dinitrate. Nitroglycerin and isosorbide dinitrate are biologically active nitrates that are metabolized into active molecules with longer hal lives than their parent compounds. Nitroglycerin is denitrated into glyceryl 1,2-dinitrate and glyceryl 1,3-dinitrate; these active metabolites have a hal -li e o approximately 40 minutes. Isosorbide dinitrate is denitrated into isosorbide 2-mononitrate and isosorbide 5-mononitrate; these active metabolites have hal -lives o 2 and 4 hours, respectively.

C h a p t e r 2 2 Pharmacology of Vascular Tone

395

is nearly 100%, allowing it to be signi cantly more e ective than equivalent amounts o isosorbide dinitrate. A ter denitration, organic nitrates are typically glucuronidated in the liver and excreted renally.

radical ormation. Because the speci c mechanisms o nitrate tolerance remain uncertain, the most e ective means o preventing tolerance is the use o a dosing strategy that includes an interval o low plasma nitrate levels every day.

Pharmacologic Tolerance

Toxicities and Contraindications

The desirable e ects o nitrates can, un ortunately, be o set by compensatory sympathetic nervous system responses (e.g., a ref ex increase in sympathetic vascular tone) and compensatory renal responses (e.g., increased salt and water retention). In addition to these mechanisms o physiologic tolerance, pharmacologic tolerance to organic nitrates is an important and clinically relevant phenomenon that signi cantly limits the e cacy o this class o vasodilators. Pharmacologic tolerance was rst documented in munitions workers exposed to volatile organic nitrates in the workplace. These workers su ered headaches at the start o the workweek, but as the week progressed, the headaches tended to disappear and remain absent or the rest o the week. Upon returning to work a ter a weekend without nitrate exposure, the headaches returned. These “Monday morning headaches” were initially ascribed to weekend intemperance, but it later became clear that the vasodilatory e ect o NTG was responsible. Development o tolerance to NTG as the workweek progressed allowed relie rom the headaches, and loss o tolerance to NTG over the weekend allowed the headaches to recur upon the workers’ return to work. Although tolerance to adverse e ects such as headaches can be desirable, tolerance to the antianginal e ects o nitrates diminishes their clinical e cacy. Tolerance to NTG does not appear to depend on the route o administration. Importantly, it is possible to minimize the development o tolerance by modulating the dosing schedule to include daily “nitrate- ree intervals.” For transdermal NTG, simple removal o the NTG patch each night can minimize the development o tolerance. In cases o severe angina that require uninterrupted nitrate therapy to manage symptoms adequately, however, patients may experience rebound angina during periods that are completely nitrate ree. The pharmacokinetic properties o oral isosorbide 5-mononitrate make this preparation an attractive solution to the dilemma o balancing nitrate tolerance and angina rebound: its high bioavailability and long hal -li e produce periods o high-therapeutic plasma concentrations ollowed by periods o low-therapeutic (rather than zero) nitrate concentrations. The examples o transdermal NTG and oral isosorbide 5-mononitrate illustrate how the pharmacokinetic properties o two mechanistically similar drugs can have a signi cant impact on their therapeutic utility. The cellular and molecular mechanisms that underlie the development o pharmacologic tolerance to organic nitrates remain unclear. There are currently two major hypotheses. First, the so-called classic (sul hydryl) hypothesis suggests that tolerance results mainly rom the intracellular depletion o sul hydryl-containing groups, such as glutathione or other cysteine-bearing species, that are involved in the ormation o NO rom organic nitrates. According to the sul hydryl hypothesis, tolerance could be attenuated or reversed by administering reduced thiol-containing compounds, such as N-acetylcysteine. Second, the ree radical (superoxide) hypothesis posits that cellular tolerance results rom the ormation o peroxynitrite, a highly reactive metabolite o NO that appears to inhibit sGC. According to the superoxide hypothesis, tolerance could be attenuated or reversed by agents that inhibit ree

Nitrates are contraindicated in patients with hypotension. Nitrates are also contraindicated in patients with elevated intracranial pressure because NO-mediated vasodilation o cerebral arteries could urther elevate intracranial pressure. Nitrates are not advised or the angina pain associated with hypertrophic cardiomyopathy because the outf ow obstruction can be exacerbated by reductions in preload. Nitrates should also be used with caution in patients with diastolic heart ailure who depend on elevated ventricular preload or optimal cardiac output. These medications should also be avoided in patients taking phosphodiesterase type V inhibitors. GF’s case provides an example o the deleterious e ects o concurrently administering organic nitrates and sildena l (see below).

cGMP Potentiators Three other classes o medications act on the NO signaling pathway to enhance smooth muscle relaxation. These agents inhibit phosphodiesterase type V or stimulate cGMP production, thereby potentiating vasodilation.

Phosphodiesterase Type V Inhibitors

The phosphodiesterase type V (PDE5) inhibitors sildenaf l, vardenaf l, tadalaf l, and avanaf l prevent the hydrolysis o cGMP to 5 -GMP, thereby potentiating the e ects o sGCinduced cGMP production on smooth muscle relaxation. PDE5 is mainly expressed in the smooth muscle o the corpus cavernosum but is also expressed in the retina and vascular smooth muscle cells. PDE5 inhibitors are most commonly prescribed or erectile dys unction, a relatively common condition in men, such as GF, who have vascular disease. Normally, NO release rom penile nerve terminals activates sGC in the smooth muscle o the corpus cavernosum, leading to increased cellular cGMP, smooth muscle relaxation, inf ow o blood, and penile erection. Through inhibition o PDE5, these drugs potentiate the e ects o endogenous NO signaling. Although PDE5 is predominantly expressed in erectile smooth muscle tissue, it is also expressed in the pulmonary vasculature, and PDE5 inhibitors at high doses have demonstrated e cacy in the management o pulmonary hypertension. The adverse e ects o PDE5 inhibitors primarily result rom drug-induced vasodilation in the systemic vasculature. Headache and f ushing are likely caused by vasodilation o cerebral and cutaneous vascular beds, respectively. Sildena l-related myocardial in arction and sudden cardiac death may also be related to its vasodilatory e ects. PDE5 inhibitors have only a nominal e ect on blood pressure, and the adverse e ects described above are relatively rare because o the small amounts o PDE5 in the systemic vasculature. However, in the presence o excess NO (e.g., when organic nitrates are used concomitantly with PDE5 inhibitors), the inhibition o cGMP degradation can markedly ampli y the vasodilatory e ect o NO. Excessive vasodilation can lead to severe, re ractory hypotension, as GF experienced a ter taking NTG and sildena l at the same time. There ore, all PDE5 inhibitors are contraindicated or patients taking organic nitrate vasodilators. Sildena l, vardena l, tadala l, and avana l all have signi cant and potentially dangerous

396 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

drug–drug interactions with nitrates. Similarly, patients taking -blockers should be monitored care ully upon the addition o PDE5 inhibitors to their medical regimen. Coadministration o PDE5 inhibitors with other antihypertensive medications (e.g., Ca2 channel blockers) is generally considered sa e; however, such patients require close monitoring. Sudden sensorineural hearing loss has been reported in a small number o patients taking PDE5 inhibitors, and medication use appears to increase the risk o hearing loss two old. Cases o vision loss due to nonarteritic ischemic optic neuropathy have been reported with PDE5 inhibitor use; however, larger studies have ailed to demonstrate an increased risk compared to placebo. The PDE5 inhibitors are metabolized by hepatic cytochrome P450 3A4.

Riociguat

Riociguat is a stimulator o sGC that has recently been ap-

proved or the treatment o chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension. Similar to PDE5 inhibitors, riociguat potentiates NO signaling and smooth muscle relaxation, but by two di erent mechanisms. First, riociguat stabilizes the interaction between NO and sGC. Second, riociguat directly stimulates sGC production o cGMP independently o NO. In contrast to the PDE5 inhibitors, riociguat has more commonly been associated with systemic hypotension and should be used with caution in patients at risk or ischemia or who are taking antihypertensive medications. Given this concern or systemic hypotension, riociguat use is contraindicated in patients taking either organic nitrates or PDE5 inhibitors. Riociguat is metabolized by cytochrome P450s 2C8 and 3A4.

Nesiritide

Nesiritide is a recombinant orm o B-type natriuretic peptide

that stimulates membrane-bound receptor guanylyl cyclases in the plasma membrane, providing an alternative route or vascular smooth muscle cell cGMP production. Nesiritide must be administered as a continuous intravenous in usion. Its primary indication is the treatment o decompensated heart ailure; recent clinical studies, however, ailed to show a change in mortality or rehospitalization rates in patients treated with nesiritide compared to controls. Nesiritide is discussed in more detail in Chapter 21, Pharmacology o Volume Regulation.

Prostacyclin Analogues Prostacyclin is a potent vasodilator through its activation o Gs-coupled IP receptors on vascular smooth muscle cells. Three prostanoids have been developed and used primarily or the treatment o pulmonary arterial hypertension: epoprostenol (a stable prostacyclin preparation), treprostinil, and iloprost. Prostacyclin not only relaxes vascular smooth muscle but also decreases smooth muscle cell proli eration, platelet aggregation, thrombosis, and extracellular matrix elaboration, all o which contribute to the pathogenesis o pulmonary arterial hypertension. Epoprostenol was the rst drug in this class and is the only drug to demonstrate a mortality bene t in patients with pulmonary arterial hypertension. Epoprostenol is administered as a continuous in usion through an indwelling central venous catheter, limiting its use to patients with severe, li estyle-limiting symptoms. An inhaled ormulation is also

available, the use o which is limited to critically ill patients with pulmonary hypertension or severe lung disease (similar to the use o inhaled NO). Epoprostenol has a hal -li e o 6 minutes; abrupt discontinuation can result in rebound pulmonary hypertension and a rapid clinical decline. Treprostinil is available in oral, subcutaneous, intravenous, and inhaled ormulations. The subcutaneous orm is delivered via continuous in usion; this modality alleviates the disadvantages associated with a chronic indwelling catheter, although there is a small risk o abscess ormation at the in usion site. Treprostinil also has a longer hal -li e than epoprostenol (4 hours), which may mitigate the adverse consequences associated with abrupt discontinuation (caused, or example, by mal unction o the in usion pump). However, clinically evident increases in pulmonary arterial pressures are seen within 1 hour o discontinuation. Intravenous treprostinil has similar e cacy to intravenous epoprostenol. Inhaled treprostinil requires less requent dosing than inhaled iloprost due to its longer hal -li e. The oral dosage orm appears to be less e ective than inhaled or parenteral therapy. Treprostinil requires dose reduction in patients with liver disease, and the oral dosage orm is contraindicated in patients with severe impairment. Iloprost is available only as an inhaled ormulation. Again, this ormulation avoids the risks o central venous catheterization. Additionally, inhalation therapy is theoretically more speci c or the pulmonary vasculature. Indeed, inhaled iloprost has been shown to increase arterial O2 saturation, which is not observed in patients receiving parenteral therapies. This is likely a consequence o selective vasodilation o ventilated alveoli and improved ventilation–per usion matching. The adverse e ects o all prostanoids include f ushing, headache, nausea, leg edema, hypotension, and syncope. Jaw pain and diarrhea are also common complaints. These symptoms tend to abate as therapy is continued. Subcutaneous iloprost can cause intense in usion site reactions and pain, which limit its administration by that route. Inhaled medications are, not surprisingly, associated with cough and throat irritation. Complications rom the intravenous delivery system can be li e-threatening and include local so t-tissue in ections, bloodstream in ections, catheter-associated thrombosis, and paradoxical embolism.

Endothelin Receptor Antagonists Bosentan is a competitive antagonist at the ETA and ETB en-

dothelin receptors that is approved or use in the treatment o pulmonary arterial hypertension. In clinical trials involving patients with severe dyspnea related to pulmonary hypertension, bosentan signi cantly improved 6-minute walk distance (i.e., the distance a patient can walk in 6 minutes) and decreased pulmonary vascular resistance relative to placebo. The major adverse e ect o bosentan is an elevation o serum transaminase levels, with approximately 10% o patients having elevations that exceed three times the upper limit o normal. It is, there ore, necessary to monitor serum transaminase levels monthly in patients taking bosentan. Ambrisentan is an ETA receptor selective antagonist. As with bosentan, patients with pulmonary hypertension have improved 6-minute walk distance and increased unctional status when taking this medication. Ambrisentan may be less hepatotoxic than bosentan. Macitentan is a recently approved nonselective ET receptor antagonist with a sa ety pro le similar to that o ambrisentan.

C h a p t e r 2 2 Pharmacology of Vascular Tone

Sympathetic Nervous System Antagonists 1-Adrenergic

Antagonists

Epinephrine and norepinephrine stimulate Gq-coupled 1adrenergic receptors on vascular smooth muscle cells to induce vasoconstriction. 1-Adrenergic antagonists, such as prazosin, block receptor activation and cause vasodilation. The e ect o these agents is greater in arterioles than in venules. These agents cause a signi cant reduction in arterial pressure and are use ul in the treatment o hypertension. Initiation o therapy with 1blockers can be associated with orthostatic hypotension. These agents are also associated with f uid retention that can be mitigated by co-administration o a diuretic. Some 1-adrenergic antagonists, such as terazosin, are used principally to inhibit the contraction o nonvascular smooth muscle (e.g., prostatic smooth muscle), but these agents also have some e ects on the vasculature. 1-Adrenergic antagonists are discussed in greater detail in Chapter 11, Adrenergic Pharmacology.

-Adrenergic Antagonists

While activation o Gs-coupled 2-adrenergic receptors on vascular smooth muscle cells leads to vasodilation, -adrenergic receptor antagonists are o major clinical importance in the treatment o hypertension, angina, cardiac arrhythmias, and other conditions through inhibition o cardiac 1-adrenergic receptors. Inhibition o 1-receptors has negative inotropic and chronotropic e ects on the heart; these actions reduce cardiac output, which is an important determinant o both myocardial O2 demand and blood pressure. Systemic inhibition o 2adrenergic receptors on vascular smooth muscle cells can lead to unopposed vasoconstriction through 1-adrenergic stimulation and, consequently, to increased systemic vascular resistance. Over time, however, the net e ect is a decrease in blood pressure owing to the cardiac e ects o these medications as well as inhibition o renin secretion and e ects o -blockers on the central nervous system. -Adrenergic antagonists are discussed in greater detail in Chapter 11.

Renin-Angiotensin-Aldosterone System Blockers Inhibition o the renin-angiotensin-aldosterone system results in signi cant vasodilation (see Chapter 21). The hypotensive e ect o ACE inhibitors may be caused, in part, by decreased catabolism o bradykinin, a vasorelaxant released in response to inf ammatory stimuli, as well as decreased stimulation o AT1 angiotensin receptors that are coupled to Gq and Gi. Antagonists o the AT1 receptor have a more direct e ect by inhibiting angiotensin II-mediated stimulation o these receptors. The direct renin inhibitor aliskiren has recently been approved or the treatment o hypertension. Mineralocorticoid receptor antagonists, such as spironolactone and eplerenone , are also e ective in the treatment o hypertension and heart ailure.

CONCLUSION AND FUTURE DIRECTIONS Vascular tone is subject to exquisite control, as would be expected or a system that must regulate blood f ow to all tissues in the body. Vascular tone represents a balance between vascular smooth muscle relaxation and contraction that is ultimately determined by the intracellular Ca2 concentration. Increases in cytoplasmic Ca2 stimulate Ca2 /CaMdependent MLCK phosphorylation, permitting actin–myosin

397

cross-bridge ormation and cellular contraction. The vascular smooth muscle cell relaxes when intracellular Ca2 concentrations decrease. Vascular smooth muscle cells integrate a variety o stimuli rom the local environment, adjacent endothelial cells, sympathetic nervous system, and humoral mediators to optimize vessel caliber. A molecular understanding o the critical signaling pathways involved in vascular smooth muscle cell biology has enabled the development o numerous targeted pharmacologic therapies or disorders o vascular tone, including systemic and pulmonary hypertension, angina, coronary artery disease, and congestive heart ailure. Indeed, new insights into novel regulators o vascular smooth muscle cell tone have identi ed novel therapeutic targets. One o these, asudil, is a RhoA-kinase (ROCK) inhibitor. In vascular smooth muscle cells, ROCK phosphorylates myosin light chain phosphatase, inhibiting its ability to dephosphorylate and inactivate myosin light chain. ROCK signaling thus potentiates vascular smooth muscle contraction, and ROCK inhibitors block this signaling pathway. In clinical studies, asudil has shown promise or treatment o pulmonary hypertension and cerebral vasospasm. Lipid signaling molecules, such as those that constitute the endothelial-derived hyperpolarizing actors and sphingosine1-phosphate, likely represent additional targets or therapeutic intervention. These compounds are known to bind G proteincoupled receptors in the plasma membrane and stimulate signal transduction pathways leading to changes in vascular smooth muscle tone. Additional research is necessary, however, to identi y and characterize the ligands, receptors, signaling pathways, and relevant tissue distributions, prior to clinical trials. These examples demonstrate how continued elucidation o the complex signaling pathways regulating vascular smooth muscle cell tone will lead to the identi cation o new targets or pharmacologic intervention in the cellular milieu o the vascular wall and will help to integrate the pharmacology o vascular tone across the spectrum o cardiovascular diseases.

Acknowledgment We thank Deborah Yeh Chong and Thomas Michel or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Abrams J. Chronic stable angina. N Engl J Med 2005;352:2524–2533. (Informative case vignette and review of the pathophysiology and pharmacotherapy of angina pectoris.) Flynn JT, Pasko DA. Calcium channel blockers: pharmacology and place in therapy o pediatric hypertension. Pediatr Nephrol 2000;15:302–316. (Overview of Ca2 channel blocker pharmacology with pharmacokinetic data, including Ca2 channel structure and function, with data from adults extrapolated for pediatric care.) Frumkin LR. The pharmacological treatment o pulmonary arterial hypertension. Pharmacol Rev 2012;64:583–620. (Comprehensive review of the current pharmacologic management of pulmonary arterial hypertension.) Gilchrist M, Shore AC, Benjamin N. Inorganic nitrate and nitrite and control o blood pressure. Cardiovasc Res 2011;89:492–498. (Detailed review of nitrate and nitrite metabolism and the role of dietary nitrate in blood pressure control.) Giles TD, Sander GE, Nossaman BD, Kadowitz PJ. Impaired vasodilation in the pathogenesis o hypertension: ocus on nitric oxide, endothelial-derived hyperpolarizing actors, and prostaglandins. J Clin Hypertens 2012;14:198–205. (Review of endothelial-derived factors regulating vascular smooth muscle tone.) Loirand G, Guérin P, Pacaud P. Rho kinases in cardiovascular physiology and pathophysiology. Circ Res 2006;98:322–334. (This review explores the functional role of Rho kinases in vascular biology and their therapeutic implications.)

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402 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

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Pharmacology of Hemostasis and Thrombosis Xa

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Eh rin J . Arm s t ro nFo g nd adnaap dpari David ndap apar ari inu nux x E. Go la n Binds to a ntith h rombi bin n II IIII (ATI (A AT II) II) via pe nt nta s a ccha ride (s ufficie nt to o ina in a cti c t va te Xa ) INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403–404 GP PIIb– IIb–II I Ia Antagonistss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 4177 Thrombin Recep epto torr (P ( A AR-1) Antag goonists . . . . . . . . . . . . . . . 4117 PHYS PH YSIO IOL LOGY OF HEMOSTASIS . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 18 Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . .Xa . . . . . . . . . . . . . . 41 Vaso a soco cons nstriction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 Warfarin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 18 Primary Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 U fractionated andd Low Un oow-M -Mol olee c ula lar ar-Wei r-Weight eightt Hep Heparinss . . . 4220 Platelet Adhesion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 4044 o22 x ban, Rivaro xab Selle cti Se tive Factor Xa Innhi hhibi bittors . . . . . . . . . . . . . . . . . . .Ri . . . . .ro 42 Platelet Granule Release Reaction. . . . . . . . . . . . . . . . . . . . 40 4044 ab apixaban Direct Thr hrom ombi binn In Inhibitors i s . . . . . . . . . . . . . . . . . . . . . .apixab .p. . . .aba 422n Plat Pl atel elet et Agg ggre rega gati tion on a nd Consoli lidda tion ion . . . . . . . . . . . . . . . 406 Recombinant Activated e Protein C (r-APC APC)) 423 Secondary Hemostasis: The Coagulationn Cascade . . . . . . . . 407 S e le ctive ly l ina i c ti tiva i te te Xa Xa Thrombolytic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 Regulation g of Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 PATHOGENESIS OF THROMBOSIS . . . . . . . . . . . . . . . . . . . . . . . . 411 4 S ubs tra teeStreptokinase re cognition cognitio Tissue P Plasminogen Activator (t-PA) . . . . . . 424 Endothelial Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 41s iite (ExosRecombinant sTenecteplase it ) ite e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Abnormal Blood Flow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 Reteplase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Hypercoagulability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 Inhibitors of Anticoagulation and Fibrinolysis . . . . . . . . . . . . . 424 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 414 Protaminee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Antiplatelet Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 Serine-Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Cyclooxygenase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 Lysine Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 Phosphodiesterase Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . 414 ADP Receptor Pathway Inhibitorss . . . . . . . . . . . . . . . . . . . . 415 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 424 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .425

INTRODUCTION Blood carries oxygen and nutrients to tissues and takes metabolic waste products away rom tissues. Humans have developed a well-regulated system o hemostasis to keep the blood f uid and clot- ree in normal vessels and to orm a localized plug rapidly in injured vessels. Thrombosis describes a pathologic state in which normal hemostatic processes are activated inappropriately. For example, a blood clot (thrombus) may orm as the result o a relatively minor vessel injury and occlude a branch o the vascular tree. This chapter presents the normal physiology o hemostasis, the pathophysiology o thrombosis, and the pharmacology o drugs that can be used to prevent or reverse a thrombotic state. Drugs introduced in this chapter are used to treat a variety o cardiovascular diseases, such as deep vein thrombosis, stroke, and myocardial in arction.

PHYSIOLOGY OF HEMOSTASIS An injured blood vessel must induce the ormation o a blood clot to prevent blood loss and to allow healing. Clot ormation

must also remain localized to prevent widespread clotting within intact vessels. The ormation o a localized clot at the site o vessel injury is accomplished in our temporally overlapping stages (Fig. 23-1). First, localized vasoconstriction occurs as a response to a ref ex neurogenic mechanism and to the secretion o endothelium-derived vasoconstrictors such as endothelin. Immediately ollowing vasoconstriction, primary hemostasis occurs. During this stage, platelets are activated and adhere to the exposed subendothelial matrix. Platelet activation involves both a change in shape o the platelet and the release o secretory granule contents rom the platelet. The secreted granule substances recruit other platelets, causing more platelets to adhere to the subendothelial matrix and to aggregate with one another at the site o vascular injury. Primary hemostasis ultimately results in the ormation o a primary hemostatic plug. The goal o the nal two stages o hemostasis is to orm a stable, permanent plug. During secondary hemostasis , also known as the coagulation cascade , the activated endothelium and other nearby cells (see below) express a membrane-bound procoagulant actor called tissue factor, which complexes 403

404 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Mr. S , a 55-ye a r-o ld m a n w ith a h is to ry o hyp e rte n s io n a n d ciga re tte s m o kin g , is a wa ke n e d in th e m id d le o th e n ig h t w ith s u b s te rn a l ch e s t p re s s u re , s w e a tin g , a n d s h o rtn e s s o b re a th . He ca lls 911 a n d is ta ke n to th e e m e rg e n cy d e p a rtm e n t. An e le ctro ca rd io g ra m s h o w s d e e p T-w a ve in ve rs io n s in le a d s V2 to V5. A ca rd ia c b io m a rke r p a n e l s h o w s a tro p o n in T le ve l o 3.2 g /L (n o rm a l, 0.01 g /L), co n s is te n t w ith m yo ca rd ia l in a rctio n . He is tre a te d w ith in tra ve n o u s n itro g lyce rin , a s p irin , u n ra ctio n a te d h e p a rin , a n d e p tif b a tid e , b u t h is ch e s t p a in p e rs is ts . He is ta ke n to th e ca rd ia c ca th e te riza tio n la b o ra to ry, w h e re h e is o u n d to h a ve a 90% m id LAD (le t a n te rio r d e s ce n d in g a rte ry) th ro m b u s w ith s lu g g is h d is ta l o w. He u n d e rg o e s s u cce s s u l a n g io p la s ty a n d s te n t p la ce m e n t. At th e tim e o s te n t p la ce m e n t, a n o ra l lo a d in g d o s e o clo p id o g re l is a d m in is te re d . Th e h e p a rin is s to p p e d , in tra ve n o u s e p tif b a tid e is co n tin u e d o r 18 m o re h o u rs , a n d h e is tra n s e rre d to th e te le m e try wa rd . S ix h o u rs la te r, Mr. S is n o te d to h a ve a n e xp a n d in g h e m a to m a (a n a re a o lo ca lize d h e m o rrh a g e ) in h is rig h t

with coagulation actor VII to initiate the coagulation cascade. The end result o this cascade is the activation o thrombin, a critical enzyme. Thrombin serves two pivotal unctions in hemostasis: (1) it converts soluble brinogen to an insoluble brin polymer that orms the matrix o the clot and (2) it induces more platelet recruitment and activation. Evidence indicates that brin clot ormation (secondary hemostasis) overlaps temporally with platelet plug ormation (primary hemostasis), and that each process rein orces the other. During the nal stage, platelet aggregation and brin polymerization lead to the ormation o a stable, permanent plug. In addition, antithrombotic mechanisms restrict the permanent plug to the site o vessel injury, ensuring that the permanent plug does not inappropriately extend to occlude the vascular tree.

Vasoconstriction Transient arteriolar vasoconstriction occurs immediately a ter vascular injury. This vasoconstriction is mediated by a poorly understood ref ex neurogenic mechanism. Local endothelial secretion o endothelin, a potent vasoconstrictor, potentiates the ref ex vasoconstriction. Because the vasoconstriction is transient, bleeding would resume i primary hemostasis were not activated.

Primary Hemostasis The goal o primary hemostasis is to orm a platelet plug that rapidly stabilizes vascular injury. Platelets play a pivotal role in primary hemostasis. Platelets are cell ragments that arise by budding rom megakaryocytes in the bone marrow; these small, membrane-bound discs contain cytoplasm but lack nuclei. Glycoprotein receptors in the platelet plasma membrane are the primary mediators by which platelets are activated. Primary hemostasis involves the trans ormation

th ig h b e lo w th e a rte ria l a cce s s s ite . Th e e p tif b a tid e is s to p p e d a n d p re s s u re is a p p lie d to th e a cce s s s ite , a n d th e h e m a to m a ce a s e s to e xp a n d . He is d is ch a rg e d 2 d a ys la te r w ith p re s crip tio n s th a t in clu d e clo p id o g re l a n d a s p irin , w h ich a re a d m in is te re d to p re ve n t th ro m b o s is o th e s te n t.

Questions 1 . How did a blood clot arise in Mr. S’s coronary artery? 2 . How do aspirin, heparin, clopidogrel, and eptif batide act in the attempt to treat Mr. S’s blood clot and to prevent recurrent thrombus ormation? 3 . What accounts or the e f cacy o eptif batide (a platelet GPIIb–IIIa antagonist) in inhibiting platelet aggregation? 4 . When the expanding hematoma was observed, could any measure other than stopping the eptif batide have been used to reverse the e ect o this agent? 5 . I low-molecular-weight heparin had been used instead o un ractionated heparin, how would the monitoring o the patient’s coagulation status during the procedure have been a ected?

o platelets into a hemostatic plug through three reactions: (1) adhesion, (2) the granule release reaction, and (3) aggregation and consolidation.

Platelet Adhesion

In the rst reaction, platelets adhere to subendothelial collagen that is exposed a ter vascular injury (Fig. 23-2). This adhesion is mediated initially by two molecular interactions. First, von Willebrand factor (vWF), a large multimeric protein that is secreted by both activated platelets and the injured endothelium, binds both to sur ace receptors (especially glycoprotein Ib [GPIb]) on the platelet membrane and to the exposed collagen. This “bridging” action mediates adhesion o platelets to the collagen. Second, platelet glycoprotein VI (GPVI) interacts directly with collagen in the exposed vessel wall. Both the GPIb:vWF:collagen interaction and the GPVI:collagen interaction are required or initiation o primary hemostasis.

Platelet Granule Release Reaction

Adherent platelets undergo a process o activation (Fig. 23-3) during which the cells’ granule contents are released. The release reaction is initiated by agonist binding to cell sur ace receptors, which activates intracellular protein phosphorylation cascades and ultimately causes release o granule contents. Speci cally, stimulation by adenosine diphosphate (ADP), epinephrine, and collagen leads to activation o platelet membrane phospholipase A2 (PLA2). PLA2 cleaves membrane phospholipids and liberates arachidonic acid, which is converted into a cyclic endoperoxide by platelet cyclooxygenase. Thromboxane synthase subsequently converts the cyclic endoperoxide into thromboxane A2 (TxA2). TxA2, via a G proteincoupled receptor, causes vasoconstriction at the site o vascular injury by inducing a decrease in cAMP levels within vascular smooth muscle cells. TxA2 also stimulates the granule release

C h a p t e r 2 3 Pharmacology o Hemostasis and Thrombosis

405

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4. S urfa ce he pa rin-like mole cule s (blocks coa gula tion ca s ca de )

reaction within platelets, thereby propagating the cascade of

platelet activation and vasoconstriction. During the release reaction, large amounts of ADP, Ca2 , adenosine triphosphate (ATP), serotonin, vWF, and platelet factor 4 are actively secreted from platelet granules. ADP is particularly important in mediating platelet aggregation, causing platelets to become “sticky” and adhere to one another (see below).

Res ting platelets

Activa te d s prea d platelet

Activated contracted platelet

FIGURE 23-1. Sequence of events in hemostasis. The hemostatic process can be divided conceptually into our stages—vasoconstriction, primary hemostasis, secondary hemostasis, and resolution—although recent evidence suggests that these stages are temporally overlapping and may be nearly simultaneous. A. Vascular injury causes endothelial denudation. Endothelin, released by activated endothelium, and neurohumoral actor(s) induce transient vasoconstriction. B. Injury-induced exposure o the subendothelial matrix (1) provides a substrate or platelet adhesion and activation (2). In the granule release reaction, activated platelets secrete thromboxane A2 (TxA2) and ADP (3). TxA2 and ADP released by activated platelets cause nearby platelets to become activated; these newly activated platelets undergo shape change (4) and are recruited to the site o injury (5). The aggregation o activated platelets at the site o injury orms a primary hemostatic plug (6). C. Tissue actor expressed on activated endothelial cells (1) and leukocyte microparticles (not shown), together with acidic phospholipids expressed on activated platelets and activated endothelial cells (2), initiate the steps o the coagulation cascade, culminating in the activation o thrombin (3). Thrombin proteolytically activates f brinogen to orm f brin, which polymerizes around the site o injury, resulting in the ormation o a def nitive (secondary) hemostatic plug (4). D. Natural anticoagulant and thrombolytic actors limit the hemostatic process to the site o vascular injury. These actors include tissue plasminogen activator (t-PA), which activates the f brinolytic system (1); thrombomodulin, which activates inhibitors o the coagulation cascade (2); prostacyclin, which inhibits both platelet activation and vasoconstriction (3); and sur ace heparin-like molecules, which catalyze the inactivation o coagulation actors (4). E. Scanning electron micrographs o resting platelets (1), a platelet undergoing cell spreading shortly a ter cell activation (2), and a ully activated platelet a ter actin f lament bundling and cross-linking and myosin contraction (3).

Although strong agonists (such as thrombin and collagen) can trigger granule secretion even when aggregation is prevented, ADP can trigger granule secretion only in the presence of platelet aggregation. Presumably, this difference is due to the set of intracellular effectors that are coupled to the various agonist receptors. Release of Ca2 ions is also important for the coagulation cascade, as discussed below. Although platelet activation can be initiated via exposure of subendothelial collagen, a separate and parallel process of platelet activation occurs without disruption of the endothelium and without the involvement of von Willebrand factor. This second pathway of platelet activation is initiated by tissue factor, a lipoprotein expressed by activated endothelial cells, activated leukocytes, and microparticles derived from activated leukocytes (see below). As in the coagulation cascade, tissue factor forms a complex with factor VIIa, and the tissue factor–factor VIIa complex activates factor IX. Factor IX activation leads to a proteolytic cascade that results in the generation of thrombin (factor IIa), a multifunctional enzyme that plays a critical role in the coagulation cascade (see below). In the tissue factor-initiated pathway of platelet activation, thrombin cleaves protease-activated receptor 1 (PAR-1) and

406 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Endothe lium Colla ge n

Endothe lium Colla ge n Re s ting pla te le t

Tis s ue fa ctor TXA2 P la te le t

Fibrinoge n

ADP

P rothrombin Thrombin

GP VI GP IIb-IIIa GP Ib von Wille bra nd fa ctor Activa te d e ndothe lium

Colla ge n (s ube ndothe lium)

von Wille bra nd fa ctor

FIGURE 23-2. Platelet adhesion and aggregation. von Willebrand actor mediates platelet adhesion to the subendothelium by binding both to platelet membrane glycoprotein GPIb and to exposed subendothelial collagen. Platelet adhesion to the subendothelial matrix also requires a direct binding interaction between platelet membrane glycoprotein GPVI and subendothelial collagen. During platelet aggregation, f brinogen cross-links platelets to one another by binding to GPIIb–IIIa receptors on platelet membranes. GPIb exists in the platelet membrane in a complex with glycoproteins GPIXand GPV(not shown).

protease-activated receptor 4 (PAR-4) on the platelet sur ace

and thereby causes the platelets to release ADP, serotonin, and TxA2. By activating other nearby platelets, these agonists ampli y the signal or thrombus ormation.

Platelet Aggregation and Consolidation

TxA2, ADP, and f brous collagen are all potent mediators o platelet aggregation. TxA2 promotes platelet aggregation through stimulation o G protein-coupled TxA2 receptors in the platelet membrane (Fig. 23-4). Binding o TxA2 to platelet TxA2 receptors leads to activation o phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) to yield inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 raises the cytosolic Ca2 concentration, and DAG activates protein kinase C (PKC), which in turn promotes the activation o PLA2. Through an incompletely understood mechanism, PLA2 activation induces the expression o unctional GPIIb–IIIa, the membrane integrin that mediates platelet aggregation. ADP triggers platelet activation by binding to G proteincoupled ADP receptors on the platelet sur ace (Fig. 23-5).

Colla ge n

FIGURE 23-3. Platelet activation. Platelet activation is initiated at the site o vascular injury when circulating platelets adhere to exposed subendothelial collagen and are activated by locally generated mediators. Activated platelets undergo shape change and granule release, and platelet aggregates are ormed as additional platelets are recruited and activated. Platelet recruitment is mediated by the release o soluble platelet actors, including ADP and thromboxane A2 (TxA2). Tissue actor, expressed on activated endothelium, is a critical initiating component in the coagulation cascade. The membranes o activated platelets provide a sur ace or a number o critical reactions in the coagulation cascade, including the conversion o prothrombin to thrombin.

The two subtypes o G protein-coupled platelet ADP receptors are termed P2Y1 receptors and P2Y(ADP) receptors . P2Y1, a Gq-coupled receptor, releases intracellular calcium stores through activation o phospholipase C. P2Y(ADP), a Gicoupled receptor, inhibits adenylyl cyclase. The P2Y(ADP) receptor is the target o the antiplatelet agents ticlopidine , clopidogrel, prasugrel, and ticagrelor (see below). Activation o ADP receptors mediates platelet shape change and expression o unctional GPIIb–IIIa. Fibrous collagen activates platelets by binding directly to platelet glycoprotein VI (GPVI). Activation o GPVI by collagen initiates signaling cascades that promote the granule release reaction and that induce con ormational changes in cell sur ace integrins (especially GPIIb–IIIa and 2 1) that promote the direct or indirect binding o these integrins to collagen. These additional binding interactions urther strengthen the adhesion o activated platelets to the subendothelial matrix. Platelets aggregate with one another through a bridging molecule, f brinogen, which has multiple binding sites or unctional GPIIb–IIIa (Fig. 23-2). Just as the vWF:GPIb

C h a p t e r 2 3 Pharmacology o Hemostasis and Thrombosis

407

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FIGURE 23-4. Platelet activation by thromboxane A2. 1. Thromboxane A2 (TxA2) is generated rom arachidonic acid in activated platelets; cyclooxygenase catalyzes the committed step in this process. 2. Secreted TxA2 binds to the cell sur ace TxA2 receptor (TxA2-R), a G protein-coupled receptor. 3. The G iso orm G q activates phospholipase C (PLC). 4. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). 5. IP3 increases the cytosolic Ca 2 concentration by promoting vesicular release o Ca 2 into the cytosol. 6. DAG activates protein kinase C (PKC). 7. PKC activates phospholipase A2 (PLA2). 8. Through an incompletely understood mechanism, activation o PLA2 leads to the activation o GPIIb–IIIa. 9. Activated GPIIb–IIIa binds to f brinogen. 10. Fibrinogen cross-links platelets by binding to GPIIb–IIIa receptors on other platelets. This crosslinking leads to platelet aggregation and ormation o a primary hemostatic plug.

interaction is important or platelet adhesion to exposed subendothelial collagen, the f brinogen:GPIIb–IIIa interaction is critical or platelet aggregation. Platelet aggregation ultimately leads to the ormation o a reversible clot, or a primary hemostatic plug. Activation o the coagulation cascade proceeds nearly simultaneously with the ormation o the primary hemostatic plug, as described below. Activation o the coagulation cascade leads to the generation o f brin, initially at the periphery o the primary hemostatic plug. Platelet pseudopods attach to the f brin strands at the periphery o the plug and contract (Fig. 23-1E). Platelet contraction yields a compact, solid, irreversible clot, or a secondary hemostatic plug.

Secondary Hemostasis: The Coagulation Cascade Secondary hemostasis is also termed the coagulation cascade . The goal o this cascade is to orm a stable f brin clot at the site o vascular injury. Details o the coagulation

cascade are presented schematically in Figure 23-6. Several general principles should be noted. First, the coagulation cascade is a sequence o enzymatic events. Most plasma coagulation actors circulate as inactive proenzymes, which are synthesized by the liver. These proenzymes are proteolytically cleaved, and thereby activated, by the activated actors that precede them in the cascade. The activation reaction is catalytic and not stoichiometric. For example, one “unit” o activated actor X can potentially generate 40 “units” o thrombin. This robust amplif cation process rapidly generates large amounts o f brin at a site o vascular injury. Second, the major activation reactions in the cascade occur at sites where a phospholipid-based protein–protein complex has ormed (Fig. 23-7). This complex is composed o a membrane sur ace (provided by activated platelets, activated endothelial cells, and possibly activated leukocyte microparticles [see below]), an enzyme (an activated coagulation actor), a substrate (the proenzyme orm o the downstream coagulation actor), and a co actor. The presence o

408 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Thrombin

ADP

4

Ade nylyl cycla s e

ADP PAR-1

P LC

1 β

αi

P 2Y(ADP ) re ce ptor

GDP

β

αi γ

αq

GTP

P 2Y1 re ce ptor

γ

αq

GDP

6

ATP cAMP

2

AMP

GTP

β

αq γ GDP

PDE

PKA

5

7

Incre a s e d P LC a ctivity le a ds to pla te le t a ctiva tion

3 De cre a s e d P KA a ctivity le a ds to pla te le t a ctiva tion

FIGURE 23-5. Platelet activation by ADP and thrombin. Left panel: 1. Binding of ADP to the P2Y(ADP) receptor activates a Gi protein, which inhibits adenylyl cyclase. 2. Inhibition of adenylyl cyclase decreases the synthesis of cAMP and hence decreases protein kinase A (PKA) activation (dashed arrow). cAMP is metabolized to AMP by phosphodiesterase (PDE). 3. PKA inhibits platelet activation through a series of incompletely understood steps. Therefore, the decreased PKA activation that results from ADP binding to the P2Y(ADP) receptor causes platelet activation. Right panel: 4. Thrombin proteolytically cleaves the extracellular domain of protease-activated receptor 1 (PAR-1). This cleavage creates a new N-terminus, which binds to an activation site on PAR-1 to activate a Gq protein. 5. ADP also activates Gq by binding to the P2Y1 receptor. 6. Gq activation (by either thrombin or ADP) activates phospholipase C (PLC). 7. PLC activation leads to platelet activation, as shown in Figure 23-4. Note that ADP can activate platelets by binding to either the P2Y(ADP) receptor or the P2Y1 receptor, although evidence suggests that full platelet activation requires the participation of both receptors. High concentrations of thrombin can also activate platelets via proteolytic cleavage of PAR-4 on platelet surfaces (not shown).

negatively charged phospholipids, especially phosphatidylserine, is critical or assembly o the complex. Phosphatidylserine, which is normally sequestered in the inner leaf et o the plasma membrane, translocates to the outer leaf et o the membrane in response to agonist stimulation o platelets, endothelial cells, or leukocytes. Calcium is required or the enzyme, substrate, and co actor to adopt the proper con ormation or the proteolytic cleavage o a coagulation actor proenzyme to its activated orm. Third, the coagulation cascade has been divided traditionally into the intrinsic and extrinsic pathways (Fig. 23-6). This division is a result o in vitro testing and is essentially arbitrary. The intrinsic pathway is activated in vitro by actor XII (Hageman actor), while the extrinsic pathway is initiated in vivo by tissue factor, activated endothelial cells, subendothelial smooth muscle cells, and subendothelial broblasts at the site o vascular injury. Although these two pathways converge at the activation o actor X, there is also much interconnection between the two pathways. Because actor VII (activated by the extrinsic pathway) can proteolytically activate actor IX (a key actor in the intrinsic pathway), the

extrinsic pathway is regarded as the primary pathway or the initiation o coagulation in vivo. Fourth, both the intrinsic and extrinsic coagulation pathways lead to the activation o actor X. In an important reaction that requires actor V, activated actor X proteolytically cleaves prothrombin ( actor II) to thrombin ( actor IIa) (Fig. 23-8). Thrombin acts in the coagulation cascade in our important ways: (1) it converts the soluble plasma protein brinogen into brin, which then orms long, insoluble polymer bers; (2) it activates actor XIII, which cross-links the brin polymers into a highly stable meshwork or clot; (3) it ampli es the clotting cascade by catalyzing the eedback activation o actors VIII and V; and (4) it strongly activates platelets, causing granule release, platelet aggregation, and platelet-derived microparticle generation. In addition to its procoagulant properties, thrombin acts to modulate the coagulation response. Thrombin activates protease-activated receptors (PARs) on the intact vascular endothelial cells adjacent to the area o vascular injury and stimulates these cells to release the platelet inhibitors prostacyclin (PGI2) and nitric oxide (NO), the pro brinolytic protein tissue plasminogen

C h a p t e r 2 3 Pharmacology o Hemostasis and Thrombosis

Tis s ue injury

XII Kallikrein

VII

IXa

P re ka llikre in

XIIa

IX

HMWK

XI

P rote olytic cle a va ge (a ctiva tion) of fa ctor X

Extrinsic pathway

Intrinsic pathway

XIa

VIIIa Ca

Thrombin (IIa)

2+

Xa

Ca 2+

Xa

X

VIIa

IXa

Ca 2+

Xa a

IX IXa

VIIIa Ca

VIIa

VIIIa

P rote olytic cle a va ge (a ctiva tion) of prothrombin

X

Thrombin (IIa) VIII

409

P rothrombin (II)

Va

Tis s ue fa ctor

2+

Ca 2+

Thrombin (IIa)

Va

Ca 2+

Ca2+2+ Ca

Common pathway Xa

FIGURE 23-7. Coagulation factor activation on phospholipid surfaces.

Thrombin (IIa) V

Va Ca 2+

Xa Ca 2+ XIII

P rothrombin (II)

Thrombin (IIa)

Ca 2+

XIIIa

Ca 2+

Fibrinoge n

Fibrin

Fibrin polyme r

Sur ace catalysis is critical or a number o the activation reactions in the coagulation cascade. Each activation reaction consists o an enzyme (e.g., actor IXa), a substrate (e.g., actor X), and a co actor or reaction accelerator (e.g., actor VIIIa), all o which are assembled on the phospholipid sur ace o activated platelets, endothelial cells, and leukocytes. Ca 2 allows the enzyme and substrate to adopt the proper con ormation in each activation reaction. In the example shown, actor VIIIa and Ca 2 act as co actors in the actor IXa-mediated cleavage o actor X to actor Xa. Factor Va and Ca 2 then act as co actors in the actor Xa-mediated cleavage o prothrombin to thrombin.

Cros s -linke d fibrin polyme r Re s ting e ndothe lia l Activa te d ce lls e ndothe lia l ce lls

FIGURE 23-6. Coagulation cascade. The coagulation cascade is arbitrarily divided into the intrinsic pathway, the extrinsic pathway, and the common pathway. The intrinsic and extrinsic pathways converge at the level o actor X activation. The intrinsic pathway is largely an in vitro pathway, while the extrinsic pathway accounts or the majority o in vivo coagulation. The extrinsic pathway is initiated at sites o vascular injury by the expression o tissue actor on several di erent cell types, including activated endothelial cells, activated leukocytes (and leukocyte microparticles), subendothelial vascular smooth muscle cells, and subendothelial f broblasts. Note that Ca 2 is a co actor in many o the steps, and that a number o the steps occur on phospholipid sur aces provided by activated platelets, activated endothelial cells, and activated leukocytes (and leukocyte microparticles). Activated coagulation actors are shown in blue and indicated with a lower case “a.” HMWK, high-molecular-weight kininogen.

V Va

VII VIIa VIII VIIIa XI

XIa

P rothrombin (II)

Va Xa Ca 2+

Re s ting pla te le ts

PL

Thrombin (IIa) Activa te d pla te le ts

Fibrinoge g n XIII

XIIIa

Fibrin n

Fibrin polyme r

activator (t-PA), and the endogenous t-PA modulator plasminogen activator inhibitor 1 (PAI-1) (see below). Thrombin binds to protease-activated receptors (PARs), which are G protein-coupled receptors expressed in the plasma membrane o platelets, vascular endothelial cells, monocytes, vascular smooth muscle cells, and f broblasts. Activation o PARs involves proteolytic cleavage o an extracellular domain o the receptor by thrombin. The new NH2terminal-tethered ligand binds intramolecularly to a discrete site within the receptor and initiates intracellular signaling. Activation o PAR-1 results in G protein-mediated activation o PLC (Fig. 23-5) and inhibition o adenylyl cyclase. Four di erent PARs have been identif ed (PARs 1–4). PAR-1 and

Cros s -linke d fibrin polyme r

FIGURE 23-8. Central role of thrombin in the coagulation cascade. In the coagulation cascade, prothrombin is cleaved to thrombin by actor Xa; actor Va and Ca 2 act as co actors in this reaction, and the reaction takes place on an activated (phosphatidylserine-expressing) phospholipid sur ace (PL). Thrombin converts the soluble plasma protein f brinogen to f brin, which spontaneously polymerizes. Thrombin also activates actor XIII, a transglutaminase that cross-links the f brin polymers into a highly stable meshwork or clot. Thrombin also activates co actors V and VIII, as well as coagulation actors VII and XI. In addition, thrombin activates both platelets and endothelial cells. Finally, thrombin stimulates the release o several antithrombotic actors—including PGI2, NO, and t-PA— rom resting (intact) endothelial cells near the site o vascular injury; these actors limit primary and secondary hemostasis to the injured site (not shown).

410 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

PAR-4 are expressed on platelets; PARs 1–4 are expressed on vascular endothelial cells; and some or all o the PARs are expressed on other cell types including monocytes and vascular smooth muscle cells. O the two protease-activated receptors expressed on platelets, PAR-1 has a higher a nity or thrombin, and PAR-1 is the target o the new antiplatelet agent vorapaxar (see below). Evidence rom intravital (in vivo) microscopy experiments suggests that microparticles also have an important role in coupling platelet plug ormation (primary hemostasis) to brin clot ormation (secondary hemostasis). Microparticles are vesicular structures derived rom leukocytes, monocytes, platelets, endothelial cells, and smooth muscle cells; they display proteins o the cells rom which they were derived. For example, a subpopulation o microparticles is released rom monocytes that are activated in the context o tissue injury and inf ammation. These microparticles express both tissue actor and P-selectin glycoprotein ligand-1 (PSGL-1). In turn, PSGL-1 on the microparticles binds to the P-selectin adhesion receptor expressed on activated platelets. By recruiting tissue actor-bearing microparticles throughout the developing platelet plug (primary hemostasis), thrombin generation and brin clot ormation (secondary hemostasis) could be greatly accelerated within the plug itsel . Indeed, both vessel-wall tissue actor (expressed by activated endothelial cells, subendothelial broblasts, and smooth muscle cells) and microparticle tissue actor are important or the ormation o a stable clot.

Regulation of Hemostasis Hemostasis is exquisitely regulated or two major reasons. First, hemostasis must be restricted to the local site o vascular injury. That is, activation o platelets and coagulation actors in the plasma should occur only at the site o endothelial damage, tissue actor expression, and procoagulant phospholipid exposure. Second, the size o the primary and secondary hemostatic plugs must be restricted so that the vascular lumen remains patent. A ter vascular injury, intact endothelium in the immediate vicinity o the injury becomes “activated.” This activated endothelium presents a set o procoagulant actors that promote hemostasis at the site o injury and anticoagulant actors that restrict propagation o the clot beyond the site o injury. The procoagulant actors, such as tissue actor and phosphatidylserine, tend to be membrane-bound and localized to the site o injury—these actors provide a sur ace on which the coagulation cascade can proceed. In contrast, the anticoagulant actors are generally secreted by the endothelium and are soluble in the blood. Thus, the activated endothelium maintains a balance of procoagulant and anticoagulant factors to limit hemostasis to the site of vascular injury. A ter vascular injury, the endothelium surrounding the injured area participates in ve separate mechanisms that limit the initiation and propagation o the hemostatic process to the immediate vicinity o the injury. These mechanisms involve prostacyclin (PGI2), antithrombin III, proteins C and S, tissue actor pathway inhibitor (TFPI), and tissue-type plasminogen activator (t-PA). Prostacyclin (PGI2) is an eicosanoid (i.e., a metabolite o arachidonic acid) that is synthesized and secreted by the endothelium. By acting through Gs protein-coupled plateletsur ace PGI2 receptors, this metabolite increases cAMP levels

within platelets and thereby inhibits platelet aggregation and platelet granule release. PGI2 also has potent vasodilatory e ects; this mediator induces vascular smooth muscle relaxation by increasing cAMP levels within the vascular smooth muscle cells. (Note that these mechanisms are physiologically antagonistic to those o TxA2, which induces platelet activation and vasoconstriction.) There ore, PGI2 both prevents platelets rom adhering to the intact endothelium that surrounds the site o vascular injury and maintains vascular patency around the site o injury. Antithrombin III inactivates thrombin and other coagulation actors (IXa, Xa, XIa, and XIIa, where “a” denotes an “activated” actor) by orming a stoichiometric complex with the coagulation actor (Fig. 23-9). These interactions are enhanced by a heparin-like molecule that is expressed at the sur ace o intact endothelial cells, ensuring that this mechanism is operative at all locations in the vascular tree except where endothelium is denuded at the site o vascular injury. (These endothelial cell sur ace proteoglycans are re erred to as heparin-like because they are the physiologic equivalent o the pharmacologic agent heparin, discussed below.) Heparin-like molecules on the endothelial cells bind to and activate antithrombin III, which is then primed to complex with (and thereby inactivate) the activated coagulation actors. Protein C and protein S are vitamin K-dependent proteins that slow the coagulation cascade by inactivating coagulation actors Va and VIIIa. Protein C and protein S are part o a eedback control mechanism, in which excess thrombin generation leads to activation o protein C, which, in turn, helps to prevent the enlarging brin clot rom occluding the vascular lumen. Speci cally, the endothelial cell sur ace protein thrombomodulin is a receptor or both thrombin and protein C in the blood. Thrombomodulin binds these proteins in such a way that thrombomodulin-bound thrombin cleaves protein C to activated protein C (also known as protein Ca). In a reaction that requires the co actor protein S, activated protein C then inhibits clotting by cleaving (and thereby inactivating) actors Va and VIIIa. Tissue factor pathway inhibitor (TFPI), as its name indicates, limits the action o tissue actor (TF). The coagulation cascade is initiated when actor VIIa complexes with TF at the site o vascular injury (Fig. 23-6). The resulting VIIa:TF complex catalyzes the activation o actors IX and X. A ter limited quantities o actors IXa and Xa are generated, the VIIa:TF complex is eedback inhibited by TFPI in a two-step reaction. First, TFPI binds to actor Xa and neutralizes its activity in a Ca2 -independent reaction. Subsequently, the TFPI:Xa complex interacts with the VIIa:TF complex via a second domain on TFPI, so that a quaternary Xa:TFPI:VIIa:TF complex is ormed. The molecular “knots” o the TFPI molecule hold the quaternary complex tightly together and thereby inactivate the VIIa:TF complex. In this manner, TFPI prevents excessive TF-mediated activation o actors IX and X. Plasmin exerts its anticoagulant e ect by proteolytically cleaving brin into brin degradation products. Because plasmin has power ul antithrombotic e ects, the formation o plasmin has intrigued researchers or many years, and a number o pharmacologic agents have been developed to target the plasmin ormation pathway (Fig. 23-10). Plasmin is generated by the proteolytic cleavage o plasminogen, a plasma protein that is synthesized in the liver. The proteolytic cleavage is catalyzed by tissue plasminogen activator (t-PA),

C h a p t e r 2 3 Pharmacology of Hemostasis and Thrombosis

411

A ATIII

+

He pa rin

Endoge nous he pa rin-like mole cule s or exogenous unfractionated heparin

Antithrombin III

ATIII He pa rin

B Active coagulation factors

Inactive coagulation factors

Thrombin

Thrombin

Thrombin

ATIII

ATIII

He pa rin

Xa

IXa

Xa

ATIII

+ He pa rin

Xa

ATIII

+

He pa rin

He pa rin

XIa

XIIa

ATIII

IXa XIa XIIa

ATIII

IXa XIa XIIa

ATIII

He pa rin

FIGURE 23-9. Antithrombin III action. Antithrombin III (ATIII) inactivates thrombin and factors IXa, Xa, XIa, and XIIa by forming a stoichiometric complex with these coagulation factors. These reactions are catalyzed physiologically by heparin-like molecules expressed on healthy endothelial cells; sites of vascular injury do not express heparin-like molecules because the endothelium is denuded or damaged. Pharmacologically, these reactions are catalyzed by exogenously administered heparin. In more detail, the binding of heparin to ATIII induces a conformational change in ATIII (A) that allows the ATIII to bind thrombin or coagulation factors IXa, Xa, XIa, or XIIa. The stoichiometric complex between ATIII and the coagulation factor is highly stable, allowing heparin to dissociate without breaking up the complex (B).

which is synthesized and secreted by the endothelium. Plasmin activity is care ully modulated by three regulatory mechanisms in order to restrict plasmin action to the site o clot ormation. First, t-PA is most e ective when it is bound to a brin meshwork. Second, t-PA activity can be inhibited by plasminogen activator inhibitor (PAI). When local concentrations o thrombin and inf ammatory cytokines (such as IL-1 and TNF- ) are high, endothelial cells increase the release o PAI, preventing t-PA rom activating plasmin. This ensures that a stable brin clot orms at the site o vascular injury. Third, 2-antiplasmin is a plasma protein that neutralizes ree plasmin in the circulation and thereby prevents systemic degradation o plasma brinogen. Plasma brinogen is important or platelet aggregation in primary hemostasis (see above), and it is also the precursor or the brin polymer that is required to orm a stable clot.

PATHOGENESIS OF THROMBOSIS Thrombosis is the pathologic extension o hemostasis. In thrombosis, coagulation reactions are inappropriately regulated so that a clot uncontrollably enlarges and occludes the lumen o a blood vessel. The pathologic clot is now termed

a thrombus . Three major actors predispose to thrombus ormation—endothelial injury, abnormal blood f ow, and hypercoagulability. These three actors inf uence one another and are collectively known as Virchow’s triad (Fig. 23-11).

Endothelial Injury Endothelial injury is the dominant inf uence on thrombus ormation in the heart and the arterial circulation. There are many possible causes o endothelial injury, including changes in shear stress associated with hypertension or turbulent f ow, hyperlipidemia, elevated blood glucose in diabetes mellitus, traumatic vascular injury, and some in ections. (Recall that Mr. S developed coronary artery thrombosis, which was probably attributable to endothelial injury secondary to hypertension and cigarette smoking.) Endothelial injury predisposes the vascular lumen to thrombus ormation through three mechanisms. First, platelet activators, such as exposed subendothelial collagen, promote platelet adhesion to the injured site. Second, exposure o tissue actor on injured endothelium initiates the coagulation cascade. Third, natural antithrombotics, such as t-PA and PGI2, become depleted at the site o vascular injury because these mechanisms rely on the unctioning o an intact endothelial cell layer.

412 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Tis s ue -type or urokina s e -type pla s minoge n a ctiva tor

Tis s ue -type or urokina s e -type pla s minoge n a ctiva tor (ina ctive )

P la s minoge n a ctiva tor inhibitor 1 or 2

P la s minoge n a ctiva tor inhibitor 1 or 2 Ina ctiva te d pla s min

P la s minoge n

+

Plasmin

C s -linke Cros li k d fibrin polyme r

Fibrin de gra da tion products

FIGURE 23-10. The f brinolytic system. Plasmin is ormed by the proteolytic cleavage o plasminogen by tissue-type or urokinase-type plasminogen activator. Plasmin ormation can be inhibited by plasminogen activator inhibitor 1 or 2, which binds to and inactivates plasminogen activators. In the f brinolytic reaction, plasmin cleaves cross-linked f brin polymers into f brin degradation products. 2-Antiplasmin, which circulates in the bloodstream, neutralizes ree plasmin in the circulation.

Abnormal Blood Flow Abnormal blood f ow re ers to a state o turbulence or stasis rather than laminar f ow. Atherosclerotic plaques commonly predispose to turbulent blood f ow in the vicinity o the plaque. Bi urcations o blood vessels can also create areas o turbulent f ow. Turbulent blood f ow causes endothelial injury, orms countercurrents, and creates local pockets o stasis. Local stasis can also result rom ormation o an aneurysm (a ocal outpouching o a vessel or a cardiac chamber) and rom myocardial in arction. In the latter condition, a region o noncontractile (in arcted) myocardium serves as a avored site or stasis. Cardiac arrhythmias, such as atrial brillation, can also generate areas o local stasis. Stasis is Endothe lia l injury

Thrombosis

Abnorma l blood flow

Hype rcoa gula bility

FIGURE 23-11. Virchow’s triad. Endothelial injury, abnormal blood ow, and hypercoagulability are three actors that predispose to thrombus ormation. These three actors are interrelated; endothelial injury predisposes to abnormal blood ow and hypercoagulability, while abnormal blood ow can cause both endothelial injury and hypercoagulability.

also the major cause or the ormation o venous thrombi, which typically occur in the deep veins o the leg. Disruption o normal blood f ow by turbulence or stasis promotes thrombosis by three major mechanisms. First, the absence o laminar blood f ow allows platelets to come into close proximity to the vessel wall. Second, stasis inhibits the f ow o resh blood into the vascular bed, so that activated coagulation actors in the region are not removed or diluted. Third, abnormal blood f ow promotes endothelial cell activation, which leads to a prothrombotic state.

Hypercoagulability Hypercoagulability is generally less important than endothelial injury and abnormal blood f ow in predisposing to thrombosis, but this condition can be an important actor in some patients. Hypercoagulability re ers to an abnormally heightened coagulation response to vascular injury, resulting rom either (1) primary (genetic) disorders or (2) secondary (acquired) disorders (see Table 23-1). (Hypocoagulable states, or hemorrhagic disorders , can also result rom primary or secondary causes; see Box 23-1 or an example.) Among the genetic causes o hypercoagulability, the most prevalent known mutation resides in the gene or coagulation actor V. It is estimated that 6% o the Caucasian population in the United States carries mutations in the actor V gene. The most common mutation is the Leiden mutation, in which glutamine is substituted or arginine at position 506. This position is important because it is part o a site in actor Va that is proteolytically cleaved by activated protein C. The mutant actor V Leiden protein is resistant to proteolytic cleavage by activated protein C. As a result o the Leiden mutation, actor Va is allowed to accumulate and thereby to promote coagulation. A second common mutation (2% incidence) is the prothrombin G20210A mutation, in which adenine (A) is substituted or guanine (G) in the 3 -untranslated region o the prothrombin gene. This mutation leads to a 30% increase in plasma prothrombin levels. Both the actor V Leiden mutation and the prothrombin G20210A mutation are associated with a signi cantly increased risk o venous thrombosis and a modestly increased risk o arterial thrombosis. Other genetic disorders that predispose some individuals to thrombosis include mutations in the brinogen, protein C, protein S, and antithrombin III genes. Although the latter disorders are relatively uncommon (less than 1% incidence), patients with a genetic de ciency o protein C, protein S, or antithrombin III o ten present with spontaneous venous thrombosis. Hypercoagulability can sometimes be acquired (secondary) rather than genetic. An example o acquired hypercoagulability is the heparin-induced thrombocytopenia syndrome. In some patients, administration o the anticoagulant heparin stimulates the immune system to generate circulating antibodies directed against a complex consisting o heparin and platelet actor 4. Because platelet actor 4 is present on platelet and endothelial cell sur aces, antibody binding to the heparin:platelet actor 4 complex results in antibody-mediated removal o platelets rom the circulation; that is, in thrombocytopenia. In some patients, however, antibody binding also causes platelet activation, endothelial injury, and a prothrombotic state. Although both un ractionated and low-molecularweight heparin (see below) can cause thrombocytopenia, low-molecular-weight heparin is associated with a lower incidence o thrombocytopenia than un ractionated heparin.

C h a p t e r 2 3 Pharmacology o Hemostasis and Thrombosis

413

TABLE 23-1 Major Causes of Hypercoagulability CONDITION

MECHANISM OF HYPERCOAGULABILITY

Primary (Genetic) Factor V Leiden mutation ( actor V R506Q) (common)

Resistance to activated protein C → excess actor Va

Hyperhomocysteinemia (common)

Endothelial damage due to accumulation o homocysteine

Prothrombin G20210A mutation (common)

Increased prothrombin level and activity

Antithrombin III def ciency (less common)

Decreased inactivation o actors IIa, IXa, and Xa

Protein C or S def ciency (less common)

Decreased proteolytic inactivation o actors VIIIa and Va

Secondary (Acquired) Antiphospholipid syndrome

Autoantibodies to negatively charged phospholipids → ↑ platelet adhesion

Heparin-induced thrombocytopenia

Antibodies to platelet actor 4 → platelet activation

Malignancy

Tumor cell induction o tissue actor expression

Myeloproli erative syndromes

Elevated blood viscosity, altered platelets

Nephrotic syndrome

Loss o antithrombin III in urine, ↑ f brinogen, ↑ platelet activation

Oral contraceptive use, estrogen replacement therapy

↑ Hepatic synthesis o coagulation actors and/or e ects o estrogen on endothelium (e ect may be more prominent in patients with underlying primary hypercoagulability)

Paroxysmal nocturnal hemoglobinuria

Lack o glycosylphosphatidylinositol-linked proteins on red blood cells, white blood cells, and platelets, leading to complement-mediated intravascular hemolysis, nitric oxide scavenging by plasma ree hemoglobin, ormation o procoagulant microvesicles, and potential disorders o f brinolysis and tissue actor pathway inhibitor unction

Postpartum period

Venous stasis, increased coagulation actors, tissue trauma

Surgery/trauma

Venous stasis, immobilization, tissue injury

B O X 2 3 - 1 He m o rrh a g ic Dis o rd e rs When the vascular endothelium is injured, the hemostatic process ensures localized, stable clot ormation without obstruction o the vascular lumen. J ust as thrombosis constitutes a pathologic variation on this otherwise orchestrated physiologic process, disorders involving insu f cient levels o unctional platelets or coagulation actors can lead to a hypocoagulable state characterized clinically by episodes o uncontrolled hemorrhage. Hemorrhagic disorders result rom a multitude o causes, including disorders o the vasculature, vitamin K def ciency, and disorders or def ciencies o platelets, coagulation actors, and von Willebrand actor. Hemophilia A serves as an example o a hemorrhagic disorder in which hypocoagulability is the underlying pathology. Hemophilia A is the most common genetic disorder o serious bleeding. The hallmark o the disorder is a reduction in the amount or activity o coagulation actor VIII. The syndrome has an X-linked mode o transmission, and the majority o patients are males or homozygous emales. Thirty percent o patients have no amily history o hemophilia A and presumably represent spontaneous mutations. The severity o the disease depends on the type o mutation in the actor VIII gene. Patients with 6–50% o normal actor VIII activity mani est a mild orm o the disease; those with 2–5% activity

mani est moderate disease; patients with less than 1% activity develop severe disease. All symptomatic patients demonstrate easy bruisability and can develop massive hemorrhage a ter trauma or surgery. Spontaneous hemorrhage can occur in body areas that are normally subjected to minor trauma, including joint spaces, where spontaneous hemorrhage leads to the ormation o hemarthroses. Petechiae (microhemorrhages involving capillaries and small vessels, especially in mucocutaneous areas), which are usually an indication o platelet disorders, are absent in patients with hemophilia. Patients with hemophilia A are currently treated with in usions o actor VIII that is either recombinant or derived rom human plasma. Factor VIII in usion therapy is sometimes complicated in patients who develop antibodies against actor VIII. HIVin ection was a serious complication o in usion therapy in patients who received actor VIII products be ore the institution o routine screening o blood or HIVin ection (be ore the mid-1980s). Some sources suggest that the entire cohort o hemophiliacs who received actor VIII concentrates ( actor VIII concentrated rom the blood o many individuals) between 1981 and 1985 has been in ected with HIV. With current blood screening practices and the development o recombinant actor VIII, the risk o contracting HIVthrough actor VIII in usions is now virtually zero. ■

414 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Microparticles bearing tissue- actor protein have been demonstrated in the blood o healthy individuals, but no detectable tissue- actor activity is present in normal blood. This observation has led to the hypothesis that such microparticles contain inactive tissue actor (in “encrypted” orm) and that the tissue actor is activated only upon recruitment o the particles to a site o vascular injury (see above). In pathologic states, circulating microparticles may contain activated tissue actor, which could predispose to the development o thrombotic events. High levels o circulating microparticles have been implicated in the thrombosis associated with a variety o disorders, such as cancer-related thrombosis, atherothrombosis, and paroxysmal nocturnal hemoglobinuria.

PHARMACOLOGIC CLASSES AND AGENTS Multiple classes o drugs have been developed to prevent and/or reverse thrombus ormation. These classes consist o antiplatelet agents, anticoagulants, and thrombolytic agents. Hemostatic agents, discussed at the end o the chapter, are occasionally used to reverse the e ects o anticoagulants or to inhibit endogenous brinolysis.

Antiplatelet Agents As described above, ormation o a localized platelet plug in response to endothelial injury is the initial step in arterial thrombosis. There ore, inhibition o platelet unction is a use ul prophylactic and therapeutic strategy against myocardial in arction and stroke caused by thrombosis in coronary and cerebral arteries, respectively. The classes o antiplatelet agents in current clinical use include cyclooxygenase (COX) inhibitors, phosphodiesterase inhibitors, ADP receptor pathway inhibitors, GPIIb–IIIa antagonists, and thrombin receptor (PAR-1) antagonists.

Cyclooxygenase Inhibitors

Aspirin inhibits the synthesis o prostaglandins, thereby in-

hibiting the platelet granule release reaction and inter ering with normal platelet aggregation. The biochemistry o prostaglandin synthesis in platelets and endothelial cells provides a basis or understanding the mechanism o action o aspirin as an antiplatelet agent. Figure 23-12 depicts the prostaglandin synthesis pathway, which is discussed in more detail in Chapter 43, Pharmacology o Eicosanoids. Brief y, activation o both platelets and endothelial cells induces phospholipase A2 (PLA2) to cleave membrane phospholipids and release arachidonic acid. Arachidonic acid is then trans ormed into a cyclic endoperoxide (also known as prostaglandin G2 or PGG2) by the enzyme COX. In platelets, the cyclic endoperoxide is converted into thromboxane A2 (TxA2). Acting through cell sur ace TxA2 receptors, TxA2 causes localized vasoconstriction and is a potent inducer o platelet aggregation and the platelet granule release reaction. In endothelial cells, the cyclic endoperoxide is converted into prostacyclin (PGI2). PGI2, in turn, causes localized vasodilation and inhibits platelet aggregation and the platelet granule release reaction. Aspirin acts by covalently acetylating a serine residue near the active site o the COX enzyme, thereby inhibiting the synthesis o the cyclic endoperoxide and the various

metabolites o the cyclic endoperoxide. In the absence o TxA2, there is a marked decrease in platelet aggregation and the platelet granule release reaction (Fig. 23-13A). Because platelets do not contain DNA or RNA, these cells cannot regenerate new COX enzyme once aspirin has permanently inactivated all of the available COX enzyme. That is, the platelets become irreversibly “poisoned” or the li etime o these cells (7–10 days). Although aspirin also inhibits the COX enzyme in endothelial cells, its action is not permanent in endothelial cells because these cells are able to synthesize new COX molecules. Thus, the endothelial cell production o prostacyclin is relatively una ected by aspirin at pharmacologically low doses (see below). Aspirin is most o ten used as an antiplatelet agent to prevent arterial thrombosis leading to transient ischemic attack, stroke, and myocardial in arction. Because the action o aspirin on platelets is permanent, it is most e ective as a selective antiplatelet agent when taken in low doses and/or at infrequent intervals. For example, aspirin is o ten used as an antiplatelet agent at a dose o 81 mg once daily, while a typical anti-inf ammatory dose o this agent could be 650 mg three to our times daily. When taken at high doses, aspirin can inhibit prostacyclin production without increasing the e ectiveness o the drug as an antiplatelet agent. A more extended discussion o the uses and toxicities o aspirin is ound in Chapter 43. Compared with aspirin, other nonsteroidal anti-inf ammatory drugs (NSAIDs) are not as widely used in the prevention o arterial thrombosis because the inhibitory action o these drugs on cyclooxygenase is not permanent. COX-1 is the predominant COX iso orm in platelets, but endothelial cells express both COX-1 and COX-2 under physiologic conditions. Because aspirin inhibits COX-1 and COX-2 nonselectively, this drug serves as an e ective antiplatelet agent. In contrast, selective COX-2 inhibitors cannot be used as antiplatelet agents because they are poor inhibitors o COX-1. Furthermore, use o the selective COX-2 inhibitors appears to be associated with increased cardiovascular risk, which has resulted in the withdrawal o most o these agents rom the market (see Chapter 43).

Phosphodiesterase Inhibitors

In platelets, an increase in the concentration o intracellular cAMP leads to a decrease in platelet aggregability. Platelet cAMP levels are regulated physiologically by TxA2 and PGI2, among other mediators (see above). The mechanism by which increased intracellular cAMP concentration leads to decreased platelet aggregability is not well understood. cAMP activates protein kinase A, which, through incompletely elucidated mechanisms, decreases availability o the intracellular Ca2 necessary or platelet aggregation (Fig. 23-13B). Inhibitors o platelet phosphodiesterase decrease platelet aggregability by inhibiting cAMP degradation, while activators o platelet adenylyl cyclase decrease platelet aggregability by increasing cAMP synthesis. (There are currently no direct adenylyl cyclase activators in clinical use.) Dipyridamole is an inhibitor o platelet phosphodiesterase that decreases platelet aggregability (Fig. 23-13B). Dipyridamole by itsel has only weak antiplatelet e ects and is there ore usually administered in combination with war arin or aspirin. The combination o dipyridamole and war arin can be used to inhibit thrombus ormation on prosthetic heart valves, while the combination o dipyridamole and aspirin

C h a p t e r 2 3 Pharmacology o Hemostasis and Thrombosis

415

Me mbra ne phos pholipids

Phospholipase A2

NSAIDs (a s pirin, othe rs )

COOH

Ara chidonic a cid

Cyclooxygenase

O

Lipoxygenase

OOH

COOH

COOH

O OOH P ros ta gla ndin G 2

5-HP ETE

Peroxidase

Dehydrase

O

O

COOH

COOH

O OH

Le ukotrie ne A4

P ros ta gla ndin H2

COOH PGI2 synthase

GlutathioneS-transferase

OH

Prostaglandin synthases

O

COOH

H

H

OH

Othe r pros ta gla ndins

S

OH

HN

P ros ta cyclin (P GI2)

PGE2 synthase

TxA2 synthase

H2 N

O

COOH

O

COOH

O

Le ukotrie ne C 4

COOH

COOH

O

H N

O OH Thromboxa ne A2

HO

OH P ros ta gla ndin E 2

FIGURE 23-12. Overview of prostaglandin synthesis. Membrane phospholipids are cleaved by phospholipase A2 to release ree arachidonic acid. Arachidonic acid can be metabolized through either o two major pathways: the cyclooxygenase pathway or the lipoxygenase pathway. The cyclooxygenase pathway, which is inhibited by aspirin and other nonsteroidal anti-inf ammatory drugs (NSAIDs), converts arachidonic acid into prostaglandins and thromboxanes. Platelets express TxA2 synthase and synthesize the pro-aggregatory mediator thromboxane A2; endothelial cells express PGI2 synthase and synthesize the anti-aggregatory mediator prostacyclin. The lipoxygenase pathway converts arachidonic acid into leukotrienes, which are potent inf ammatory mediators. (See Chapter 43, Pharmacology o Eicosanoids, or a detailed discussion o the lipoxygenase and cyclooxygenase pathways.) Aspirin inhibits cyclooxygenase by covalent acetylation o the enzyme near its active site. Because platelets lack the capability to synthesize new proteins, aspirin inhibits thromboxane synthesis or the li e o the platelet.

can be used to reduce the likelihood of thrombosis in patients with a thrombotic diathesis. Dipyridamole also has vasodilatory properties. It may paradoxically induce angina in patients with coronary artery disease by causing the coronary artery steal, which involves intense dilation of coronary arterioles (see Chapter 22, Pharmacology of Vascular Tone).

ADP Receptor Pathway Inhibitors

Ticlopidine , clopidogrel, and prasugrel are derivatives of

thienopyridine. These agents, which irreversibly inhibit the ADP-dependent pathway of platelet activation, have antiplatelet effects in vitro and in vivo. Ticlopidine, clopidogrel, and prasugrel act by covalently modifying and inactivating

416 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY A Ara chidonic a cid NSAIDs (aspirin, others)

Cyclooxygenase

TXA2 (re le a s e d by a ctiva te d pla te le ts ) P LC

PIP2

TXA2 -R

β

αq

DAG

αq

γ

P KC

(a ctive )

GTP

GDP P KC IP 3 Ca 2+ Ca 2 + PLA2

GP

IIb -II Ia

Abciximab, eptifibatide, tirofiban

Fibrinoge n

Thrombin

Vorapaxar

B Ticlopidine, clopidogrel, prasugrel, ticagrelor

ADP

PAR-1

β P 2Y(ADP ) re ce ptor

ADP

Ade nylyl cycla s e

αi GDP

β

αi γ

αq

GTP

GDP

ATP

γ

P LC

P 2Y1 re ce ptor

αq GTP

cAMP

Dipyridamole

β αq γ GDP

PDE

PKA AMP

P la te le t a ctiva tion

P la te le t a ctiva tion

FIGURE 23-13. Mechanism of action of antiplatelet agents. A. NSAIDs and GPIIb–IIIa antagonists inhibit steps in thromboxane A2 (TxA2)-mediated platelet activation. Aspirin inhibits cyclooxygenase by covalent acetylation o the enzyme near its active site, leading to decreased TxA2 production. The e ect is pro ound because platelets lack the ability to synthesize new enzyme molecules. GPIIb–IIIa antagonists, such as the monoclonal antibody abciximab and the small-molecule antagonists eptif batide and tirof ban, inhibit platelet aggregation by preventing activation o GpIIb–IIIa (dashed line), leading to decreased platelet cross-linking by f brinogen. B. Ticlopidine, clopidogrel, prasugrel, ticagrelor, and dipyridamole inhibit steps in ADP-mediated platelet activation. Ticlopidine, clopidogrel, prasugrel, and ticagrelor are antagonists o the P2Y(ADP) receptor. Dipyridamole inhibits phosphodiesterase (PDE), thereby preventing the breakdown o cAMP and increasing cytoplasmic cAMP concentration. Vorapaxar inhibits PAR-1, thereby decreasing phospholipase C (PLC)-mediated platelet activation.

C h a p t e r 2 3 Pharmacology of Hemostasis and Thrombosis

the platelet P2Y(ADP) receptor (also called P2Y12), which is physiologically coupled to the inhibition o adenylyl cyclase (Fig. 23-13B). Ticlopidine, a f rst-generation thienopyridine, is a prodrug that requires conversion to active thiol metabolites in the liver. Maximal platelet inhibition is observed 8–11 days a ter initiating therapy with the drug; when used in combination with aspirin, 4–7 days are needed to achieve maximal platelet inhibition. Administration o a loading dose can produce a more rapid antiplatelet response. Ticlopidine is approved in the United States or two indications: (1) secondary prevention o thrombotic strokes in patients intolerant o aspirin, and (2) in combination with aspirin, prevention o stent thrombosis a ter placement o coronary artery stents. The use o ticlopidine has occasionally been associated with neutropenia, thrombocytopenia, and thrombotic thrombocytopenic purpura (TTP); or this reason, blood counts must be monitored requently when using ticlopidine. Ticlopidine has largely been replaced by clopidogrel because o the latter drug’s more avorable adverse e ect prof le and more rapid onset o action. Clopidogrel, a second-generation thienopyridine closely related to ticlopidine, has been used widely in combination with aspirin or improved platelet inhibition during and a ter percutaneous coronary intervention. Clopidogrel is a prodrug that must undergo oxidation by hepatic P450 enzymes to the active drug orm; it may there ore interact with statins, proton pump inhibitors, and other drugs metabolized by these P450 iso orms (see Chapter 4, Drug Metabolism). Clopidogrel is approved or secondary prevention in patients with recent myocardial in arction, stroke, or peripheral vascular disease. It is also approved or use in acute coronary syndromes that are treated with either percutaneous coronary intervention or coronary artery bypass gra ting. Like ticlopidine, clopidogrel requires a loading dose to achieve a maximal antiplatelet e ect rapidly. For this reason, Mr. S was given an oral loading dose o clopidogrel in the context o his myocardial in arction. The adverse e ect prof le o clopidogrel is more acceptable than that o ticlopidine: the gastrointestinal e ects o clopidogrel are similar to those o aspirin, and clopidogrel lacks the signif cant bone marrow toxicity associated with ticlopidine. Prasugrel, a third-generation thienopyridine, is an irreversible antagonist o the P2Y12 ADP receptor. This drug is approved or use in acute coronary syndromes treated with percutaneous coronary intervention. Like clopidogrel, prasugrel is a prodrug and is used in combination with aspirin. Prasugrel is more e f ciently metabolized than clopidogrel, resulting in higher concentrations o the active drug and more complete inhibition o the P2Y12 ADP receptor. Because o its more complete platelet inhibition, prasugrel may increase the risk o bleeding relative to clopidogrel, especially in patients over 75 years o age or weighing less than 60 kilograms. It is also contraindicated or use in patients with a prior history o stroke or transient ischemic attack because o an increased risk o intracranial hemorrhage. Ticagrelor is a competitive antagonist at the P2Y12 ADP receptor. Unlike the other agents in this class, ticagrelor is a cyclopentyltriazolopyrimidine. Ticagrelor is approved or use in patients with acute coronary syndromes. It is an orally active drug that does not require hepatic activation. Compared to other agents in its class, ticagrelor inhibits platelet activation more than clopidogrel but slightly less than prasugrel. Adverse e ects specif c to ticagrelor may include dyspnea and bradycardia. Similar to prasugrel, ticagrelor is

417

contraindicated or use in patients with a history o intracranial hemorrhage.

GPIIb–IIIa Antagonists

As noted above, platelet membrane GPIIb–IIIa receptors constitute the f nal common pathway o platelet aggregation, serving to bind f brinogen molecules that bridge platelets to one another. A variety o stimuli (e.g., TxA2, ADP, epinephrine, collagen, and thrombin), acting through diverse signaling pathways, are capable o inducing the expression o unctional GPIIb–IIIa on the platelet sur ace. It could thereore be predicted that antagonists o GPIIb–IIIa would serve as power ul inhibitors o platelet aggregation by preventing f brinogen binding to the GPIIb–IIIa receptor (Fig. 23-13A). Eptif batide , the GPIIb–IIIa receptor antagonist used in the opening case, is a highly e f cacious inhibitor o platelet aggregation. A synthetic peptide, eptif batide antagonizes the platelet GPIIb–IIIa receptor with high a f nity. This drug is used to reduce ischemic events in patients undergoing percutaneous coronary intervention and to treat unstable angina and non-ST elevation myocardial in arction. Abciximab is a chimeric mouse–human monoclonal antibody directed against the human GPIIb–IIIa receptor. Experiments in vitro have shown that occupation o 50% o platelet GPIIb–IIIa receptors by abciximab signif cantly reduces platelet aggregation. The binding o abciximab to GPIIb–IIIa is essentially irreversible, with a dissociation hal -time o 18–24 hours. In clinical trials, adding abciximab to conventional antithrombotic therapy reduces both longterm and short-term ischemic events in patients undergoing high-risk percutaneous coronary intervention. Tirof ban is a nonpeptide tyrosine analogue that reversibly antagonizes f brinogen binding to the platelet GPIIb–IIIa receptor. Both in vitro and in vivo studies have demonstrated the ability o tirof ban to inhibit platelet aggregation. Tirof ban has been approved or use in patients with acute coronary syndromes. Because o their mechanism o action as antiplatelet agents, all o the GPIIb–IIIa antagonists can cause bleeding as an adverse e ect. In the opening case, Mr. S developed a hematoma in his right thigh near the arterial access site. The expanding hematoma was caused by the excessive antiplatelet e ect o eptif batide. Importantly, the ability to reverse the e ect o GPIIb–IIIa receptor antagonists di ers or the di erent agents. Because abciximab is an irreversible inhibitor o platelet unction, and all the abciximab previously inused is already bound to platelets, in usion o resh platelets a ter the drug has been stopped can reverse the antiplatelet e ect. In contrast, because the two small-molecule antagonists (eptif batide and tirof ban) bind the receptor reversibly and are in used in great stoichiometric excess o receptor number, in usion o resh platelets simply o ers new sites to which the drug can bind, and it is not practical to deliver a su f cient number o platelets to overwhelm the vast excess o drug present. There ore, one must stop the drug in usion and wait or platelet unction to return to normal as the drug is cleared. In the case o Mr. S, no other measure could have been taken to reverse the e ect o eptif batide at the time his hematoma was recognized.

Thrombin Receptor (PAR-1) Antagonists

Vorapaxar is a reversible antagonist o protease-activated

receptor 1 (PAR-1), one o two major thrombin receptors

418 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

expressed on platelets (Fig. 23-13B). Inhibition o PAR-1 inhibits platelet activation with minimal e ect on the coagulation cascade. Vorapaxar is approved or secondary prevention o myocardial in arction, death, and stroke in patients with a prior myocardial in arction or with peripheral artery disease. It is orally available and metabolized by hepatic CYP3A4 enzymes, with primarily hepatic clearance. Vorapaxar should not be administered to patients with a prior history o stroke, transient ischemic attack, or intracranial hemorrhage.

Anticoagulants As with antiplatelet agents, anticoagulants are used both to prevent and treat thrombotic disease. There are our classes o anticoagulants: war arin, un ractionated and low-molecularweight heparins, selective actor Xa inhibitors, and direct thrombin inhibitors. Anticoagulants target various actors in the coagulation cascade, thereby interrupting the cascade and preventing the ormation o a stable f brin meshwork (secondary hemostatic plug). In this section, the our classes o anticoagulants are discussed in order o selectivity, rom the least selective agents (war arin and un ractionated heparin) to the most selective agents (selective actor Xa inhibitors and direct thrombin inhibitors). Recombinant activated protein C also has anticoagulant activity, although its clinical indication is severe sepsis. Because o the mechanisms o action o these drugs, bleeding is an adverse e ect common to all anticoagulants.

Warfarin

In the early 1900s, armers in Canada and the North Dakota plains adopted the practice o planting sweet clover instead o corn or odder. In the winter months o 1921 to 1922, a atal hemorrhagic disease was reported in cattle that had oraged on the sweet clover. In almost every case, it was ound that the a ected cattle had oraged on sweet clover that had been spoiled by the curing process. A ter an intensive investigation, scientist K. P. Link reported that the spoiled clover contained the natural anticoagulant 3,3 -methylenebis-(4-hydroxycoumarin) or dicumarol. Dicumarol and warfarin (a potent synthetic congener) were introduced during the 1940s as rodenticides and as oral anticoagulants. Because the oral anticoagulants act by a ecting vitamin K-dependent reactions, it is important to understand how vitamin K unctions. Mechanism of Action of Vitamin K

Vitamin K (“K” is derived rom the German word “Koagulation”) is required or the normal hepatic synthesis o our coagulation actors (II, VII, IX, and X), protein C, and protein S. The coagulation actors, protein C, and protein S are biologically inactive as unmodif ed polypeptides ollowing protein synthesis on ribosomes. These proteins gain biological activity by post-translational carboxylation o their 9 to 12 amino-terminal glutamic acid residues. The -carboxylated glutamate residues (but not the unmodif ed glutamate residues) are capable o binding Ca2 ions. Ca2 binding induces a con ormational change in these proteins that is required or e f cient binding o the proteins to phospholipid sur aces. The binding o Ca2 to the -carboxylated molecules increases the enzymatic activity o coagulation actors IIa, VIIa, IXa, Xa, and protein Ca by approximately 1,000- old. Thus, vitamin K-dependent carboxylation is crucial or the enzymatic activity o the our coagulation actors and protein C and or the co actor unction o protein S.

The carboxylation reaction requires (1) a precursor orm o the target protein with its 9 to 12 amino-terminal glutamic acid residues, (2) carbon dioxide, (3) molecular oxygen, and (4) reduced vitamin K. The carboxylation reaction is schematically presented in Figure 23-14. During this reaction, vitamin K is oxidized to the inactive 2,3-epoxide. An enzyme, vitamin K epoxide reductase (also called VKORC1), is then required to convert the inactive 2,3-epoxide into the active, reduced orm o vitamin K. Thus, the regeneration of reduced vitamin K is essential for the sustained synthesis of biologically functional clotting factors II, VII, IX, and X, all of which are critical components of the coagulation cascade. Mechanism of Action of Warfarin

War arin acts on the carboxylation pathway, not by inhibiting the carboxylase directly but by blocking the epoxide reductase that mediates the regeneration o reduced vitamin K (Fig. 23-14). Because depletion o reduced vitamin K in the liver prevents the -carboxylation reaction that is required or the synthesis o biologically active coagulation actors, the onset o action o the oral anticoagulants parallels the hal -li e o these coagulation actors in the circulation. O the our a ected clotting actors (II, VII, IX, and X), actor VII has the shortest hal -li e (6 hours). Thus, the pharmacologic e ect o a single dose o war arin is not mani ested or approximately 18–24 hours (i.e., 3–4 actor VII hal -lives). This delayed action is one pharmacologic property that distinguishes the war arin class o anticoagulants rom all the other classes o anticoagulants. Evidence rom studies o long-term rodenticide use and o anticoagulant use supports the hypothesis that the epoxide reductase is the molecular target o oral anticoagulant action. The use o oral anticoagulants as rodenticides has been a widespread practice in arming communities. In some areas o the United States, heavy rodenticide use has selected or a population o wild rodents that is resistant to 4-hydroxycoumarins. In vitro studies o tissues rom these rodents have demonstrated a mutation in the rodent epoxide reductase that renders the enzyme resistant to inhibition by the anticoagulant. Similarly, a small population o patients is genetically resistant to war arin because o mutations in their epoxide reductase gene. These patients require 10–20 times the usual dose o war arin to achieve the desired anticoagulant e ect. More generally, genetic variation in the VKORC1 gene has been associated with approximately 25–30% o the variance in war arin maintenance dose in patients taking this drug (see Chapter 7, Pharmacogenomics). Clinical Uses of Warfarin

War arin is o ten administered to complete a course o anticoagulation that has been initiated with heparin (see below) and to prevent thrombosis in predisposed patients. Orally administered war arin is nearly 100% bioavailable, and its levels in the blood peak at 0.5–4 hours a ter administration. In the plasma, 99% of racemic warfarin is bound to plasma protein (albumin). War arin has a relatively long elimination hal -li e (approximately 36 hours). The drug is hydroxylated by the cytochrome P450 system in the liver to inactive metabolites that are subsequently eliminated in the urine (see Table 4-3). Drug–drug interactions must be carefully considered in patients taking warfarin. Because war arin is highly albumin-bound in the plasma, co-administration o war arin with other albumin-bound drugs can increase the ree

C h a p t e r 2 3 Pharmacology of Hemostasis and Thrombosis

FIGURE 23-14. Mechanism of action of warfarin. Vitamin K

O H N

O H N

N H COOH

N H COOH

COOH

γ-Ca rboxygluta ma te re s idue in

Gluta ma te re s idue in coa gula tion fa ctor

coa gula tion fa ctor

CO 2 Vitamin K-dependent carboxylase

is a necessary cofactor in the post-translational carboxylation of glutamate residues on factors II, VII, IX, and X. During the carboxylation reaction, vitamin Kis oxidized to the inactive 2,3-epoxide. The enzyme vitamin K epoxide reductase (also called VKORC1) converts the inactive vitamin K 2,3-epoxide into the active, reduced form of vitamin K. The regeneration of reduced vitamin K is essential for the sustained synthesis of biologically functional coagulation factors II, VII, IX, and X. Warfarin acts on the carboxylation pathway by inhibiting the epoxide reductase that is required for the regeneration of reduced (active) vitamin K. Dicumarol is the natural anticoagulant formed in spoiled clover. Both warfarin and dicumarol are orally bioavailable.

O2 O

OH R

R O

OH

O

Vita min K-re duce d (a ctive form)

Vita min K 2,3-e poxide (ina ctive form)

Epoxide reductase

NAD+

NADH Warfarin

OH

O

O O Dicumarol

O

OH

OH

O

O

O

Warfarin

(unbound) plasma concentrations of both drugs. In addition, because warfarin is metabolized by P450 enzymes in the liver, co-administration of warfarin with drugs that induce and/or compete for P450 metabolism can affect the plasma concentrations of both drugs. Tables 23-2 and 23-3 list some of the major interactions between warfarin and other drugs. Among the adverse effects of warfarin, bleeding is the most serious and predictable toxicity. Withdrawal of the drug may be recommended for patients who suffer from repeated bleeding episodes at otherwise therapeutic drug concentrations. For severe hemorrhage, patients should promptly receive fresh frozen plasma, which contains biologically functional clotting factors II, VII, IX, and X. Warfarin should never be administered to pregnant women because it can cross the placenta and cause a hemorrhagic disorder in

419

TABLE 23-2 Examples of Drugs That Diminish Warfarin’s Anticoagulant Effect DRUG OR DRUG CLASS

MECHANISM

Cholestyramine

Inhibits warfarin absorption in the gastrointestinal tract

Barbiturates, carbamazepine, phenytoin, rifampin

Accelerate warfarin metabolism by inducing hepatic P450 enzymes (especially P450 2C9)

Vitamin K (reduced)

Bypasses warfarin’s inhibition of epoxide reductase

420 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

TABLE 23-3 Examples of Drugs That Enhance Warfarin’s Anticoagulant Effect DRUG OR DRUG CLASS

MECHANISM

Chloral hydrate

Displaces war arin rom plasma albumin

Amiodarone, clopidogrel, ethanol (intoxicating dose), f uconazole, f uoxetine, metronidazole, sul amethoxazole

Decrease war arin metabolism by inhibiting hepatic P450 enzymes (especially P450 2C9)

Broad-spectrum antibiotics

Eliminate gut bacteria and thereby reduce availability o vitamin K in the gastrointestinal tract

Anabolic steroids (testosterone)

Inhibit synthesis and increase degradation o coagulation actors

heparin is the strongest organic acid in the human body. Commercial preparations o heparin are quite heterogeneous, with molecular weights ranging rom 1 to 30 kDa. Conventionally, commercially prepared heparins have been categorized into un ractionated (standard) heparin and low-molecular-weight (LMW) heparin. Unfractionated heparin, which is o ten prepared rom bovine lung and porcine intestinal mucosa, ranges in molecular weight rom 5 to 30 kDa. LMW heparins are prepared rom standard heparin by gel-f ltration chromatography; their molecular weights range rom 1 to 5 kDa. Mechanism of Action of Heparin

the etus. In addition, newborns exposed to war arin in utero may have serious congenital de ects characterized by abnormal bone ormation (note that certain bone matrix proteins are -carboxylated). Rarely, war arin causes skin necrosis as a result o widespread thrombosis in the microvasculature. The act that war arin can cause thrombosis may seem paradoxical. Recall that, in addition to inhibiting the synthesis o biologically active coagulation actors II, VII, IX, and X, war arin also prevents the synthesis o biologically active proteins C and S, which are natural anticoagulants. In patients who are genetically def cient in protein C or protein S (most commonly, patients who are heterozygous or protein C def ciency), an imbalance between war arin’s e ects on coagulation actors and its e ects on proteins C and S may lead to microvascular thrombosis and skin necrosis. Because war arin has a narrow therapeutic index and participates in numerous drug–drug interactions, the pharmacodynamic ( unctional) e ect o chronic war arin therapy must be monitored regularly (on the order o every 2–4 weeks). Monitoring is most easily per ormed using the prothrombin time (PT), which is a simple test o the extrinsic and common pathways o coagulation. In this test, the patient’s plasma is added to a crude preparation o tissue actor (called thromboplastin), and the time or ormation o a f brin clot is measured. War arin prolongs the PT mainly because it decreases the amount o biologically unctional actor VII in the plasma. (Recall that actor VII is the vitamin K-dependent coagulation actor with the shortest hal -li e.) Measurement o the PT has been standardized worldwide and is expressed as the international normalized ratio (INR) o the prothrombin time in the patient sample to that in a control sample, normalized or the international sensitivity index (ISI) o the laboratory’s thromboplastin preparation compared to the World Health Organization’s re erence thromboplastin preparation. The ormula used to calculate the INR is as ollows: INR [PTpatient / PTcontrol]ISI.

Unfractionated and Low-Molecular-Weight Heparins Structure of Heparin Heparin is a sul ated mucopolysaccharide stored in the se-

cretory granules o mast cells. It is a highly sul ated polymer o alternating uronic acid and D-glucosamine. Heparin molecules are highly negatively charged; indeed, endogenous

Heparin’s mechanism o action depends on the presence o a specif c plasma protease inhibitor, antithrombin III (Fig. 23-9). Antithrombin III is actually a misnomer because, in addition to inactivating thrombin, antithrombin III inactivates other serine proteases including actors IXa, Xa, XIa, and XIIa. Antithrombin III can be considered as a stoichiometric “suicide trap” or these serine proteases. When one o the proteases encounters an antithrombin III molecule, the serine residue at the active site o the protease attacks a specif c Arg–Ser peptide bond in the reactive site o the antithrombin. The result o this nucleophilic attack is the ormation o a covalent ester bond between the serine residue on the protease and the arginine residue on the antithrombin III. This results in a stable 1:1 complex between the protease and antithrombin molecules, which prevents the protease rom urther participation in the coagulation cascade. In the absence o heparin, the binding reaction between the proteases and antithrombin III proceeds slowly. Heparin, acting as a co actor, accelerates the reaction by 1,000- old. Heparin has two important physiologic unctions: (1) it serves as a catalytic sur ace to which both antithrombin III and the serine proteases bind and (2) it induces a con ormational change in antithrombin III that makes the reactive site o this molecule more accessible to the attacking protease. The f rst step o the reaction involves the binding o the negatively charged heparin to a lysine-rich region (a region o positive charge) on antithrombin III. Thus, the interaction between heparin and antithrombin III is partly electrostatic. During the conjugation reaction between the protease and the antithrombin, heparin may be released rom antithrombin III and become available to catalyze additional protease– antithrombin III interactions (i.e., heparin is not consumed by the conjugation reaction). In practice, however, heparin’s high negative charge o ten causes this “sticky” molecule to remain electrostatically bound to protease, antithrombin, or another nearby molecule in the vicinity o a thrombus. Interestingly, heparins o di erent molecular weights have divergent anticoagulant activities. These divergent activities derive rom the di erential requirements or heparin binding exhibited by the inactivation o thrombin and actor Xa by antithrombin III (Fig. 23-15). To catalyze most e f ciently the inactivation o thrombin by antithrombin III, a single molecule o heparin must bind simultaneously to both thrombin and antithrombin. This “sca olding” unction is required in addition to the heparin-induced con ormational change in antithrombin III that renders the antithrombin susceptible to conjugation with thrombin. In contrast, to catalyze the inactivation o actor Xa by antithrombin III, the heparin molecule must bind only to the antithrombin, because the con ormational change in antithrombin III induced by heparin binding is su f cient by itsel to render the antithrombin susceptible

C h a p t e r 2 3 Pharmacology o Hemostasis and Thrombosis Anticoagulant class

Effect on Thrombin

421

Effect on Factor Xa

Unfra ctiona te d he pa rin (a bout 45 s a ccha ride units , MW ~ 13,500)

Xa

Thrombin

ATIII

ATIII He pa rin

He pa rin

Binds to a ntithrombin III (ATIII) a nd thrombin (ina ctiva te s thrombin)

Binds to a ntithrombin III (ATIII) via pe nta s a ccha ride (s ufficie nt to ina ctiva te Xa )

Low mole cula r we ight (LMW) he pa rins (a bout 15 s a ccha ride units , MW ~ 4,500)

Xa

Thrombin ATIII

ATIII

LMWH

Binds to a ntithrombin III (ATIII) but not to thrombin (poorly ina ctiva te s thrombin)

LMWH

Binds to a ntithrombin III (ATIII) via pe nta s a ccha ride (s ufficie nt to ina ctiva te Xa )

Xa

S e le ctive fa ctor Xa inhibitors

ATIII

No e ffe ct on thrombin

Fondaparinux Binds to a ntithrombin III (ATIII) via pe nta s a ccha ride (s ufficie nt to ina ctiva te Xa )

Xa

Rivaro Rivaroxaban, apixaban apixab S e le ctive ly ina ctiva te Xa S ubs tra te re cognition s ite (Exos ite ) Thrombin

Dire ct thrombin inhibitors

He pa rinbinding s ite

Ca ta lytic s ite (Active s ite ) No e ffe ct on Xa Argatroban, dabigatran

Thrombin

Lepirudin, desirudin, bivalirudin Thrombin

S e le ctive ly ina ctiva te thrombin

FIGURE 23-15. Differential effects of unfractionated heparin, low-molecular-weight heparins, selective factor Xa inhibitors, and direct thrombin inhibitors on coagulation factor inactivation. Effect on thrombin: To catalyze the inactivation o thrombin, heparin must bind both to antithrombin III via a higha f nity pentasaccharide unit and to thrombin via an additional 13-saccharide unit. Low-molecular-weight heparin (LMWH) does not contain a su f cient number o saccharide units to bind thrombin and there ore is a poor catalyst or thrombin inactivation. Selective actor Xa inhibitors do not inactivate thrombin, while direct thrombin inhibitors selectively inactivate thrombin. Argatroban and dabigatran bind only to the active (catalytic) site o thrombin, while lepirudin, desirudin, and bivalirudin bind to both the active site and the substrate-recognition site o thrombin. Effect on factor Xa: Inactivation o actor Xa requires only the binding o antithrombin III to the high-a f nity pentasaccharide unit. Since un ractionated heparin, low-molecular-weight heparin, and ondaparinux all contain this pentasaccharide, these agents are all able to catalyze the inactivation o actor Xa. Rivaroxaban and apixaban competitively inhibit actor Xa by binding to the active site o the enzyme; these agents bind actor Xa that is complexed with actor Va and Ca 2 on phospholipid sur aces, as shown in Figure 23-7. Direct thrombin inhibitors have no e ect on actor Xa.

422 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

to conjugation with actor Xa. Thus, LMW heparins, which have an average molecular weight o 3–4 kDa and contain ewer than 18 monosaccharide units, e f ciently catalyze the inactivation o actor Xa by antithrombin III but less e f ciently catalyze the inactivation o thrombin by antithrombin III. In contrast, un ractionated heparin, which has an average molecular weight o 20 kDa and contains more than 18 monosaccharide units, is o su f cient length to bind simultaneously to thrombin and antithrombin III and there ore e f ciently catalyzes the inactivation o both thrombin and actor Xa by antithrombin III. Quantitatively, LMW heparin has a three old higher ratio o anti-Xa to anti-thrombin (anti-IIa) activity than does un ractionated heparin. LMW heparin is there ore a more selective therapeutic agent than un ractionated heparin. Both LMW heparin and un ractionated heparin use a pentasaccharide structure o high negative charge to bind antithrombin III and to induce the con ormational change in antithrombin required or the conjugation reactions. This pentasaccharide is approved or use as a highly selective inhibitor o actor Xa (fondaparinux; see below). Clinical Uses of Heparins

Heparins are used or both prophylaxis and treatment o thromboembolic diseases. Both un ractionated and LMW heparins are used to prevent propagation o established thromboembolic disease such as deep vein thrombosis and pulmonary embolism. For prophylaxis against thrombosis, heparins are used at much lower doses than those indicated or the treatment o established thromboembolic disease. Because the enzymatic coagulation cascade unctions as an amplif cation system (as noted above, 1 unit o actor Xa generates 40 units o thrombin), the administration o relatively small amounts o circulating heparin at the f rst generation o actor Xa is highly e ective. Heparins are highly negatively charged, and neither un ractionated heparin nor LMW heparin can cross the epithelial cell layer o the gastrointestinal tract. Hence, heparin must be administered parenterally, usually via intravenous or subcutaneous routes. Unfractionated heparin is o ten used in combination with antiplatelet agents in the treatment o acute coronary syndromes. For example, Mr. S was treated with the antiplatelet agents aspirin and eptif batide and with un ractionated heparin in an attempt to limit the extent o his myocardial in arction. Monitoring o un ractionated heparin therapy is important or maintaining the anticoagulant e ect within the therapeutic range because excessive heparin administration signif cantly increases the risk o bleeding. Monitoring is usually per ormed using the activated partial thromboplastin time (aPTT) assay. The aPTT is a simple test o the intrinsic and common pathways o coagulation. The patient’s plasma is added to an excess o phospholipid, and f brin orms at a normal rate only i the actors in the intrinsic and common pathways are present at normal levels. Increasing amounts o un ractionated heparin in the plasma prolong the time required or the ormation o a f brin clot. As is true o the other anticoagulants, the major adverse e ect o heparin is bleeding. Thus, it is critical to maintain the anticoagulant e ect o un ractionated heparin within the therapeutic range in order to prevent the rare, devastating adverse e ect o intracranial hemorrhage. As discussed above, a small raction o patients taking heparin develop heparin-induced thrombocytopenia (HIT). In this syndrome, patients develop antibodies to a hapten created when heparin molecules bind

to the platelet sur ace. In HIT type 1, the antibody-coated platelets are targeted or removal rom the circulation, and the platelet count decreases by 50–75% approximately 5 days into the course o heparin therapy. The thrombocytopenia in HIT type 1 is transient and rapidly reversible upon heparin withdrawal. In HIT type 2, however, the heparin-induced antibodies not only target the platelets or destruction but also act as agonists to activate the platelets, leading to platelet aggregation, endothelial injury, and potentially atal thrombosis. There is a higher incidence o HIT in patients receiving unractionated heparin than in those receiving LMW heparin. The LMW heparins enoxaparin, dalteparin, and tinzaparin are each ractionated heparins o low molecular weight. As discussed above, these agents are relatively selective or anti-Xa compared to anti-IIa (anti-thrombin) activity. All LMW heparins are approved or use in the prevention and treatment o deep vein thrombosis. Additionally, enoxaparin and dalteparin have been studied in the treatment o acute myocardial in arction and as adjuncts to percutaneous coronary intervention. LMW heparins have a higher therapeutic index than un ractionated heparin, especially when used or prophylaxis. For this reason, it is generally not necessary to monitor blood activity levels o LMW heparins. Accurate measurement o the anticoagulant e ect o LMW heparins requires a specialized assay or anti- actor Xa activity. Because LMW heparins are excreted via the kidneys, care should be taken to avoid excessive anticoagulation in patients with renal insu f ciency.

Selective Factor Xa Inhibitors

Fondaparinux is a synthetic pentasaccharide that contains the

sequence o f ve essential carbohydrates necessary or binding to antithrombin III and inducing the con ormational change in antithrombin required or conjugation to actor Xa (Fig. 23-15; see above). This agent is there ore a specif c, indirect inhibitor o actor Xa, with negligible anti-IIa (anti-thrombin) activity. Fondaparinux is approved or prevention and treatment o deep vein thrombosis and is available as a once-daily subcutaneous injection. It is excreted via the kidneys and should not be administered to patients with renal insu f ciency. Rivaroxaban and apixaban directly inhibit actor Xa by binding to its active site and competitively inhibiting the enzyme (Fig. 23-15). Both o these agents bind actor Xa that is complexed with actor Va and calcium ions on phospholipid sur aces. As a result, these agents do not require antithrombin III as a co actor or inhibition o actor Xa. Both rivaroxaban and apixaban are administered orally. Both agents are approved or prevention o stroke in patients with non-valvular atrial f brillation (i.e., atrial f brillation not related to mitral stenosis or a prosthetic heart valve) and or prevention o deep vein thrombosis a ter hip or knee orthopedic surgery. Additionally, rivaroxaban is approved at a higher initial dose or treatment o deep vein thrombosis or pulmonary embolism. Rivaroxaban and apixaban are cleared by both metabolism in the liver and excretion in the kidney and should be administered at reduced doses in patients with renal impairment.

Direct Thrombin Inhibitors

As discussed above, thrombin has a number o critical roles in the hemostatic process (Fig. 23-8). Among other e ects, this clotting actor (1) proteolytically converts f brinogen to f brin; (2) activates actor XIII, which cross-links f brin polymers to

C h a p t e r 2 3 Pharmacology of Hemostasis and Thrombosis

orm a stable clot; (3) activates platelets; and (4) induces endothelial release o PGI2, t-PA, and PAI-1. Thus, direct thrombin inhibitors would be expected to have pro ound e ects on coagulation. The currently approved direct thrombin inhibitors include lepirudin, desirudin, bivalirudin, argatroban, and dabigatran. These agents are speci c inhibitors o thrombin, with negligible anti- actor Xa activity (Fig. 23-15). Lepirudin, a recombinant 65-amino-acid polypeptide derived rom the medicinal leech protein hirudin, is the prototypical direct thrombin inhibitor. For years, surgeons have used medicinal leeches to prevent thrombosis in the ne vessels o reattached digits. Lepirudin binds with high a nity to two sites on the thrombin molecule—the enzymatic active site and the “exosite,” a region o the thrombin protein that orients substrate proteins. Lepirudin binding to thrombin prevents the thrombin-mediated activation o brinogen and actor XIII. Lepirudin is a highly e ective anticoagulant because it can inhibit both ree and f brin-bound thrombin in developing clots and because lepirudin binding to thrombin is essentially irreversible. It is approved or use in the treatment o heparin-induced thrombocytopenia. Lepirudin has a short hal -li e, is available parenterally, and is renally excreted. It can be administered with relative sa ety to patients with hepatic insu ciency. As with all direct thrombin inhibitors, bleeding is the major adverse e ect o lepirudin, and clotting times must be monitored closely. A small percentage o patients may develop anti-hirudin antibodies, limiting the long-term e ectiveness o this agent as an anticoagulant. Another recombinant ormulation o hirudin, desirudin, has been approved or prophylaxis against deep vein thrombosis in patients undergoing hip replacement. Bivalirudin is a synthetic 20-amino-acid peptide that, like lepirudin and desirudin, binds to both the active site and exosite o thrombin and thereby inhibits thrombin activity. Thrombin slowly cleaves an arginine–proline bond in bivalirudin, leading to reactivation o the thrombin. Bivalirudin is approved or anticoagulation in patients undergoing coronary angiography and angioplasty and may reduce rates o bleeding relative to heparin or this indication. The drug is excreted renally and has a short hal -li e (25 minutes). Argatroban is a small-molecule inhibitor o thrombin that is approved or the treatment o patients with heparin-induced thrombocytopenia. Unlike the polypeptide-based direct thrombin inhibitors (i.e., lepirudin, desirudin, and bivalirudin), argatroban binds only to the active site o thrombin (i.e., it does not interact with the exosite). Also unlike the polypeptide-based direct thrombin inhibitors, argatroban is excreted by biliary secretion and can there ore be administered with relative sa ety to patients with renal insu ciency. Argatroban has a short hal li e and is administered by continuous intravenous in usion. Dabigatran is an orally available direct thrombin inhibitor that is approved or prevention o thromboembolism in patients with non-valvular atrial brillation. It is also indicated or the treatment o established deep vein thrombosis and pulmonary embolism. Dabigatran is a prodrug that is metabolized to an active species that, like argatroban, binds competitively to the active site o thrombin. Like other anticoagulants, dabigatran may cause signi cant bleeding. One advantage relative to war arin is that plasma levels o dabigatran do not need to be monitored. Dabigatran should not be administered as an alternative to war arin in patients with a mechanical heart valve, because such patients have been ound to have an increased risk o valve thrombosis.

423

Recombinant Activated Protein C (r-APC)

As described above, endogenously activated protein C (APC) exerts an anticoagulant e ect by proteolytically cleaving actors Va and VIIIa. APC also reduces the amount o circulating plasminogen activator inhibitor 1, thereby enhancing brinolysis. Finally, APC reduces inf ammation by inhibiting the release o tumor necrosis actor (TNF- ) by monocytes. Because enhanced coagulability and inf ammation are both hallmarks o septic shock, APC has been tested both in animal models o this disorder and in humans. Recombinant activated protein C (r-APC) has been ound to signi cantly reduce mortality in patients at high risk o death rom septic shock, and the US Food and Drug Administration (FDA) has approved r-APC or the treatment o patients with severe sepsis who demonstrate evidence o acute organ dys unction, shock, oliguria, acidosis, and hypoxemia. r-APC is not indicated or the treatment o patients with severe sepsis and a lower risk o death, however. As is the case with other anticoagulants, r-APC increases the risk o bleeding. This agent is there ore contraindicated in patients who have recently undergone a surgical procedure and in those with chronic liver ailure, kidney ailure, or thrombocytopenia.

Thrombolytic Agents Although war arin, un ractionated and low-molecular-weight heparins, selective actor Xa inhibitors, and direct thrombin inhibitors are e ective in preventing the ormation and propagation o thrombi, these agents are generally ine ective against preexisting clots. Thrombolytic agents are used to lyse already- ormed clots and thereby to restore the patency o an obstructed vessel be ore distal tissue necrosis occurs. Thrombolytic agents act by converting the inactive zymogen plasminogen to the active protease plasmin (Fig. 23-10). As noted above, plasmin is a relatively nonspeci c protease that digests brin to brin degradation products. Un ortunately, thrombolytic therapy has the potential to dissolve not only pathologic thrombi but also physiologically appropriate brin clots that have ormed in response to vascular injury (systemic brinolysis). Thus, the use o thrombolytic agents can lead to hemorrhage o varying severity.

Streptokinase

Streptokinase is a protein produced by -hemolytic strepto-

cocci as a component o that organism’s tissue-destroying machinery. The pharmacologic action o streptokinase involves two steps—complexation and cleavage. In the complexation reaction, streptokinase orms a stable, noncovalent 1:1 complex with plasminogen. The complexation reaction produces a con ormational change in plasminogen that exposes this protein’s proteolytically active site. Streptokinasecomplexed plasminogen, with its active site exposed and available, can then proteolytically cleave other plasminogen molecules to plasmin. In act, the thermodynamically stable streptokinase:plasminogen complex is the most catalytically e cient plasminogen activator in vitro. Although streptokinase exerts its most dramatic and potentially bene cial e ects in resh thrombi, its use has been limited by two actors. First, streptokinase is a oreign protein that is capable o eliciting antigenic responses in humans upon repeated administration. Previous administration o streptokinase is a contraindication to its use because o the risk o anaphylaxis. Second, the thrombolytic actions o streptokinase are relatively nonspeci c and can result in

424 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

systemic brinolysis. Streptokinase is approved or treatment o ST elevation myocardial in arction and or treatment o li e-threatening pulmonary embolism.

Recombinant Tissue Plasminogen Activator (t-PA)

An ideal thrombolytic agent would be nonantigenic and would cause local brinolysis only at the site o a pathologic thrombus. Tissue plasminogen activator (t-PA) approximates these goals. t-PA is a serine protease produced by human endothelial cells; there ore, t-PA is not antigenic. t-PA binds to newly ormed ( resh) thrombi with high a nity, causing brinolysis at the site o a thrombus. Once bound to the resh thrombus, t-PA undergoes a con ormational change that renders it a potent activator o plasminogen. In contrast, t-PA is a poor activator o plasminogen in the absence o brin-binding. Recombinant DNA technology has allowed the production o recombinant t-PA, generically re erred to as alteplase . Recombinant t-PA is e ective at recanalizing occluded coronary arteries, limiting cardiac dys unction, and reducing mortality ollowing an ST elevation myocardial in arction. At pharmacologic doses, however, recombinant t-PA can generate a systemic lytic state and (as with other thrombolytic agents) cause unwanted bleeding, including cerebral hemorrhage. Thus, its use is contraindicated in patients who have had a recent hemorrhagic stroke. Like streptokinase, t-PA is approved or use in the treatment o patients with ST elevation myocardial in arction or li e-threatening pulmonary embolism. It is also approved or the treatment o acute ischemic stroke.

Tenecteplase

Tenecteplase is a genetically engineered variant o t-PA.

The molecular modi cations in tenecteplase increase its brin speci city relative to t-PA and make tenecteplase more resistant to plasminogen activator inhibitor 1. Large trials have shown that tenecteplase is identical in e cacy to t-PA, with similar (and possibly decreased) risk o bleeding. Additionally, tenecteplase has a longer hal -li e than t-PA. This pharmacokinetic property allows tenecteplase to be administered as a single weight-based bolus, thus simpli ying administration.

Reteplase

Similar to tenecteplase, reteplase is a genetically engineered variant o t-PA with longer hal -li e and increased speci city or brin. Its e cacy and adverse-e ect pro le are similar to those o streptokinase and t-PA. Because o its longer hal li e, reteplase can be administered as a “double bolus” (two boluses, 30 minutes apart).

Inhibitors of Anticoagulation and Fibrinolysis Protamine

Protamine , a low-molecular-weight polycationic protein, is

a chemical antagonist o heparin. This agent rapidly orms a stable complex with the negatively charged heparin molecule through multiple electrostatic interactions. Protamine is administered intravenously to reverse the e ects o heparin in situations o li e-threatening hemorrhage or great heparin excess (e.g., at the conclusion o coronary artery bypass gra t surgery). Protamine is most active against the large heparin molecules in un ractionated heparin and it can partially reverse the anticoagulant e ects o low-molecularweight heparins, but it is inactive against ondaparinux.

Serine-Protease Inhibitors

Aprotinin, a naturally occurring polypeptide, is an inhibi-

tor o the serine proteases plasmin, t-PA, and thrombin. By inhibiting brinolysis, aprotinin promotes clot stabilization. Inhibition o thrombin may also promote platelet activity by preventing platelet hyperstimulation. At higher doses, aprotinin may also inhibit kallikrein and thereby (paradoxically) inhibit the coagulation cascade. Although clinical trials demonstrated decreased perioperative bleeding and erythrocyte trans usion requirement in patients treated with aprotinin during cardiac surgery, these positive ndings were tempered by evidence suggesting that, compared to other anti brinolytic agents, aprotinin may increase the risk o postoperative acute renal ailure. Aprotinin has also been associated with atal anaphylactic reactions. Aprotinin was removed rom the US market in 2008 because o an increased risk o mortality.

Lysine Analogues

Aminocaproic acid and tranexamic acid are analogues o ly-

sine that bind to and inhibit plasminogen and plasmin. These agents are used to promote hemostasis in situations where brinolysis contributes to bleeding. Like aprotinin, these agents are also used to reduce perioperative bleeding during coronary artery bypass gra ting. Unlike aprotinin, these agents may not increase the risk o postoperative acute renal ailure.

CONCLUSION AND FUTURE DIRECTIONS Hemostasis is a highly regulated process that maintains the f uidity o blood in normal vessels and initiates rapid ormation o a stable brin-based clot in response to vascular injury. Pathologic thrombosis results rom endothelial injury, abnormal blood f ow, and hypercoagulability. Antiplatelet agents, anticoagulants, and thrombolytic agents target di erent stages o thrombosis and thrombolysis. Antiplatelet agents inter ere with platelet adhesion, the platelet release reaction, and platelet aggregation; these agents can provide power ul prophylaxis against thrombosis in susceptible individuals. Anticoagulants primarily target plasma coagulation actors and disrupt the coagulation cascade by inhibiting crucial intermediates. A ter a brin clot has been established, thrombolytic agents mediate dissolution o the clot by promoting the conversion o plasminogen to plasmin. These classes o pharmacologic agents can be administered, individually or in combination, to prevent or disrupt thrombosis and to restore the patency o blood vessels occluded by thrombus. Future development o new antiplatelet, anticoagulant, and thrombolytic agents will be orced to contend with two major constraints. First, or many clinical indications in this eld, highly e ective, orally bioavailable, and inexpensive therapeutic agents are already available: these include the antiplatelet drug aspirin and the anticoagulant war arin. Second, virtually every antithrombotic and thrombolytic agent is associated with the mechanism-based toxicity o bleeding, and this adverse e ect is likely to plague new agents under development. Nonetheless, opportunities remain or the development o sa er and more e ective therapies. It is likely that pharmacogenomic techniques (see Chapter 7) will

C h a p t e r 2 3 Pharmacology of Hemostasis and Thrombosis

be capable o identi ying individuals in the population who carry an increased genetic risk o thrombosis, and such individuals may benef t rom long-term antithrombotic treatment. Combinations o antiplatelet agents, low-molecular-weight heparins, orally bioavailable direct thrombin inhibitors, and new agents that target currently unexploited components o hemostasis (such as inhibitors o the actor VIIa/tissue actor pathway) could all be use ul in these settings. At the other end o the spectrum, there remains a great need or new agents that can achieve rapid, noninvasive, convenient, and selective lysis o acute thromboses associated with li ethreatening emergencies such as ST elevation myocardial in arction and stroke. Care ully designed clinical trials will be critical to optimize the indications, dose, and duration o treatment or such drugs and drug combinations.

Acknowledgment We thank April W. Armstrong or her valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

425

Suggested Reading Abrams CS, Plow EF. Molecular basis or platelet unction. In: Ho man R, Benz EJ Jr, Silberstein LE, Heslop H, Weitz J, Anastasi J, eds. Hematology: basic principles and practice. 6th ed. Philadelphia: Churchill Livingstone; 2012:1809–1820. (Detailed and mechanistic description of platelet activation.) Angiolillo DJ. The evolution o antiplatelet therapy in the treatment o acute coronary syndromes: rom aspirin to the present day. Drugs 2012;72:2087–2116. (Reviews the clinical evidence supporting the use of antiplatelet agents.) Furie B, Furie BC. Mechanisms o thrombus ormation. N Engl J Med 2008;359:938–949. (Reviews mechanisms of hemostasis and thrombosis, with an emphasis on in vivo coagulation.) Owens AP III, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 2011;108:1284–1297. (Reviews biology of microparticles in regulating thrombosis.) Perzborn E, Roehrig S, Straub A, Kubitza D, Misselwitz F. The discovery and development o rivaroxaban, an oral, direct actor Xa inhibitor. Nat Rev Drug Discov 2011;10:61–75. (Reviews the development of anticoagulants with a focus on rivaroxaban.) Yeh CH, Hogg K, Weitz JI. Overview o the new oral anticoagulants: opportunities and challenges. Arterioscler Thromb Vasc Biol 2015;35:1056–1065. (Reviews mechanisms of action and clinical indications for direct thrombin inhibitors and factor Xa inhibitors.)

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432 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

pote ntia l A Norma l co o nduction

a 1

Nonn-ee xc xcit itaa ble b lee a re a

24

2

Pharmacology of Cardiac Rhythm Ca rrd rdi dia ia c a ctioon on pote ntiaa l B

R e -e ntr traa nt n t circuit

Eh rin J . Arm s t ro n g a n d David E. Cla p h a m Unidir iree ctiona c l cond d uc ucti tioo n b lock

Abnorma lly s low lloo w INTRODUCTION 4333 43 434 ACO COLO LOGI GIC C CLASSES S AND AN AGENTS . . . . . . . . . . . . . . . . 440 ogra de4 PHARMA trogra R e -e ntra Re t& CASE ntt . . . . . . . . . a. . . . . . . . . . . . . . . . .re tr General Mechanisms of Action of An A tiiar arrh rhyt ythm hmic i Age g nts . . 440 ELECTRICAL PHYSIO OLOGY O Y OF THE EH HEAR EA ART . . . . . . . . . .co conduc tion conduction cconduction cond co o nd nduc n uction ucti tion

Classes of Antiarrhythmic Agents . . . . . . . . . . . . . . . . . . . . . . . 4411 Pacemaker and No Nonp npac aace cemaker ma r C Cel ells . . . . . . . . . . . . . . . . . . . 433 (daa m a gee d or pa rtia i Clas llllyasss I Antiarrhythmic Agents: Fast Na Channel Block ker erss . . .44 441 1e ntiaa lss . . . . . . . . . . . . . . . . . . . .2. . . (d Cardiac Action Pote 433 de po pola laa riz ize z e d c e lls lls ) Class II Ant ntia iarr rrhy hythmic Agents: -Adrenergic Antagonists. . . 444 Dete De term rmin inatiion of F Fiiringg Rate te . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437 Class III Antiarrhythm h ic Agents: ge Inhibitors of PATHOPHYSIOLOGY OGY OF ELECTRICAL ECTRI RICA CAL L DY DYS YS SF S FUNC U CTION UN C . . . . . . . . 438 Repolarizationn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445 Defects D f s iin Imp mpul ulse see F Formation i (SA N Node) d ) . . . . . . . . . . . . . . . . . 438 2 Cl lass IV Antiarrhythmic Ag g ents: : Ca Channel ha Blockers . . . . 44 4477 Altered Alte tee red Autom Automaticity aticityy . . . . . . . . . . . . . . . . . . .b. . . . . . . . . . . . . 43 438 38 Other Ot he r Ag Agen e nts ts Tha hatt Mo Modu dula late te Cardiac Rhythm or Triggered Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4388 Ion Cha hann nnels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4477 Defects in Imp mpul ulse se Con ondu duct ctio ionn . . . . . . . . . . . . . . . . . . . . . . . . . 439 4488 Re-entry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 44 Conduction Block. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .448 Accessory Tract Pathwayss . . . . . . . . . . . . . . . . . . . . . . . . . . 440

INTRODUCTION The human heart is both a mechanical and an electrical organ. To per use the body adequately with blood, the mechanical and electrical components o the heart must work in precise concert with each other. The mechanical component pumps the blood; the electrical component controls the rhythm o the pump. When the mechanical component ails despite a normal rhythm, heart ailure can result (see Chapter 26, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure). When the electrical component goes awry (called an arrhythmia), cardiac myocytes ail to contract in synchrony, and e ective pumping is compromised. Changes in the membrane potential o cardiac cells directly a ect cardiac rhythm, and most antiarrhythmic drugs act by modulating the activity o ion channels in the plasma membrane. This chapter discusses the ionic basis o electric rhythm ormation and conduction in the heart, the pathophysiology o electrical dys unction, and the pharmacologic agents used to restore a normal cardiac rhythm.

ELECTRICALPHYSIOLOGY OF THE HEART Electrical activity in the heart, leading to rhythmic cardiac contraction, is a mani estation o the heart’s exquisite control o cell depolarization and impulse conduction. Once initiated, a cardiac action potential is a spontaneous event that proceeds based on the characteristic responses o ion channels to changes in membrane voltage. At the completion o

a cycle, the spontaneous depolarization o pacemaker cells ensures that the process repeats without interruption.

Pacemaker and Nonpacemaker Cells The heart contains cardiac myocytes that can spontaneously initiate action potentials and myocytes that cannot. Cells possessing the ability to initiate spontaneous action potentials are termed pacemaker cells . All pacemaker cells possess automaticity, the ability to depolarize above a threshold voltage in a rhythmic ashion. Automaticity results in the generation o spontaneous action potentials. Pacemaker cells are ound in the sinoatrial node (SA node), the atrioventricular node (AV node), and the ventricular conducting system (bundle o His, bundle branches, and Purkinje f bers). Together, the pacemaker cells constitute the specialized conducting system that governs the electrical activity o the heart. The second type o cardiac cells, the nonpacemaker cells , includes the atrial and ventricular myocytes. The nonpacemaker cells contract in response to depolarization and are responsible or the majority o cardiac contraction. In pathologic conditions, these nonpacemaker cells can acquire automaticity and thereby also act as pacemaker cells.

Cardiac Action Potentials Ions are not distributed equally across cell membranes. Transporters (pumps) drive K into cells while pumping Na and Ca2 out, giving rise to electrical and chemical gradients across the membrane. These gradients ultimately determine the membrane potential o a cardiac cell. The Nernst equilibrium 433

434 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

On e w in te r m o rn in g , Dr. J , a 74-ye a r-o ld p ro e s s o r, is le ctu rin g o n th e tre a tm e n t o ca rd io m yo p a th ie s to th e s e co n d -ye a r m e d ica l s ch o o l cla s s . He e e ls h is h e a rt b e a tin g irre g u la rly a n d b e co m e s n a u s e a te d . He is a b le to f n is h h is le ctu re , b u t h e co n tin u e s to e e l s ig n if ca n tly s h o rt o b re a th th ro u g h o u t th e m o rn in g . His p e rs is te n t s ym p to m s p ro m p t h im to w a lk d o w n th e s tre e t to th e lo ca l e m e rg e n cy d e p a rtm e n t. Phys ica l e xa m in a tio n re ve a ls a n irre g u la r h e a rtb e a t ra n g in g ro m 120 to 140 b e a ts /m in . Dr. J ’s b lo o d p re s s u re is s ta b le (132/76 m m Hg ), a n d h is oxyg e n s a tu ra tio n is 10 0% o n ro o m a ir. An e le ctro ca rd io g ra m (ECG) co n f rm s th a t Dr. J h a s a tria l f b rilla tio n , w ith o u t a ny e vid e n ce o is ch e m ia . Se ve ra l in tra ve n o u s b o lu s e s o d iltia ze m a re a d m in is te re d , a n d h is h e a rt ra te d e cre a s e s to 80–10 0 b e a ts /m in , b u t h is rhyth m re m a in s irre g u la r. Fu rth e r la b o ra to ry s tu d ie s , a n e ch o ca rd io g ra m , a n d a ch e s t x-ra y d o n o t re ve a l a n u n d e rlyin g ca u s e o r Dr. J ’s a tria l f b rilla tio n . Du rin g o b s e rva tio n ove r th e n e xt 12 h o u rs , Dr. J re m a in s in a tria l f b rilla tio n . Alth o u g h h is h e a rt ra te is u n d e r b e tte r co n tro l, h e co n tin u e s to e xp e rie n ce p a lp ita tio n s . Un d e r co n tin u o u s ECG m o n ito rin g , a

potential or each ion (ENa

70 mV, EK 94 mV, and ECa 150 mV) depends on the relative concentrations o ions inside and outside the cell. The di erence between an ion’s Nernst potential and the cell’s membrane potential determines the driving orce or ions into or out o the cell. Re er to Chapter 8, Principles o Cellular Excitability and Electrochemical Transmission, or a detailed discussion o the Nernst equilibrium potential. When an ion-selective channel opens, the membrane potential approaches the equilibrium potential or that ion. For example, opening a K -selective channel drives the membrane potential toward EK ( 94 mV). When a Na -selective channel opens, the membrane potential is driven toward ENa ( 70 mV), and opening a Ca2 -selective channel drives the membrane potential toward ECa ( 150 mV). Note that the reversal potential or a nonselective ion channel (e.g., a channel that passes all cations nonselectively) is 0 mV. The f nal membrane potential depends on the number o channels o each type, their conductances (i.e., the ability o each channel to pass ions), and the duration or which each channel remains open. The resting membrane of the cardiac myocyte is relatively permeable to K (because some types of K -selective channels are open) but not to Na or Ca2 ; hence, the resting membrane potential is close to the equilibrium potential or K . (The actual cardiac myocyte membrane potential is a bit more positive than the equilibrium potential or K , due to the contribution o other ion channels to the resting membrane potential.) Changing the membrane potential requires the movement o relatively ew ions across the membrane. There ore, despite the opening and closing o ion channels, the ionic concentration gradients across the membrane remain relatively

ca rd io lo g is t a d m in is te rs a n in tra ve n o u s in u s io n o ib u tilid e . Tw e n ty m in u te s a te r re ce ivin g th e ib u tilid e , Dr. J ’s ECG s h o w s a re tu rn to n o rm a l s in u s rhyth m . At th e tim e o d is ch a rg e ro m th e h o s p ita l, h e is s ta rte d o n wa r a rin in o rd e r to re d u ce h is ris k o s tro ke . Dr. J e e ls f n e a t f rs t, b u t h e d e ve lo p s re cu rre n t p a lp ita tio n s w ith in 3 w e e ks o h is in itia l e ve n t. A te r d is cu s s io n w ith h is ca rd io lo g is t, h e e le cts to s ta rt a m io d a ro n e a t a m a in te n a n ce d o s e o 200 m g /d a y, in a d d itio n to co n tin u in g h is wa r a rin . Dr. J to le ra te s th e a m io d a ro n e w e ll a n d re p o rts n o d i f cu lty b re a th in g . He re m a in s s ym p to m - re e d u rin g th e re s t o h is ca rd io lo g y le ctu re s .

Questions 1 . Why were ibutilide and amiodarone e ective in converting Dr. J ’s heart rhythm to normal sinus rhythm? 2 . Why should ibutilide be administered only under care ully monitored circumstances? 3 . What adverse e ects o amiodarone could develop at higher daily doses? 4 . Why did diltiazem slow Dr. J ’s heart rate without a ecting his underlying heart rhythm, atrial f brillation?

stable, and the Nernst potential or each ion remains relatively constant. Cardiac action potentials are strikingly longer than those o nerve or skeletal muscle, lasting or almost hal a second. Prolonged cardiac action potentials provide the sustained depolarization and contraction needed to empty the heart’s chambers. Sinoatrial (SA) nodal cells pace the heart at normal resting heart rates between 60 and 100 beats/min, while ventricular muscle cells orchestrate the contraction that ejects blood rom the heart (Fig. 24-1). SA nodal cells f re spontaneously in a cycle def ned by three phases, re erred to as phase 4, phase 0, and phase 3 (Fig. 24-2 and Table 24-1). Phase 4 consists o a slow, spontaneous depolarization that is caused by an inward pacemaker current (If –encoded by HCN). This spontaneous depolarization accounts or the automaticity o the SA node. The channels that carry the If current are activated during the repolarization phase o the previous action potential. The If channels are relatively nonselective cation channels. Phase 0 consists o a more rapid depolarization mediated by highly selective voltage-gated Ca2 channels that, upon opening, drive the membrane potential toward ECa ( 150 mV). In phase 3, the Ca2 channels slowly close and K -selective channels open, resulting in membrane repolarization. Once the membrane potential repolarizes to approximately 60 mV, the opening o If channels is triggered and the cycle begins again. Although the If (inward pacemaker) current is responsible or the slow spontaneous depolarization in phase 4 o the SA-node action potential, the kinetics o this depolarization are modulated by voltage-gated Na channels that are also expressed in the SA node. There are gradients o expression

C h a p t e r 2 4 Pharmacology o Cardiac Rhythm 435

A S A node ce ll E Ca +150 mV

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The f ve phases o the ventricular myocyte action potential result rom an intricately woven cascade o channel openings and closings; the phases are numbered rom 0 to 4 (Fig. 24-3 and Table 24-1). In phase 0, an action potential upstroke o very rapid depolarization is caused by a transient increase in inward

0

100

200

300

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500

Ma jor Curre nts

P ha s e 4

If = P a ce ma ke r curre nt, re la tive ly nons e le ctive . IK1 = Inwa rd re ctifie r, outwa rd K+ curre nt

P ha s e 0

ICa = Inwa rd Ca 2+ curre nt

P ha s e 3

IK = De la ye d re ctifie r, outwa rd K+ curre nt

EK -94 mV

Time (ms ) B Ve ntricula r mus cle ce ll E Ca +150 mV

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FIGURE 24-1. SA node and ventricular muscle cell action potentials. The

IK1

P ha s e 3

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Time (ms )

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o If channels and o the more selective voltage-gated Na and Ca2 channels within the SA node, such that cells at the border o the node express relatively more voltage-gated Na channels and cells in the center o the node express relatively more If and voltage-gated Ca2 channels. The expression o voltage-gated Na channels in the SA node is partly responsible or the e ect o certain antiarrhythmics on the automaticity o SA nodal cells (see below). Unlike SA nodal cells, ventricular myocytes do not depolarize spontaneously under physiologic conditions. As a result, the membrane potential o the resting ventricular myocyte remains near EK until the cell is stimulated by a wave o depolarization that is initiated by nearby pacemaker cells.

-60

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P ha s e 0

r

Time (ms )

resting membrane potential o a sinoatrial (SA) node cell is approximately 55 mV, while that o a ventricular muscle cell is 85 mV. The shaded areas represent the approximate depolarization required to trigger an action potential in each cell type. Together, the cardiac action potentials last or approximately hal a second. SA node cells (A) depolarize to a peak o 10 mV, and ventricular muscle cells (B) depolarize to a peak o 45 mV. Note that the ventricular action potential has a much longer plateau phase. This long plateau ensures that ventricular myocytes have adequate time to contract be ore the onset o the next action potential. The Nernst equilibrium potentials o the major ions (ECa , ENa , EK) are shown as dashed horizontal lines. Em, membrane potential.

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Time (ms) FIGURE 24-2. SA-node action potential and ion currents. A. SA nodal cells are depolarized slowly by the pacemaker current (If ) (phase 4), which consists o an inward f ow o sodium (mostly) and calcium ions. Depolarization to the threshold potential opens highly selective voltage-gated calcium channels, which drive the membrane potential toward ECa (phase 0). As the calcium channels close and potassium channels open (phase 3), the membrane potential repolarizes. B. The f ux o each ion species correlates roughly with each phase o the action potential. Positive currents indicate an outward f ow o ions (blue and purple), while negative currents are inward (gray and black).

436 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

TABLE 24-1 Major Characteristics of Action Potential Phases for SA Nodal Cells and Ventricular Myocytes

P ha s e s of Ve ntricula r Action P ote ntia l Ma jor Curre nts

SA Nodal Cells

P ha s e 4

IK = Inwa rd re ctifie r, outwa rd K+ curre nt 1 INa /Ca = Inwa rd Na + a nd Ca 2+ curre nt

P ha s e 0

INa = Fa s t inwa rd Na + curre nt

P ha s e 1

Ito = Tra ns ie nt outwa rd K+ curre nt

P ha s e 2

ICa = Inwa rd Ca 2+ curre nt IK = De la ye d re ctifie r, outwa rd K+ curre nt IK = Inwa rd re ctifie r, outwa rd K+ curre nt 1 Ito = Tra ns ie nt outwa rd K+ curre nt

P ha s e 3

IK = De la ye d re ctifie r, outwa rd K+ curre nt

SEGMENT

CHARACTERISTICS

MAJ OR UNDERLYING CURRENT

Phase 4

Slow depolarization

Inward If current (carried mainly by Na )

Phase 0

Action potential upstroke

Inward Ca 2 current through voltage-sensitive Ca 2 channels (ICa )

Phase 3

Repolarization

Outward K current through K channels (IK)

Ca 2+ K+

Na +

Ventricular Myocytes SEGMENT

CHARACTERISTICS

MAJ OR UNDERLYING CURRENT

Phase 4

Resting membrane potential

Inward and outward currents are equal

K+ Na +/Ca 2+ A

60 ) V (

m

Inward Na current through Na channels (INa )

Ito

P ha s e 1

ICa

30

IK, IK , Ito 1

l

Rapid depolarization

i

P ha s e 2

t n

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Late phase o rapid repolarization

e t o

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-30

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r

Balance between inward Ca 2 current through Ca 2 channels (ICa.T, ICa.L) and outward K current through K channels (IK, IK1, Ito)

P ha s e 3 IK

-60 -90

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IK

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INa /Ca

Time (ms )

2

Decrease in inward Ca current and large increase in outward K current

B Ion curre nts of ve ntricula r a ction pote ntia l 4 IK IK 1 Ito 2 F s µ s / o

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Na current through voltage-gated Na channels. (Note that currents in phase 0 o the SA nodal and ventricular myocyte action potentials are carried by di erent ions—Ca2 and Na , respectively.) The opening o Na channels leads to a rapid in ux o Na (INa), which accounts or the depolarization and drives the membrane potential toward ENa ( 70 mV). Although large, the increase in Na conductance during phase 0 lasts or only 1–2 milliseconds because the Na channels inactivate as a unction o time and voltage. Inactivation o the ast Na channels causes a dramatic decrease in the inward Na current. The time it takes or Na channels to recover rom their voltage-dependent and timedependent inactivation determines the refractory period o the myocyte. The re ractory period is the time during which another action potential cannot f re. This serves as a protective mechanism to ensure that the heart has su f cient time to eject blood rom its chambers. The re ractory period lasts rom the initiation o the action potential upstroke until the repolarization phase. INa is the major determinant o the velocity o impulse conduction throughout the ventricle. The threshold-dependent activation o INa quickly depolarizes the membrane. The upstroke terminates be ore

IK

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p e

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-6

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0

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Time (ms )

FIGURE 24-3. Ventricular action potential and ion currents. A. At the resting membrane potential (phase 4), the inward and outward currents are equal and the membrane potential approaches the K equilibrium potential (EK). During the action potential upstroke (phase 0), a large transient increase in Na conductance occurs. This event is ollowed by a brie period o initial repolarization (phase 1), which is mediated by a transient outward K current. The plateau o the action potential (phase 2) results rom the opposition o an inward Ca 2 current and an outward K current. The membrane repolarizes (phase 3) when the inward Ca 2 current decreases and the outward K current predominates. B. The ion f uxes that give rise to the ventricular action potential consist o a complex pattern o changing ion permeabilities that are separated in time. Note especially that the Na current in phase 0 is very large but extremely brie .

C h a p t e r 2 4 Pharmacology o Cardiac Rhythm 437

B O X 2 4 - 1 Th e Ele ctro ca rd io g ra m The electrocardiogram (ECG or EKG) is used to in er changes in cardiac impulses by recording electrical potentials at various locations on the sur ace o the body. An ECG recording ref ects changes in the excitation o the myocardium. A basic understanding o the ECG is use ul or discussions o the clinical applications o the various antiarrhythmic agents. A normal electrocardiogram contains three electrical waveorms: the P wave, the QRS complex, and the T wave (Fig. 24-4). The P wave represents atrial depolarization; the QRS complex represents ventricular depolarization; and the T wave represents ventricular repolarization. The ECG does not show atrial repolarization explicitly because the atrial repolarization is “drowned out” by the QRS complex. The ECG also contains two intervals and one

reaching ENa, however, and is ollowed by an early phase o rapid repolarization to about 20 mV. This phase 1 repolarization is a consequence o two events: (1) the rapid voltage-dependent inactivation o INa and (2) the activation o transient K currents (transient outward; Ito). Phase 2, the plateau phase o the ventricular action potential, is unique to cardiac cell electrophysiology. The plateau is maintained by a f nely tuned balance between an inward Ca2 current through two types o Ca2 channels (ICa.T, ICa.L) and an outward K current through several types o K channels (IK, IK1, Ito). Remarkably, only a ew hundred channels per cell are used to maintain this f ne balance. Because only a small number o channels are open, the total membrane conductance is low. The high membrane resistance during the plateau phase insulates the cardiac cells electrically, allowing rapid propagation o the action potential with little current dissipation. During the plateau phase, two distinct Ca2 currents—the transient Ca2 current, ICa.T, and the long-lasting Ca2 current, ICa.L—mediate the in ux o Ca2 needed to initiate cardiac myocyte contraction. T-type Ca2 channels inactivate with time and are insensitive to block by dihydropyridines such as ni edipine. Current through the L-type Ca2 channels (ICa.L) provides the dominant Ca2 current in virtually all cardiac cells. ICa.L is activated at 30 mV and inactivates slowly (hundreds o milliseconds). It is sensitive to block by dihydropyridines (nifedipine ), benzothiazepines (diltiazem), and phenylalkylamines (verapamil), as discussed below. L-type Ca2 channels carry inward current throughout the plateau phase; because Ca2 stimulates the contraction o cardiac myocytes, these channels are crucial or coupling membrane excitability to myocardial contraction. Opposing the inward Ca2 currents are outward currents through the K channels that are activated during the plateau phase. As the time-dependent inward Ca2 currents inactivate, the outward K currents (mostly IK) rapidly drive the membrane potential toward EK, thus repolarizing the cell in phase 3. However, these channels are unable to drive the membrane potential all the way to EK because they deactivate at 40 mV. In phase 4, the resting membrane potential is reestablished by the activation o time-independent K currents (IK1), which drive the membrane potential close to the K equilibrium potential.

segment: the PR interval, the QT interval, and the ST segment. The PR interval spans rom the beginning o the P wave (initial depolarization o the atria) to the beginning o the Q wave (initial depolarization o the ventricles). Hence, the length o the PR interval varies with conduction velocity through the AV node. For example, i a patient has an electrical block in the AVnode, then the conduction velocity through the AV node decreases and the PR interval increases. The QT interval spans rom the beginning o the Q wave to the end o the T wave, representing the entire sequence o ventricular depolarization and repolarization. The ST segmentt extends rom the end o the S wave to the beginning o the T wave; this segment, which represents the period during which the ventricles are depolarized, corresponds to the plateau phase o the ventricular action potential. ■

In clinical practice, the overall electrical activity o the heart is measured rather than the ionic changes that occur at a single-cell level. This overall activity is reported in the electrocardiogram, or ECG (Box 24-1 and Fig. 24-4).

Determination of Firing Rate The specialized conduction system o the heart consists o the SA node, AV node, bundle o His, and Purkinje system. These di erent populations o cells have di erent intrinsic rates o f ring. Three actors determine the f ring rate. First, as the rate o spontaneous depolarization in phase 4 increases, the rate o f ring increases because the threshold potential (the minimum potential necessary to trigger an action potential) is reached more quickly at the end o phase 4. Second, i the threshold potential becomes more negative,

PR

QT R

5 mm = 0.5 mV T

P Q

S ST

QRS 5 mm = 0.2 s e cond

FIGURE 24-4. Electrocardiogram. The electrocardiogram (ECG or EKG) measures the body sur ace potentials induced by cardiac electrical activity. The P wave ref ects atrial depolarization, the QRS complex represents ventricular depolarization, and the T wave indicates ventricular repolarization. The PR interval spans rom the beginning o the P wave (initial depolarization o the atria) to the beginning o the Q wave (initial depolarization o the ventricles). The QT interval spans rom the beginning o the Q wave to the end o the T wave, representing the entire interval o ventricular depolarization and repolarization. The ST segment extends rom the end o the S wave to the beginning o the T wave, representing the period during which the ventricles are depolarized (i.e., the plateau phase o the action potential).

438 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

the rate o f ring increases because the threshold potential is reached more quickly at the end o phase 4. Third, i the maximum diastolic potential (the resting membrane potential) becomes more positive, the rate o f ring increases because less time is needed to repolarize the membrane ully at the end o phase 3. Because the various populations o pacemaker cells possess di erent intrinsic rates o f ring, the pacemaker population with the astest f ring rate sets the heart rate. The SA node possesses the astest intrinsic f ring rate (60–100 times per minute) and is the native pacemaker o the heart. The cells o the atrioventricular (AV) node and bundle o His f re intrinsically between 50 and 60 times per minute, and the cells o the Purkinje system have the slowest intrinsic f ring rate at 30–40 times per minute. The cells o the AV node, bundle o His, and Purkinje system are termed latent pacemakers because their intrinsic rhythm is overridden by the aster SA-node automaticity. In a mechanism termed overdrive suppression, the SA node suppresses the intrinsic rhythm o the other pacemaker populations and entrains them to f re at the SA nodal f ring rate.

PATHOPHYSIOLOGY OF ELECTRICAL DYSFUNCTION Causes o electrical dys unction in the heart can be divided into de ects in impulse ormation and de ects in impulse conduction. In the ormer case, SA-node automaticity is interrupted or altered, leading to missed beats or ectopic beats, respectively. In the latter case, impulse conduction is altered (e.g., in the case o reentrant rhythms), and sustained arrhythmias can result.

Defects in Impulse Formation (SA Node) As the native pacemaker o the heart, the SA node has a pivotal role in normal impulse ormation. Electrical events that alter SA nodal unction or disturb overdrive suppression can lead to impaired impulse ormation. Two mechanisms commonly associated with de ective impulse ormation are altered automaticity and triggered activity.

Altered Automaticity

Some mechanisms that alter automaticity o the SA node are physiologic. In particular, the autonomic nervous system o ten modulates automaticity o the SA node as part o a physiologic response. In sympathetic stimulation during exercise, an increased concentration o catecholamines leads to greater 1-adrenergic receptor activation. Activation o 1 receptors causes the opening o a greater number o pacemaker channels (I channels); a larger pacemaker current is then conducted through these channels; and aster phase 4 depolarization results. Sympathetic stimulation also causes the opening o a greater number o Ca2 channels and thereby shi ts the threshold to more negative potentials. Both o these mechanisms increase heart rate. The parasympathetic vagus nerve a ects the SA node by a number o mechanisms that oppose the sympathetic regulation o heart rate. Vagus nerve release o acetylcholine initiates an intracellular signaling cascade that (1) reduces the pacemaker current by decreasing pacemaker channel opening, (2) shi ts the threshold to more positive potentials by reducing Ca2 channel opening, and (3) makes the maximum diastolic potential (analogous

to the resting membrane potential in these spontaneously f ring cells) more negative by increasing K channel opening. The SA node, atria, and AV node are highly innervated and are thus more sensitive than the ventricular conducting system to the e ects o vagal stimulation. In pathologic conditions, automaticity can be altered when latent pacemaker cells take over the SA node’s role as the pacemaker o the heart. When the SA nodal f ring rate becomes pathologically slow or when conduction o the SA impulse is impaired, an escape beat may occur as a latent pacemaker initiates an impulse. A series o escape beats, known as an escape rhythm, may result rom prolonged SA nodal dys unction. On the other hand, an ectopic beat occurs when latent pacemaker cells develop an intrinsic rate o f ring that is aster than the SA nodal rate, in some cases despite the presence o a normally unctioning SA node. A series o ectopic beats, termed an ectopic rhythm, can result rom ischemia, electrolyte abnormalities, or heightened sympathetic tone. Direct tissue damage (such as can occur a ter a myocardial in arction) also results in altered automaticity. Tissue injury can cause structural disruption o the cell membrane. Disrupted membranes are unable to maintain ion gradients, which are critical or maintaining appropriate membrane potentials. I the resting membrane potential becomes su f ciently positive (more positive than 60 mV), nonpacemaker cells may begin to depolarize spontaneously. Another mechanism by which tissue damage leads to altered automaticity is through the loss o gap junction connectivity. Direct electrical connectivity is important or the e ective delivery o overdrive suppression rom the SA node to the rest o the cardiac myocytes. When connectivity is disrupted due to tissue injury, overdrive suppression is not e f ciently relayed, and the unsuppressed cells can initiate their own rhythm. This abnormal rhythm can lead to cardiac arrhythmia.

Triggered Activity

Afterdepolarizations occur when a normal action potential

triggers extra abnormal depolarizations. That is, the f rst (normal) action potential triggers additional oscillations o membrane potential, which may lead to arrhythmia. There are two types o a terdepolarizations—early a terdepolarizations and delayed a terdepolarizations. I the a terdepolarization occurs during the inciting action potential, it is termed an early afterdepolarization (Fig. 24-5). Conditions that prolong the action potential (e.g., drugs that prolong the QT interval, such as procainamide and ibutilide) tend to trigger early a terdepolarizations. Specif cally, an early a terdepolarization can occur during the plateau phase (phase 2) or the rapid repolarization phase (phase 3). During the plateau phase, because most o the Na channels are inactivated, an inward Ca2 current is responsible or the early a terdepolarization. On the other hand, during the rapid repolarization phase, partially recovered Na channels can conduct an inward Na current that contributes to the early a terdepolarization. I an early a terdepolarization is sustained, it can lead to a type o ventricular arrhythmia termed torsades de pointes . Torsades de pointes, French or “twisting o the points,” is characterized by QRS complexes o varying amplitudes as they “twist” along the baseline; this rhythm is a medical emergency that can lead to death i not treated immediately. In contrast to early a terdepolarizations, delayed afterdepolarizations occur shortly a ter the completion o repolarization

C h a p t e r 2 4 Pharmacology o Cardiac Rhythm 439

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Normal cardiac conduction is initiated at the SA node and propagated to the AV node, bundle of His, Purkinje system, and myocardium in an orderly fashion. The cellular refractory period ensures that stimulated regions of the myocardium depolarize only once during propagation of an impulse. Figure 24-7A depicts normal impulse conduction, in which an impulse arriving at point a travels synchronously down two parallel pathways, 1 and 2. Re-entry of an electrical impulse occurs when a selfsustaining electrical circuit stimulates an area of the myocardium repeatedly and rapidly. Two conditions must be present for a re-entrant electrical circuit to occur: (1) unidirectional block (anterograde conduction is prohibited, but retrograde conduction is permitted) and (2) slowed retrograde conduction velocity. Figure 24-7B shows a re-entrant electrical circuit. As the impulse arrives at point a, it can travel only down pathway 1 (the left branch) because pathway 2 (the right branch) is blocked unidirectionally in the anterograde direction. The impulse

ally occur during the repolarizing phase o the action potential, although they can also occur during the plateau phase. Repetitive a terdepolarizations can trigger an arrhythmia.

(Fig. 24-6). The mechanism of delayed afterdepolarizations is not well understood; it has been proposed that high intracellular Ca2 concentrations (such as in digoxin toxicity) lead to an inward Na current, which, in turn, triggers the delayed afterdepolarization.

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Defects in Impulse Conduction The second type of electrical disturbance of the heart involves defects in impulse conduction. Normal cardiac function requires unobstructed and timely propagation of an electrical impulse through the cardiac myocytes. In pathologic conditions, altered impulse conduction can result from one or a combination of three mechanisms: re-entry, conduction block, and accessory tract pathways.

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FIGURE 24-7. Normal and re-entrant electrical pathways. A. In normal impulse conduction, an impulse traveling down a pathway arrives at point a, where it is able to travel down two alternate pathways, 1 and 2. In the absence o re-entry, the impulses continue on and depolarize di erent areas o the ventricle. B. A re-entrant circuit can develop i one o the branch pathways is pathologically disrupted. When the impulse arrives at point a, it can travel only down pathway 1 because pathway 2 is blocked unidirectionally (i.e., the e ective re ractory period o the cells in pathway 2 is prolonged to such an extent that anterograde conduction is prohibited). The impulse conducts through pathway 1 and proceeds to point b. At this point, the cells in pathway 2 are no longer re ractory, and the impulse conducts in a retrograde ashion up pathway 2 toward point a. When the retrograde impulse arrives at point a, it can initiate re-entry. Re-entry can result in a sustained pattern o rapid depolarizations that trigger tachyarrhythmias. This mechanism can occur over small or large regions o the heart.

440 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

conducts through pathway 1 and travels to point b. At this junction, the impulse travels in a retrograde ashion up pathway 2 toward point a. The conduction time rom point b to point a is slowed because o cell damage or the presence o cells that are still in the re ractory state. By the time the impulse reaches point a, the cells in pathway 1 have had adequate time to repolarize, and these cells are stimulated to continue conducting the action potential toward point b. In this manner, tachyarrhythmias result rom the combination o unidirectional block and decreased conduction velocity in the abnormal pathway.

Conduction Block

Conduction block occurs when an impulse ails to propagate because o the presence o an area o inexcitable cardiac tissue. This area o inexcitable tissue could consist o normal tissue that is still re ractory, or it could represent tissue that has been damaged by trauma, ischemia, or scarring. In either case, the myocardium is unable to conduct an impulse. Because conduction block removes overdrive suppression by the SA node, the cardiac myocytes are ree to beat at their intrinsically slower requency. For this reason, conduction block can be mani ested clinically as bradycardia.

Accessory Tract Pathways

During the normal cardiac cycle, the SA node initiates an impulse that travels quickly through the atrial myocardium and arrives at the AV node. Impulse conduction then slows through the AV node, allowing su f cient time or f lling o the ventricles with blood be ore ventricular contraction is initiated. A ter the impulse travels through the AV node, it again propagates quickly throughout the ventricles to trigger ventricular contraction. Some individuals possess accessory electrical pathways that bypass the AV node. One common accessory pathway is the bundle of Kent, a band o myocardium that conducts impulses directly rom the atria to the ventricles, bypassing the AV node (Fig. 24-8). In these individuals, an impulse

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FIGURE 24-8. Bundle of Kent. The bundle of Kent is an accessory electrical pathway that conducts impulses directly from the atria to the ventricles, bypassing the AV node. Impulse conduction through this accessory tract is more rapid than conduction through the AV node, setting up the conditions for re-entrant tachyarrhythmias.

originating in the SA node is conducted through the bundle o Kent to the ventricles more rapidly than the same impulse would be conducted through the AV node. Because the bundle o Kent is an accessory pathway, the ventricular tissue receives impulses rom both the normal conduction pathway and the accessory pathway. As a result, electrocardiograms rom these individuals typically exhibit a wider-than-normal QRS complex and an earlier-than-normal ventricular upstroke. More importantly, because the two conduction tracts have di erent conduction velocities, the presence o an accessory tract can set up the conditions or a re-entrant loop and thereby predispose the individual to tachyarrhythmias.

PHARMACOLOGIC CLASSES AND AGENTS Ion currents across the plasma membrane induce changes in the membrane potential o cells. Changes in the membrane potential o cardiac pacemaker cells underlie the timely contraction o cardiac myocytes. De ects in impulse ormation and altered impulse conduction can lead to disturbances in cardiac rhythm. Antiarrhythmic agents are used to restore normal cardiac rhythm by targeting proarrhythmic regions o the heart.

General Mechanisms of Action of Antiarrhythmic Agents Although there are many di erent antiarrhythmic agents, there are surprisingly ew mechanisms o antiarrhythmic action. In general, drugs that a ect cardiac rhythm act by altering (1) the maximum diastolic potential in pacemaker cells (and/or the resting membrane potential in ventricular cells), (2) the rate o phase 4 depolarization, (3) the threshold potential, or (4) the action potential duration. The specif c e ect o a particular channel blocker ollows directly rom the role o the current carried by that channel in the cardiac action potential. For example, Na and Ca2 channel blockers typically alter the threshold potential, while K channel blockers tend to prolong action potential duration. These drugs generally block the pore rom inside the cell; they can access their sites o action by either traversing the pore o the channel or di using across the lipid bilayer within which the channel is embedded. State-dependent ion channel block is an important concept in antiarrhythmic drug action. Ion channels are capable o switching among various con ormational states, and changes in the permeability o the membrane to a particular ion are mediated by con ormational changes in the channels that conduct that ion. Antiarrhythmic drugs o ten have di erent a f nities or di erent con ormational states o the ion channel; that is, these drugs bind to one con ormation o the channel with higher a f nity than they do to other con ormations o the channel. This type o binding is re erred to as state-dependent. Na channel blockers serve as an excellent example to illustrate the concept o state-dependent ion channel block. The Na channel undergoes three major state changes (open–closed–inactivated) throughout the course o an action potential. During the upstroke, the channel is in the open con ormation. The channel becomes inactivated during the plateau phase, and it changes again to the resting (closed) con ormation as the membrane is repolarized to its

C h a p t e r 2 4 Pharmacology o Cardiac Rhythm 441

resting potential. Most Na channel blockers bind pre erentially to the open and inactivated states o the Na channel, not to the resting (closed) state o the channel. In this way, the drugs tend to block the channels during the action potential (cardiac systole) and to dissociate rom the channels during diastole. The unblocking rate (dissociation rate) o the various Na channel blockers is an important determinant o the steadystate block o Na channels. For example, when heart rate increases, the time available or unblocking (dissociation o the drug rom its binding site on the channel) decreases and the degree o steady-state Na channel block increases. The action o Na channel blockers on ischemic tissue illustrates the therapeutic utility o state-dependent block. It has been observed that Na channel blockers depress Na conduction in ischemic tissue to a much greater extent than in normal tissue. In ischemic tissue, cardiac myocytes are depolarized or a longer period o time. This increase in action potential duration prolongs the inactivation state o the Na channels, thereby making the inactivated Na channels accessible to Na channel blockers or a longer period o time. The rate o channel recovery rom block is also decreased in depolarized ischemic myocytes because o the prolonged action potential. Thus, the higher a f nity o Na channel blockers or open and inactivated states o the channel allows these agents to act pre erentially on ischemic tissue and thereby to block an arrhythmogenic ocus at its source. See Chapter 12, Local Anesthetic Pharmacology, or more discussion on the concept o state-dependent Na channel block. Developing and using e ective antiarrhythmic treatments is o ten complicated by the possibility that the antiarrhythmic agent can also cause arrhythmias. For example, many e orts have been directed at the treatment o re-entry, a mechanism responsible or a large proportion o arrhythmias. One way to treat re-entry is to block action potential propagation. I the

retrograde impulse in the re-entrant circuit is completely extinguished by an antiarrhythmic agent, then the impulse will be unable to repeatedly depolarize the cardiac tissue in the re-entrant circuit. I the impulse is not completely extinguished, however, then the antiarrhythmic-induced slowing o conduction can actually promote re-entry arrhythmia. The “surviving” impulse may use the original re-entrant pathway to propagate the arrhythmia, or it may f nd other pathways and create new re-entrant circuits.

Classes o Antiarrhythmic Agents Antiarrhythmic agents have traditionally been organized into our classes based on their mechanism o action (known as the Vaughn-Williams classif cation). Class I antiarrhythmics are Na channel blockers; class II antiarrhythmics are -adrenergic receptor antagonists; class III antiarrhythmics are K channel blockers; and class IV antiarrhythmics are Ca2 channel blockers. It is important to realize, however, that many antiarrhythmic agents are not entirely selective blockers o Na , K , or Ca2 channels; rather, many o these agents block more than one channel type. This section presents some use ul def nitions o common cardiac electrical disturbances (Box 24-2) and describes the mechanism o drug action or each class o antiarrhythmic agent.

Class I Antiarrhythmic Agents: Fast Na Channel Blockers

Na channel blockers decrease automaticity in SA nodal cells by (1) shi ting the threshold to more positive potentials and (2) decreasing the slope o phase 4 depolarization (Fig. 24-9). The block o Na channels leaves ewer channels available to open in response to membrane depolarization, thereby raising the threshold or action potential f ring and slowing the rate o depolarization. Both o these e ects

B O X 2 4 - 2 De f n itio n s o Co m m o n Ca rd ia c Ele ctrica l Dis tu rb a n ce s To appreciate the clinical applications o the various antiarrhythmic agents, it is help ul to understand the basic def nitions o terms that describe common electrical abnormalities o the heart. E ective re ractory period: The period during which a region o cardiac tissue cannot be excited by an electrical impulse. Sinus tachycardia: The SA node f res between 100 and 180 times per minute, and the ECG shows normal P waves and QRS complexes. Sinus tachycardia can be a normal physiologic response (e.g., during exercise) or a pathologic condition that results rom altered SA-node automaticity. Paroxysmal supraventricular tachycardia (PSVT): PSVT is characterized by atrial f ring rates o 140–250 beats per minute, but it is usually transient and sel -limited. In 90% o cases, PSVT is caused by re-entry involving the AV node, SA node, or atrial tissue. Atrial f utter: The atrial rate is between 280 and 300 beats per minute, and the ECG shows a rapid, “saw-tooth” appearance o atrial electrical activity. Because the pace o atrial

f ring is so rapid, some impulses rom the atria reach the AV node during its re ractory period. These impulses are not transmitted to the ventricles and, there ore, the ventricular rate is slower than the atrial rate. The ratio o atrial to ventricular f ring rate is typically 2:1. Atrial or ventricular ibrillation: These arrhythmias are characterized by chaotic, re-entrant impulse conduction through the atrium or ventricle. Ventricular f brillation (VF) is invariably atal i the arrhythmia is not converted, while atrial f brillation (AF) can be tolerated or many years. Ventricular tachycardia (VT): A series o three or more ventricular extrasystoles at rates between 100 and 250 beats per minute. Torsades de pointes: This arrhythmia is o ten generated by a terdepolarizations in individuals with prolonged QT syndrome. The varying amplitudes o the QRS complex are described as a “twisting o points” along the baseline o an ECG tracing. Torsades is o ten transient and sel -limited but can lead to more li e-threatening arrhythmias. ■

442 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY A

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myocytes in a re-entrant pathway. However, i conduction velocity is not su f ciently decreased, and the impulse is not extinguished, then the slowed impulse can support re-entry as it reaches cells that are no longer re ractory (see above) and thereby precipitate an arrhythmia. In addition to decreasing phase 0 upstroke velocity, class IA Na channel blockers prolong repolarization. Prolonged repolarization increases the e ective re ractory period, so that cells in a re-entrant circuit cannot be depolarized by the re-entrant action potential. In summary, Na channel blockers decrease the likelihood o re-entry, and thereby prevent arrhythmia, by (1) decreasing conduction velocity and (2) increasing the re ractory period o ventricular myocytes. Although the three subclasses o class I antiarrhythmics (class IA, IB, and IC) have similar e ects on the action potential in the SA node, there are important di erences in their e ects on the ventricular action potential.

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Time (ms ) FIGURE 24-9. Effects of class I antiarrhythmics and natural agonists on the SA-node action potential. A. The normal SA-node action potential is shown as a solid curve. Class I antiarrhythmics (Na channel blockers) alter SA-node automaticity by a ecting two aspects o the SA nodal action potential: (1) the threshold is shi ted to more positive potentials and (2) the slope o phase 4 depolarization is decreased. B. Acetylcholine (ACh) and adenosine slow the SA nodal f ring rate by opening K channels that hyperpolarize the cell and decrease the slope o phase 4 depolarization.

extend the duration o phase 4 and thereby decrease heart rate. Furthermore, the shi t in threshold potential means that, in patients with implanted def brillators who are treated with Na channel blockers, a higher voltage is needed to def brillate the heart. There ore, it is important to take into account the e ect o Na channel blockers when choosing appropriate settings or implanted def brillators. In addition to decreasing SA-node automaticity, Na channel blockers act on ventricular myocytes to decrease reentry. This is achieved mainly by decreasing the upstroke velocity o phase 0 and, or some Na channel blockers, by prolonging repolarization (Fig. 24-10). By decreasing phase 0 upstroke velocity, Na channel blockers decrease the conduction velocity through cardiac tissue. Ideally, conduction velocity is reduced to such an extent that the propagating wave ront is extinguished be ore it is able to restimulate

Class IA antiarrhythmics exert a moderate block on Na channels and prolong the repolarization o both SA nodal cells and ventricular myocytes. By blocking Na channels, these agents decrease the phase 0 upstroke velocity, which decreases conduction velocity through the myocardium. Class IA antiarrhythmics also block K channels and thereby reduce the outward K current responsible or repolarization o the membrane. This prolongation o repolarization increases the e ective re ractory period o the cells. Together, the decreased conduction velocity and increased e ective re ractory period decrease re-entry. Quinidine is o ten considered the prototypical drug among the class IA antiarrhythmics, but it is becoming less requently used due to its adverse e ects. In addition to the pharmacologic actions described above or all class IA antiarrhythmics, quinidine exerts an anticholinergic (vagolytic) e ect, most likely by blocking the K channels that are opened upon vagal stimulation o M2 muscarinic receptors in the AV node (see Fig. 24-9B, Fig. 10-1). The anticholinergic e ect is signif cant clinically because it can increase conduction velocity through the AV node. For this reason, an agent that slows AV nodal conduction—such as a -adrenergic antagonist or verapamil (a Ca2 channel blocker)—should be used in conjunction with quinidine to prevent an excessively rapid ventricular response in patients with atrial utter or other supraventricular tachycardias. The most common adverse e ects o quinidine are diarrhea, nausea, headache, and dizziness. These e ects make it di f cult or patients to tolerate chronic therapy with quinidine. Quinidine is contraindicated in patients with QT prolongation and in patients who are taking medications that predispose to QT prolongation because o the increased risk o torsades de pointes. Relative contraindications to quinidine use include sick sinus syndrome, bundle branch block, myasthenia gravis (because o quinidine’s anticholinergic action), and liver ailure. Quinidine is administered orally and metabolized by cytochrome P450 enzymes in the liver. Quinidine increases plasma levels o digoxin (an inotropic agent), most likely by competing or the P450 enzymes that are responsible or digoxin metabolism. Because digoxin has a narrow therapeutic index (see Chapter 25, Pharmacology o Cardiac Contractility), quinidine-induced digoxin toxicity occurs in a signif cant raction o patients. The plasma potassium

C h a p t e r 2 4 Pharmacology of Cardiac Rhythm 443

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level must be care ully monitored in patients treated with quinidine because hypokalemia decreases quinidine e f cacy, exacerbates QT prolongation, and, most importantly, predisposes to torsades de pointes. It is hypothesized that torsades de pointes is the mechanism most likely responsible for quinidine-induced syncope. Because o quinidine’s numerous adverse e ects and contraindications, this drug has largely been replaced by class III agents—such as ibutilide and amiodarone— or the pharmacologic conversion o atrial utter or atrial f brillation to normal sinus rhythm. Procainamide is a class IA antiarrhythmic agent that is e ective in the treatment o many types o supraventricular and ventricular arrhythmias. Procainamide can be used in the pharmacologic conversion o new-onset atrial f brillation to normal sinus rhythm, although with lower e f cacy than intravenous ibutilide. Procainamide can also be used sa ely to decrease the likelihood o re-entrant arrhythmias in the setting o acute myocardial in arction, even in the presence o decreased cardiac output. Procainamide can be administered by slow intravenous in usion to treat acute ventricular tachycardia. Unlike quinidine, procainamide has ew anticholinergic e ects and does not alter plasma levels o digoxin. Procainamide can cause peripheral vasodilation via inhibition o neurotransmission at sympathetic ganglia. With chronic therapy, almost all patients develop a lupus-like syndrome and positive antinuclear antibodies; the precise mechanism o this reaction is not known, but it remits i the drug is discontinued. Procainamide is acetylated in the liver to N-acetyl-procainamide (NAPA); this active metabolite produces the pure class III antiarrhythmic e ects o prolonging the re ractory period and lengthening the QT interval. NAPA does not appear to cause the lupus-like adverse e ects o procainamide. Disopyramide is similar to quinidine in its electrophysiologic and antiarrhythmic e ects; the di erence between the two drugs lies in their adverse e ects. Disopyramide causes ewer gastrointestinal problems but has even more pro ound anticholinergic e ects than quinidine, producing such adverse e ects as urinary retention and dry mouth. The pro ound anticholinergic e ects o disopyramide appear to be related to the drug’s action as an antagonist at muscarinic

acetylcholine receptors. Disopyramide is contraindicated in patients with obstructive uropathy or glaucoma. Disopyramide is also contraindicated in patients with conduction block between the atria and ventricles and in patients with sinus-node dys unction. Disopyramide has the prominent but unexplained e ect that it depresses cardiac contractility, which has led to its use in the treatment o hypertrophic obstructive cardiomyopathy and neurocardiogenic syncope. Because o its negative inotropic e ects, disopyramide is absolutely contraindicated in patients with decompensated heart ailure. Oral disopyramide is approved only or the treatment o li e-threatening ventricular arrhythmias; oral or intravenous disopyramide is sometimes used to convert supraventricular tachycardia to normal sinus rhythm. The current trend in the treatment o li e-threatening arrhythmias, however, is away rom class I antiarrhythmic agents and toward class III agents and electrical devices such as implantable def brillators. Class IB Antiarrhythmics

Class IB antiarrhythmics include lidocaine , mexiletine , and phenytoin. Lidocaine is the prototypical class IB agent. These drugs alter the ventricular action potential by blocking Na channels and sometimes by shortening repolarization; the latter e ect may be mediated by the drugs’ ability to block the ew Na channels that inactivate late during phase 2 o the cardiac action potential (Fig. 24-10). In comparison to class IA antiarrhythmics, which pre erentially bind to open Na channels, class IB drugs bind to both open and inactivated Na channels. There ore, the more time Na channels spend in the open or inactivated state, the more blockade the class IB antiarrhythmics can exert. The major distinguishing characteristic o the class IB antiarrhythmics is their fast dissociation rom Na channels. Because Na channels recover quickly rom class IB blockade, these drugs are most e ective in blocking depolarized or rapidly driven tissues, where there is a higher likelihood o the Na channels being in the open or inactivated state. Thus, class IB antiarrhythmics exhibit use-dependent block in diseased myocardium, where the cells have a tendency to f re more requently; these antiarrhythmics have relatively little e ect on normal cardiac tissue.

444 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Myocardial ischemia provides an example o the therapeutic utility o the use-dependent block exerted by class IB antiarrhythmics. The increase in extracellular H concentration in ischemic tissue activates membrane pumps that cause an increase in the extracellular K concentration. This increase in extracellular K shi ts EK to a more depolarized (more positive) value; or example, EK may shi t rom 94 mV to 85 mV. The altered electrochemical K gradient provides a smaller driving orce or K ions to ow out o cells, and depolarization o the membrane leads to a higher likelihood o action potential f ring. Because ischemic cardiac myocytes tend to f re more requently, the Na channels spend more time in the open or inactivated state, serving as a better target or blockade by class IB antiarrhythmics. Lidocaine (or more commonly amiodarone ) is used to treat ventricular arrhythmias in emergency situations. This drug is not e ective in treating supraventricular arrhythmias. In hemodynamically stable patients, lidocaine is reserved or treatment o ventricular tachyarrhythmias or requent premature ventricular contractions (PVCs) that are bothersome or hemodynamically signif cant. Lidocaine has a short plasma hal -li e (approximately 20 minutes), and it is metabolically de-ethylated in the liver. Its metabolism is governed by two actors: liver blood ow and liver cytochrome P450 activity. For patients whose liver blood ow is decreased by old age or heart ailure, or whose P450 enzymes are acutely inhibited, or example, by cimetidine (see Chapter 4, Drug Metabolism), a lower dose o lidocaine should be considered. For patients whose P450 enzymes are induced by drugs such as barbiturates, phenytoin, or ri ampin, the dose o lidocaine should be increased. Because lidocaine shortens repolarization, possibly by blocking the ew Na channels that inactivate late during phase 2 o the cardiac action potential, it does not prolong the QT interval. There ore, the drug is sa e or use in patients with long QT syndrome. However, because lidocaine also blocks Na channels in the central nervous system (CNS), it can produce CNS adverse e ects such as con usion, dizziness, and seizures. In addition to its use as an acute intravenous therapy or ventricular arrhythmias, lidocaine is used as a local anesthetic (see Chapter 12). Mexiletine , an analogue o lidocaine, is available in oral ormulation. While the e f cacy o mexiletine is similar to that o quinidine, mexiletine does not prolong the QT interval and it lacks vagolytic e ects. In addition, little hemodynamic depression has been reported with the use o mexiletine. The primary indication or mexiletine is li e-threatening ventricular arrhythmia. In practice, however, mexiletine is o ten used as an adjunct to other antiarrhythmic agents. For example, mexiletine is used in combination with amiodarone in patients with implantable cardioverter-def brillators (ICDs) and in patients with recurrent ventricular tachycardia. Mexiletine is also used in combination with quinidine or sotalol to increase antiarrhythmic e f cacy while reducing adverse e ects. There are no data supporting reduced mortality with the use o mexiletine or any o the other class IB antiarrhythmic agents. Major adverse e ects o mexiletine include doserelated nausea and tremor, which can be ameliorated when the drug is taken with ood. Mexiletine undergoes hepatic metabolism, and its plasma levels may be altered by inducers o hepatic P450 enzymes such as phenytoin and ri ampin. While phenytoin is usually considered an antiepileptic medication, its e ects on the myocardium also allow it to

be classif ed as a class IB antiarrhythmic agent. The pharmacologic properties o phenytoin are discussed in detail in Chapter 16, Pharmacology o Abnormal Electrical Neurotransmission in the Central Nervous System. Although the use o phenytoin as an antiarrhythmic agent is limited, it has been ound to be e ective in ventricular tachycardia o young children. Specif cally, phenytoin has been used in the treatment o congenital prolonged QT syndrome when therapy with -adrenergic antagonists alone has ailed; it is also used to treat ventricular tachycardia a ter congenital heart surgery. Phenytoin maintains AV conduction in digoxintoxic arrhythmias, and it is especially use ul in the rare patient who has concurrent epilepsy and cardiac arrhythmia. Phenytoin is an inducer o hepatic enzymes including P450 3A4 and thus a ects plasma levels o other antiarrhythmic agents such as mexiletine, lidocaine, and quinidine. Class IC Antiarrhythmics

Class IC antiarrhythmics are the most potent Na channel blockers, and they have little or no e ect on action potential duration (Fig. 24-10). By markedly decreasing the rate o phase 0 upstroke o ventricular cells, these drugs suppress premature ventricular contractions. Class IC antiarrhythmics also prevent paroxysmal supraventricular tachycardia and atrial f brillation. However, these drugs have marked depressive e ects on cardiac unction and, thus, must be used with discretion. In addition, the CAST (Cardiac Arrhythmia Suppression Trial) and other studies have brought attention to the proarrhythmic e ects o these agents. Flecainide is the prototypical class IC drug; other members o this class include encainide , moricizine , and propafenone . Flecainide illustrates the principle that antiarrhythmic agents can also cause arrhythmia. When ecainide is administered to patients with preexisting ventricular tachyarrhythmias and to those with a history o myocardial in arction, it can worsen the arrhythmia even at normal doses. Because ecainide is pro-arrhythmic in patients with ischemic or structural heart disease, its major use is or prevention o atrial arrhythmias (e.g., atrial f brillation) in patients with structurally normal hearts. Flecainide is eliminated very slowly rom the body; it has a plasma hal -li e o 12–30 hours. Because o its marked blockade o Na channels and its suppressive e ects on cardiac unction, ecainide use is associated with adverse e ects that include sinus-node dys unction, a marked decrease in conduction velocity, and conduction block.

Class II Antiarrhythmic Agents: -Adrenergic Antagonists

Class II antiarrhythmic agents are -adrenergic antagonists (also called -blockers). These agents act by inhibiting sympathetic input to the pacing regions o the heart. ( -Adrenergic antagonists are more extensively discussed in Chapter 11, Adrenergic Pharmacology.) Although the heart is capable o beating on its own without innervation rom the autonomic nervous system, both sympathetic and parasympathetic f bers innervate the SA node and the AV node and thereby alter the rate o automaticity. Sympathetic stimulation releases norepinephrine, which binds to 1-adrenergic receptors in the nodal tissues. ( 1-Adrenergic receptors are the adrenergic subtype pre erentially expressed in cardiac tissue.) Activation o 1-adrenergic receptors in the SA node triggers an increase in the pacemaker current (If), which increases the rate o phase 4 depolarization and, consequently,

C h a p t e r 2 4 Pharmacology of Cardiac Rhythm 445

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leads to more requent f ring o the node. Stimulation o 2 and 1-adrenergic receptors in the AV node increases Ca K currents, thereby increasing the conduction velocity and decreasing the re ractory period o the node. 1-Antagonists block the sympathetic stimulation o 1-adrenergic receptors in the SA and AV nodes (Fig. 24-11). The AV node is more sensitive than the SA node to the e ects o 1-antagonists. 1-Antagonists a ect the action potentials o SA and AV nodal cells by (1) decreasing the rate o phase 4 depolarization and (2) prolonging repolarization. Decreasing the rate o phase 4 depolarization results in decreased automaticity, and this, in turn, reduces myocardial oxygen demand. Prolonged repolarization at the AV node increases the e ective re ractory period, which decreases the incidence o re-entry. 1-Antagonists are the most requently used agents in the treatment o supraventricular and ventricular arrhythmias precipitated by sympathetic stimulation. 1-Adrenergic antagonists have been shown to reduce mortality a ter myocardial in arction, even in patients with relative contraindications to this therapy such as severe diabetes mellitus or asthma. Because o their wide spectrum o clinical application and established sa ety record, -adrenergic antagonists are the most use ul antiarrhythmic agents currently available. There are several generations o -antagonists, each characterized by slightly di erent pharmacologic properties. First-generation -antagonists, such as propranolol, are nonselective -adrenergic antagonists that antagonize both 1-adrenergic and 2-adrenergic receptors. They are widely used to treat tachyarrhythmias caused by catecholamine stimulation during exercise or emotional stress. Because propranolol does not prolong repolarization in ventricular tissue, it can be used in patients with long QT syndrome. Second-generation agents, including atenolol, metoprolol, acebutolol, and bisoprolol, are relatively selective or 1adrenergic receptors when administered in low doses.

Class III antiarrhythmic agents block K channels. Two types o currents determine the duration o the plateau phase o the cardiac action potential: inward, depolarizing Ca2 currents, and outward, hyperpolarizing K currents. During a normal action potential, the hyperpolarizing K currents eventually dominate, returning the membrane potential to more hyperpolarized values. Larger hyperpolarizing K currents shorten plateau duration, returning the membrane potential to its resting value more rapidly, while smaller hyperpolarizing K currents lengthen plateau duration and delay return o the membrane potential to its resting value. When K channels are blocked, a smaller hyperpolarizing K current is generated. There ore, K channel blockers cause a longer plateau and prolong repolarization (Fig. 24-12). The ability o K channel blockers to lengthen plateau duration is responsible or both their pharmacologic uses and their adverse e ects. On the benef cial side, prolongation o the plateau duration increases the e ective

(

action potentials. Class II antiarrhythmics ( -antagonists) reverse the tonic sympathetic stimulation of cardiac 1-adrenergic receptors. By blocking the adrenergic effects on the SA and AV nodal action potentials, these agents decrease the slope of phase 4 depolarization (especially important at the SA node) and prolong repolarization (especially important at the AV node). These agents are useful in the treatment of supraventricular and ventricular arrhythmias that are precipitated by sympathetic stimulation.

Class III Antiarrhythmic Agents: Inhibitors of Repolarization

l

FIGURE 24-11. Effects of class II antiarrhythmics on pacemaker cell

a

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Third-generation -antagonists cause vasodilation in addition to 1-receptor antagonism. Labetalol and carvedilol induce vasodilation by antagonizing -adrenergic receptormediated vasoconstriction; pindolol is a partial agonist at the 2-adrenergic receptor; and nebivolol stimulates endothelial production o nitric oxide. The di erent generations o -antagonists produce varying degrees o adverse e ects. Three general mechanisms are responsible or the adverse e ects o -blockers. First, antagonism at 2-adrenergic receptors causes smooth muscle spasm, leading to bronchospasm, cold extremities, and impotence. These e ects are more commonly caused by the nonselective f rst-generation -antagonists. Second, exaggeration o the therapeutic e ects o 1-receptor antagonism can lead to excessive negative inotropic e ects, heart block, and bradycardia. Third, drug penetration into the CNS can produce insomnia and depression.

-100

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Time (s e c) FIGURE 24-12. Effects of class III antiarrhythmics on the ventricular action potential. Class III antiarrhythmics (K channel blockers) decrease the magnitude of the repolarizing K currents during phase 2 of the action potential and thereby prolong action potential duration. This prolongation of the plateau phase decreases re-entry, but it can also predispose to early afterdepolarizations.

446 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

re ractory period, which, in turn, decreases the incidence o re-entry. On the toxic side, prolongation o the plateau duration increases the likelihood o developing early a terdepolarizations and torsades de pointes. With the exception o amiodarone, K channel blockers also exhibit the undesirable property o “reverse use-dependency”: action potential prolongation is most pronounced at slow rates (undesirable) and least pronounced at ast rates (desirable). K channel blockers have little or no e ect on the upstroke phase or conduction velocity o the impulse. Ibutilide is a class III agent that prolongs repolarization by inhibiting the delayed rectif er K current. This agent also enhances a slow inward Na current that urther prolongs repolarization. Ibutilide is used to terminate atrial f brillation and utter, as exemplif ed in the introductory case. The major adverse e ect o ibutilide results rom its prolongation o the QT interval; the serious arrhythmia torsades de pointes may result, requiring electrical cardioversion (delivery o an electrical shock to resynchronize the heart) in almost 2% o patients taking the drug. For this reason, Dr. J was closely monitored by a cardiologist during his ibutilide in usion. Ibutilide is generally not administered to patients with preexisting long QT syndrome. Dofetilide is a class III agent that is only available orally. It inhibits exclusively the rapid component o the delayed rectif er K current and has no e ect on the inward Na current. Do etilide increases the action potential duration and prolongs the QT interval in a dose-dependent manner. Because do etilide has the potential o inducing ventricular arrhythmias, it is reserved or patients with highly symptomatic atrial f brillation and/or atrial utter. Do etilide is used in the cardioversion o atrial f brillation and atrial utter to normal sinus rhythm, and it is e ective in the maintenance o sinus rhythm in such patients a ter cardioversion. Because do etilide has no negative inotropic e ects, it can be used in patients with depressed ejection unction. Similar to ibutilide, the major adverse e ect o do etilide is torsades de pointes, which occurs in 1–3% o patients taking the drug. Because do etilide is excreted by the kidneys, patients with renal dys unction must have their dose o drug reduced based on their creatinine clearance. Sotalol is a mixed class II and class III antiarrhythmic agent. This drug nonselectively antagonizes -adrenergic receptors (class II action), and it also increases action potential duration by blocking K channels (class III action). Sotalol exists in two isomeric orms: the l-isomer and d-isomer. While the two isomeric orms are equipotent in blocking K channels, the l- orm is a more potent -antagonist. Sotalol is used to treat severe ventricular arrhythmias, especially in patients who cannot tolerate the adverse e ects o amiodarone. Sotalol is also used to prevent recurrent atrial utter or f brillation and thereby to maintain normal sinus rhythm. Like other -antagonists, sotalol can cause atigue and bradycardia; like other class III antiarrhythmic agents, it can induce torsades de pointes. Amiodarone is mainly a class III antiarrhythmic agent, but it also acts as a class I, class II, and class IV antiarrhythmic. The ability o amiodarone to exert such a diverse range o e ects can be explained by its mechanism o action: alteration of the lipid membrane in which ion channels and receptors are located. In all cardiac tissues, amiodarone lengthens the e ective re ractory period by inhibiting the K channels responsible or repolarization; this prolongation o action

potential duration decreases re-entry. As a potent class I agent, amiodarone blocks Na channels and thereby decreases the rate o f ring in pacemaker cells; it exhibits usedependent Na channel blockade by binding pre erentially to channels in the inactivated con ormation. Amiodarone exerts class II antiarrhythmic activity by noncompetitively antagonizing -adrenergic and -adrenergic receptors. Finally, as a Ca2 channel blocker (class IV), amiodarone can cause signif cant AV nodal block and bradycardia, although ortunately its use is associated with a relatively low incidence o torsades de pointes. In recent years, the results o several clinical trials have caused the popularity o amiodarone to increase rom a last-resort agent to one that is requently used to treat li ethreatening arrhythmias, including ventricular tachycardia and ventricular f brillation. Amiodarone is also highly e ective in the prevention o recurrent paroxysmal atrial f brillation or utter, as exemplif ed in the introductory case. The wide spectrum o action o amiodarone is accompanied by a panoply o serious adverse e ects when the drug is used or long periods or in high doses. These e ects include cardiac, pulmonary, thyroid, hepatic, neurological, and idiosyncratic complications (Table 24-2). In the heart, amiodarone can decrease AV- or SA-node unction by blocking Ca2 channels. Amiodarone can exert a negative inotropic e ect by inhibiting -adrenergic receptors, especially when the drug is used chronically. As an -adrenergic antagonist, amiodarone can cause hypotension. Severe pulmonary complications can occur in patients taking high doses o amiodarone (400 mg daily or higher). Pneumonitis leading to pulmonary f brosis is the most dreaded o all complications associated with amiodarone use. Fortunately, such complications occur rarely in patients taking prophylactic doses (200 mg daily) or prevention o ventricular or atrial arrhythmias. Because o its structural similarity to thyroxine, amiodarone a ects thyroid hormone metabolism by inhibiting peripheral conversion o thyroxine (T4) to triiodothyronine (T3). Either hyperthyroidism or hypothyroidism can occur as a consequence o this dysregulation o thyroid hormone metabolism (see Chapter 28, Pharmacology o the Thyroid Gland). O patients taking amiodarone, 10–20% mani est an abnormal increase in liver enzymes, although this e ect is reversible when the dose o the drug is reduced.

TABLE 24-2 Major Adverse Effects of Amiodarone, Particularly at High Doses CATEGORY

ADVERSE EFFECT

Cardiovascular

↓ AV- or SA-node unction ↓ Cardiac contractility Hypotension

Pulmonary

Pneumonitis leading to pulmonary f brosis

Thyroid

Hyperthyroidism or hypothyroidism

Hepatic

Elevated liver enzymes

Neurological

Peripheral neuropathy, headache, ataxia, tremors

Other

Corneal microdeposits Testicular dys unction Skin discoloration

C h a p t e r 2 4 Pharmacology of Cardiac Rhythm 447

Class IV Antiarrhythmic Agents: Ca Blockers

2

Channel

Drugs that block cardiac Ca2 channels act pre erentially on SA and AV nodal tissues because these pacemaker tissues depend on Ca2 currents or the depolarization phase o the action potential (Fig. 24-2). In contrast, Ca2 channel blockers have little e ect on ast Na channel-dependent tissues, such as Purkinje f bers and atrial and ventricular muscle. The major therapeutic action of the class IV antiarrhythmics is to slow the action potential upstroke in AV nodal cells, leading to slowed conduction velocity through the AV node (Fig. 24-13). This also blocks re-entrant arrhythmias in which the AV node is part o the re-entry circuit. In the introductory case, however, the re-entry circuit responsible or atrial f brillation was isolated to the atria. This is why diltiazem, a Ca2 channel blocker, slowed Dr. J’s heart rate but did not change his underlying heart rhythm. (Re er to Chapter 22, Pharmacology o Vascular Tone, or a more extended discussion o Ca2 channel blockers.) Because di erent tissues express di erent subtypes o 2 Ca channels, and di erent subclasses o Ca2 channel blockers interact pre erentially with di erent Ca2 channel subtypes, the various Ca2 channel blockers have di erential e ects in di erent tissues. Dihydropyridines (such as nifedipine ) have a relatively greater e ect on the Ca2 current in vascular smooth muscle, while verapamil and diltiazem are relatively more selective or cardiac tissues. Verapamil and diltiazem are used to treat re-entrant paroxysmal supraventricular tachycardias because these are o ten re-entrant arrhythmias that involve the AV node. Verapamil and diltiazem are rarely used in ventricular tachycardia. In act, the only indications or these agents in ventricular arrhythmias are idiopathic right ventricular out ow tract tachycardia and ascicular tachycardias. Verapamil is also used to treat hypertension and vasospastic (Prinzmetal’s) angina. Class IV

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Neurological symptoms can include peripheral neuropathy, headache, ataxia, and tremors. Patients taking amiodarone should be monitored or abnormal pulmonary, thyroid, and liver unction. Amiodarone is contraindicated in patients with cardiogenic shock, second-degree or third-degree heart block, or severe SA-node dys unction with marked sinus bradycardia or syncope. Dronedarone is a class III agent that is structurally similar to amiodarone. It was developed in an attempt to create a drug with the antiarrhythmic e ects o amiodarone, while limiting the adverse e ects. Compared to amiodarone, dronedarone is less lipophilic (resulting in a shorter hal -li e) and lacks iodine moieties in its structure (thereby reducing the incidence o thyroid toxicity). In clinical studies o patients with atrial f brillation, dronedarone reduced the incidence o recurrent atrial f brillation when compared with placebo, with relatively ew adverse e ects. However, a separate study suggested that dronedarone is associated with increased mortality in a subgroup o patients with systolic heart ailure. Thus, dronedarone should be used with caution, i at all, in patients with systolic heart ailure. Dronedarone is also contraindicated in patients with permanent atrial f brillation (i.e., atrial f brillation that cannot be cardioverted to normal sinus rhythm), due to increased mortality. Recent reports have suggested an association between dronedarone and rare but severe hepatotoxicity. Liver unction tests should there ore be monitored periodically.

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Time (ms ) FIGURE 24-13. Effects of class IV antiarrhythmics on pacemaker cell action potentials. Class IV antiarrhythmics (Ca 2 channel blockers) decrease excitability of SA nodal cells and prolong AV nodal conduction, primarily by slowing the action potential upstroke in nodal tissue. Class IV antiarrhythmics are useful in the treatment of arrhythmias that involve reentry through the AV node, but high doses of Ca 2 channel blockers can prolong AV nodal conduction to such an extent that heart block results.

agents can cause AV nodal block by reducing the conduction velocity excessively. Administration o intravenous verapamil to patients taking -blockers can precipitate severe heart ailure and lead to irreversible electromechanical dissociation. Verapamil and diltiazem increase plasma digoxin levels by competing with digoxin or renal excretion.

Other Agents That Modulate Cardiac Rhythm or Ion Channels

Adenosine , while not considered a classical antiarrhythmic

agent, has important e ects on cardiac electrophysiology. Ranolazine is a recently approved agent or the treatment o chronic stable angina; its mechanism o action appears to involve inhibition o the late Na current. Ivabradine is an investigational agent that slows the heart rate by modulating the If current. Adenosine

The nucleoside adenosine is naturally present throughout the body. By stimulating the P1 class o purinergic receptors, adenosine opens a G protein-coupled K channel (IKACh) and thereby inhibits SA nodal, atrial, and AV nodal conduction (Fig. 24-9B). The AV node is more sensitive than the SA node to the e ects o adenosine. Adenosine also inhibits the potentiation o Ca2 channel activity by cAMP and thereby suppresses Ca2 -dependent action potentials. Adenosine has a plasma hal -li e o less than 10 seconds and is o ten used as the f rst-line agent or converting narrow-complex paroxysmal supraventricular tachycardia to normal sinus rhythm. For this indication, it is e f cacious in 90% o cases. Most adverse e ects o adenosine are transient, including headache, ushing, chest pain, and excessive AV or SA nodal inhibition. Adenosine can also cause bronchoconstriction lasting or up to 30 minutes in patients with asthma. In many patients, a transient new arrhythmia occurs at the onset o adenosine administration.

448 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

Ranolazine

Some patients with chronic stable angina have chest pain with exertion despite mechanical revascularization and use o -adrenergic antagonists or calcium channel blockers. Ranolazine is a recently approved agent that improves exercise capacity and reduces anginal events in patients with chronic stable angina. Despite extensive evaluation, the exact mechanism o action o ranolazine is uncertain. Proposed mechanisms o action include inhibition o cardiac myocyte atty acid -oxidation, inhibition o the delayed rectif er K current, and inhibition o the late Na current. Inhibition o atty acid -oxidation may improve myocardial ATP utilization, while inhibition o Na channel activity may reduce the energy required or myocardial repolarization. Ranolazine has been generally well tolerated in clinical trials; its most common adverse e ects are nausea, constipation, and dizziness. Ranolazine also prolongs the QT interval. It is currently approved as a second-line treatment or patients with chronic stable angina. Ivabradine Ivabradine inhibits the I current responsible or phase 4 de-

polarization in SA nodal cells. By inhibiting phase 4 depolarization, ivabradine slows the heart rate, thereby decreasing myocardial oxygen demand. Ivabradine is currently approved in Europe or use in patients with chronic stable angina but remains investigational in the United States. It is also occasionally used to treat inappropriate sinus tachycardia, a syndrome where the pacemaker cells f re more than 100 times per minute without any secondary cause. Ivabradine is contraindicated in patients with preexisting bradycardia.

CONCLUSION AND FUTURE DIRECTIONS Cardiac arrhythmias arise rom de ects in impulse ormation, de ects in impulse conduction, or a combination o the two mechanisms. Because derangements in ion conductance lead to arrhythmias, antiarrhythmic agents act directly or indirectly to alter the con ormational states o ion channels and thereby change the membrane permeability to ions. The pharmacologic property o use-dependent ion channel blockade allows many antiarrhythmic agents to target diseased cardiac tissues pre erentially based on the altered electrophysiology o these tissues. In general, class I antiarrhythmics block Na channels; class II antiarrhythmics ( -blockers) inhibit sympathetic stimulation and thereby decrease automaticity; class III agents block K channels; and class IV agents block Ca2 channels. Despite continuing developments in antiarrhythmic drugs, the paradox still exists that antiarrhythmic drugs can also generate arrhythmias. Nonetheless, judicious use o antiarrhythmic agents can reduce mortality in certain clinical circumstances, and care ul tailoring o a drug regimen to the individual patient’s clinical status can reduce the adverse e ects o these agents. The most important new directions in the pharmacology o cardiac rhythm involve identi ying specif c genes or ion channels in the human heart (Table 24-3). Currently, animal models are used or the majority o ion channel

TABLE 24-3 Molecular Identity of Known Cardiac Ion Currents ION CURRENT

CHANNEL PROTEIN

INa

Na V1.5

ICa.L (dihydropyridine-sensitive)

Ca V1.2

ICa.T

Ca V3.1

If

HCN2, HCN4

Ito

KV4.3

IKs *

KV7.1 (KvLQT1)

IKr*

KV11, HERG

IK1

Kir2.1 (inward rectif er)

IKACh

Kir3.1 Kir3.4 (G protein-gated)

*Collectively re erred to as IK.

research; comparatively little is known about the clinical pharmacology o ion channels expressed in humans. With the mouse and human genomes now completely sequenced, researchers will be able to investigate the possibility that newly identif ed gene products can serve as selective targets or new therapeutic agents. The identif cation o ion channel gene expression in the various tissues o the human heart (SA node, AV node, atrial conduction pathways, endocardium, ventricular conduction pathways, etc.), both during development and in response to injury, may provide new targets that are not now known. Many o the genes are likely to encode channels that orm heteromultimers, and there are likely to be many genetic variants within the population. This enormous complexity will likely represent a boon to drug development because it will allow more tailored strategies to be employed. For example, current research in atrial f brillation has ocused on the development o antiarrhythmics selective or ion channels that are expressed selectively in the atria. In parallel, the development o implantable computers, stimulators, and def brillators will constitute an alternative strategy to prevent or terminate arrhythmias.

Suggested Reading Ackerman MJ, Clapham DE. Ion channels—basic science and clinical disease. N Engl J Med 1997;336:1575–1586. (Broad review o ion channels.) Dobrev D, Nattel S. New antiarrhythmic drugs or treatment o atrial f brillation. Lancet 2010;375:1212–1223. (Future directions in drug development or treatment o atrial f brillation.) Link MS. Clinical practice. Evaluation and initial management o supraventricular tachycardia. N Engl J Med 2012;367:1438–1448. (Discussion o the clinical uses o antiarrhythmic agents in treating supraventricular tachycardia.) Rudy Y, Silva JR. Computational biology in the study o cardiac ion channels and cell electrophysiology. Q Rev Biophys 2006;39:57–116. (Summarizes the known cardiac ion channels in models o cardiac action potentials.) Swedberg K, Komajda M, Böhm M, et al. Ivabradine and outcomes in chronic heart ailure (SHIFT): a randomised placebo-controlled study. Lancet 2010;376:875–885. (Large trial suggesting that ivabradine may benef t patients with heart ailure.)

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C h a p t e r 2 4 Pharmacology o Cardiac Rhythm 453

2

S a rc rcoole o le mma m maa

25

1

xtruu s ion C a 2+ e xt

C a 2+

3Na +

3Naa + 3N

Naa + extrus ioo n 2K K+ Dig oxiin

Pharmacology of Cardiac Contractility ha ngg e r Naa +/Ca 2+ excha

Ca 2 +

Na +/K / + -A ATP T ase

3Na + 3N

33Na Na +

2K+

Eh rin J . Arm s t ro n g 3

Ca 2+ s tore s

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . ADP . . . P. . . . . . . 454––455 AD 2+ CaN . . . . . . . . . . . .P. . . . . 45 PHYSIOLOGY OF CARD RDIA IAC C CO CONT TRA ACT CTIO ION 454 54 Myocyte Anat atom omyy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4555 Myoc My ocyt yte Contraction . . . . . . . . . .ATP . . . . . . . . . . . . . . . . . . . . . . . . 455 Regulation of Contractility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456 The Sodium Pump and 2+ Sodiu odium– m Ca Calc lciu ium Exchange . . . . . . . . . . . . . . . Ca . . . . . . . . . 456 Callciium Storage annd Re Rele leaa s e . . . . . . . . . . . . . . . . . . . . . . . . 457 Adrenergic Receppto or S Signal gnalin ingg and Calcium Cyclingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 Sensitivityy off Con ontr trac acti tile le Protei ro ins to Calcium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 PATHOPHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Cellular Pathophysiology of Contractile Dysfunction. . . . . . . 459

INTRODUCTION In 1785, Dr. William Withering described the cardiovascular benef ts o a preparation rom the oxglove plant called digitalis. He used this preparation to treat patients su ering rom “dropsy,” a condition in which accumulation o extravascular uid leads to dyspnea (di f culty breathing) and peripheral edema. These symptoms are now recognized as characteristic mani estations o heart failure (HF), a clinical syndrome most commonly caused by systolic dys unction o the le t ventricle (LV). In this condition, the LV is unable to maintain adequate stroke volume despite normal f lling volumes, and the LV end-diastolic volume increases in an e ort to preserve stroke output. However, beyond a certain enddiastolic volume, LV diastolic pressures begin to increase, o ten precipitously. This increase in LV diastolic pressure results in increased le t atrial and pulmonary capillary pressures, which, in turn, lead to interstitial and alveolar pulmonary edema and to increased right heart and pulmonary artery pressures. The elevated right heart pressures result in systemic venous hypertension and peripheral edema. Dr. Withering’s use o digitalis presaged the current use o digoxin, a member o the cardiac glycoside amily o drugs, to treat conditions in which myocardial contractility is impaired. Cardiac glycosides are positive inotropes , def ned as agents that increase the contractile force of cardiac myocytes. Since the advent o digitalis, elucidation o the cellular mechanism o cardiac contraction has acilitated the development o other inotropic agents. A ter reviewing the physiology o 454

4

Myofibril contraa ccti tion on

PHARMA PHAR MAC COLOGIC CLASSES S AN AND D AG AGEN E TS . . . . . . . . . . . . . . . . 461 EN Cardiac di c Glycosides Glycos o2+ iddes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 s tore toor tor ore re e d C Ca a Digo Di Digoxin g xinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 goxi Digitoxin t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 -Adrenergic e g Receptor Agoni en nist stss . . . . . . . . . . . . . . . . . . . . . . . 463 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 re leeDopamine ased C Caa 2+ Dobutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464 Epinepphrrin inee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464 Norepinephrine i h i e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464 Isop Is opro rote tere reno noll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464 Phosphodiesterase (PDE) Inhibitors . . . . . . . . . . . . . . . . . . . . . 46 4644 Calcium-Sensitizing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 465 Sugg Su gges este tedd Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46 4655

cardiac contraction and the cellular pathophysiology o contractile dys unction, this chapter describes our classes o positive inotropic drugs that are either approved or use or under investigation in clinical trials. An integrated discussion o therapeutic strategies or HF can be ound in Chapter 26, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure.

PHYSIOLOGY OF CARDIAC CONTRACTION The heart is responsible or receiving deoxygenated blood rom the periphery, propelling this blood through the pulmonary circulation (where the hemoglobin is reoxygenated), and ultimately distributing the oxygenated blood to peripheral tissues. To accomplish the latter task, the le t ventricle (LV) must develop su f cient tension to overcome the impedance to ejection that resides in the peripheral circulation. The relationship between the tension generated during the systolic phase o the cardiac cycle and the extent o LV f lling during diastole is reerred to as the contractile state o the myocardium. Together with preload (intraventricular blood volume), afterload (the resistance against which the le t ventricle ejects), and heart rate , myocardial contractility is a primary determinant o cardiac output. Cardiac pump per ormance at the organ level has been studied by cardiac physiologists or many years, but now the major cellular and molecular mechanisms o cardiac contraction are understood as well.

C h a p t e r 2 5 Pharmacology of Cardiac Contractility 455

GW, a 68-ye a r-o ld m a n w ith kn o w n s ys to lic d ys u n ctio n a n d h e a rt a ilu re , is a d m itte d to th e h o s p ita l w ith s h o rtn e s s o b re a th a n d n a u s e a . GW’s ca rd ia c h is to ry is n o ta b le o r tw o p rio r m yo ca rd ia l in a rctio n s , th e m o re re ce n t o ccu rrin g a b o u t 2 ye a rs a g o . S in ce th e s e co n d in a rctio n , h e h a s h a d s ig n if ca n t lim ita tio n o h is e xe rcis e ca pa city. A tw o -d im e n s io n a l e ch o ca rd io g ra m is n o ta b le o r a n LV e je ctio n ra ctio n o 25% (n o rm a l, 55%) a n d m o d e ra te m itra l va lve re g u rg ita tio n . GW h a s b e e n tre a te d with as p irin , ca rve d ilo l (a -a d re n e rg ic re ce p to r a n ta g o n is t), ca p to p ril (a n a n g io te n s in co nve rtin g e n zym e in h ib ito r), d ig o xin (a ca rd ia c g lyco s id e ), u ro s e m id e (a lo o p d iu re tic), a n d s p iro n o la cto n e (an a ld o s teron e re cep to r a n tag o nis t). He h as also h ad an a u to m a tic in te rn al card iove rte r-d ef b rillato r (AICD) p la ce d to p re ve n t s u s ta in e d ve n tricu la r a rrhyth m ia a n d s u d d e n ca rd ia c d e a th . Phys ica l e xa m in a tio n in th e e m e rg e n cy d e p a rtm e n t is n o ta b le o r a b lo o d p re s s u re o 90/50 m m Hg a n d a n irre g u la r h e a rt ra te o 120 b e a ts /m in . An e le ctro ca rd io g ra m in d ica te s th a t th e u n d e rlyin g ca rd ia c rhyth m is a tria l f b rilla tio n . GW is s ta rte d o n a m io d a ro n e (a cla s s III a n tia rrhyth m ic), a n d h is h e a rt ra te d e cre a s e s to a p p ro xim a te ly 80 b e a ts /m in . La b o ra to ry te s ts a re n o ta b le o r s e ru m Na 148 m Eq /L (n o rm a l, 135–145), BUN 56 m g /d L (n o rm a l, 7–19), K 2.9 m Eq /L (n o rm a l, 3.5–5.1), a n d cre a tin in e

Myocyte Anatomy Like skeletal muscle, cardiac muscle contracts when action potentials depolarize the plasma membranes o cardiac muscle cells. The process o excitation–contraction (EC) coupling, in which the intracellular machinery transduces an electrochemical signal into mechanical orce, involves the ollowing cascade o events: voltage-gated calcium channels open, intracellular calcium increases, contractile proteins are activated, and actin–myosin interactions shorten the contractile elements. The cellular anatomy o ventricular myocytes is well suited to the excitation and regulation o cardiac contraction (Fig. 25-1). Specialized components o the ventricular myocyte include the sarcolemma, or myocyte plasma membrane; the sarcoplasmic reticulum (SR), a large internal membrane system that encircles the myof brils; and the myof brils themselves. Myof brils are rope-like units containing precisely organized contractile proteins; the coordinated interaction o these proteins is responsible or the physical shortening o the cardiac muscle. These anatomic specializations are illustrated in Figures 25-1 and 25-2, and summarized in Table 25-1.

Myocyte Contraction Increased cytosolic Ca2 is the link between excitation and contraction. During the ventricular action potential (see Chapter 24, Pharmacology o Cardiac Rhythm), Ca2 in ux through L-type Ca2 channels in the sarcolemma causes an

4.8 m g /d L (n o rm a l, 0.6–1.2). Th e s e ru m d ig oxin le ve l is 3.2 n g /m L (th e ra p e u tic co n ce n tra tio n , typ ica lly 1 n g /m L). Ba s e d o n th e s e f n d in g s , GW is a d m itte d to th e ca rd io lo g y in te n s ive ca re u n it (ICU). His o ra l d ig oxin d o s e is h e ld , a n d h e is g ive n in tra ve n o u s K to in cre a s e h is s e ru m p o ta s s iu m co n ce n tra tio n . Ba s e d o n th e s e ve rity o th is clin ica l d e co m p e n s a tio n , a p u lm o n a ry a rte ry (PA) ca th e te r is p la ce d to m o n ito r ca rd ia c p re s s u re s . GW is a ls o s ta rte d o n d o b u ta m in e , a n d h is ca rve d ilo l is h e ld . A te r in itia tio n o in tra ve n o u s d o b u ta m in e , h e h a s in cre a s e d u rin e o u tp u t a n d b e g in s to e e l s ym p to m a tica lly im p rove d . He re m a in s in th e ca rd io lo g y ICU o r 7 d a ys , a n d h is d ig o xin le ve l d e cre a s e s to th e th e ra p e u tic ra n g e .

Questions 1 . What are the major cellular mechanisms that contribute to the pathophysiology of systolic heart failure? 2 . What is the mechanism of action of digoxin? 3 . What factors (including drug interactions) have contributed to digoxin toxicity in this patient? 4 . Why is GW being treated with a -adrenergic receptor antagonist and a positive inotrope (digoxin) at the same time? 5 . What is the mechanism of action of dobutamine?

increase in the cytosolic Ca2 concentration. This “trigger calcium” stimulates the ryanodine receptor in the SR membrane, causing release o stored Ca2 rom the SR into the cytosol. When the Ca2 concentration in the cytoplasm reaches approximately 10 5 M, calcium binds to troponin C and induces a con ormational change in tropomyosin that releases the inhibitory protein troponin I. This release o troponin I exposes an interaction site or myosin on the actin f lament, and the binding o myosin to actin initiates the contraction cycle. Figure 25-2 illustrates the cycle by which actin–myosin interactions physically shorten the sarcomere. Each myosin f lament is studded with protruding exible heads that orm reversible cross-bridges with actin f laments. Formation o actin–myosin cross-bridges, bending o the myosin heads at their exible hinges, and detachment o the cross-bridges together allow the myosin f lament to “walk up” the actin f lament in both directions and thereby to pull the two ends o the sarcomere together. The normal unction o the sarcomeric cross-bridge cycle is critically dependent on adenosine triphosphate (ATP). The ATP hydrolase (ATPase) activity o myosin provides the energy used both to drive contraction and to reset the contractile proteins, leading to relaxation. I an insu f cient amount o ATP is available or cross-bridge cycling, myosin and actin remain “locked” in the associated state and the myocardium is unable to relax. This ATP dependence explains the pro ound impact o ischemia on both systolic

456 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY T-tubule

S a rcotubula r ne twork Te rmina l cis te rna

Ca 2+

T-tubule

Ca 2+

S a rcole mma Myofibrils

Mitochondrion

Ca 2+

S a rcopla s mic re ticulum

S a rcome re

I ba nd

A ba nd

Z line

Z line

A ba nd Actin

Z line Myos in

FIGURE 25-1. Cardiac myocyte structure. Each cardiac myocyte contains myof brils and mitochondria surrounded by a specialized plasma membrane termed the sarcolemma. Invaginations o the sarcolemma, called T-tubules, provide conduits or Ca 2 in ux. Within the cell, an extensive sarcoplasmic reticulum stores Ca 2 or use in contraction. Extracellular Ca 2 enters through the sarcolemma and its T-tubules during phase 2 o the action potential. This trigger Ca 2 binds to channels on the sarcoplasmic reticulum membrane, causing release into the cytosol o a large pool o so-called activation Ca 2 . Increased cytosolic Ca 2 initiates myof bril contraction. The sarcomere is the unctional unit o the myof bril. Each sarcomere consists o interdigitating bands o actin and myosin. These bands orm distinctive structures that can be visualized using an electron microscope. The A bands correspond to regions o overlapping actin and myosin. The Z lines demarcate the borders o each sarcomere. The I bands span neighboring sarcomeres and correspond to regions o actin without overlapping myosin. During cardiac myocyte contraction, the I bands become shorter (i.e., the Z lines move closer to one another), but the A bands maintain a constant length.

contraction (the contraction cycle cannot proceed) and diastolic relaxation (actin and myosin cannot dissociate) o the myocardium. The organization o the sarcomere and the physical mechanism o contraction explain the undamental relationship between muscle length and tension development. Increased stretch (length) o the muscle exposes additional sites or calcium binding and or actin–myosin interaction; increased stretch also e ects greater release o calcium rom the SR. These cellular events provide the mechanistic explanation or the Frank-Starling law : an increase in the end-diastolic volume of the left ventricle leads to an increase in ventricular stroke volume during systole. Chapter 26 describes the organlevel implications o the Frank-Starling law in more detail.

Regulation of Contractility Three major control mechanisms regulate calcium cycling and myocardial contractility in cardiac myocytes. At the sarcolemma, calcium f ux is mediated by interactions between the sodium pump and sodium–calcium exchanger. At the sarcoplasmic reticulum, calcium channels and pumps regulate the extent o calcium release and reuptake. Neurohumoral inf uences, especially the -adrenergic signaling pathway,

urther modulate calcium cycling through these channels and transporters.

The Sodium Pump and Sodium–Calcium Exchange

In the sarcolemma, three key proteins are involved in calcium regulation: the Na /K -ATPase, herea ter re erred to as the sodium pump; the sodium–calcium exchanger; and the calcium–ATPase or calcium pump (Fig. 25-3). The activity o the sodium pump is crucial to maintain both the resting membrane potential and the concentration gradients o sodium and potassium across the sarcolemma ([Na ]out 145 mM, [Na ]in 15 mM, [K ]out 5 mM, [K ]in 150 mM). Sodium pump activity is closely linked to the intracellular calcium concentration via the sodium–calcium exchanger; this antiporter exchanges sodium and calcium in both directions across the sarcolemma. Changes in the concentration o either sodium or calcium ions inside or outside the cell a ect the direction and magnitude o sodium–calcium exchange. Under normal conditions, the low intracellular sodium concentration avors sodium inf ux and calcium e f ux. Some drugs take advantage o the unctional coupling between the sodium pump and the sodium–calcium exchanger to exert their e ect as positive inotropes. Digoxin, discussed in the

C h a p t e r 2 5 Pharmacology o Cardiac Contractility 457

receptor) and the calcium pump (sarcoendoplasmic reticulum calcium ATPase, SERCA) are critical to the regulation o con-

Tropomyos in Actin filament TN-I

TN-T

TN-C

Troponin comple x 1 ATP hydrolys is

ADP

ATP

P

Myos in Ca 2+

Re la xe d

Re la xe d, e ne rgize d

4

2 Dis s ocia tion of a ctin a nd myos in

Forma tion of a ctive comple x Ca 2+

ATP

Ca 2+

Ca 2+

3 P roduct dis s ocia tion

Rigor comple x

ADP P

tractility (Fig. 25-3). Proper contraction requires both that Ca2 release into the cytoplasm is adequate to stimulate contraction and that Ca2 reuptake into the SR is su f cient to permit relaxation and to replenish calcium stores. Cytoplasmic concentrations o both calcium and ATP regulate the activity o both the ryanodine receptor and SERCA. As noted above, trigger calcium opens the ryanodine receptor. Cytoplasmic calcium concentration is directly related to the number o receptors that open. A sa ety mechanism also exists whereby high calcium levels lead to calcium– calmodulin complex ormation: this complex inhibits calcium release by decreasing the open time o the ryanodine receptor. High concentrations o ATP avor the open channel con ormation and thereby acilitate SR calcium release into the cytosol. In addition to opening the ryanodine receptor, cytoplasmic calcium also stimulates SERCA, which pumps calcium back into the SR. This pump provides another control mechanism to prevent a positive eedback cycle that could irreversibly deplete the SR o calcium. As calcium pumps ref ll the SR, the rate o Ca2 reuptake slows because o the declining cytoplasmic calcium concentration. ATP also avors SERCA activity; conversely, decreased ATP impairs calcium reuptake. The latter mechanism causes the rate and extent o diastolic relaxation to decrease in ischemic myocardium.

Active comple x ADP + P

FIGURE 25-2. Cardiac contractile proteins and the contraction cycle. During contraction, myosin ratchets along actin f laments, resulting in an overall shortening o sarcomere length. Actin f laments (top panel) consist o two actin polymers wound around one another, three troponin proteins (TN-I, TN-C, and TN-T), and tropomyosin. In the absence o Ca 2 , tropomyosin is oriented on actin so that it inhibits the interaction o actin with myosin. The contraction cycle (bottom panel) occurs in a our-step process. 1. Cardiac myocyte contraction begins with hydrolysis o ATP to ADP by myosin; this reaction energizes the myosin head. 2. Ca 2 released rom the sarcoplasmic reticulum binds to TN-C; this reaction causes a con ormational change in tropomyosin that allows myosin to orm an active complex with actin. 3. Dissociation o ADP rom myosin allows the myosin head to bend; this bending pulls the Z lines closer together and thus shortens the I band (not shown). This contracted state is o ten re erred to as a rigor complex because muscle remains in the contracted state until there is su f cient ATP available to displace the myosin heads rom actin. 4. Binding o a new ATP molecule to myosin allows the actin–myosin complex to dissociate. Ca 2 also dissociates rom TN-C, and the contraction cycle is repeated.

introductory case and described in detail below, is the prototype o an inotropic agent that acts by inhibiting the sodium pump. A sarcolemmal calcium pump also helps to maintain calcium homeostasis by actively extruding calcium rom the cytoplasm a ter cardiac contraction. A high concentration o ATP avors calcium removal (relaxation), both directly via the calcium pump and indirectly via the sodium pump.

Calcium Storage and Release

As described above, Ca2 signaling is central to both cardiac contraction and relaxation. As such, the cardiac myocyte has well-developed systems to regulate Ca2 ux during the cardiac cycle. In the SR, the calcium release channel (ryanodine

TABLE 25-1 Functional Anatomy o Cardiac Myocyte Contraction Sarcolemma T-tubules

Invaginations o sarcolemma, acilitate ion ux across the cell membrane

Voltage-gated L-type Ca 2 channels

Mediate in ux o trigger Ca 2 ions when sarcolemma is depolarized

Sarcoplasmic Reticulum (SR) Ca 2 release channels

Stimulated by trigger Ca 2 , release internal Ca 2 stores

Ca 2 -ATPase pumps

Sequester intracellular Ca 2 in SR to terminate contraction

Terminal cisternae

Sacs at distal branches o SR that store Ca 2

Myof bril Sarcomere

Basic contractile unit o the myof bril

Myosin

Thick f lament, hydrolyzes ATP or energy

Actin

Thin f lament, provides sca olding or myosin binding

Tropomyosin

Coils around actin, preventing actin–myosin binding at rest

Troponin complex:

Complex o three proteins that regulate actin–myosin binding: Binds troponin complex to tropomyosin Inhibits actin–myosin binding at rest Binds Ca 2 , displacing troponin I rom actin–myosin binding site

Troponin T Troponin I Troponin C

458 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY A

S a rcole mma Ca 2+ 2 Ca 2+ - induce d Ca 2+ re le a s e

1

o relaxation by regulating calcium reuptake into the SR: unphosphorylated phospholamban slows relaxation, while phosphorylated phospholamban accelerates relaxation.

Adrenergic Receptor Signaling and Calcium Cycling

T-tubule

1-Adrenoceptor

Ca 2+

Ca 2+

Ca Ca2+2+

Ca 2+

3 Myofibril contra ction fre e Ca 2+

stimulation supports cardiac per ormance in several ways. First, -receptor agonists increase 1-adrenoceptormediated increases in Ca2 entry during systole; increased Ca2 entry increases ractional shortening o cardiac muscle during contraction. This positive inotropic e ect results in a higher stroke volume or any given end-diastolic volume. -Agonists also have a positive chronotropic e ect, increasing heart rate in a relatively linear dose-dependent manner. The net e ect o these inotropic and chronotropic e ects is to increase cardiac output: CO

B

4 Na +/Ca 2+ e xcha nge Ca 2+

5 Na +/K+ ATP a s e

3Na +

3Na +

2K+

Na +/K+ ATP a s e

NCX ATP

Ca 2+

3Na +

ADP

3Na +

2K+

6 Cytopla s m

P hos phola mba n

ADP

Ca 2+

Ca 2+ PKA

S ERCA

S a rcopla s mic re ticulum

P

ATP

Ca 2+

FIGURE 25-3. Regulation o cardiac myocyte Ca 2 f ux. A. During contraction: 1. Extracellular Ca 2 enters the cardiac myocyte through Ca 2 channels in the sarcolemma. 2. This trigger Ca 2 induces release o Ca 2 rom the sarcoplasmic reticulum into the cytosol (so-called Ca 2 -induced Ca 2 release). 3. The increased cytosolic Ca 2 acilitates myof bril contraction. B. During relaxation: 4. The Na /Ca 2 exchanger (NCX) extrudes Ca 2 rom the cytosol, using the Na gradient as a driving orce. 5. The Na /K ATPase (sodium pump) maintains the Na gradient, thus keeping the cardiac myocyte hyperpolarized. The sodium pump is tonically inhibited by phospholemman; phosphorylation o phospholemman by protein kinase A (PKA) disinhibits the pump, thereby increasing sodium extrusion and indirectly enhancing Na /Ca 2 exchange (not shown). 6. The sarcoendoplasmic reticulum Ca 2 ATPase (SERCA) in the sarcoplasmic reticulum membrane is tonically inhibited by phospholamban. Phosphorylation o phospholamban by PKA disinhibits the Ca 2 ATPase, allowing sequestration o cytosolic Ca 2 in the sarcoplasmic reticulum. A sarcolemmal Ca 2 ATPase (calcium pump) also helps to maintain calcium homeostasis by actively extruding calcium rom the cytoplasm (not shown).

A third mediator o SERCA activity is phospholamban, an SR membrane protein that inhibits SERCA. High levels o intracellular cAMP stimulate protein kinase A to phosphorylate phospholamban, which reverses its inhibition o SERCA (Fig. 25-3). Phospholamban thus controls the rate

HR

SV

Equation 25-1

where CO is cardiac output, HR is heart rate, and SV is stroke volume. A third, but less widely appreciated, mechanism by which -agonists support cardiac per ormance is by enhancing the rate and extent o diastolic relaxation (sometimes called the positive lusitropic e ect). This is a critical permissive e ect o 1-receptor stimulation because it acilitates maintenance o adequate LV f lling (i.e., preservation o LV end-diastolic volume), despite the reduction in diastolic f lling time that occurs as heart rate increases. In the peripheral circulation, the e ects o sympathetic stimulation are more complex. Activation o peripheral 2-receptors dilates vascular smooth muscle, but 1-receptor stimulation constricts vascular smooth muscle. Thus, 2-receptor stimulation typically decreases systemic vascular resistance (SVR) and afterload, while 1-receptor stimulation increases SVR and afterload. Dopamine receptors in the splanchnic and renal circulations also modulate the resistance vessels in these vascular beds, as discussed below. As noted above, the cardiostimulatory actions o the sympathetic nervous system are mediated by activation o 1-, 2-, and 1-adrenergic receptor subtypes located in the heart and peripheral vasculature. Stimulation o these G protein-coupled receptors induces con ormational changes in the receptors and their associated G proteins that activate adenylyl cyclase and thereby elevate intracellular cAMP levels (Fig. 25-4). Higher levels o cAMP activate protein kinase A, which phosphorylates multiple targets in the cell. These targets include L-type calcium channels in the sarcolemma and phospholamban in the SR membrane. As discussed above, the phosphorylation o phospholamban releases its inhibition o SERCA, allowing calcium to be pumped rom the cytosol back into the SR; this is one o the molecular mechanisms o enhanced diastolic relaxation induced by 1-adrenoceptor stimulation.

Sensitivity of Contractile Proteins to Calcium

As mentioned above, the tension developed by cardiac myocytes during contraction is directly related to the precontraction length o the sarcomere units. Increased stretch o the sarcomeres exposes more calcium binding sites on troponin C, making more sites available or actin–myosin cross-bridge ormation and thereby increasing the sensitivity o the contractile proteins to calcium. Several other mechanisms also regulate contractile protein sensitivity. Phosphorylation o troponin I by protein kinase A (a process that, like phospholamban phosphorylation, depends on cAMP levels) decreases contractile protein sensitivity to calcium. Expression o various iso orms o the contractile proteins,

C h a p t e r 2 5 Pharmacology o Cardiac Contractility 459

particularly troponin T, has also been linked to altered calcium sensitivity. Pharmacologic agents that sensitize contractile proteins to calcium are under investigation.

Ca rdia c myocyte

PATHOPHYSIOLOGY β1 -re ce ptor

β1-agonist

Ade nylyl cycla s e

β

Ca 2+

P

αs

γ

Ca 2+

GTP Amrinone

ATP

Phosphodiesterase AMP

cAMP

P KA

P KA

ina ctive

a ctive

P hos phola mba n ADP

P

Ca 2+ ATP

Ca 2+ S a rcopla s mic re ticulum

FIGURE 25-4. Regulation of cardiac contractility by

-adrenergic receptors. -Adrenergic receptors both increase cardiac myocyte contractility and enhance relaxation. Binding o an endogenous or exogenous agonist to 1-adrenergic receptors on the sur ace o cardiac myocytes causes G s proteins to activate adenylyl cyclase, which in turn catalyzes the conversion o ATP to cAMP. cAMP activates multiple protein kinases, including protein kinase A (PKA). PKA phosphorylates and activates sarcolemmal Ca 2 channels and thereby increases cardiac myocyte contractility. PKA also phosphorylates phospholamban and thereby disinhibits the SERCA pump, which pumps Ca 2 into the sarcoplasmic reticulum; the increased rate o Ca 2 sequestration enhances cardiac myocyte relaxation. Finally, PKA phosphorylates phospholemman, thereby disinhibiting the sarcolemmal sodium pump and enhancing sarcolemmal Na /Ca 2 exchange (not shown). The conversion o cAMP to AMP by phosphodiesterase terminates 1-adrenergic receptor-mediated actions. Phosphodiesterase is inhibited by amrinone (also known as inamrinone), a drug that can be used in the treatment o heart ailure.

TABLE 25-2 Effects of Increased Intracellular cAMP in Cardiac Cells Sarcolemma

↑ Phosphorylation o voltage-gated Ca 2 channel → ↑ contractility, heart rate, and AV conduction ↑ Phosphorylation o phospholemman → ↑ Ca 2 e f ux rom cytoplasm via Na /Ca 2 exchange

Sarcoplasmic reticulum

↑ Phosphorylation o phospholamban → ↑ Ca 2 uptake and release

Contractile proteins

↑ Phosphorylation o troponin I → ↓ Ca 2 sensitivity

Energy production

↑ Glycogenolysis → ↑ ATP availability

Many disease processes can lead to myocyte dys unction or death, causing replacement o myocardium with f brous tissue and leading to impaired contractility. The most common etiology o contractile dys unction in the United States is coronary artery disease (CAD) resulting in myocardial inarction; other common etiologies o contractile dys unction include systemic hypertension and valvular heart disease. In each o the a orementioned disease states, dys unction o the cardiac myocyte occurs as a consequence o a nonmyocardial disease process. A less common cause o LV dys unction is idiopathic cardiomyopathy, in which the principal abnormality occurs at the level o the cardiac myocyte. Irrespective o underlying etiology, progressive contractile dys unction o the myocardium leads ultimately to the syndrome o systolic heart failure (HF). It is important to note, however, that HF can occur in the absence o contractile dys unction. For example, several common cardiovascular disease states—such as acute myocardial ischemia and restrictive cardiomyopathy—are associated with abnormalities o LV relaxation and/or f lling, leading to decreased chamber compliance and elevated LV diastolic pressure. This abnormal elevation o intraventricular pressure can occur even in the presence o normal systolic unction, leading to a syndrome re erred to as diastolic heart failure (also known as heart failure with preserved ejection fraction). The organlevel pathology and treatment o HF are discussed in detail in Chapter 26. Here, we ocus on the salient cellular and molecular aspects o normal and abnormal contractile unction. The clinical expression o HF o ten re ects the impact o neurohumoral systems that are activated by inadequate orward cardiac output. In advanced stages o the disease, it may be di f cult to determine whether the cellular abnormalities observed in ailing cardiac myocytes re ect primary cellular de ects or secondary responses to extracardiac stimuli (such as circulating cytokines and neuroendocrine peptides). Nonetheless, the cellular and molecular alterations in the ailing myocardium can be contrasted with the events o normal contraction in an e ort to obtain mechanistic insight, and many o these changes are active areas o investigation. Study o these alterations also promises to identi y potential new molecular targets or pharmacologic intervention.

Cellular Pathophysiology of Contractile Dysfunction At the cellular level, pathophysiologic mechanisms associated with decreased cardiac contractility include dysregulation o calcium homeostasis, changes in the regulation and expression pattern o contractile proteins, and alterations in -adrenoceptor signal transduction pathways (Fig. 25-5). As noted above, some o these alterations may result rom local myocardial pathology, whereas others likely represent responses to circulating hormonal and in ammatory signals. Altered calcium homeostasis in ailing cardiac myocytes results in prolongation o the action potential and o the Ca2 transient associated with each contraction. Mechanisms that increase the cytosolic concentration o Ca2 and deplete

460 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Normal myocardium

Failing myocardium

A Ca lcium home os ta s is Ca 2+

3Na +

Ca 2+

Ca 2+

3Na +

Ca 2+

NCX

Ca 2+

NCX

Ca 2+

3Na +

Ca 2+ P hos phola mba n ADP

Ca 2+

3Na +

Ca 2+ P hos phola mba n

P

ATP S ERCA

S ERCA

Ca 2+ S a rcopla s mic re ticulum

S a rcopla s mic re ticulum

B Contra ctile e le me nts ATP

Myos in

ADP

ATP

Actin

Myos in

ADP

Actin

P

TN-I

TN-I

TN-T

TN-C

TN-T

TN-C

C Ade nylyl cycla s e s igna ling pa thwa y β-AR β1-agonist

β-AR Ade nylyl cycla s e

β

γ

Ade nylyl cycla s e

αs β

αs

P P

GTP

P P

ATP

cAMP

γ

GDP

ATP

cAMP

β-a rre s tin P KA

P KA

P KA

P KA

ina ctive

a ctive

ina ctive

a ctive

FIGURE 25-5. Cellular mechanisms of contractile pathophysiology. The ailing myocardium exhibits derangements in Ca 2 homeostasis, the contractile elements, and the adenylyl cyclase signaling pathway. In each panel (A, B, and C), normal myocardium is shown on the left and ailing myocardium on the right. A. In the normal myocardium, Ca 2 homeostasis is tightly controlled by Ca 2 pumps and channels, including the Na /Ca 2 exchanger (NCX) and the Ca 2 ATPase (SERCA). Operation o these pathways allows the myocardium to relax during diastole. In the ailing myocardium, diastolic Ca 2 remains elevated because phospholamban is not phosphorylated and there ore tonically inhibits SERCA. Also, the expression o NCXincreases (thick arrows), so that cytosolic Ca 2 is extruded rom the cardiac myocyte rather than stored in the sarcoplasmic reticulum. B. In the normal myocardium, phosphorylation o troponin-I (TN-I) exposes the actin–myosin interaction site, and myosin e ectively hydrolyzes ATP during each contraction cycle. In the ailing myocardium, there is decreased phosphorylation o TN-I, resulting in less e f cient actin–myosin cross-linking. Myosin does not hydrolyze ATP as e f ciently (dashed arrow), urther reducing the e ectiveness o each contraction cycle. The ailing myocardium also exhibits increased expression o the etal iso orm o TN-T; the signif cance o this alteration is uncertain. C. In the normal myocardium, -agonists stimulate cAMP ormation and subsequent activation o protein kinase A (PKA) (arrows). In the ailing myocardium, -arrestin binds to and inhibits the activity o -adrenergic receptors ( -AR), leading to decreased stimulation o adenylyl cyclase (dashed arrows). Expression o the inhibitory G iso orm G i is also induced in the ailing myocardium (not shown).

C h a p t e r 2 5 Pharmacology o Cardiac Contractility 461

SR Ca2 stores include reduced SR Ca2 reuptake and an increased number o sodium–calcium exchangers in the sarcolemma. As described above, e f cient sequestration o calcium by the SR is essential or the termination o contraction. Thus, the inability o the myocyte to regulate intracellular calcium impairs both systolic contraction and diastolic relaxation. Changes in the transcription o various genes in ailing cardiac myocytes result in the synthesis o dys unctional contractile proteins. The available data suggest that myocytes enter a maladaptive growth phase, reverting to production o the etal iso orms o some proteins. For example, ailing myocytes exhibit increased expression o the etal iso orm o troponin T, which is potentially a more e f cient contractile protein. Other contractile protein alterations identif ed in heart ailure include a reduction in phosphorylation o troponin I and diminished ATP hydrolysis by myosin; each o these changes results in a slower rate o cross-bridge cycling. In addition, activation o collagenase and matrix metalloproteinases may disrupt the stromal ramework that maintains the structural and unctional integrity o the myocardium. Desensitization o the -adrenergic receptor–G protein– adenylyl cyclase signaling pathway is the third major abnormal f nding in cardiac myocytes o patients with systolic HF. Failing myocytes down-regulate the number o -adrenergic receptors expressed at the cell sur ace, possibly as an adaptive response to the presence o increased neurohormonal stimulation. Sympathetic stimulation o the remaining receptors results in a smaller increase in cAMP than would occur in the presence o a normal number o receptors. The reduction in -adrenergic signaling may also re ect increased expression o both -adrenergic receptor kinase (which phosphorylates and thereby inhibits -adrenoceptors) and the inhibitory G protein (G i). Another contributor to the reduction o -adrenergic signaling may be inducible nitric oxide synthase (iNOS), the expression o which is increased in HF. The diminished response o ailing myocytes to adrenergic stimulation causes decreased phosphorylation o phospholamban, which impairs SR Ca2 uptake capacity. Decreased cAMP levels also result in a decreased ability to produce and use ATP. Together, the impaired calcium regulation, altered contractile elements, and decreased cAMP levels in ailing cardiac myocytes attenuate many o the steps o myocyte contraction and relaxation.

PHARMACOLOGIC CLASSES AND AGENTS The central roles o intracellular calcium and cAMP in cardiac myocyte contraction provide a basis or the classif cation o inotropic agents. The cardiac glycosides elevate intracellular Ca2 concentration via inhibition o the sarcolemmal Na /K -ATPase (sodium pump), while -agonists and phosphodiesterase inhibitors increase intracellular levels o cAMP. Calcium-sensitizing agents , a class o drugs under active investigation, are also discussed brie y.

Cardiac Glycosides The cardiac glycosides include the digitalis derivatives digoxin and digitoxin and nondigitalis agents such as ouabain. Glycosides are def ned by a common chemical sca old that includes a steroid nucleus, an unsaturated lactone ring, and

one or more sugar residues. This common structural substrate underlies the common mechanism o action o these agents. In clinical practice, digoxin is both the most requently used cardiac glycoside and the most widely used inotropic agent, although its overall use in the treatment o heart ailure has declined in recent years.

Digoxin

Digoxin is a selective inhibitor o the plasma membrane so-

dium pump (Fig. 25-6). Cardiac myocytes exposed to digoxin extrude less sodium, leading to a rise in intracellular sodium concentration. In turn, the increase in intracellular sodium concentration alters the equilibrium o the sodium– calcium exchanger: calcium e ux is decreased because the gradient or sodium entry is decreased, while calcium inux is increased because the gradient or sodium e ux is increased. The net result is an increase in the intracellular calcium concentration. In response to this increase, the SR o the digoxin-treated cell sequesters more calcium. When the digoxin-treated cell depolarizes in response to an action potential, more Ca2 is available to bind troponin C, and tension development during contraction is acilitated. In addition to its e ects on myocardial contractility, digoxin exerts autonomic e ects through its binding to sodium pumps in the plasma membranes o neurons in the central and peripheral nervous systems. These e ects include inhibition o sympathetic nervous out ow, sensitization o baroreceptors, and increased parasympathetic (vagal) tone. Digoxin also alters the electrophysiologic properties o the heart by a direct action on the cardiac conduction system. At therapeutic doses, digoxin decreases automaticity at the atrioventricular (AV) node, prolonging the e ective re ractory period 2

S a rcole mma

Ca 2+

Ca 2+ e xtrus ion 3Na +

1 3Na +

Ca 2+

Na +/K+ -ATP a s e 3Na +

Ca 2+ s tore s Ca 2+

ADP

2K+ Digoxin

Na +/Ca 2+ e xcha nge r

3

Na + e xtrus ion

P

3Na +

4

2K+

Myofibril contra ction

s tore d Ca 2+

ATP

Ca 2+

re le a s e d Ca 2+

FIGURE 25-6. Positive inotropic mechanism of digoxin. 1. Digoxin selectively binds to and inhibits the Na /K -ATPase. Decreased Na extrusion (dashed arrows) leads to an increased concentration o cytosolic Na . 2. The increased intracellular Na decreases the driving orce or the Na /Ca 2 exchanger (dashed arrows), leading to decreased extrusion o Ca 2 rom the cardiac myocyte into the extracellular space and to increased cytosolic Ca 2 . 3. The increased cytosolic Ca 2 is pumped by the SERCA Ca 2 -ATPase into the sarcoplasmic reticulum (thick arrow), increasing the Ca 2 that is available or release rom the SR during subsequent contractions. 4. During each contraction, the increased Ca 2 released rom the sarcoplasmic reticulum leads to increased myof bril contraction and there ore to increased cardiac inotropy.

462 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

TABLE 25-3 Pharmacokinetics of Digoxin Oral bioavailability

75%

Onset of action (intravenous)

30 minutes

Peak effect (intravenous)

1–5 hours

Half-life

36 hours

Elimination Volume of distribution

70% renal excretion, proportional to glomerular f ltration rate Large ( 640 L/70 kg): binds to skeletal muscle

o AV nodal tissue and slowing conduction velocity through the node. These combined vagotonic and electrophysiologic properties underlie the use o digoxin in the treatment o patients with atrial f brillation and rapid ventricular response rates; both the decreased automaticity o AV nodal tissue and the decreased conduction velocity through the node increase the degree o AV block and thereby decrease the ventricular response rate. In contrast to its e ects at the AV node, digoxin enhances automaticity o the in ranodal (His–Purkinje) conduction system. These divergent e ects at the AV node and HisPurkinje system explain the characteristic electrophysiologic disturbance o complete heart block with accelerated junctional or accelerated idioventricular escape rhythm (re erred to as regularized atrial f brillation) in patients with atrial f brillation and digoxin toxicity. Digoxin has a narrow therapeutic window, and prevention o digoxin toxicity depends on a complete understanding o the pharmacokinetics o this agent (Table 25-3). Orally administered digoxin has a bioavailability o approximately 75%. A minority o patients harbor gut ora that metabolize digoxin to the inactive metabolite dihydrodigoxin. In these patients, it is sometimes necessary to co-administer antibiotics with digoxin in order to decontaminate the gut and thereby acilitate oral absorption o digoxin. Digoxin has a large volume o distribution; the primary binding reservoir consists o Na / K -ATPase molecules in skeletal muscle. Approximately 70% o the drug is excreted unchanged by the kidney; the rest is excreted in the gut or via hepatic metabolism. Several specif c aspects o digoxin pharmacokinetics merit emphasis. Chronic kidney disease reduces both the volume o distribution and the clearance o digoxin, obligating a reduction in both the loading dose and the maintenance dose o the drug (see Chapter 3, Pharmacokinetics). (The reduced volume o distribution appears to be related to reduced tissue binding o the drug.) Hypokalemia increases the myocardial localization o digoxin. Reductions in extracellular K concentration appear to result in increased phosphorylation o the sodium pump and/or its regulator phospholemman, and digoxin may have a higher binding a f nity or the phosphorylated orms o these proteins than or the dephosphorylated orms. (Note that increasing plasma K concentration can help to relieve symptoms o digoxin toxicity by promoting dephosphorylation o these proteins.) Digoxin also interacts with many drugs. These interactions can be divided into pharmacodynamic and pharmacokinetic interactions. Co-administration o digoxin with -adrenergic

antagonists, Ca2 channel blockers, or K -wasting diuretics can result in pharmacodynamic drug–drug interactions. -Adrenergic antagonists decrease AV nodal conduction, and the combined use o -antagonists and digoxin can increase the risk o developing high-grade AV block. Both -antagonists and Ca2 channel blockers can decrease cardiac contractility and potentially attenuate the inotropic e ects o digoxin. K -wasting diuretics (e.g., urosemide) can decrease plasma potassium concentration, which can increase the a f nity o digoxin or the Na /K -ATPase and thereby predispose to digoxin toxicity (see above). Pharmacokinetic interactions can result rom changes in the absorption, volume o distribution, or renal clearance o digoxin (Table 25-3). Many antibiotics, such as erythromycin, can increase digoxin absorption by killing the enteric bacteria that would ordinarily metabolize a raction o orally administered digoxin be ore its absorption. Co-administration o digoxin with verapamil (a calcium channel blocker), quinidine (a class IA antiarrhythmic), or amiodarone (a class III antiarrhythmic) can increase digoxin levels because o the impact o these drugs on the volume o distribution and/or renal clearance o digoxin. In the introductory case, multiple actors likely contributed to the marked increase in GW’s serum digoxin level. The glomerular f ltration rate (GFR) was reduced (indicated by the elevated creatinine), resulting in decreased digoxin clearance. Administration o a loop diuretic likely contributed to the reduction in GFR. This reduction o GFR could have been exacerbated by co-administration o an angiotensin converting enzyme inhibitor via inter erence with angiotensin II-mediated autoregulation o glomerular hydrostatic pressure. Together, these actors likely contributed to the elevated serum digoxin concentration (3.2 ng/mL). To put this value into perspective, toxic e ects, such as ventricular ectopy, begin to appear at digoxin concentrations o 2–3 ng/mL. Treatment o digoxin toxicity relies on normalizing plasma K levels and minimizing the potential or ventricular arrhythmias. In addition, li e-threatening digoxin toxicity can be treated with antidigoxin antibodies . These polyclonal antibodies orm 1:1 complexes with digoxin that are rapidly cleared rom the body. Fab ragments o these antibodies (i.e., the portion o the antibody that interacts with antigen) have been shown to be less immunogenic than antidigoxin IgG and to have a larger volume o distribution, more rapid onset o action, and higher clearance than the intact IgG. It may seem counterintuitive to co-administer digoxin (a positive inotrope) with the -antagonist carvedilol (a negative inotrope). However, both agents have been shown to provide benef t in patients with HF. -Antagonists reduce mortality by 30% or more in patients with HF. It is postulated that -receptor antagonists counteract the cardiotoxic e ects o the chronic sympathetic stimulation that can occur in patients with contractile dys unction. -Antagonists have been shown to e ect changes in cellular morphology and chamber remodeling. The mechanism underlying the benef t o digoxin in HF is not ully understood; it is thought to be related to both digoxin’s positive e ect on contractile unction and its neurohumoral e ects. This issue is discussed in greater detail in Chapter 26. Several large randomized trials provide a consistent picture o the clinical e f cacy and limitations o digoxin. These trials indicate that withdrawal o digoxin in patients with HF leads to a decline in clinical status compared to patients

C h a p t e r 2 5 Pharmacology of Cardiac Contractility 463

who continue digoxin therapy. For example, withdrawal o digoxin is associated with deterioration in exercise capacity and increased requency o hospitalization or worsening heart ailure. However, the use o digoxin in patients with heart ailure does not have a signif cant impact on survival. In short, while digoxin has not been shown to improve survival, it does palliate symptoms, improve unctional status, and reduce hospitalization rates. These clinical benef ts can provide signif cant improvement in quality o li e or patients with HF; in all such patients, the circulating levels o digoxin should be monitored closely. Digoxin is also used to control ventricular rate in patients with long-standing atrial f brillation. The combined bradycardic and inotropic e ects o digoxin make it an especially use ul agent or patients with both HF and atrial f brillation.

Digitoxin

Digitoxin is a less requently used digitalis preparation that may

be pre erable to digoxin in selected clinical circumstances. Digitoxin is structurally identical to digoxin except or the presence (digoxin) or absence (digitoxin) o a hydroxyl group at position 12 o the steroid nucleus. This structural modif cation renders digitoxin less hydrophilic than digoxin and signif cantly alters the pharmacokinetics o the drug—in particular, digitoxin is metabolized and excreted primarily by the liver. The act that its clearance does not depend on renal excretion makes digitoxin a suitable alternative to digoxin or the treatment o patients with HF and chronic kidney disease. Dosing regimens need to account or the very long hal -li e o digitoxin (approximately 7 days) compared to digoxin (approximately 36 hours).

-Adrenergic Receptor Agonists -Adrenoceptor agonists are a heterogeneous group o drugs that have di erential specif city or adrenergic receptor subtypes. Inhaled ormulations o these medications are also used requently in the treatment o asthma, as discussed in Chapter 48, Integrative In ammation Pharmacology: Asthma. For all these agents, it merits emphasis that the di erential activation o receptor subtypes is inf uenced both by the agent selected and by the dose at which that agent is administered. For example, dopamine administered at low in usion rates (2–5 g/kg/min) has an overall cardiostimulatory e ect (caused by increased contractility and decreased systemic vascular resistance), while the same drug in used at higher rates ( 10 g/kg/min) has an overall impact that is largely related to 1-receptor activation.

Thus, the pharmacodynamic e ects o the agent (Table 25-4) must be considered in the context o the patient’s overall hemodynamic prof le; this o ten requires placement o hemodynamic monitoring catheters to quanti y intracardiac f lling pressures, systemic vascular resistance (SVR), and cardiac output. For this reason, in the introductory case, GW’s physicians placed a PA catheter be ore starting the dobutamine in usion. The clinical use o sympathomimetic inotropes is generally reserved or short-term support o the ailing circulation. This is attributable to the adverse e ect prof le o these agents and to their pharmacodynamic and pharmacokinetic properties. In general, sympathomimetic agents that stimulate myocardial -adrenergic receptors share the adverse e ect prof le o tachycardia, arrhythmia, and increased myocardial oxygen consumption. These agents also induce tolerance via rapid down-regulation and desensitization o adrenergic receptors. In addition, the sympathomimetic amines have low oral bioavailability and must typically be administered by continuous intravenous in usion.

Dopamine

Dopamine (DA) is an endogenous sympathomimetic amine

that unctions as a neurotransmitter; it is also a biosynthetic precursor o norepinephrine and epinephrine (see Chapter 11, Adrenergic Pharmacology). At low doses, dopamine has a vasodilatory e ect in the periphery by stimulating dopaminergic D1 receptors in the renal and mesenteric vascular beds. This regional vascular dilation reduces the impedance to le t ventricular ejection (a terload). At intermediate doses, DA causes vasodilation via stimulation o 2-adrenergic receptors; at these doses, DA also activates 1-receptors, thereby increasing contractility and heart rate. At higher doses, activation o 1-receptors predominates in the periphery, leading to generalized vasoconstriction and increased a terload. Dopamine must be administered intravenously in a closely monitored setting. It is metabolized rapidly by monoamine oxidase (MAO) and dopamine -hydroxylase to inactive metabolites that are excreted by the kidney. Patients receiving dopamine and MAO inhibitors concomitantly have decreased metabolism o dopamine; in these patients, dopamine can cause signif cant tachycardia, arrhythmia, and increased myocardial oxygen consumption. Despite its complex pharmacology, DA f nds wide clinical application in patients with sepsis and anaphylaxis, syndromes in which peripheral vasodilation is a major contributor to circulatory ailure. At low and intermediate doses, DA is

TABLE 25-4 Receptor Selectivity of Sympathomimetics RECEPTOR TYPE 1

AGENT

VASOCONSTRICTS PERIPHERAL VESSELS

Dopamine Dobutamine Epinephrine Norepinephrine

/

2

PRESYNAPTIC INHIBITION AT NE SYNAPSE

1

INCREASES HEART RATE, CONTRACTILITY, DIASTOLIC RELAXATION

2

VASODILATES PERIPHERAL VESSELS

D1 LOW DOSES VASODILATE RENAL VESSELS

464 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

used occasionally in patients with cardiogenic shock or HF. However, its use in cardiogenic circulatory ailure has largely been supplanted by alternative agents (such as dobutamine and the phosphodiesterase inhibitors) that have a more predictable vasodilator e ect in the periphery and/or are less likely to induce tachycardia and ventricular arrhythmia.

and chronotropic e ects o Epi supersede concerns related to its adverse peripheral vasomotor e ects. Noncardiovascular indications or Epi include relie o bronchospasm (via 2-mediated bronchial relaxation), potentiation o the e ect o local anesthetics (via local 1-mediated vasoconstriction), and treatment o allergic hypersensitivity reactions.

Dobutamine

Norepinephrine

developed in an attempt to optimize the overall hemodynamic benef ts o -adrenergic receptor activation or patients with acute cardiogenic circulatory ailure. Overall, dobutamine approximates the desirable hemodynamic prof le o a “pure” 1 agonist. However, this prof le is not the result o selective activation o 1-receptors but rather derives rom the act that the clinically available ormulation is a racemic mixture o enantiomers that have di erential e ects on adrenergic receptor subtypes. Both the ( ) and ( ) enantiomers stimulate 1-receptors and, to a lesser degree, 2-receptors, but the ( ) enantiomer acts as an 1 antagonist, whereas the ( ) enantiomer is an 1 agonist. Because the clinical ormulation includes both enantiomers, the opposing hemodynamic responses produced by these enantiomers at the 1-receptor e ectively negate one another. The predominant overall e ect is that o an agonist at cardiac 1-receptors, with modest peripheral vasodilation mediated by agonist action at peripheral 2-receptors. Dobutamine is administered by continuous intravenous in usion and titrated to achieve the desired clinical e ect. Catechol-O-methyl trans erase rapidly metabolizes dobutamine, so that the circulating hal -li e is only about 2.5 minutes. As with all sympathomimetic amines with -agonist e ects, dobutamine has the potential to induce cardiac arrhythmias. In clinical practice, supraventricular tachycardia and highgrade ventricular arrhythmia occur less requently with dobutamine than with dopamine. On the basis o this constellation o clinical e ects, dobutamine has become the sympathomimetic inotrope o choice or patients with acute cardiogenic circulatory ailure.

leased at sympathetic nerve terminals. NE is a potent 1receptor agonist, and there ore, it supports both systolic and diastolic cardiac per ormance. NE is also a potent 1-receptor agonist in the peripheral vessels, and thus, it increases systemic vascular resistance. During exercise, the release o NE increases heart rate and contractility, enhances diastolic relaxation, and, via its 1 agonist-mediated vasoconstriction, supports redistribution o the cardiac output away rom noncritical vascular beds. Intravenous NE is rapidly metabolized by the liver to inactive metabolites. At therapeutic doses, NE may precipitate tachycardia, arrhythmia, and increased myocardial oxygen consumption. When administered to patients with contractile dys unction, NE has a tendency to cause tachycardias involving both the sinoatrial (SA) node and ectopic sites in the atria and ventricles. Furthermore, the peripheral vasoconstriction induced by NE increases a terload and thereby limits the inotropic benef t o this agent. The increase in a terload occurs most requently in patients who have already recruited compensatory vasoconstrictive responses via sympathoadrenal and renin-angiotensin-aldosterone system activation. NE is, however, requently used or acute hemodynamic support in patients with distributive shock (e.g., Gram-negative bacterial sepsis) in the absence o underlying heart disease.

Dobutamine is a synthetic sympathomimetic amine that was

Epinephrine

Epinephrine (Epi) is a nonselective adrenergic agonist that

is endogenously released by the adrenal glands to support the circulation. Exogenously administered Epi stimulates 1-, 2-, 1-, and 2-receptors; the net e ect depends on the dose. At all dose levels, Epi is a potent 1 agonist with positive inotropic, chronotropic, and lusitropic e ects. Low-dose Epi predominantly stimulates peripheral 2-receptors, causing vasodilation. At higher Epi doses, however, stimulation o 1-receptors causes vasoconstriction and increased a terload. These e ects make high-dose Epi a suboptimal agent or patients with HF. As with other adrenergic agonists, epinephrine is primarily administered intravenously, although it can also be administered as an inhaled agent ( or treatment o asthma) or subcutaneously ( or treatment o anaphylaxis). Epinephrine is rapidly metabolized to metabolites that are excreted by the kidney. At high doses, epinephrine can cause tachycardia and li e-threatening ventricular arrhythmias. The primary clinical application o Epi is in the setting o resuscitation rom cardiac arrest, a situation in which rapid restoration o spontaneous circulatory unction is the immediate treatment goal. In this clinical setting, the potent inotropic

Norepinephrine (NE) is the endogenous neurotransmitter re-

Isoproterenol

Isoproterenol is a synthetic

-adrenergic agonist with relative selectivity or 1-receptors. The hemodynamic e ects o isoproterenol are dominated by a signif cant chronotropic response. The 2 e ects o isoproterenol can cause peripheral vasodilation and hypotension. Isoproterenol should not be administered to patients with active coronary artery disease, as it can worsen ischemia. Isoproterenol is used in requently, but it may be indicated in patients with re ractory bradycardia not responsive to atropine. It may also be administered in the treatment o -antagonist overdose.

Phosphodiesterase (PDE) Inhibitors Like -adrenergic receptor agonists, phosphodiesterase (PDE) inhibitors increase cardiac contractility by raising intracellular cAMP levels (Fig. 25-4). PDE inhibitors inhibit the enzyme that hydrolyzes cAMP, thereby increasing intracellular cAMP and indirectly increasing intracellular calcium concentration. There are multiple iso orms o PDE, each o which is linked to a distinct signal transduction pathway. Nonspecif c PDE inhibitors, such as theophylline , have been studied since the 1960s. Theophylline was initially used to treat asthma (see Chapter 48) but was later observed to have possible inotropic benef ts. Although cardiac muscle expresses multiple PDE isoenzymes, selective inhibition o PDE3 has been shown to have benef cial cardiovascular e ects. The relatively selective PDE3 inhibitors inamrinone (also known as amrinone) and milrinone increase contractility and enhance the rate and

C h a p t e r 2 5 Pharmacology of Cardiac Contractility 465

extent o diastolic relaxation. PDE3 inhibitors also have important vasoactive e ects in the peripheral circulation. These peripheral actions occur through cAMP-mediated e ects on intracellular calcium handling in vascular smooth muscle and result in decreased arterial and venous tone. In the systemic arterial circulation, vasodilation leads to a decrease in systemic vascular resistance (decreased a terload); in the systemic venous circulation, an increase in venous capacitance results in a decrease in venous return to the heart (decreased preload). The combination o positive inotropy and mixed arterial and venous dilation has led to the designation o PDE inhibitors as “ino-dilators.” Similar to -agonists, PDE inhibitors have ound clinical utility in short-term support o the severely ailing circulation. Widespread application o inamrinone has been limited by the adverse e ect o clinically signif cant thrombocytopenia in about 10% o patients. Both intravenous and oral ormulations o PDE3 inhibitors have been developed. Un ortunately, long-term use o these agents has been limited by data demonstrating increased mortality.

Calcium-Sensitizing Agents Calcium-sensitizing drugs, such as levosimendan, are a novel class o positive inotropes that are under investigation as possible therapeutic agents. Calcium sensitizers, which have the same “ino-dilator” actions as PDE inhibitors, augment myocardial contractility by enhancing the sensitivity o troponin C to calcium. This potentiating e ect increases the extent o actin–myosin interactions at any given concentration o intracellular calcium, without a substantial increase in myocardial oxygen consumption. In the peripheral circulation, levosimendan activates ATP-sensitive K channels, leading to peripheral vasodilation. Preliminary clinical trial data suggest that levosimendan improves cardiac hemodynamics in severe systolic HF and may reduce short-term mortality. Levosimendan is available in some European countries but is not currently approved or use in the United States.

CONCLUSION AND FUTURE DIRECTIONS Knowledge o the cellular and molecular bases or myocardial contraction has provided several pharmacologic strategies designed to increase myocardial contractility in patients with heart ailure attributable to le t ventricular systolic dysunction. By inhibiting the sodium pump, digoxin raises intracellular calcium levels and thereby increases contractile orce. This drug is the only oral inotropic agent in wide clinical use today. Although digoxin has no demonstrable impact on the mortality o patients with heart ailure, it helps alleviate symptoms and improves unctional capacity. Digoxin also slows AV nodal conduction, an e ect that is use ul in treating patients with atrial f brillation and rapid ventricular response rates. The -adrenergic receptor agonists— including the endogenous amines dopamine, norepinephrine, and epinephrine and the synthetic agents dobutamine and

isoproterenol—act through G protein-mediated elevation o intracellular cAMP to enhance both myocardial contractility and diastolic relaxation. The latter e ect allows the le t ventricle to f ll adequately during diastole, despite the increase in heart rate that is stimulated by these agents. -Agonists are administered intravenously, and they provide short-term hemodynamic support to patients with cardiogenic circulatory ailure. The longer term utility o these agents has been limited both by the lack o an oral ormulation with acceptable bioavailability and by the adverse e ect prof le o these drugs. PDE inhibitors, including inamrinone and milrinone, act as positive inotropes and as mixed arterial and venous dilators by increasing the levels o cyclic AMP in the heart and vascular smooth muscle. The increased mortality associated with longer term use o these agents has similarly restricted their role to the short-term management o severe HF. New classes o pharmacologic agents are under investigation or their ability to augment myocardial contractility. These agents are directed at a variety o biochemical targets, including the e f ciency o actin–myosin interactions (e.g., cardiac myosin activators ) and the synthesis o contractile proteins (e.g., cardiac neuregulins ). These approaches may improve cardiac contractility without increasing myocardial oxygen demand or signif cantly altering calcium signaling. Alternative strategies attempt to preserve myocardial contractility by inhibiting the e ects o proin ammatory cytokines associated with HF, but recent trials o these agents, such as endothelin receptor antagonists, have met with limited success. Finally, gene therapy methods are being investigated to increase contractility, including the delivery o genes with cardiac-specif c promoters that alter the production o contractile proteins, pumps, channels, and regulators in the heart. At the present time, the most promising candidates or gene therapy include the SR calcium pump, phospholamban, and cardiac troponin I.

Acknowledgment We thank Thomas P. Rocco or his valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Aronson D, Krum H. Novel therapies in acute and chronic heart ailure. Pharmacol Ther 2013;135:1–17. (Pathophysiology of acute and chronic heart failure, with a focus on emerging treatment strategies.) Endoh M. Cardiac calcium signaling and calcium sensitizers. Circ J 2008;72:1915–1925. (Physiology of excitation–contraction coupling and pharmacology of investigational agents for treatment of heart failure.) Gheorghiade M, Adams KF, Colucci WS. Digoxin in the management o cardiovascular disorders. Circulation 2004;109:2959–2964. (Reviews the clinical pharmacology of digoxin.) Hasen uss G, Teerlink JR. Cardiac inotropes: current agents and uture directions. Eur Heart J 2011;32:1838–1845. (Excellent review of contractile physiology and inotrope pharmacology.) Teerlink JR. A novel approach to improve cardiac per ormance: cardiac myosin activators. Heart Fail Rev 2009;14:289–298. (One of the possible future approaches to treatment of acute heart failure.)

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468 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

4

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Ma xim a l coroo n a r y flow 3

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Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 1

Re s ting coroo n a r y flo w

J a m e s M . M c Ca b e a n d Eh rin J . Arm s t ro n g

INTRODUCTION Lipid-Lowering Lipid p Loweringg Agents g s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4844 0 ONN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470 Meta Me tabo boli licc Mo Modu dula du lato tors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484 CASE, PART I: HYPERTENSION 4700 47 0 20 2 40 6 0 Unst 8 0 and Non-ST 1 0Eleva 100 0 tioi n nstab able Angina PATHOPHYSIOLOGY OF HYPERTENSION . . . . . . . . . . . . . . . . . . 470 Myocardial Infar farct ctio ionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 Cardiac Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472 Antianginal Drugss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4866 Vascular Function. . . 472 Heparin and Aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486 Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472 Glycoprotein IIb–IIIa Antagonists . . . . . . . . . . . . . . . . . . . . . 486 Neuroendocrine Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 ADP Receptor Antagonistss . . . . . . . . . . . . . . . . . . . . . . . . . . 486 CLINICAL MANAGEMENT OF HYPERTENSION . . . . . . . . . . . . . 473 Direct Thrombin Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . . . . 486 Reduction of Intravascular Volume . . . . . . . . . . . . . . . . . . . . . . 474 ST Elevation Myocardial Infarction. . . . . . . . . . . . . . . . . . . . . . 486 Diureticss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474 Thrombolytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 Down-Regulation of Sympathetic Tone . . . . . . . . . . . . . . . . . . 475 Primary Percutaneous Intervention . . . . . . . . . . . . . . . . . . . 487 -Adrenoceptor Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . 475 Postmyocardial Infarction Management . . . . . . . . . . . . . . . . . 487 -Adrenoceptor Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . 475 Central Sympatholytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 CASE, PART III: HEART FAILURE . . . . . . . . . . . . . . . . . . . . . . . . . . 488 Modulation of Vascular Smooth Muscle Tone . . . . . . . . . . . . 476 PATHOPHYSIOLOGY OF HEART FAILURE. . . . . . . . . . . . . . . . . . . 488 Etiologies of Contractile Dysfunction . . . . . . . . . . . . . . . . . . . . 489 Ca 2 Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 Cardiac Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 K Channel Openerss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 Frank-Starling Mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . 490 Modulation of the Renin-AngiotensinCardiac Remodeling and Hypertrophy . . . . . . . . . . . . . . . . . 491 Aldosterone System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 Neurohumoral Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491 Renin Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 Angiotensin Converting Enzyme Inhibitorss . . . . . . . . . . . . . 476 CLINICAL MANAGEMENT OF HEART FAILURE. . . . . . . . . . . . . . 492 AT T1 Antagonists (Angiotensin Receptor Blockers)) . . . . . . 477 Preload Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 Monotherapy and Stepped Care . . . . . . . . . . . . . . . . . . . . . . . . 477 Diureticss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 Possible Demographic Factors . . . . . . . . . . . . . . . . . . . . . . . . . 478 Aquaretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494 Hypertensive Crisis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478 Aldosterone Receptor Antagonistss . . . . . . . . . . . . . . . . . . . 494 Venodilatorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494 CASE, PART II: ISCHEMIC HEART DISEASE. . . . . . . . . . . . . . . . . 479 Afterload Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494 PATHOPHYSIOLOGY OF ISCHEMIC HEART DISEASE . . . . . . . . 479 ACE Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494 Chronic Coronary Artery Disease . . . . . . . . . . . . . . . . . . . . . . . 479 -Adrenoceptor Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . 495 Coronary Flow Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480 Vasodilatorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Endothelial Dysfunctionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 Inotropic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Acute Coronary Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 Cardiac Glycosidess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 CLINICAL MANAGEMENT OF ISCHEMIC HEART DISEASE . . . 482 Sympathomimetic Aminess . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Chronic Coronary Artery Disease . . . . . . . . . . . . . . . . . . . . . . . 482 Phosphodiesterase Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . 495 -Adrenoceptor Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . 483 Combination Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Ca 2 Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 Nitratess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 495 Aspirinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .496

Pee rce nt occlus ion of co P corona c a ry a rte r te t ry

469

470 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

PART I: HYPERTENSION Th o m a s N, a 45-ye a r-o ld m a n a g e r a t a te le co m m u n ica tio n s co m p a ny, p re s e n ts to th e ca rd io lo g y clin ic o r e va lu a tio n o e xe rtio n a l s h o rtn e s s o b re a th . Mr. N h a d a lwa ys b e e n ze a lo u s in m a in ta in in g a e ro b ic f tn e s s , b u t a b o u t 6 m o n th s b e o re h is ca rd io lo g y clin ic vis it, h e b e ga n to n o te s e ve re b re a th le s s n e s s a s h e a p p ro a ch e d th e co m p le tio n o h is d a ily ru n , w h ich co n clu d e s w ith a lo n g b u t g e n tle u p h ill clim b . Du rin g th e in te rve n in g 6 m o n th s , th e p a tie n t re p o rts a p ro g re s s io n in h is s ym p to m s to th e p o in t th a t, n o w, h e ra re ly co m p le te s th e f rs t h a l o h is d a ily ru n w ith o u t re s tin g . He d e n ie s ch e s t d is co m o rt a t re s t o r w ith e xe rcis e . His a m ily h is to ry is n o ta b le o r hyp e rte n s io n a n d p re m a tu re a th e ro s cle ro s is . Mr. N h a s n e ve r u s e d to b a cco p ro d u cts . On e xa m in a tio n , th e p a tie n t is h yp e rte n s ive (b lo o d p re s s u re , 160/102 m m Hg ), a n d a p ro m in e n t p re s ys to lic S 4 is h e a rd a t th e le t ve n tricu la r a p e x. Th e e xa m is o th e rw is e u n re m a rka b le . Th e ch e s t x-ra y is re p o rte d a s n o rm a l. Th e e le ctro ca rd io g ra m (ECG) re ve a ls n o rm a l s in u s rh yth m w ith vo lta g e crite ria o r le t ve n tricu la r hyp e rtro p hy. Mr. N is re e rre d o r n o n inva s ive ca rd ia c e va lu a tio n , in clu d in g a tre a d m ill e xe rcis e te s t (ETT) a n d a tra n s th o ra cic e ch o ca rd io g ra m . On th e ETT, h e re a ch e s a p e a k h e a rt ra te o 170 b e a ts /m in d u rin g e xe rcis e a n d h a s

INTRODUCTION In Chapters 20–25, cardiovascular pharmacology is considered in the context o individual physiologic systems. For example, diuretics are discussed in the context o volume regulation, while inhibitors o angiotensin converting enzyme (ACE) are discussed in the context o vascular tone. However, the clinical presentation o cardiovascular diseases o ten involves interactions among these individual systems. As a result, pharmacologic management o ten necessitates the use o agents rom several drug classes. This chapter presents three common cardiovascular disease states—hypertension, ischemic heart disease, and heart ailure—in a single, longitudinal clinical case. For each disease, an understanding o the disease pathophysiology underscores the rationale or pharmacologic interventions and may also highlight the potential or adverse e ects (such as serious drug–drug interactions). This chapter aims to integrate pathophysiology with pharmacology to provide a thorough and mechanistic understanding o the contemporary management o these common cardiovascular disease states.

PATHOPHYSIOLOGY OF HYPERTENSION Hypertension is a widely prevalent disease and a major risk actor or adverse cardiovascular events including stroke, coronary artery disease, peripheral vascular disease, heart ailure, and chronic kidney disease. In primary prevention studies, there is a continuous relationship between blood

to te rm in a te th e te s t b e ca u s e o s e ve re d ys p n e a a t a w o rklo a d o 7 METS . (METS a re m e ta b o lic e q u iva le n ts , a m e a s u re o e n e rg y co n s u m p tio n ; a va lu e o 7 METS is b e lo w n o rm a l o r th is p a tie n t’s a g e .) His b lo o d p re s s u re a t p e a k e xe rcis e is 240/120 m m Hg . Th e re is n o e vid e n ce o m yo ca rd ia l is ch e m ia b y ECG crite ria . Th e tw o -d im e n s io n a l e ch o ca rd io g ra m re ve a ls co n ce n tric-p a tte rn le t ve n tricu la r hyp e rtro p hy, a n e n la rg e d le t a triu m , a n d n o rm a l a o rtic a n d m itra l va lve s . Glo b a l a n d re g io n a l le t ve n tricu la r s ys to lic u n ctio n a re n o rm a l. Le t ve n tricu la r d ia s to lic f llin g is a b n o rm a l, w ith a re d u ce d ra te o e a rly ra p id f llin g a n d a s ig n if ca n t in cre a s e in th e e xte n t o f llin g d u rin g a tria l s ys to le .

Questions 1 . What are the current recommendations or initiation o antihypertensive drug therapy, and what are the therapeutic goals? 2 . Thiazide diuretics have been used or many years as f rstline therapy in patients with hypertension. What specif c clinical circumstances might avor use o another agent, such as an angiotensin converting enzyme inhibitor? 3 . Given the severity o hypertension in this case, Mr. N will likely require at least two drugs to achieve adequate control o his blood pressure. When is multidrug therapy required?

pressure and adverse cardiovascular outcomes including death. Although elevated diastolic blood pressure had long been the main indication or initiating antihypertensive treatment, it is now appreciated that elevated systolic blood pressure alone (isolated systolic hypertension) is also su f cient indication or treatment, particularly in elderly patients. Recommendations or treatment o hypertension have recently undergone revision, with di erential treatment thresholds based on age and presence o chronic kidney disease or diabetes (Fig. 26-1). The current recommendations re ect the results o recent randomized trials, which did not show a benef t o intensive blood pressure control in some populations and which also ound that patients with chronic kidney disease or diabetes are at higher risk o cardiovascular events. One o the main obstacles in the treatment o hypertension is the largely asymptomatic nature o the disease, even in patients with marked elevation in systemic blood pressure. This disconnect between symptoms and long-term adverse consequences has earned hypertension the designation “silent killer.” For example, Mr. N began to exhibit symptoms only a ter exercising. Nonetheless, the severity o his hypertension puts him at major risk or developing coronary artery disease, stroke, and heart ailure. Thus, e ective strategies or detection and management o hypertension are critical elements in the primary and secondary prevention o cardiovascular disease. Fortunately, the number and spectrum o agents available to treat patients with hypertension have expanded dramatically over the past two decades. These drugs can be administered

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Life s tyle inte rve ntions to b e a p p lie d throug hout tre a tme nt a lg orithm

Se t b lood p re s s ure g oa l a nd initia te b lood p re s s ure -lowe ring me d ic a tion b a s e d on a g e , d ia b e te s , a nd CKD.

Age ≥ 60 ye a rs a nd no dia be te s , no CKD

Age < 60 ye a rs a nd no dia be te s , no CKD

All a ge s with dia be te s a nd no CKD

All a ge s with CKD

Goa l S BP < 150 mm Hg Goa l DBP < 90 mm Hg

Goa l S BP < 140 mm Hg Goa l DBP < 90 mm Hg

Goa l S BP < 140 mm Hg Goa l DBP < 90 mm Hg

Goa l S BP < 140 mm Hg Goa l DBP < 90 mm Hg

Bla ck Initia te thia zide -type diure tic or CCB a lone or in combina tion.

Nonbla ck

All ra ce s

Initia te thia zide -type diure tic or ACEI or ARB or CCB a lone or in combina tion.

Initia te ACEI or ARB a lone or in combina tion with othe r cla s s .

S e le ct a tre a tme nt titra tion s tra te gy: A: ma ximize firs t me dica tion be fore a dding s e cond, B: a dd s e cond me dica tion be fore re a ching ma ximum dos e of firs t, or C: s ta rt with two me dica tion cla s s e s s e pa ra te ly or a s fixe d dos e combina tion. At goa l blood pre s s ure ?

YES

NO

Re inforce me dica tion a nd life s tyle a dhe re nce . For s tra te gie s A a nd B, a dd a nd titra te thia zide -type diure tic or ACEI or ARB or CCB (us e me dica tion cla s s not pre vious ly s e le cte d, a nd a void combine d us e of ACEI a nd ARB). For s tra te gy C, titra te dos e s of initia l me dica tions to ma ximum. At goa l blood pre s s ure ?

YES

NO

Continue curre nt tre a tme nt a nd monitoring.

Re inforce me dica tion a nd life s tyle a dhe re nce . Add a nd titra te thia zide -type diure tic or ACEI or ARB or CCB (us e me dica tion cla s s not pre vious ly s e le cte d, a nd a void combine d us e of ACEI a nd ARB). At goa l blood pre s s ure ?

YES

NO

Re inforce me dica tion a nd life s tyle a dhe re nce . Add fourth me dica tion cla s s (e .g., be ta blocke r, a ldos te rone a nta gonis t, or othe rs ) a nd/or re fe r to phys icia n with e xpe rtis e in hype rte ns ion ma na ge me nt.

FIGURE 26-1. Current recommendations for treatment of hypertension. Current treatment guidelines have incorporated di erent treatment thresholds and blood pressure goals based on the patient’s age and presence o chronic kidney disease (CKD) or diabetes. Patients over the age o 60 who do not have diabetes or chronic kidney disease can be treated with a more liberal blood pressure threshold o 150/90 mm Hg. Patients under the age o 60 without diabetes or chronic kidney disease should be treated to a target blood pressure o 140/90 mm Hg. All patients with diabetes or chronic kidney disease should be treated to a target blood pressure o 140/90 mm Hg, regardless o age. Among patients without chronic kidney disease, initial treatment should consist o a thiazide-type diuretic or calcium channel blocker (CCB); angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) may also be considered as initial therapy in nonblack patients. The subsequent treatment algorithm emphasizes continued li estyle modif cation and addition o multiple agents to achieve the goal systolic and diastolic blood pressure (SBP and DBP, respectively). (Adapted, with permission, rom J ames PA, Oparil S, Carter BL, et al. 2014 evidence-based guidelines or the management o high blood pressure in adults. J AMA 2014;311:507–520.)

472 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

initially as single agents (monotherapy). However, the progressive nature o hypertension characteristically leads to the use o a multidrug regimen. Although the clinical endpoints o therapy can vary somewhat rom patient to patient, the principal goal o treatment is to reduce the measured blood pressure, typically to levels less than 140 mm Hg systolic and less than 90 mm Hg diastolic. Hypertension is typically categorized as either primary (essential) or secondary hypertension. Essential hypertension, in which the cause o the elevation in blood pressure is unknown, a ects 90–95% o the hypertensive population. The etiology o essential hypertension is likely multi actorial, including both genetic actors and environmental actors such as alcohol use, obesity, and salt consumption. A more complete understanding o the pathophysiology o primary hypertension awaits the elucidation o underlying genetic predispositions and/or molecular mechanisms. Secondary hypertension re ers to patients in whom elevated blood pressure can be attributed to a def ned cause. Examples o secondary hypertension include primary hyperaldosteronism, oral contraceptive use, intrinsic renal disease, and renovascular disease. The principal determinants o blood pressure are discussed in Chapter 22, Pharmacology o Vascular Tone. Brie y, blood pressure is determined by the product of heart rate, stroke volume, and systemic vascular resistance (Fig. 26-2). Heart rate is determined largely by sympathetic activity. Stroke volume depends on loading conditions (preload and a terload) and contractility. Systemic vascular resistance re ects the aggregate vascular tone o the arteriolar subdivisions o the systemic circulation. A rational pharmacologic approach to the treatment o both primary and secondary hypertension requires an understanding o the physiology o normal blood pressure regulation and the mechanisms that could be responsible or hypertension in individual patients.

Cardiac Function One potential mechanism or persistent blood pressure elevation is a primary elevation in cardiac output (“high-output” hypertension). A “hyperkinetic” circulation can result rom excessive sympathoadrenal activity and/or increased sensitivity o the heart to basal levels o neurohumoral regulators. The hemodynamic pattern o pump-based hypertension (i.e., increased cardiac output [CO] with normal systemic vascular resistance [SVR]) is most o ten seen in younger patients with essential hypertension; this pattern can evolve over time into a hemodynamic prof le in which the principal locus o disease appears to shi t to the peripheral vasculature (see below). The underlying mechanism o high-output hypertension makes treatment with -adrenoceptor antagonists attractive in this population.

Vascular Function Vascular resistance-based hypertension (i.e., normal CO with

increased SVR) is a common mechanism underlying hypertension in the elderly. In individuals with this orm o hypertension, it is hypothesized that the vasculature is abnormally responsive to sympathetic stimulation, circulating actors, or local regulators o vascular tone. The abnormal responsiveness o the vasculature may be mediated in part by endothelial damage or dys unction, which is known to disrupt the normal equilibrium between local vasodilatory (e.g., nitric oxide) and vasoconstrictive (e.g., endothelin) actors. In addition, ion channel de ects in vascular smooth muscle can

BP CO

HR

S VR

SV

Dire ct inne rva tion

P S NS S NS Ca te chola mine s

Circula ting re gula tors Ca te chola mine s ATII

Contra ctility

P re loa d

Loca l re gula tors NO P ros ta cyclin Endothe lin ATII O2 H+ Ade nos ine

S NS Ca te chola mine s

Ve nous tone

Intrava s cula r volume

S NS Ca te chola mine s Thirs t

Na +/H2 O re te ntion S NS Aldos te rone ADH Na triure tic pe ptide s

FIGURE 26-2. Determinants of systemic blood pressure. Blood pressure (BP ) is the product of cardiac output (CO) and systemic vascular resistance (SVR), and CO is the product of heart rate (HR) and stroke volume (SV). These determinants are altered by a number of homeostatic mechanisms. Heart rate is increased by the sympathetic nervous system (SNS ) and catecholamines and decreased by the parasympathetic nervous system (PSNS). Stroke volume is increased by contractility and preload and decreased by afterload (not shown); all of these determinants are important parameters for cardiac function. Preload is altered by changes in venous tone and intravascular volume. The SNS and hormones, including aldosterone, antidiuretic hormone (ADH), and natriuretic peptides, are the major factors affecting intravascular volume. Systemic vascular resistance is a function of direct innervation, circulating regulators, and local regulators. Direct innervation comprises 1-adrenergic receptors ( 1-AR), which increase SVR. Circulating regulators include catecholamines and angiotensin II (AT II), both of which increase SVR. A number of local regulators alter SVR. These include endothelial-derived signaling molecules such as nitric oxide (NO), prostacyclin, endothelin, and AT II and local metabolic regulators such as O2, H , and adenosine. SVR is the major component of afterload, which is inversely related to stroke volume. The combination of a direct effect of SVR on blood pressure and an inverse effect of afterload on stroke volume illustrates the complexity of the system. ↑ indicates a stimulatory effect; ↓ indicates an inhibitory effect on the boxed variable.

cause abnormal elevations in basal vasomotor tone that result in increased systemic vascular resistance. Vascular resistance-based hypertension may present as a predominant elevation o systolic blood pressure. Studies have demonstrated the e ectiveness o thiazide diuretics in this population, making such agents the pre erred initial treatment.

Renal Function Abnormalities o renal unction can also contribute to the development o systemic hypertension. Excessive Na and H2O retention by the kidney is responsible or volume-based hypertension. Renal parenchymal disease, caused by glomerular injury with reduction o unctional nephron mass and/or excessive secretion o renin, can lead to an abnormal

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TABLE 26-1 Major Classes of Antihypertensive Agents RENIN–ANGIOTENSIN SYSTEM BLOCKERS

DIURETICS

SYMPATHOLYTICS

VASODILATORS

Thiazide diuretics Loop diuretics K -sparing diuretics

CNS sympathetic outf ow blockers Ganglionic blockers Postganglionic adrenergic nerve terminal antagonists 1-Adrenergic antagonists 1-Adrenergic antagonists Mixed -adrenergic/ -adrenergic antagonists

Calcium channel blockers Minoxidil Hydralazine Sodium nitroprusside

increase in intravascular volume. Alternatively, ion channel mutations can impair normal Na excretion. Renovascular disease (e.g., renal artery stenosis caused by atherosclerotic plaques, f bromuscular dysplasia, emboli, vasculitis, or external compression) can result in decreased renal blood ow. In response to this decrease in per usion pressure, juxtaglomerular cells increase the secretion o renin, which in turn leads to increased production o angiotensin II and aldosterone. The latter mediators increase both vasomotor tone and Na and H2O retention, leading to a hemodynamic prof le in which both CO and SVR are elevated.

Neuroendocrine Function Dys unction o the neuroendocrine system—including abnormal central regulation o basal sympathetic tone, atypical stress responses, abnormal responses to signals rom baroreceptors and intravascular volume receptors, and excessive production o hormones that act to regulate the circulation— can alter cardiac, vascular, and/or renal unction, leading to increased systemic blood pressure. Examples o endocrine abnormalities associated with systemic hypertension include excessive secretion o catecholamines (pheochromocytoma), excessive secretion o aldosterone by the adrenal cortex (primary aldosteronism), and excessive production o thyroid hormones (hyperthyroidism).

CLINICAL MANAGEMENT OF HYPERTENSION As discussed above, hypertension presents a complex clinical challenge, since blood pressure elevation may be asymptomatic or many years even as substantial end-organ damage occurs. As a result, the e ective treatment o hypertension requires strategies to identi y asymptomatic patients, especially those at high risk or the adverse end-organ e ects o the disease. Because antihypertensive drugs can add inconvenience to the li e o a patient who is asymptomatic, long-term treatment o the hypertensive patient requires the use o drug regimens that are individualized or optimal adherence and e f cacy. This requires consideration o sa ety prof le, dosing schedule, and cost. The f rst line in hypertension treatment is counseling regarding the importance o li estyle modif cations. Li estyle modif cations associated with avorable results in hypertensive patients include weight loss, increased physical activity, smoking cessation, and a low- at, low-sodium diet. Reduction or elimination o exogenous agents that can induce hypertension—such as ethanol, oral contraceptives, glucocorticoids, and stimulant drugs—can also have demonstrable clinical benef t. While nonpharmacologic therapies alone

Renin inhibitors ACE inhibitors AT1 antagonists Aldosterone antagonists

may not achieve a su f cient reduction in blood pressure, they remain critical adjuncts to pharmacologic treatment. An extensive armamentarium o drugs is used to treat systemic hypertension. Ultimately, though, these agents all exert their e ects on blood pressure through a reduction in cardiac output and/or systemic vascular resistance. Strategies currently used to treat hypertension include reduction o intravascular volume with concomitant vasodilation (diuretics), down-regulation o sympathetic tone ( -antagonists, 1-antagonists, central sympatholytics), modulation o vascular smooth muscle tone (calcium channel blockers, K channel openers), and inhibition o the neurohumoral regulators o the circulation (renin inhibitors, ACE inhibitors, AT1 antagonists [angiotensin II type 1 receptor antagonists]) (Table 26-1 and Fig. 26-3). The reduction in blood CCB Dire ct a rte ria l va s odila tors

BP CO

HR

S VR

SV

Dire ct inne rva tion Anta gonis ts

-Anta gonis ts

Circula ting re gula tors Anta gonis ts

Re nin inhibitors

Contractility

Loca l re gula tors Endothe lin a nta gonis ts S odium nitroprus s ide Re nin inhibitors ACE inhibitors AT1 a nta gonis ts

P re loa d

-Anta gonis ts

Ve nous tone

Intrava s cula r volume

Anta gonis ts

Re nin inhibitors

Na +/H2 O re te ntion Diure tics Re nin inhibitors ACE inhibitors AT1 a nta gonis ts

FIGURE 26-3. Pharmacologic effects of commonly used antihypertensive agents. Antihypertensive agents modulate blood pressure by inter ering with the determinants o blood pressure. Many o these antihypertensive drugs have multiple actions. For example, renin-angiotensin system blockers, such as ACE inhibitors and AT1 antagonists, alter the levels o local regulators and circulating regulators and a ect renal Na retention and venous tone. BP, blood pressure; CO, cardiac output; SVR, systemic vascular resistance; HR, heart rate; SV, stroke volume; CCB, Ca 2 channel blockers; ACE, angiotensin converting enzyme.

474 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

pressure caused by these agents is sensed by baroreceptors and renal juxtaglomerular cells, which can activate counter-regulatory responses that attenuate the magnitude o blood pressure reduction. These compensatory responses can be substantial, necessitating dose adjustments and/or the use o more than one agent to achieve long-term blood pressure control (Fig. 26-4).

TABLE 26-2 Diuretics Used in the Treatment of Hypertension DRUG

DURATION OF ACTION (HOURS)

Thiazide Diuretics Chlorothiazide

6–12

Reduction of Intravascular Volume

Chlorthalidone

48–72

Diuretics

Hydrochlorothiazide

16–24

Although diuretics have long been a cornerstone o antihypertensive therapy, the mechanism o action o diuretics in hypertension is incompletely understood. As discussed in Chapter 21, Pharmacology o Volume Regulation, diuretics decrease intravascular volume by increasing renal excretion o Na and H2O. However, volume depletion alone is unlikely to ully explain the antihypertensive e ect o diuretics. Thiazide diuretics (e.g., hydrochlorothiazide ) are the natriuretic drugs most commonly prescribed or the treatment o hypertension (Table 26-2). The pharmacokinetic Hype rte ns ion Pharmacologic interventions BP

Indapamide

24

Metolazone

24

Loop Diuretics Bumetanide

4–5

Ethacrynic acid

4–5

Furosemide

4–5

Torsemide

6–8

Potassium-Sparing Diuretics Amiloride Eplerenone Spironolactone

1

-antagonists

Ba rore ce ptor re flex

Re na l pe rfus ion

S ympa the tic ouflow

Re nin re le a s e

( -Antagonists) Na +/H2 O re te ntion

Ta chyca rdia , contra ctility

Va s ocons triction

Triamterene

Diuretics Renin inhibitors ACE inhibitors AT1 antagonists

BP

FIGURE 26-4. Compensatory homeostatic responses to antihypertensive treatment. When blood pressure is lowered by pharmacologic interventions, homeostatic responses are activated to increase blood pressure. These homeostatic responses can be divided broadly into baroreceptor ref exes and renal per usion ref exes. Baroreceptor ref exes originating in the aortic arch and carotid sinus increase sympathetic outf ow, leading to tachycardia, increased contractility, and vasoconstriction; these e ects all increase blood pressure. Sympatholytics, such as -antagonists, blunt the tachycardia and contractility responses by interrupting the sympathetic nervous system. 1-Antagonists inhibit vasoconstriction but have minimal e ects on tachycardia or contractility. Decreased renal per usion causes increased release o renin rom juxtaglomerular cells o the kidney. Renin then cleaves angiotensinogen to angiotensin I, which, in turn, is activated to the potent vasoconstrictor angiotensin II (not shown). Angiotensin II increases adrenal secretion o aldosterone, which acts on principal cells o the collecting duct to increase Na (and, there ore, water) reabsorption. The increased Na reabsorption increases intravascular volume and thereby results in increased blood pressure. Diuretics interrupt this homeostatic response by decreasing Na reabsorption rom the nephron; renin inhibitors prevent the generation o angiotensin I; angiotensin converting enzyme (ACE) inhibitors interrupt the ormation o angiotensin II; and AT1 antagonists prevent the target-organ signaling o angiotensin II.

6–24 24 72–96 8–12

and pharmacodynamic characteristics o the thiazides make them especially use ul agents in the treatment o chronic hypertension. Thiazides have high oral availability and long duration o action. The initial antihypertensive e ect seems to be mediated by decreasing intravascular volume. There ore, thiazides are particularly effective in patients with volume-based hypertension, such as patients with primary renal disease and African American patients. Thiazides induce an initial decrease in intravascular volume that decreases blood pressure by lowering cardiac output. However, the decrease in cardiac output stimulates the renin-angiotensin system, which leads to volume retention and attenuation o the e ect o the thiazide on volume status. It is hypothesized that a vasodilatory e ect o the thiazides complements the compensated volume depletion, leading to a sustained decrease in blood pressure. This hypothesis is supported by the observation that the maximal antihypertensive e ect o the thiazides is requently achieved at doses lower than those needed to achieve a maximal diuretic e ect. There ore, thiazides achieve their blood pressure e ect by in uencing both cardiac output and systemic vascular resistance. The Joint National Commission (JNC) treatment algorithm suggests thiazide diuretics as a potential f rst-line agent or many patients and a pre erred f rst-line agent or A rican American patients (Fig. 26-1). This recommendation arises rom the results o large-scale trials, which ound avorable outcomes and decreased cost associated with thiazide therapy. The practice at present is to initiate thiazide therapy at low doses (e.g., 12.5–25 mg/day).

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

Loop diuretics (e.g., furosemide ) are in requently pre-

scribed or the treatment o mild or moderate hypertension. These agents typically have a relatively short duration o action (4–6 hours), and despite the brisk diuresis that ollows their administration, their antihypertensive e f cacy is o ten modest. It is thought that this modest impact on blood pressure is due to activation o compensatory responses involving the neurohumoral regulators o intravascular volume and systemic vascular resistance. There are, however, several well-recognized clinical situations in which loop diuretics are preferable to thiazides, including malignant hypertension (see below) and volume-based hypertension in patients with advanced chronic kidney disease. K -sparing diuretics (e.g., spironolactone , triamterene , amiloride ) are less e f cacious than thiazide and loop diuretics and are used primarily in combination with other diuretics to attenuate or correct drug-induced kaliuresis (K excretion) and the resultant hypokalemia. An exception is spironolactone, an aldosterone receptor antagonist that is especially effective in the treatment of secondary hypertension caused by hyperaldosteronism. Hypokalemia is a common metabolic adverse e ect o the thiazide and loop diuretics, which inhibit Na reabsorption in proximal segments o the nephron and thereby increase delivery o Na and water to distal segments o the nephron. Increased distal Na delivery results in a compensatory increase in Na reabsorption in the distal tubule, which is coupled to an increase in K excretion. Because the latter e ect is mediated by aldosterone (see Chapter 21), the K -sparing diuretics attenuate this e ect and thereby help to maintain normal serum potassium levels. It should be emphasized that both ACE inhibitors (which decrease aldosterone activity and K excretion) and K supplements may need to be decreased or eliminated in patients taking K -sparing diuretics, because li e-threatening hyperkalemia has been reported in association with clinical use o the K -sparing agents. Use o these agents should be undertaken with great caution in patients with even mild degrees o renal insu f ciency.

Down-Regulation of Sympathetic Tone Drugs that modulate adrenergic activity are discussed in detail in Chapter 11, Adrenergic Pharmacology; re er to that chapter or descriptions o the tissue distribution o - and -adrenergic receptors and the cardiovascular e ects mediated by these receptors. Sympatholytic drugs treat hypertension via two major mechanisms: reduction of systemic vascular resistance and/or reduction of cardiac output. Clinically, these agents are broadly divided into -adrenoceptor antagonists, -adrenoceptor antagonists, and central sympatholytics.

-Adrenoceptor Antagonists

-Adrenoceptor antagonists (e.g., propranolol, metoprolol, atenolol, nebivolol) are commonly prescribed agents in the treatment o hypertension. The negative chronotropic and inotropic e ects o these agents (and the reductions in heart rate, stroke volume, and cardiac output that ollow) account or the initial antihypertensive e ect o the -antagonists. Decreased vasomotor tone, with a consequent decrease in systemic vascular resistance, has also been reported with longer term therapy. The -antagonist-induced reduction in vasomotor tone may seem paradoxical, given that 2-adrenergic receptors

475

in the peripheral vasculature mediate vasodilation. However, antagonism o 1-adrenergic receptors in the kidney decreases secretion o renin and thereby decreases production o the potent vasoconstrictor, angiotensin II. The latter e ect likely predominates, even when nonselective -receptor antagonists are administered. Although -antagonists e ectively reduce blood pressure in hypertensive patients, these agents typically do not cause hypotension in individuals with normal blood pressure. Increased baseline sympathetic activity in hypertensive patients may in part explain the e f cacy o -antagonists in lowering blood pressure in these individuals. In contrast, basal activation o -receptors in normal individuals may be su f ciently low that receptor antagonists have little hemodynamic e ect. -Antagonist therapy has been associated with both elevation o serum triglyceride levels and reduction o high-density lipoprotein (HDL) levels; the clinical signif cance o these potentially harm ul metabolic e ects remains unclear. Noncardiac adverse e ects o -antagonist therapy may include exacerbation o glucose intolerance (hyperglycemia), sedation, impotence, depression, and bronchoconstriction. Mixed – antagonists (e.g., labetalol) are available in both oral and parenteral ormulations. Intravenous administration o labetalol causes a substantial reduction in blood pressure and has ound wide use in the treatment o hypertensive emergencies. Oral labetalol is also used in the longterm treatment o hypertension. One potential advantage o this drug is that the decrease in blood pressure achieved by reduction o systemic vascular resistance (because labetalol antagonizes 1-receptors) is not associated with the re ex increase in heart rate or cardiac output that can occur when pure vasodilator drugs are used as monotherapy (because labetalol also antagonizes cardiac 1-receptors). In recent years, -adrenoceptor antagonists have been used less requently in the initial treatment o hypertension, due to clinical data suggesting that they may not be as e f cacious as diuretics or inhibitors o the renin-angiotensinaldosterone system. However, these agents are still important in the treatment o hypertension when there are other clinical indications or a -adrenoceptor antagonist, such as coronary artery disease or heart ailure. -Receptor antagonists are generally e f cacious in the treatment o hypertension in younger patients.

-Adrenoceptor Antagonists

1-Adrenergic

antagonists (e.g., prazosin, terazosin, doxazosin) are also used in the treatment o high blood pressure. 1-Adrenergic antagonists inhibit peripheral vasomotor tone, reducing vasoconstriction and decreasing systemic vascular resistance. The absence o adverse e ects on the serum lipid prof le during long-term treatment with 1-adrenergic antagonists is o ten cited as a distinctive advantage o these agents relative to other antihypertensive medications. However, the long-term benef t o this advantage, i any, remains to be determined in randomized clinical trials. Furthermore, in a large trial comparing di erent antihypertensives, an increased incidence o heart ailure was observed in the group randomized to doxazosin. Nonselective -adrenergic antagonists (e.g., phenoxybenzamine , phentolamine ) are not employed in the long-term treatment o hypertension because excessive compensatory responses can result rom their long-term use. For example, antagonism o central 2-adrenergic receptors disinhibits

476 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

sympathetic out ow, resulting in unopposed re ex tachycardia. However, these agents are indicated for the medical treatment of pheochromocytoma.

Central Sympatholytics The

2-adrenergic

agonists methyldopa , clonidine , and guanabenz reduce sympathetic out ow rom the medulla, leading to decreases in heart rate, contractility, and vasomotor tone. These drugs are available in oral ormulations (clonidine is also available as a transdermal patch) and were widely used in the past despite their un avorable adverse e ect prof le. The availability o multiple alternative agents, as well as the current trend toward the use o multidrug regimens at submaximal doses, have substantially diminished the clinical role o 2-agonists in the treatment o hypertension. Ganglionic blockers (e.g., trimethaphan, hexamethonium) inhibit nicotinic cholinergic activity at sympathetic ganglia. These agents are extremely e ective at lowering blood pressure. However, the severe adverse e ects o combined parasympathetic and sympathetic blockade (e.g., constipation, blurred vision, sexual dys unction, and orthostatic hypotension) have made ganglionic blockers o historic interest only. Some sympatholytic agents (e.g., reserpine , guanethidine ) are taken up into the terminals o postganglionic adrenergic neurons, where they induce long-term depletion o neurotransmitter rom norepinephrine-containing synaptic vesicles (see Chapter 11). These agents lower blood pressure by decreasing the activity o the sympathetic nervous system. However, reserpine and guanethidine have little role in the contemporary treatment o hypertension because o their signif cant adverse-e ect prof les, which include severe depression (reserpine) and orthostatic hypotension and sexual dys unction (guanethidine).

Modulation of Vascular Smooth Muscle Tone As discussed in Chapter 22, vascular tone is dependent on the degree o vascular smooth muscle contraction. Vasodilators reduce systemic vascular resistance by acting on arteriolar smooth muscle and/or the vascular endothelium. The major mechanisms o action o the arterial vasodilators include blockade o Ca2 channels and opening o metabotropic K channels.

Ca 2 Channel Blockers

Ca2 channel blockers (e.g., verapamil, diltiazem, nifedipine , amlodipine ) are oral agents that are widely used in the longterm treatment o hypertension. Calcium channel blockers (CCBs) have a variety o hemodynamic e ects, re ecting the multiple sites at which calcium is involved in the electrical and mechanical events o the cardiac cycle and in vascular regulation. These agents can act as arterial vasodilators, negative inotropes, and/or negative chronotropes. The dihydropyridine agents ni edipine and amlodipine act primarily as vasodilators. In contrast, the nondihydropyridine drugs verapamil and diltiazem act principally as negative inotropes and chronotropes, thereby decreasing myocardial contractility, heart rate, and impulse conduction. Thus, CCBs can lower blood pressure through reduction of both systemic vascular resistance and cardiac output. CCBs are o ten used in combination with other cardioactive drugs, either as components o a multidrug antihypertensive regimen or or combined antihypertensive and antianginal treatment in patients with ischemic heart disease (IHD).

Given the distinctive pharmacodynamic e ects o the di erent CCBs, the potential adverse e ects o CCB therapy (including adverse interactions with other cardiovascular therapies) are agent specif c. The nondihydropyridine agents verapamil and diltiazem should be used with caution in patients who have impaired le t ventricular (LV) systolic unction, as these agents can exacerbate systolic heart ailure (see below). These agents should also be used with caution in patients with conduction system disease, as these drugs can potentiate unctional abnormalities o the sinoatrial (SA) and atrioventricular (AV) nodes. Both o these cautions are particularly relevant in patients receiving concomitant -antagonist therapy.

K Channel Openers

Minoxidil and hydralazine are orally available arterial vaso-

dilators that are occasionally used in the long-term treatment o hypertension. Minoxidil is a metabotropic K channel opener that hyperpolarizes vascular smooth muscle cells and thereby attenuates the cellular response to depolarizing stimuli. Hydralazine is a less power ul vasodilator with an uncertain mechanism o action. Both minoxidil and hydralazine can cause compensatory retention o Na and H2O as well as re ex tachycardia; these adverse e ects are more requent and more severe with minoxidil than with hydralazine. Concomitant use o a -antagonist and a diuretic can mitigate these adverse e ects. The use o hydralazine is limited by the requent occurrence o tolerance and tachyphylaxis to the drug. In addition, increases in the total daily dose o hydralazine can be associated with a drug-induced lupus syndrome. Given the more avorable sa ety prof le o the Ca2 channel blockers, the use o minoxidil is now largely restricted to patients with severe hypertension that is re ractory to other pharmacologic therapies. O note, hydralazine (in combination with isosorbide dinitrate) has now emerged as an adjunctive therapy (i.e., in patients who are already receiving an ACE inhibitor and a -antagonist) in the treatment o systolic heart ailure in A rican American patients.

Modulation of the Renin-AngiotensinAldosterone System Renin-angiotensin-aldosterone system blockers include the renin inhibitor aliskiren, the ACE inhibitors (e.g., captopril, enalapril, lisinopril), and the angiotensin receptor (AT1) antagonists (e.g., losartan, valsartan). These agents are increasingly used in the treatment o hypertension.

Renin Inhibitors

Aliskiren is a competitive inhibitor o renin, the enzyme that cleaves angiotensinogen to angiotensin I. This earlystage blockade o the renin-angiotensin-aldosterone system may theoretically result in more e ective reduction o blood pressure and regression o le t ventricular hypertrophy than that achieved by angiotensin converting enzyme inhibitors or angiotensin receptor blockers. Recent clinical trials have suggested that increased renal ailure and hypotension may occur when aliskiren is prescribed in combination with ACE inhibitors or ARBs. For this reason, aliskiren is less commonly used in the treatment o hypertension.

Angiotensin Converting Enzyme Inhibitors

ACE inhibitors prevent the ACE-mediated conversion o angiotensin I to angiotensin II, leading to decreased circulating

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

levels o angiotensin II and aldosterone. By decreasing angiotensin II levels, ACE inhibitors decrease systemic vascular resistance and thereby decrease the impedance to LV ejection. By decreasing aldosterone levels, these agents promote natriuresis and thereby reduce intravascular volume. ACE inhibitors also decrease bradykinin degradation, and the resulting increase in circulating bradykinin causes vasodilation. ACE inhibitors are effective in patients with hyperreninemic hypertension, but these agents also reduce blood pressure in patients with low-to-normal circulating renin levels. The antihypertensive e ectiveness o ACE inhibitors in patients with low-to-normal plasma renin activity may be due to potentiation o the vasodilatory e ects o bradykinin, although this hypothesis is unproven. Therapy with ACE inhibitors is as e ective as therapy with thiazide diuretics or -antagonists in the treatment o hypertension. ACE inhibitors are attractive antihypertensive agents because these drugs seem to have unique benef ts (e.g., a decrease in the loss o renal unction in patients with chronic kidney disease) and relatively ew adverse e ects (ACE inhibitors do not increase the risk o hypokalemia or cause elevated serum glucose or lipid levels). Despite these attractive eatures, it merits emphasis that, in at least one large comparison trial, thiazide diuretics were more cardioprotective than ACE inhibitors. ACE inhibitors should be administered with caution in patients with intravascular volume depletion. Such patients may have reduced renal per usion at baseline, leading to a compensatory increase in renin and angiotensin II; this increase in angiotensin II is one o the physiologic mechanisms by which glomerular f ltration rate (GFR) is maintained in the ace o relative renal hypoper usion. Administration o ACE inhibitors to such patients can disrupt this autoregulatory mechanism, leading to renal insu f ciency. The same autoregulatory mechanism is the basis or the contraindication to ACE inhibitors in patients with bilateral renal artery stenosis (or unilateral stenosis in patients with a single kidney). Despite these cautionary notes, it should be emphasized that ACE inhibitors are considered the preferred therapy in the hypertensive diabetic patient, as these agents have been shown to delay the onset and progression o diabetic

477

glomerular disease through avorable e ects on intraglomerular pressure.

AT1 Antagonists (Angiotensin Receptor Blockers)

Angiotensin II receptor (AT1) antagonists (also known as angiotensin receptor blockers , or ARBs ) are oral antihypertensive agents that competitively antagonize the binding o angiotensin II to its cognate AT1 receptors; examples include losartan, valsartan, and irbesartan. In addition to their antihypertensive e ect, these agents may also reduce reactive arteriolar intimal proli eration. Like ACE inhibitors, AT1 antagonists are e ective in lowering blood pressure and are sometimes substituted or ACE inhibitors in patients with ACE inhibitor-induced cough. Cough, a common adverse e ect o ACE inhibitor therapy, results rom drug-induced increases in bradykinin levels; this e ect o ten leads to nonadherence or discontinuation o the drug. Because AT1 antagonists do not a ect the activity o the converting enzyme responsible or bradykinin degradation, cough is not an adverse e ect o therapy with ARBs.

Monotherapy and Stepped Care Monotherapy (treatment with a single drug) is o ten su f cient

to normalize blood pressure in patients with mild hypertension; this approach may improve patient adherence and avoid the risk o potential drug interactions. Controversy exists as to which antihypertensive agents are pre erred as initial therapy. Thiazide diuretics, ACE inhibitors, AT1 antagonists, and calcium channel blockers (CCBs) are similar in terms o e f cacy in lowering blood pressure (each e ectively lowers blood pressure in 30–50% o patients). Ultimately, the ideal agent is that which reduces a patient’s blood pressure to the optimal range with the least severe adverse e ects. Drug toxicities are o ten related to drug dose, and there ore, the clinician must also consider the use o a combination o “synergistic” agents at lower doses, especially i blood pressure control is marginal or inadequate. Certain clinical circumstances avor initiating a specif c class o antihypertensive medication (Table 26-3). -Antagonists are the agents o choice in patients with a

TABLE 26-3 Relative Indications and Contraindications for Antihypertensive Agents DRUG CLASS

INDICATIONS

CONTRAINDICATIONS

Diuretics

Heart failure Systolic hypertension

Gout

-Antagonists

Coronary artery disease Heart failure Migraine Tachyarrhythmias

Asthma Heart block

-Antagonists

Prostatic hypertrophy

Heart failure

Calcium channel blockers

Systolic hypertension

Heart block

ACE inhibitors

Diabetic or other nephropathy Heart failure Previous myocardial infarction

Bilateral renal artery stenosis Hyperkalemia Pregnancy

AT1 antagonists

ACE inhibitor-associated cough Diabetic or other nephropathy Heart failure

Bilateral renal artery stenosis Hyperkalemia Pregnancy

478 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

history o myocardial in arction (MI). ACE inhibitors are recommended in patients with le t ventricular dys unction, diabetes, and/or chronic kidney disease. Diuretics are e ective in treating hypertension associated with volume retention in nephrotic syndrome. ACE inhibitors are also used in nephrotic syndrome to attenuate the degree o proteinuria. Stepped care re ers to the progressive, step-by-step addition o drugs to a therapeutic regimen or hypertension. Combination therapy is based on the use o agents with distinct mechanisms o action; it also emphasizes the use o submaximal doses o drugs in an attempt to minimize potential adverse e ects and toxicities. Two examples o combination therapy are the use o an ACE inhibitor with either a diuretic or a calcium channel blocker. These combinations have several potential mechanistic advantages. By inducing a mild degree o volume depletion, thiazide diuretics activate the renin-angiotensin system. I this response is blocked by an ACE inhibitor, then the antihypertensive e ect o the thiazide is potentiated. Furthermore, inhibition o the renin-angiotensin system by itsel promotes natriuresis. Finally, the combination o a thiazide and an ACE inhibitor decreases systemic vascular resistance. When an ACE inhibitor is used with a calcium channel blocker, the combination may have an additive e ect on regression o le t ventricular hypertrophy. The addition o a calcium channel blocker may also potentiate ACE inhibitormediated peripheral vasodilation. A recent study (ACCOMPLISH trial) has suggested that the combination o an ACE inhibitor with a calcium channel blocker may reduce the incidence o cardiovascular events more than the combination o an ACE inhibitor with a thiazide diuretic, despite similar reductions in blood pressure.

Possible Demographic Factors Certain classes o antihypertensive drugs have been reported to be more e ective than others in special populations. Some data also suggest that distinct etiologies o hypertension may be more or less prevalent in di erent populations. Elderly patients tend to respond more avorably to diuretics and dihydropyridine Ca2 channel blockers than to other antihypertensive agents. -Antagonists are more likely to cause SA or AV node dys unction or to impair myocardial unction in elderly patients; these e ects are likely related to the higher prevalence o conduction system disease and LV systolic dys unction in such patients. Elderly patients also tend to have decreased circulating levels o renin and have been reported to be less responsive to ACE inhibitors. Hypertension in patients o A rican descent seems to be more responsive to diuretics and Ca2 channel blockers than to -antagonists and ACE inhibitors. (A notable exception is the avorable response o young A rican Americans to -antagonist therapy.) Reports indicate that some A rican Americans may have lower circulating renin levels, and this could account or the observation that ACE inhibitors are less e ective in these patients. Reports have also suggested that the prevalence o Na sensitivity is substantially increased in some A rican Americans, including both the hypertensive and the normotensive cohort. Although less well studied, there is some evidence o di erential responsiveness to the various classes o antihypertensive agents in hypertensive Asian and Hispanic cohorts. Despite these demographic observations, the clinical benef t o drug selection on the basis o di erential responsiveness

to specif c drug classes has not been evaluated systematically. For example, although elderly patients are reportedly less responsive to -antagonists, the results o the Systolic Hypertension in the Elderly Project (SHEP Trial) indicate that both -antagonists and diuretics are, in act, associated with mortality reduction, and this avorable treatment e ect is demonstrated within several years o treatment initiation. Similarly, although reports have suggested that A rican Americans are less responsive to -antagonists and ACE inhibitors, it would be di f cult to apply these observations to the treatment o a hypertensive, diabetic A rican American with chronic kidney disease or to advocate or the use o a thiazide diuretic in a hypertensive A rican American with a history o previous MI. Finally, it should again be emphasized that the risk o disease complications related to hypertension cannot be explained by the degree o blood pressure elevation alone. Conversely, the ull spectrum o treatment benef ts cannot be explained by the degree o blood pressure reduction alone. For these reasons, the empirical observation that some antihypertensive agents do not lower blood pressure as e ectively in some patients does not necessarily mean that these drugs will be less e ective in preventing uture cardiovascular disease morbidity and mortality in these patients. These questions remain the ocus o active research.

Hypertensive Crisis The term hypertensive crisis re ers to clinical syndromes characterized by severe (typically acute) elevations in blood pressure. This abrupt increase in blood pressure can cause acute vascular injury and derivative end-organ damage. Although most cases o severe hypertension were, at one time, designated as “hypertensive crisis” or “malignant hypertension,” current practice attempts to distinguish those patients in whom the blood pressure elevation and vascular injury are acute (hypertensive emergency) rom the patient cohort in which the temporal course o blood pressure elevation is more gradual and the end-organ damage is chronic and slowly progressive. A true hypertensive emergency is a li e-threatening condition in which severe and acute blood pressure elevation is associated with acute vascular injury. The vascular injury can mani est clinically as retinal hemorrhages, papilledema, encephalopathy, and acute (or acute superimposed on chronic) renal insu f ciency; this syndrome is o ten associated with acute le t ventricular ailure. The pathogenesis o malignant hypertension remains unclear. However, it is likely that f brinoid arteriolar necrosis contributes to the signs and symptoms o this syndrome. Fibrinoid arteriolar necrosis o specif c vascular beds can result in acute vascular injury and end-organ hypoper usion (e.g., renal ailure, stroke). Fibrinoid arteriolar necrosis can also lead to microangiopathic hemolytic anemia. Treatment o patients with hypertensive emergency necessitates rapid reduction o blood pressure to prevent endorgan damage. Drug classes used to treat this condition include parenteral vasodilators (e.g., clevidipine, nitroprusside, enoldopam, nicardipine), diuretics (e.g., urosemide), and/or -antagonists (e.g., labetalol). Because o the acuity o the syndrome and the need to titrate these power ul antihypertensive agents care ully, patients are hospitalized or treatment. A ter the acute episode has been controlled, subsequent lowering o blood pressure to the normal range o the patient is then attempted more cautiously over a longer

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

PARTII: ISCHEMICHEARTDISEASE Mr. N is tre a te d o r hyp e rte n s io n w ith lo w-d o s e h yd ro ch lo ro th ia zid e a n d a n ACE in h ib ito r. He re tu rn s o r o llo w-u p vis its a t 1 m o n th a n d 6 m o n th s , a n d re p o rts th a t h e is d o in g w e ll. He a ith u lly a d h e re s to h is p re s crib e d m e d ica l re g im e n a n d n o te s a d e f n ite im p rove m e n t in e xe rcis e ca p a city. His re g u la r b lo o d p re s s u re m e a s u re m e n ts n o w s h o w re a d in g s o 130 to 150/86 to 90 m m Hg . A s e ru m lip id p ro f le is n o ta b le o r in cre a s e d to ta l ch o le s te ro l, w ith a m o d e ra te ly e le va te d LDL. Lo w-d o s e a s p irin is a d d e d to h is re g im e n . Tre a tm e n t w ith a lip id -lo w e rin g a g e n t is a ls o a d vis e d , b u t Mr. N d e clin e s , in s te a d re q u e s tin g th a t h is lip id p ro f le b e re ch e cke d a te r a p e rio d o d ie t a n d li e s tyle m o d if ca tio n s . An e xe rcis e to le ra n ce te s t 1 ye a r a te r h is in itia l vis it is n o ta b le o r im p ro ve d e xe rcis e ca p a city (10 MET w o rklo a d ), w ith b lu n tin g o th e h e a rt ra te a n d re d u ctio n o th e b lo o d p re s s u re a t p e a k e xe rcis e co m p a re d to th e o rig in a l s tu d y (120/m in a n d 190/90 m m Hg , re s p e ctive ly); th e re is n o e vid e n ce o m yo ca rd ia l is ch e m ia b y ECG crite ria . A re p e a t LDL ch o le s te ro l d e te rm in a tio n is w ith in th e n o rm a l ra n g e (128 m g /d L). His m e d ica tio n s (a s p irin , hyd ro ch lo ro th ia zid e , a n d ACE in h ib ito r) a re co n tin u e d , a n d ro u tin e o llo w-u p is e s ta b lis h e d . One week later, Mr. N experien ces the abrupt onset o severe retrosternal chest pressure. He is visibly diap ho retic a nd dyspn eic. He calls 911 and is tran s ported

period o time (12–24 hours), in an e ort to decrease the risk o critical-organ hypoper usion and extension o vascular injury. Although malignant hypertension is a li e-threatening medical emergency, it is an uncommon expression o hypertensive disease that occurs in ar less than 1% o hypertensive patients. More common are cases o hypertensive urgency, in which the blood pressure elevation is less acute and the target organ disease has been present or some time. Conditions illustrative o hypertensive urgency include a stroke or MI that is accompanied by severe blood pressure elevation or acute le t heart ailure with severe hypertension.

PATHOPHYSIOLOGY OF ISCHEMIC HEART DISEASE Ischemic heart disease (IHD), the leading cause o mortality in the United States, accounts or more than 500,000 deaths each year. Since the advent o cardiac intensive care units in the early 1960s, an improved understanding o the biology o IHD has resulted in a spectrum o diagnostic and therapeutic advances. These advances, coupled with increased public awareness, healthier li estyles, and sustained e orts to improve both primary and secondary prevention

479

to th e lo ca l e m e rg e n cy d e p a rtm e n t, w h e re a n ECG s hows sinus tachycardia and ST se gm ent e leva tion in th e in e rio r lea d s . Em e rg en cy card ia c ca th eteriza tio n is p e r o rm e d , co n f rm in g to ta l o cclu s io n o a d o m in a n t rig h t co ro n a ry a rte ry, a n d p e rcu ta n e o u s co ro n a ry in te rvention (PCI) is p er orm e d with pla cem e nt o a co ro n ary s tent. The p ro ce dure is s u cce s s u l, a nd h e re m a in s re e o ch e s t p a in a n d is h e m o d yn a m ica lly s ta b le . ECG a n d s e ru m e nzym e ch a n g e s (p e a k cre atine kin as e [CK], 2,400 IU/L [n o rm al, 60–400 IU/L]; ca rd ia c is o o rm [MB] ra ctio n , p o s itive ) a re co n s is te n t with a n e vo lvin g m yo card ial in a rctio n . A re p e a t e ch o ca rd io g ra m im m e d ia te ly b e o re Mr. N’s d is charge rom the hospital dem ons trates concentric le t ventricu lar hyp ertrop hy with a le t ven tricular ejection ractio n o 40% (no rm al, 55%); the in erior wall ro m th e b a s e to th e a p e x is a kin e tic, with th in n in g o th e m yo ca rdium in this akin etic reg ion.

Questions 4 . Which class of lipid-lowering agent is appropriate for this patient? 5 . Which pharmacologic interventions are appropriate during the interval between the patient’s emergency department evaluation and his cardiac catheterization? 6 . What are the critical drug components of a postmyocardial infarction treatment regimen in the setting of left ventricular dysfunction?

strategies, have resulted in signif cant mortality reduction or patients with IHD. With respect to pharmacotherapy, IHD can be considered in two broad categories: chronic coronary artery disease (CAD) and acute coronary syndromes (ACS). Each o these clinical presentations o IHD has a distinct pathogenesis, and as a result, the pharmacologic strategies employed to treat these distinct clinical entities di er in emphasis. The therapeutic goal in patients with chronic CAD is to maintain the balance between myocardial oxygen supply and demand; in patients with ACS, the goal is to restore and/or maintain patency of the coronary artery lumen (Fig. 26-5).

Chronic Coronary Artery Disease Chronic CAD is characterized by impaired coronary vasodilator reserve. Under conditions o hyperemic stress (i.e., stress requiring increased coronary blood ow), this can result in an imbalance between myocardial oxygen supply and demand, leading to unctional cardiac abnormalities (poor contraction o the ischemic portion o the myocardium) as well as clinical symptoms o CAD. The basic physiology o myocardial oxygen supply and demand is discussed in Chapter 22. Imbalances in myocardial oxygen supply and demand occur mainly as a result o coronary ow reduction and endothelial dys unction.

480 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Is che mic he a rt dis e a s e

A Norma l

Endothe lia l ce ll

Pa te nt lume n Norma l e ndothe lia l function P la te le t a ggre ga tion inhibite d

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P la que

Lume n na rrowe d by pla que Ina ppropria te va s ocons triction

FIGURE 26-5. Classif cation o ischemic heart disease. Ischemic heart disease is divided into two broad categories: chronic coronary artery disease and acute coronary syndromes. Stable angina is the prototypical mani estation o chronic coronary artery disease. Acute coronary syndromes constitute a series (not necessarily a linear progression) o clinical presentations, including unstable angina, non-ST elevation myocardial in arction, and ST elevation myocardial in arction.

C Uns ta ble a ngina Rupture d pla que

Coronary Flow Reduction

The coronary vasculature is composed o two types o vessels: large, proximal epicardial vessels and small, distal endocardial vessels. The epicardial vessels are the more requent sites o atheroma ormation; in disease states, total coronary artery blood ow is limited by the extent o epicardial vessel stenosis. In comparison, endocardial vessels regulate intrinsic coronary vascular resistance in response to local metabolic changes. When myocardial oxygen demand is increased, endocardial vessels dilate in response to local metabolic actors, resulting in a regional increase in myocardial blood ow and thereby providing increased oxygen to these metabolically active tissues. Angina pectoris (Fig. 26-6) is the principal clinical maniestation o chronic CAD. This symptom is characterized by precordial pressure-like discom ort resulting rom myocardial ischemia. Most patients with chronic CAD experience stable angina , a clinical syndrome in which ischemic chest pain occurs at characteristic and reproducible workloads (e.g., walking up a ight o stairs). Pathologically, chronic CAD is associated with subintimal deposition o atheroma in the epicardial coronary arteries. In general, atherosclerotic plaques in patients with chronic stable angina are characterized by an overlying f brous cap that is thick and resistant to disruption. The immediate cause o angina pectoris is an imbalance between myocardial oxygen supply and demand. Under normal physiologic conditions, coronary blood ow is modulated care ully to ensure adequate tissue per usion in response to varying levels o myocardial oxygen demand. This ability to modulate blood ow is re erred to as the coronary ow reserve : CFR

maximal CBF/resting CBF

where CFR is coronary ow reserve and CBF is coronary blood ow. In healthy individuals, the maximal CBF is approximately f ve old greater than the resting CBF.

P la te le t P la que rupture d P la te le t a ggre ga tion Thrombus forma tion Unoppos e d va s ocons triction Thrombus

D Va ria nt a ngina

No ove rt pla que s Inte ns e va s os pa s m

FIGURE 26-6. Pathophysiology o anginal syndromes. A. Normal coronary arteries are widely patent, the endothelium unctions normally, and platelet aggregation is inhibited. B. In stable angina, atherosclerotic plaque and inappropriate vasoconstriction (caused by endothelial damage) reduce the vessel lumen diameter and hence decrease coronary blood f ow. C. In unstable angina, rupture o the plaque triggers platelet aggregation, thrombus ormation, and vasoconstriction. Depending on the anatomic site o plaque rupture, this process can progress to non-Q wave (non-ST elevation) or Q wave (ST elevation) myocardial in arction. D. In variant angina, atherosclerotic plaques are absent, and ischemia is caused by intense vasospasm.

Because o this wide sa ety margin, the resting CBF does not decrease until an epicardial stenosis exceeds 80–90% o the original arterial diameter. Changes in maximal CBF can be observed more readily with exercise, as maximal CBF begins to decrease during exercise when an epicardial stenosis exceeds 50–70% o the original arterial diameter. In patients with chronic CAD, the decrease in CFR is directly related to the severity o epicardial artery stenosis; the coronary ow reserve may be urther impaired as a consequence o endothelial dys unction (discussed below), resulting in a urther reduction in CBF. Demand-related ischemia occurs during periods in which myocardial oxygen demand exceeds myocardial oxygen delivery, and the patient experiences angina pectoris. The degree o epicardial artery stenosis and the degree o compensatory endocardial artery dilation determine the

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

hemodynamic consequence o an atherosclerotic plaque (Fig. 26-7). I the endocardial arteries are normal, an epicardial stenosis that narrows the diameter o the arterial lumen by less than 50% does not signif cantly reduce maximal coronary blood ow. However, i the stenosis narrows the arterial lumen diameter by more than 80%, then the endocardial vessels must dilate to provide adequate per usion to the myocardium, even at rest. The need or endocardial vessels to dilate at rest attenuates coronary ow reserve, because the endocardial vessels cannot then dilate urther during exercise. This reduction in coronary ow reserve leads to inadequate myocardial blood ow during hyperemic stress. Myocardial ischemia can occur at rest when the epicardial artery stenosis exceeds 90% o the lumen diameter: under these conditions, endocardial vessels cannot maintain adequate myocardial per usion even at maximal dilation.

Endothelial Dysfunction

Endothelial dys unction is a general term or pathologic endothelial cell regulation. Clinically, endothelial dys unction is mani ested by abnormal vascular tone and prothrombotic properties. Abnormal vascular tone is a result o dysregulated endothelial control o smooth muscle contraction: arterial beds with endothelial dys unction cannot dilate in response to hyperemic stimuli. For example, when mental stress or physical exertion triggers activation o the sympathetic nervous system (SNS), two opposing orces act on the coronary vascular endothelium: catecholamine-mediated vasoconstriction and nitric oxide (NO)-mediated vasodilation. Normally, endothelial release o NO is stimulated by the shear stress on the

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Pe rce nt occlus ion of corona ry a rte ry FIGURE 26-7. E ect o coronary artery occlusion on resting and maximal coronary blood f ow. The dotted line depicts resting coronary blood f ow, and the solid line represents maximal blood f ow when there is ull dilation o distal coronary arteries. Comparison o these two lines shows that maximal coronary blood f ow is compromised when the lesion occludes more than about 50% o the arterial lumen, whereas resting coronary blood f ow is relatively una ected until the lesion exceeds about 80% o the arterial diameter. The y-axis represents coronary artery blood f ow relative to the f ow in a resting coronary artery with 0% occlusion.

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coronary vascular endothelium that results rom increased blood ow. Eventually, the vasodilator e ects o NO predominate over the vasoconstrictor e ects o SNS activation, and the overall e ect is coronary vasodilation. However, when the vascular endothelium is damaged, the production o endothelial vasodilators is decreased and catecholaminemediated vasoconstriction predominates. Because the endothelium also plays a crucial role in regulating platelet activation and the coagulation cascade, endothelial dys unction can promote blood coagulation (thrombosis) at the site o endothelial injury. Endothelialderived NO and prostacyclin exert signif cant antiplatelet e ects, and molecules on the sur ace o healthy endothelial cells have signif cant anticoagulant properties (see Chapter 23, Pharmacology o Hemostasis and Thrombosis). Endothelial damage decreases the ability o the endothelium to utilize these endogenous antiplatelet and anticoagulant mechanisms, leading to a local predominance o procoagulant actors and increasing the likelihood o platelet and coagulation actor activation.

Acute Coronary Syndromes Acute coronary syndromes (ACS) are most o ten caused by the f ssuring or rupture o atherosclerotic plaques. These socalled unstable or vulnerable plaques are characterized by thin f brous caps that are prone to rupture. Plaque rupture results in the exposure o procoagulant actors, such as subendothelial collagen (Fig. 26-8), that activate platelets and the coagulation cascade. Under physiologic circumstances, hemostasis at a site o vascular injury is sel -limited by endogenous anticoagulant mechanisms (see Chapter 23). However, the dys unctional endothelium overlying the atherosclerotic plaque cannot elaborate su f cient anticoagulant actors to control the extent o clot ormation. Dysregulated coagulation can then result in intraluminal thrombus ormation, which leads to myocardial ischemia and potentially to irreversible myocardial injury. The three subtypes o acute coronary syndromes are unstable angina, non-ST elevation MI, and ST elevation MI. In unstable angina , patients experience either acceleration in the requency or severity o chest pain, new-onset anginal pain, or characteristic anginal chest pain that abruptly occurs at rest. Enzymatic evidence o tissue in arction (e.g., elevated troponin levels) is absent in unstable angina, but patients are at high risk or MI because o the presence o an active prothrombotic sur ace at the site o plaque rupture. Non-ST elevation myocardial in arction occurs when an unstable plaque abruptly ruptures and signif cantly compromises (but does not completely occlude) the lumen o an epicardial coronary artery. Because the artery is partially occluded and there is a persistent prothrombotic sur ace at the site o plaque rupture, patients with non-ST elevation MI are at high risk or recurrence o ischemia. The pathophysiology and clinical management o unstable angina and non-ST elevation MI are very similar, and these two syndromes are o ten re erred to by the combined acronym unstable angina/ non-ST elevation MI (UA/NSTEMI). I the intraluminal thrombus completely occludes the epicardial coronary artery at the site o plaque rupture, then blood ow ceases downstream rom the locus o obstruction. Persistent, total epicardial artery occlusion provides the substrate or acute myocardial injury (ST elevation myocardial in arction; STEMI), which progresses inexorably to transmural

482 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY A

B

C

D

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FIGURE 26-8. Pathogenesis of acute coronary syndromes. A. A normal coronary artery has an intact endothelium surrounded by smooth muscle cells. B. Endothelial cell activation or injury recruits monocytes and T lymphocytes to the site o injury, leading to development o a atty streak. C. Continued oxidative stress within a atty streak leads to development o an atherosclerotic plaque. D. Macrophage apoptosis and continued cholesterol deposition cause urther plaque organization and may induce the expression o additional in ammatory proteins and matrix metalloproteinases. At this stage, the cap o the f broatheroma remains intact. E. Continued in ammation within an atherosclerotic plaque leads to thinning o the f brous cap and, eventually, to plaque erosion or rupture. Exposure o plaque constituents to the bloodstream activates platelets and the coagulation cascade, with resulting coronary artery occlusion.

in arction unless per usion is reestablished. This clinical syndrome can also present as out-o -hospital sudden cardiac death ( 30% o patients); in these cases, death is usually caused by ischemia-induced electrical instability o the myocardium. In the absence o atal electrical instability, ST elevation MI typically presents with unremitting chest pain that is o ten accompanied by dyspnea and ischemic le t heart ailure. Mortality in STEMI is signif cantly reduced by prompt relie o the complete epicardial obstruction. There ore, the principal management goal in STEMI is expeditious reper usion o the occluded artery. The extent o myocardial necrosis a ter ischemic injury depends on the mass o myocardium supplied by the occluded artery, the amount o time over which the artery is totally occluded, and the degree o collateral circulation. Regions o the myocardium that are supplied directly and exclusively by the occluded artery sustain extensive ischemic injury. Cell death occurs in a “wave ront” that progresses both spatially and temporally rom the subendocardial region to the epicardial sur ace o the myocardium. As a result, the extent o “transmurality” o an MI bears a direct relationship to the duration o coronary artery occlusion. Adjacent to the region o transmural necrosis, a border zone o myocardium receives nutrients and oxygen rom collateral vessels; this collateral per usion can maintain the viability o border zone cells or

some period o time. However, in the absence o reper usion o the occluded (in arct-causing) artery, lethal cardiomyocyte injury eventually occurs in these border zones as well.

CLINICAL MANAGEMENT OF ISCHEMIC HEART DISEASE As noted above, both the pathophysiology and the clinical management o ischemic heart disease are di erent in patients with chronic coronary artery disease compared to patients with acute coronary syndromes. Chronic CAD results rom an imbalance between myocardial oxygen supply and demand, and the treatment o chronic CAD ocuses on modulating this balance, usually by reduction o oxygen demand. In comparison, treatment o ACS relies on reestablishing and maintaining the patency o the occluded epicardial coronary artery as rapidly as possible. All patients with CAD, irrespective o clinical presentation, also require modif cation o underlying risk actors, including aggressive lipid-lowering therapy and blood pressure control.

Chronic Coronary Artery Disease The treatment goal in chronic CAD is to restore the balance between myocardial oxygen supply (coronary artery blood

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

ow) and myocardial oxygen demand (myocardial oxygen consumption). Pharmacologic therapies concentrate on the reduction of myocardial oxygen demand, which is governed by heart rate, contractility, and ventricular wall stress (see Chapter 22). Antianginal drugs can be categorized on the basis o their impact on these parameters.

-Adrenoceptor Antagonists

Activation o 1-adrenergic receptors by the sympathoadrenal system leads to an increase in heart rate, contractility, and conduction through the AV node. It ollows that antagonists acting at 1-adrenergic receptors decrease sinus rate, reduce inotropic state, and slow AV nodal conduction. 1-Adrenoceptor antagonists (also re erred to as -blockers) are the cornerstone o medical treatment regimens in patients with chronic stable angina. -Antagonists reduce myocardial oxygen demand by decreasing heart rate and contractility, and the drug-induced decrease in heart rate may also increase myocardial per usion via prolongation o the diastolic f lling time. When used in chronic angina, -antagonists decrease both the resting heart rate and the peak heart rate achieved during exercise and delay the time to onset o angina. Dosing regimens or -antagonists are drug-specif c, re ecting the characteristic pharmacokinetics o each individual agent. As a general rule, the dose o drug is calibrated to maintain the resting heart rate at approximately 50 beats/min and to maintain the peak heart rate during exertion at approximately 110 to 120 beats/min. -Antagonists are requently co-administered with organic nitrates in patients with stable angina. This combination is o ten more e ective than either agent used alone. -Antagonists are also requently combined with CCBs— typically, with agents o the dihydropyridine class (see below). (In early clinical trials, short-acting ormulations o the dihydropyridine CCB ni edipine were associated with re ex tachycardia when administered as monotherapy; this tachycardia was attenuated when ni edipine was coadministered with a -antagonist. In current practice, the availability o long-acting dihydropyridine agents has e ectively diminished this adverse e ect.) Although -antagonists are generally well tolerated in patients with stable angina, certain clinical scenarios require caution. Combining -antagonists with CCBs o the nondihydropyridine classes (e.g., diltiazem or verapamil) can result in synergistic suppression o SA-node automaticity (leading to extreme sinus bradycardia) and/ or AV-node conduction (leading to high-grade AV conduction block). Likewise, because o their depressant e ects on nodal tissues, -antagonists may exacerbate preexisting bradycardia and/or high-grade AV block. However, given the clear and consistent mortality benef t associated with -antagonists in secondary prevention trials, it is currently standard clinical practice to implant a permanent transvenous pacing device i such rhythm abnormalities are the major contraindication to the use o -antagonists and then to administer the -antagonist. (Secondary prevention trials test the e f cacy o pharmacologic interventions to reduce adverse cardiovascular events in patients with known CAD.) -Antagonists are now also used in patients with clinically stable heart ailure (see the ollowing discussion). It must be emphasized that the survival benef t demonstrated in HF treatment trials occurred when these agents were initiated

483

during periods o clinical stability. -Antagonists must not be administered to patients with decompensated HF. When used in an attempt to treat the rare patient with pure vasospastic or variant angina (i.e., angina in the absence o epicardial artery obstruction; see Fig. 26-6), -antagonists can induce coronary vasospasm as a consequence o unopposed -receptor-mediated vasoconstriction. -Antagonists can also exacerbate bronchospasm in patients with asthma and chronic airway obstruction. However, in such patients, the decision to avoid -antagonists should be based on objective documentation o exacerbation o air ow obstruction during -antagonist therapy. Peripheral vascular disease is another relative contraindication to -antagonist therapy; the concern in this circumstance is the potential or antagonism o the 2-adrenergic receptors that mediate dilation o peripheral vessels. In clinical practice, however, this concern is rarely justif ed. Furthermore, patients with peripheral arterial disease have an extremely high risk o concomitant CAD and are there ore likely to benef t signif cantly rom -antagonist therapy. Common adverse e ects o -antagonists include atigue, lethargy, insomnia, and impotence. Although the precise mechanism o atigue is unclear, decreased exercise capacity is directly related to drug-induced blunting o the physiologic tachycardia o exertion. The impotence reported by 1% o patients treated with -antagonists is due to inhibition o 2-adrenoceptor-mediated peripheral vasodilation.

Ca 2 Channel Blockers

Calcium channel blockers (CCBs) decrease the in ux o calcium through voltage-gated L-type calcium channels in the plasma membrane. The resulting decrease in intracellular calcium concentration leads to reduced contraction o both cardiac myocytes and vascular smooth muscle cells (see Chapter 22). Calcium channel blockers decrease myocardial oxygen demand and may also increase myocardial oxygen supply. Calcium channel blockers decrease myocardial oxygen demand by decreasing systemic vascular resistance and by decreasing cardiac contractility. In the periphery, calcium entry into vascular smooth muscle cells is required or contraction o the cells and is there ore a central determinant o resting vasomotor tone. By blocking calcium entry, CCBs cause relaxation o vascular smooth muscle and thereby reduce systemic vascular resistance. Calcium channel blockers can theoretically increase myocardial oxygen supply by blocking calcium-mediated increases in coronary vasomotor tone; the resulting dilation o epicardial vessels and arteriolar resistance vessels would, in theory, increase coronary blood ow. However, the contribution o this coronary vasodilator mechanism to the clinical e ects o the CCBs is controversial, because regional metabolic abnormalities that result rom myocardial ischemia should e ect a maximal vasodilator response in the absence o pharmacologic modulation. The various classes o calcium channel blockers have distinctive inotropic e ects on cardiac myocytes. Compared to verapamil and diltiazem, dihydropyridines (such as ni edipine) are more selective or calcium channels in the peripheral vasculature. Nonetheless, all CCBs do have the potential to impair contractile unction by reducing intracellular calcium levels in cardiac myocytes. There ore, decompensated heart ailure is a contraindication to the use o certain CCBs because o their negative inotropic e ects. However, newer

484 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

generation vasoselective dihydropyridines, such as amlodipine and elodipine, are typically tolerated by patients with reduced LV ejection ractions and can there ore be administered to patients with LV dys unction and re ractory angina. Calcium channel blockers are reported to be as e ective as -antagonists in the treatment o chronic stable angina. I the initial treatment o angina with -antagonists alone is not success ul, CCBs can be used either in combination with -antagonists or as monotherapy. Calcium channel blockers appear to produce a greater antianginal e ect when coadministered with -antagonists than when administered alone, although combination therapy can induce bradyarrhythmias (see above). Despite the proven e f cacy o CCBs in reducing symptoms in patients with chronic CAD, there are no data to support a mortality benef t associated with CCB therapy in either primary or secondary prevention in patients with CAD. Unlike -antagonists, CCBs can be effective in the treatment of vasospastic angina. Calcium channel blockers relieve the vasospasm o coronary vessels by dilating both epicardial coronary arteries and arteriolar resistance vessels. It is common practice to use nitrates in combination with CCBs when treating vasospastic angina.

Nitrates

Organic nitrates exert their principal therapeutic e ect by dilation o peripheral capacitance veins, thereby decreasing preload and reducing myocardial oxygen demand (see Chapter 22). Some investigators argue that nitrates also increase myocardial blood ow by reducing coronary vasomotor tone, although the magnitude o the incremental vasodilator e ect is debated in patients with regional myocardial ischemia. Nitrates do have a coronary vasodilator e ect in patients with vasospastic angina. Nitrates also have anti-aggregatory e ects on platelets. In patients with stable exertional angina, nitrates improve exercise tolerance when used as monotherapy and act synergistically with -antagonists or CCBs. Sublingual nitroglycerin tablets or nitroglycerin sprays are e ective or immediate relie o exertional angina. Provided that su f cient nitrate- ree intervals are allowed to attenuate the development o tolerance, long-acting nitrates (e.g., isosorbide dinitrate and mononitrate) are also e ective or prophylaxis and treatment o exertional angina. Nitrates are also e ective in the treatment o both acute and chronic LV ailure. This treatment e ect is related to the power ul venodilator action o the nitrates, which causes peripheral redistribution o intravascular volume and marked reduction o preload. The anti-ischemic e ect o nitrates may be o particular benef t in patients with ischemia-related diastolic dys unction. In this clinical setting, nitrates may e ect both preload reduction and restoration o normal diastolic chamber compliance and f lling. The development o tolerance is the major obstacle to long-term use o nitrates. Through uncertain mechanisms (see Chapter 22), tolerance develops to both vasodilator and antiplatelet e ects o these drugs. Dosing regimens that are punctuated by su f ciently long nitrate- ree intervals (8–12 hours) may prevent nitrate tolerance. Headache, the most common adverse e ect o nitrate therapy, can develop as a result o cerebral vessel dilation.

Aspirin

Platelet activation is critically important in the initiation o thrombus ormation (see Chapter 23), and antiplatelet

agents play a central role in the treatment o patients with CAD. Aspirin irreversibly inhibits platelet cyclooxygenase, an enzyme required or generation o the pro-aggregatory compound thromboxane A2 (TxA2). There ore, the platelet inhibition that ollows aspirin administration persists or the li espan o the platelet (approximately 10 days). Unless specif c contraindications are present, aspirin is an essential therapy or patients with chronic CAD. Aspirin is used to prevent arterial thrombosis leading to MI, transient ischemic attack, and stroke. Aspirin is most effective as a selective antiplatelet agent when taken at low doses and/ or infrequent intervals (see Chapter 23). Clinical data have demonstrated a signif cant treatment benef t or aspirin in patients with unstable angina ( 50% reduction in death and non atal MI). Aspirin is contraindicated in patients with a known allergy to the drug; in this setting, clopidogrel is indicated as an alternative. Aspirin and other antiplatelet agents should be used cautiously in patients with compromised liver unction, because such patients may have a bleeding diathesis due to decreased circulating levels o hepatically synthesized coagulation actors. Aspirin use also predisposes to gastrointestinal adverse e ects such as gastritis and peptic ulcer disease; these e ects can o ten be alleviated by co-administration o agents that decrease gastric acid production (see Chapter 47, Integrative In ammation Pharmacology: Peptic Ulcer Disease).

Lipid-Lowering Agents

Clinical studies indicate that, in patients with known CAD, the administration o drugs that lower serum LDL cholesterol decreases the risk o ischemic cardiovascular events. (Re er to Chapter 20, Pharmacology o Cholesterol and Lipoprotein Metabolism, or a detailed discussion o lipidlowering agents.) The selection o a specif c lipid-lowering agent is based on both clinical trial data and the patient’s lipid phenotype. HMG-CoA reductase inhibitors (statins) are the most requently used and best-studied lipid-lowering agents. Because HMG-CoA reductase mediates the f rst committed step in sterol biosynthesis, inhibitors o HMG-CoA reductase dramatically reduce the extent o hepatic cholesterol synthesis. This reduction in cholesterol synthesis results in increased hepatic LDL receptor expression and thereby increases clearance o cholesterol-containing lipoprotein particles rom the bloodstream. Clinical trials (e.g., the Scandinavian Simvastatin Survival Study and the Cholesterol and Recurrent Events Study) demonstrate that lipid-lowering therapy reduces cardiovascular event rates in patients with CAD. All patients with a myocardial in arction should be treated with a statin, with goal LDL cholesterol targets o 70 mg/dL or lower (note that current guidelines do not speci y exact target values). Dietary and other li estyle modif cations should also be included as part o a comprehensive approach to primary and secondary prevention. HMG-CoA reductase inhibitors are contraindicated in women who are or may become pregnant or who are nursing.

Metabolic Modulators

Some patients with stable angina continue to experience requent angina despite maximal attempts at medical management and revascularization. In these cases, metabolic modulators that increase the e f ciency o ATP utilization may be clinically use ul. In this class o drugs, ranolazine is approved or the second-line treatment o re ractory angina (i.e., angina that occurs despite otherwise maximal therapy).

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

Clinical trials o ranolazine in stable angina have demonstrated improved e ort tolerance and decreased requency o anginal symptoms relative to placebo. There may also be gender-specif c e ects, with women deriving greater benef t rom ranolazine than men. Other metabolic modulators remain an active area o investigation and drug development.

485

UA have a 15–20% risk o progression to acute MI over a period o 4–6 weeks. Aggressive treatment can reduce this risk by more than 50%. Patients with UA have no overt evidence o myocardial damage, whereas patients with NSTEMI have elevated biomarkers that re ect cardiomyocte necrosis. Untreated UA may progress to NSTEMI, or NSTEMI may be the initial result o plaque rupture with extensive in ammation and coagulation at the rupture site. Goals o treatment in UA/NSTEMI are to relieve ischemic symptoms and to prevent additional thrombus ormation at the site o plaque rupture. UA/NSTEMI is typically treated with aspirin, heparin, and -antagonists. Other antiplatelet agents (GPIIb–IIIa antagonists and ADP receptor antagonists) and/or direct thrombin inhibitors (bivalirudin) are indicated in high-risk patients to prevent additional thrombus ormation (Fig. 26-9). Although conventional

Unstable Angina and Non-ST Elevation Myocardial Infarction Unstable angina (UA) and non-ST elevation myocardial inarction (NSTEMI) may occur as the f rst presentation o CAD or in patients with a history o stable CAD. (In the latter circumstance, management strategies appropriate or unstable angina take precedence over those or stable CAD.) It is estimated that, in the absence o treatment, patients with Is che mic he a rt dis e a s e Chronic corona ry a rte ry dis e a s e (s ta ble a ngina )

Acute corona ry s yndrome s

Aspirin -Antagonists Nitrates Ca2+ channel blockers ACE inhibitors Ranolazine

Aspirin -Antagonists Nitrates

No S T e leva tion on ECG: Uns table a ngina or non-S T e leva tion myoca rdia l infa rction

S T e leva tion on ECG: S T e leva tion myoca rdia l infa rction

Thrombolys is

Add: Heparin GPIIb-IIIa antagonist ADP receptor antagonist or Bivalirudin ADP receptor antagonist

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Pos t-myoca rdia l infa rction ma na ge me nt

Pos s ible a ddition of: Statin ACE inhibitor Aldosterone receptor antagonist Continue : Aspirin ADP receptor antagonist

FIGURE 26-9. Pharmacologic management of acute coronary syndromes. All patients with chronic coronary artery disease are given aspirin unless a li e-threatening contraindication is present. -Antagonists, nitrates, calcium channel blockers, ACE inhibitors, and ranolazine are primarily used to reduce myocardial oxygen demand. All patients with symptoms that raise concerns about a possible acute coronary syndrome are given aspirin and, i tolerated, a -antagonist. Sublingual or intravenous nitrates can also be administered to relieve chest discom ort and minimize ischemia. Electrocardiographic (ECG) f ndings o ST elevation should prompt emergency measures to open the occluded artery, either with a thrombolytic agent (thrombolysis) or mechanical revascularization (angioplasty). Additional adjunctive pharmacologic therapies or ST elevation myocardial in arction may include aspirin, -antagonists, nitrates, heparin, ADP receptor antagonists, and GPIIb–IIIa antagonists or bivalirudin. For patients with acute coronary syndrome but no ST elevation on the electrocardiogram, laboratory assays o myocyte damage (e.g., troponin I or troponin T) determine whether the patient is classif ed as experiencing unstable angina or non-ST elevation myocardial in arction. In either case, management generally includes administration o aspirin, -antagonists, nitrates, ADP receptor antagonists, and bivalirudin or heparin with GPIIb–IIIa antagonists. For all patients with acute coronary syndrome, postmyocardial in arction management should include modif cation o risk actors; possible addition o lipid-lowering agents (statins), ACE inhibitors, and aldosterone receptor antagonists; and continuation o aspirin and ADP receptor antagonists.

486 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

antianginal drugs have no demonstrable impact on mortality in UA/NSTEMI, these “demand-based” agents are also used empirically or symptom relie . Thrombolytic agents are contraindicated in patients with UA/NSTEMI: use o these agents in UA/NSTEMI has been associated with a signif cant increase in morbidity and a trend toward increased mortality. I ischemic chest discom ort recurs a ter initiation o treatment, or i a patient presents with certain high-risk eatures, urgent coronary angiography is warranted (with revascularization guided by the angiographic data).

Antianginal Drugs

Intravenous nitroglycerin is o ten administered or the f rst 24 hours a ter the onset o UA/NSTEMI. The intravenous ormulation is used to achieve and maintain predictable blood levels o the drug. A ter 24 hours, the asymptomatic patient can be switched to a long-acting oral nitrate preparation. Myocardial oxygen demand should also be reduced by co-administration o a -adrenergic antagonist. Even without symptoms o chest pain, a -antagonist should be administered empirically because o the mortality benef t associated with -antagonist use in the setting o MI. Although Ca2 channel blockers such as verapamil and diltiazem also reduce myocardial oxygen demand, their use is purely palliative; unlike -antagonists, these agents have not been shown to reduce the risk o recurrent MI or cardiac death in patients with UA/NSTEMI.

Heparin and Aspirin

In patients with UA/NSTEMI, heparin and aspirin reduce the risk o recurrent, li e-threatening cardiovascular events by 50%. Although these agents also increase the risk o bleeding, the clinical benef ts outweigh the potential adverse e ects. The combination o heparin and aspirin appears to be more e ective than either agent alone in reducing cardiac mortality and recurrent ischemia.

Glycoprotein IIb–IIIa Antagonists

Glycoprotein IIb–IIIa (GPIIb–IIIa) antagonists are highly e f cacious antiplatelet agents. In the process o platelet aggregation, GPIIb–IIIa receptors on activated platelets bind the bridging molecule f brinogen. GPIIb–IIIa antagonists inter ere with this critical step o platelet aggregation and thereby limit the size o the platelet plug (see Chapter 23). GPIIb–IIIa antagonists have been used requently both in the cardiac catheterization laboratory (during percutaneous revascularization procedures) and in the pharmacologic treatment o UA/NSTEMI, although recently their use has declined in the United States (see below). GPIIb– IIIa antagonists reduce the risk o atal and non atal MI in patients with UA, and these agents reduce the risk o recurrent MI and urgent revascularization in patients with NSTEMI. In UA/NSTEMI patients with ongoing ischemia or certain high-risk eatures, a GPIIb–IIIa antagonist may be administered in addition to aspirin and heparin; both eptif batide and tirof ban have been approved or this use. The use o abciximab has been restricted largely to the periprocedural setting (i.e., in preparation or and immediately ollowing percutaneous coronary intervention). Studies have shown that GPIIb–IIIa antagonist use is associated with increased bleeding outcomes compared to the use o direct thrombin inhibitors such as bivalirudin;

consequently, the use o the GPIIb–IIIa antagonists has waned recently.

ADP Receptor Antagonists

Antagonists at the platelet ADP receptor P2Y12 are used in the treatment o many patients with ACS. The thienopyridines clopidogrel, prasugrel, and the rarely used ticlopidine are irreversible ADP receptor antagonists. Ticagrelor is a reversible P2Y12 antagonist that is also used in the treatment o patients with acute coronary syndromes. Because these are all power ul antiplatelet agents, ADP receptor antagonists are routinely used in the setting o ACS. Clopidogrel is indicated in all patients with ACS who have true aspirin allergy. Clopidogrel reduces recurrent coronary events in patients with UA/NSTEMI who undergo percutaneous coronary intervention and in patients with UA/NSTEMI who are treated with a noninvasive approach (e.g., patients who do not undergo cardiac catheterization and target-vessel revascularization). Importantly, although the combination o clopidogrel, aspirin, and a GPIIb–IIIa antagonist signif cantly increases the risk or major bleeding, the overall reduction in cardiovascular morbidity and mortality outweighs the increased risk o bleeding in selected groups o patients. Unlike clopidogrel, prasugrel does not require conversion rom a prodrug orm in the liver. There ore, compared to clopidogrel, prasugrel has ewer “non-responders,” results in higher antiplatelet activity, and is more rapidly bioavailable a ter oral administration. In clinical trials comparing prasugrel to clopidogrel in patients with recent myocardial in arction who underwent angioplasty, prasugrel was associated with improved overall clinical outcomes. However, prasugrel also has an increased risk o bleeding in certain patient populations, including those with a history o stroke, patients over 75 years o age, and patients weighing less than 60 kilograms. Ticagrelor is an ADP receptor antagonist that may demonstrate a mortality benef t over clopidogrel in the setting o STEMI (see below). Patients who are prescribed both ticagrelor and aspirin must be administered low-dose aspirin ( 100 mg daily), because clinical trials have suggested the possibility o increased mortality among patients prescribed higher doses o aspirin with ticagrelor. Intravenous ormulations o thienopyridines have recently been investigated, but these agents are not yet commercially available.

Direct Thrombin Inhibitors

Direct thrombin inhibitors such as bivalirudin have been increasingly used in the treatment o patients with UA/ NSTEMI. The main use o these agents is as adjunctive antithrombotic therapy during percutaneous intervention in the cardiac catheterization laboratory. In these cases, direct thrombin inhibitors can be administered instead o heparin and a GPIIb–IIIa antagonist. Compared to the dual use o heparin and a GPIIb–IIIa antagonist, the administration o a direct thrombin inhibitor or this indication may result in ewer adverse bleeding events, although compared to heparin alone, there may be clinical equipoise.

ST Elevation Myocardial Infarction The treatment o STEMI is aimed at expeditious reper usion o the occluded epicardial coronary artery. As with UA/NSTEMI, aspirin and heparin are standards o care or STEMI; used alone, however, these agents are o ten

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

not su f cient to recanalize an occluded coronary artery (Fig. 26-9). Two approaches are used to open an occluded coronary artery: pharmacologic (thrombolysis) and mechanical (angioplasty or emergency coronary artery bypass). When thrombolysis is used, clopidogrel co-administration increases the likelihood that the in arct vessel will remain patent. GPIIb–IIIa antagonists are not utilized with thrombolytics because this combination con ers a signif cantly increased risk o bleeding, including hemorrhagic stroke. When angioplasty is per ormed, a GPIIb–IIIa antagonist may be employed together with clopidogrel as adjunctive treatment.

Thrombolytics

Four thrombolytic agents are currently used in the pharmacologic management o STEMI: streptokinase, alteplase, tenecteplase, and reteplase. (All are discussed in greater detail in Chapter 23.) The timeliness o administration is a crucial actor in determining the success o thrombolytic therapy in acute MI. Patients who receive thrombolytic therapy within 2 hours of the onset of symptoms have a twofold improvement in survival rate compared to patients who receive thrombolytic therapy more than 6 hours after the onset of symptoms. This observation is consistent with the known relationship between the duration o vessel occlusion and the extent o in arction. Several important contraindications to thrombolysis, primarily related to increased bleeding risk, may also limit use o this intervention. Streptokinase

The pharmacologic action o streptokinase involves two steps: complexation and cleavage. In the complexation reaction, streptokinase orms a stable, noncovalent 1:1 complex with plasminogen (either ree plasminogen or f brin-bound plasminogen). The complexation reaction produces a conormational change that exposes the active site on plasminogen. Plasminogen, now with its active site exposed, can e ect proteolytic cleavage o other plasminogen molecules (again, either ree plasminogen or f brin-bound plasminogen) to plasmin and thereby initiate thrombolysis. In the treatment o STEMI, streptokinase is administered as an intravenous loading dose ollowed by a continuous intravenous in usion. A ter 90 minutes o administration, streptokinase produces reper usion in 60% o acutely occluded vessels. However, the use ulness o streptokinase is limited by two actors. First, streptokinase is a oreign protein that is capable o eliciting antigenic reactions upon repeated administration. Patients with antibodies against streptokinase ( rom either a previous streptococcal in ection or previous treatment with streptokinase) can develop an allergic reaction and ever. Second, because the streptokinase:plasminogen complex activates both f brin-bound and ree plasminogen molecules, its relatively nonspecif c antithrombotic activity can result in systemic f brinolysis. Alteplase Alteplase is the generic name or recombinant tissue plas-

minogen activator (t-PA). Alteplase is e ective in restoring the patency o occluded coronary arteries, limiting cardiac dys unction, and reducing mortality ollowing STEMI. As with endogenously produced t-PA, recombinant t-PA binds to newly ormed thrombi with high a f nity, causing f brinolysis at the site o a thrombus. Once bound to the nascent

487

thrombus, t-PA undergoes a con ormational change that enhances plasminogen activation. t-PA is a poor activator o plasminogen in the absence o f brin binding. Recombinant t-PA is typically administered intravenously at a high dose rate or 1 hour and then at a lower dose rate or the next 2 hours. Despite its high a f nity or f brin-bound plasminogen, recombinant t-PA at pharmacologic doses can generate a systemic lytic state (as can other thrombolytic agents) and cause undesirable bleeding, including cerebral hemorrhage. Thus, this agent is contraindicated in patients who have had a recent stroke or other major bleeding event. Tenecteplase Tenecteplase is a genetically engineered variant o t-PA.

The molecular modif cations in tenecteplase increase its f brin specif city relative to t-PA and make it more resistant to plasminogen activator inhibitor 1. Large trials have shown that tenecteplase is identical in e f cacy to t-PA, with similar (and possibly decreased) risk o bleeding. Additionally, tenecteplase has a longer hal -li e than t-PA. This pharmacokinetic property allows tenecteplase to be administered as a single, weight-based bolus, thus simpli ying administration.

Reteplase

Similar to tenecteplase, reteplase is a genetically engineered variant o t-PA with increased hal -li e and increased specif city or f brin relative to t-PA. Its e f cacy and adverse e ect prof le are similar to those o t-PA. Because o its longer hal -li e, reteplase can be administered as a “double bolus” (two boluses, 30 minutes apart).

Primary Percutaneous Intervention

In the United States, the majority o patients with STEMI are treated with thrombolytics. Multiple studies have shown, however, that primary angioplasty, i per ormed within 90 minutes o presentation to the emergency department, yields a mortality benef t compared to thrombolysis. Increasingly, primary angioplasty includes placement o a drug-eluting stent. The our currently approved devices consist o a stainless steel or cobalt chromium alloy stent coated with sirolimus , everolimus , zotarolimus , or paclitaxel. Each o these agents decreases early restenosis by interrupting cell cycle progression (see Chapter 46, Pharmacology o Immunosuppression). Although drug-eluting stents were originally approved or the treatment o stable coronary artery disease, these devices are now o ten used in the treatment o acute coronary syndromes. Recent evidence has suggested that patients with drug-eluting stents may be at increased risk or late stent thrombosis (i.e., thrombosis occurring within the stent more than 30 days a ter initial stent placement), and dual antiplatelet therapy with aspirin and an ADP receptor antagonist or up to 1 year is indicated to prevent this complication in such patients.

Postmyocardial Infarction Management Patients must be care ully managed a ter an MI to prevent rein arction. The goals o any post-MI medical regimen are two old: (1) to prevent and treat residual ischemia and (2) to identi y and treat major risk actors such as hypertension, smoking, hyperlipidemia, and diabetes. Because the extent o the MI and its unctional consequences vary greatly among patients, the medical regimen must be individualized. The American College o Cardiology and the American

488 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

PART III: HEART FAILURE Mr. N is d is ch a rg e d ro m th e h o s p ita l o n a m u ltid ru g re g im e n th a t in clu d e s a s p irin , clo p id o g re l, m e to p ro lo l, a to rva s ta tin , lis in o p ril, a n d e p le re n o n e . He d o e s w e ll a s h e in cre a s e s h is a ctivity le ve l d u rin g th e f rs t 4–6 w e e ks a te r th e in a rctio n . At th a t p o in t, h o w e ve r, h e o n ce a ga in e xp e rie n ce s b re a th le s s n e s s a t m o d e ra te le ve ls o e xe rtio n . He in itia lly a ttrib u te s th is to d e co n d itio n in g , b u t h e b e co m e s co n ce rn e d e a rly o n e m o rn in g w h e n h e a w a ke n s ro m s le e p w ith s e ve re b re a th le s s n e s s . He s ch e d u le s a n a p p o in tm e n t w ith h is p hys icia n o r la te r th a t d a y. On e xa m in a tio n in th e p h ys icia n ’s o f ce , Mr. N a p p e a rs co m o rta b le s e a te d in th e u p rig h t p o s itio n . His h e a rt ra te is 64 b e a ts /m in , a n d h is b lo o d p re s s u re is 168/10 0 m m Hg . Th e p u lm o n ic co m p o n e n t o S 2 is p ro m in e n t (re p re s e n tin g a ch a n g e ro m h is p re vio u s e xa m s ), a n d th e a p ica l S 4 is a ga in n o te d ; th e re is a g ra d e III/VI a p ica l h o lo s ys to lic m u rm u r w ith ra d ia tio n to th e le t a xilla . An e ch o ca rd io g ra m re ve a ls a kin e s is o th e b a s a l s e g m e n t o th e in e rio r wa ll o th e le t ve n tricle , w ith p ro m in e n t th in n in g a n d a n e u rys m a l re m o d e lin g o th e s e g m e n t. Th e LV e je ctio n ra ctio n is a ga in q u a n tif e d a t 40%. Alth o u g h th e m itra l va lve le a e ts a n d th e s u p p o rtin g s tru ctu re s o th e va lve a p p e a r s tru ctu ra lly n o rm a l, th e re is a d e g re e o p o s te rio r le a e t p ro la p s e (LV → LA) d u rin g ve n tricu la r s ys to le . A Do p p le r s tu d y co n f rm s th e p re s e n ce o m itra l re g u rg ita tio n

Heart Association have made the ollowing general recommendations or the management o post-MI patients: 1. Aspirin (75–325 mg/d), in the absence o contraindications, or clopidogrel or patients with a contraindication to aspirin 2. -Antagonists 3. Lipid-lowering agents (targeting reductions in LDL cholesterol) 4. ACE inhibitors or patients with heart ailure, le t ventricular dys unction (ejection raction, 40%), hypertension, or diabetes 5. Spironolactone or eplerenone or patients with le t ventricular dys unction (ejection raction, 40%) 6. Clopidogrel, prasugrel, or ticagrelor in addition to aspirin, or a designated period, in patients who have undergone percutaneous coronary intervention In addition to designing an individualized drug regimen, the physician must also educate the patient about risk actors or the recurrence o MI. A use ul mnemonic or guiding overall treatment in post-MI patients is ABCDE: Antiplatelet agents, ACE inhibitors, antianginals, and aldosterone antagonists; Beta-antagonists and blood pressure control; Cholesterol-lowering and quitting cigarettes; Diet and diabetes control; Education and exercise.

th a t is a t le a s t m o d e ra te in s e ve rity. Th e rig h t ve n tricle is d ila te d a n d hyp e rtro p h ic, w ith re la tive p re s e rva tio n o s ys to lic u n ctio n . Re p e a t ca th e te riza tio n is p e r o rm e d to a s s e s s th e e tio lo g y o th e p a tie n t’s n e w b ive n tricu la r h e a rt a ilu re . An g io g ra p hy s h o w s w id e p a te n cy o th e rig h t co ro n a ry a rte ry a t th e s ite o th e p re vio u s p e rcu ta n e o u s co ro n a ry in te rve n tio n , a n d th e le t co ro n a ry s ys te m is re e o o b s tru ctio n . He m o d yn a m ic d a ta d e m o n s tra te in cre a s e d p u lm o n a ry a rte ry a n d rig h t ve n tricu la r p re s s u re s a s w e ll a s a n e le va te d p u lm o n a ry ca p illa ry w e d g e p re s s u re . Mr. N is p re s crib e d o ra l u ro s e m id e a t a s u f cie n t d o s e th a t h e lo s e s a p p roxim a te ly 5 p o u n d s in th e n e xt 3 d a ys . Be ca u s e h is b lo o d p re s s u re re m a in s e le va te d d e s p ite n e a r m a xim a l d o s e s o lis in o p ril a n d m e to p ro lo l, h e is a ls o p re s crib e d ca n d e s a rta n . Ove r th e co u rs e o th e n e xt w e e k, Mr. N n o te s a n im p rove m e n t in h is e xe rcis e to le ra n ce . He ch e cks h is w e ig h t re q u e n tly a n d , i it in cre a s e s b y m o re th a n 2 p o u n d s a b o ve h is u s u a l le ve l, h e ta ke s a n e xtra d o s e o u ro s e m id e .

Questions 7 . How does the addition of furosemide improve Mr. N’s symptoms? 8 . Which drug interactions are of concern when Mr. N adds candesartan to his regimen? 9 . Which parenteral inotropic agents would be available if Mr. N’s heart failure were to decompensate acutely?

PATHOPHYSIOLOGY OF HEART FAILURE Heart ailure is a common clinical problem. As many as 5 million patients in the United States carry this diagnosis, with approximately 500,000 new cases diagnosed each year. The syndrome o HF has a grave prognosis: the mortality rate at 5 years approximates 50%, and in the subset o patients with the most severe clinical symptoms, the annual mortality is as high as 30–50%. Because the impairment o cardiac unction that underlies this syndrome is o ten irreversible, HF is typically a chronic illness with intercurrent episodes o acute decompensation. Acute exacerbations are o ten multi actorial in etiology, with contributions rom dietary indiscretion (excess sodium or uid intake), nonadherence to prescribed medications, and concomitant noncardiac illness. Myocardial ischemia, progression o the proximate cause o cardiac disease, and activation o neurohumoral regulatory systems may also lead to clinical decompensation. The management o HF requires the clinician to construct, evaluate, and periodically modi y a treatment regimen that includes multiple drugs, some o which may carry signif cant risk or adverse interactions. Although the discussion that ollows emphasizes cardiogenic circulatory ailure, it should be noted that circulatory ailure can occur in the absence o contractile

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

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Le t ventricular contractile dys unction (systolic heart ailure ) is the primary cause o heart ailure. Although multiple disease states can result in contractile dys unction, the majority o cases o le t HF ( 70%) are attributed to CAD. Additional causes o systolic HF include chronic abnormalities o the loading conditions imposed on the heart, such as systemic arterial hypertension (pressure loading) and valvular heart disease (volume loading rom mitral regurgitation or aortic insu f ciency; pressure loading rom aortic stenosis). The contractile per ormance o the myocardium is initially preserved in disease states associated with abnormal loading conditions, but cardiomyocyte injury and whole-organ contractile dys unction supervene i the abnormal loading conditions are not corrected. The latter phase o cardiac pump dys unction has been re erred to as cardiomyopathy of chronic overload. Systolic dys unction can also result rom diverse conditions in which the proximate pathologic abnormality is cardiomyocyte injury or dys unction. These conditions are re erred to as dilated cardiomyopathies , because the heart characteristically remodels to produce LV chamber dilation (with or without wall thinning) in states o primary myocyte dys unction. Symptomatic HF can also occur in patients with normal or near-normal LV systolic unction (i.e., preserved LV ejection raction). In such cases, the symptoms o le t HF are caused by abnormalities o LV relaxation and/or f lling (diastolic heart ailure ). Impaired relaxation results in the elevation o LV diastolic pressure at any given f lling volume. This elevation o LV diastolic pressure causes elevation o le t atrial and pulmonary capillary pressures, leading to transudation o uid into the pulmonary interstitium (as well as secondary, or passive, elevation o pulmonary artery

m

Etiologies o Contractile Dys unction

(

dys unction (Table 26-4). Common examples include abnormalities o cardiac f lling (e.g., hypovolemia), cardiac rhythm (e.g., bradycardia or tachycardia), or the peripheral circulation (e.g., distributive shock related to sepsis). As always, treatment should be tailored to the pathophysiology in each individual case.

e

SVR, systemic vascular resistance; MAP, mean arterial pressure.

r

Hypertensive crisis (↑ SVR → ↑ impedance to LV ejection → ↓ stroke volume) Distributive shock (↓ SVR → ↓ MAP → organ hypoper usion)

u

Abnormal peripheral circulation

s

Bradycardia (↓ rate → ↓ orward output) Tachycardia (↑ rate → ↓ duration o diastolic f lling interval)

S troke volume

Re la xa tion

Contra ction

s

Abnormal cardiac rhythm

e

Hypovolemia (e.g., hemorrhage) Cardiac tamponade (compression by pericardial uid prevents normal diastolic f lling)

r

Abnormal cardiac f lling

P

MECHANISM

V

CAUSE OF CIRCULATORY FAILURE

and right heart pressures). The most common acute cause o isolated diastolic HF is acute myocardial ischemia. In the setting o acute reversible ischemia (i.e., ischemia not associated with MI), LV diastolic pressures increase as a consequence o incomplete LV relaxation. (Recall rom the discussion in Chapter 25, Pharmacology o Cardiac Contractility, that both contraction and relaxation o cardiomyocytes depend on adequate levels o intracellular ATP.) Both systolic and diastolic HF can be understood by considering the determinants o cardiac per ormance and the pathophysiologic conditions that a ect these parameters. Although diastolic dys unction is now appreciated as a common cause o clinical heart ailure, the balance o this section will deal principally with heart ailure due to systolic dys unction. Each o the major actors a ecting stroke volume— preload, a terload, and contractility—can be described by its e ect on cardiac unction curves. Figure 26-10 illustrates a normal LV pressure-volume loop. In the normal cycle, LV volume increases when the mitral valve opens during diastole. Isovolumetric contraction begins when LV pressure exceeds le t atrial pressure and the mitral valve closes; during this segment o the cardiac cycle, intraventricular pressure increases while intracavitary volume remains constant. Ejection begins when the impedance to LV ejection is exceeded and the aortic valve opens; ejected blood is then transmitted to the systemic circulation by the elastic properties o the aorta. The aortic valve closes when LV pressure alls below aortic pressure; at this point, intraventricular pressure decreases rapidly (isovolumetric relaxation), up to (and perhaps beyond) the point at which the mitral valve opens, and the cycle is repeated.

L

TABLE 26-4 Causes o Circulatory Failure in the Absence o Cardiac Pump Dys unction

489

EDP

MV clos e s MV ope ns

Ve ntricula r filling

ES V

EDV

LV Volume (ml) FIGURE 26-10. Normal le t ventricular pressure-volume loop. Mitral valve (MV) opening allows the le t ventricular (LV) volume to increase as the chamber f lls with blood during diastole. When ventricular pressure exceeds le t atrial pressure, the mitral valve closes. During the isovolumetric phase o systolic contraction, the le t ventricle generates a high pressure, which eventually orces open the aortic valve (AV). Ejection o the stroke volume ensues, and the aortic valve closes when aortic pressure exceeds LV pressure. Isovolumetric relaxation returns the ventricle to its lowest pressure state, and the cycle is repeated. Stroke volume (i.e., the volume o blood ejected with each contraction cycle) is the di erence between end-diastolic volume (EDV) and end-systolic volume (ESV). EDP, end-diastolic pressure; ESP, end-systolic pressure.

490 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

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LV Volume (ml) FIGURE 26-11. Determinants of cardiac output. Changes in preload, afterload, and myocardial contractility alter the pressure-volume relationship of the cardiac cycle. A. Increases in preload (lines 1, 2, 3 ) result in greater stretch of ventricular myocytes, development of greater ventricular end-diastolic pressure, and ejection of greater stroke volume (the Frank-Starling mechanism). Note that the end-systolic volume (ESV) is the same in each case, because the contractility of the heart has not changed. B. Increases in afterload (points 1, 2, 3 ) create greater impedance to left ventricular output and result in proportionately decreased stroke volume (the difference between end-diastolic volume [EDV] and ESV). The end-systolic pressure is linearly related to ESV; this linear relationship is called the end-systolic pressure-volume relationship (ESPVR). C. Increases in myocardial contractility (lines 1, 2 ), as occurs after administration of a positive inotrope, shift the ESPVR up and to the left, resulting in increased stroke volume.

As illustrated in Figure 26-11A, the orward stroke volume ejected by the LV depends on the degree o LV f lling during diastole, or preload. This undamental relationship between preload and stroke volume is the Frank-Starling law ; it derives rom the relationship between muscle length and degree o muscle shortening, as described in Chapter 25. In brie , increased diastolic volume increases myocardial f ber length. As a result, a higher raction o the actin f lament length is exposed in each sarcomere and is thereby available or myosin cross-bridge ormation when the cardiomyocyte is depolarized. Impedance to LV ejection, or afterload, is the second determinant o stroke volume (Fig. 26-11B). As impedance to ejection (a terload) increases, the stroke output o the ventricle alls. This characteristic o the intact heart derives rom the act that increasing the resistance against which cardiac muscle must contract leads to a decrease in the extent o shortening (i.e., to reduced stroke volume). Because the sensitivity o stroke volume to out ow resistance is accentuated in the ailing ventricle, agents that decrease a terload are able to increase LV stroke volume in patients with systolic HF (see below). The third determinant o cardiac per ormance is contractility, also described in Chapter 25. The contractile state o the LV is described by the end-systolic pressure-volume relationship (ESPVR, Fig. 26-11C). The ESPVR is, in e ect, a variant o the Frank-Starling law. While the Frank-Starling law def nes the relationship between LV diastolic volume (or preload) and LV stroke volume (or cardiac output), the ESPVR describes the relationship between diastolic f lling volume and LV tension development during isovolumetric contraction. As shown in Figure 26-11C, an increase in the contractile state o the LV, re ected by an upward shi t o the ESPVR, results in a greater degree o tension development or any given end-diastolic volume. In the presence o a f xed a terload, increased contractility results in a greater degree o muscle shortening and an increase in LV stroke volume.

The f nal determinant o cardiac pump per ormance is heart rate . Heart rate can be an important determinant o cardiac output in patients with systolic contractile dys unction. However, i LV contractile per ormance is preserved, then impairment o cardiac output occurs as a consequence o abnormal heart rate only at extreme rates outside the physiologic range.

Cardiac Compensation As the ability o the myocardium to maintain normal orward output ails, compensatory mechanisms are activated to preserve circulatory unction. The Frank-Starling mechanism increases stroke volume in direct response to increased preload. This recruitment o preload reserve is the f rst response o the system to hemodynamic stress. Hemodynamic stress that cannot be ully compensated by the Frank-Starling mechanism stimulates signaling systems that initiate structural changes at the cellular level, a process re erred to as remodeling o the myocardium. Although the underlying stimuli or remodeling remain an active area o investigation, it has been noted that the specif c pattern o remodeling is determined by the nature o the applied stress. I the Frank-Starling mechanism and remodeling mechanisms are unable to reestablish adequate orward cardiac output, neurohumoral systems are then activated. These systems modulate intravascular volume and vasomotor tone to maintain oxygen delivery to critical organs. Although each o these compensatory mechanisms contributes to the maintenance o circulatory unction, each may also contribute to the development and progression o pump dys unction and circulatory ailure, as described below.

Frank-Starling Mechanism

In the intact heart, increased preload leads to increased stroke volume via the Frank-Starling mechanism. This mechanism remains operative in the ailing heart; importantly, though, the relationship between end-diastolic volume and stroke

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure S ymptoms of high e nd-dias tolic pre s s ure

S ymptoms of high e nd-dia s tolic pre s s ure

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Afte rloa d re duction

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HF with pos itive inotrope

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Norma l

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Untre a te d HF

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Untre a te d HF

P re loa d re duction

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Ve ntricula r e nd-dia s tolic pre s s ure

S ymptoms of low ca rdia c output

Ve ntricula r e nd-dia s tolic pre s s ure

S ymptoms of low ca rdia c output

FIGURE 26-12. The Frank-Starling relationship in heart failure (HF). Left panel: The normal Frank-Starling relationship shows a steep increase in cardiac output with increasing ventricular end-diastolic pressure (preload). Point A describes the end-diastolic pressure and cardiac output o a normal heart under resting conditions. With contractile dys unction (untreated HF), cardiac output alls (B) and the Frank-Starling curve attens, so that increasing preload translates to only a modest increase in cardiac output (C). This increase in cardiac output is accompanied by symptoms o high end-diastolic pressure, such as dyspnea. Treatment with a positive inotrope, such as digitalis, shi ts the Frank-Starling curve upward, and cardiac output increases (D). The improvement in myocardial contractility supports a su f cient reduction in preload that the venous congestion is relieved (E). Right panel: Two o the principal pharmacologic treatments o HF are a terload reduction (e.g., ACE inhibitors) and preload reduction (e.g., diuretics). A terload reduction (F) increases cardiac output at any given preload and thereby elevates the Frank-Starling relationship. Preload reduction (G) alleviates congestive symptoms by decreasing ventricular end-diastolic pressure along the same Frank-Starling curve.

volume is altered. In patients with systolic dys unction, the relationship between end-diastolic volume and stroke volume is characterized by a f atter plateau (Fig. 26-12). Thereore, unlike normal individuals who are operating on the ascending limb o the Frank-Starling curve, where volume expansion can be a use ul strategy or increasing stroke volume, the majority o patients with heart ailure operate with elevated intravascular volume. This increased intravascular volume re ects the end result o neurohumoral activation (i.e., the sympathoadrenal axis and the renin-angiotensin-aldosterone system; see below). Thus, the treatment o cardiogenic circulatory ailure rarely involves volume expansion. It also merits emphasis that preload expansion can result in signif cant LV dilation, thereby increasing LV systolic and diastolic wall stress and exacerbating pulmonary congestion.

Cardiac Remodeling and Hypertrophy

In the setting o increased myocardial wall stress, cardiac hypertrophy develops in order to maintain ventricular systolic per ormance. Because LV ejection raction is inversely proportional to wall stress, adaptations that decrease systolic wall stress increase LV ejection raction. Laplace’s law states that wall stress ( ) is directly proportional to the pressure (P) and radius (R) o a chamber and inversely proportional to wall thickness (h): P

R/2h

Equation 26-1

In cases o chronic pressure overload, such as aortic stenosis or systemic hypertension, the LV develops a concentric pattern o hypertrophy as contractile proteins and new sarcomeres are added in parallel to the existing myof laments. Concentric hypertrophy simultaneously increases wall thickness (h) and decreases cavity size (R), resulting in

a net reduction in systolic wall stress and thereby preserving systolic per ormance. The disadvantage o concentric remodeling derives rom the decrease in LV compliance that occurs as a consequence o this pattern o hypertrophy. In a ventricle with reduced compliance, diastolic pressure in the chamber is increased at any given f lling volume. This in turn leads to elevation o LA and pulmonary capillary pressures, thereby predisposing to congestive symptoms. In conditions o chronic volume overload, such as mitral or aortic regurgitation, the LV develops an eccentric pattern o hypertrophy as contractile proteins and new sarcomeres are added in series to the existing myof laments. Eccentric hypertrophy helps to maintain cardiac per ormance via modulation o diastolic wall stress. In contrast to the situation that occurs a ter concentric remodeling, eccentric hypertrophy is associated with increased LV compliance. The increase in compliance allows LV end-diastolic volume to increase without a signif cant elevation in le t ventricular and le t atrial diastolic pressures. This attenuation o the rise in chamber pressure allows the system to maintain orward cardiac output by a volume-driven increase in total stroke volume. During the compensated phase o eccentric hypertrophy, LV wall thickness increases in approximate proportion to the increase in chamber radius.

Neurohumoral Activation

Failure o the heart to provide adequate orward output activates several neurohumoral systems, o ten with deleterious consequences (Fig. 26-13). Decreased arterial pressure activates the baroreceptor re ex, stimulating release o catecholamines; in turn, the catecholamines produce tachycardia (via 1-receptors) and vasoconstriction (via peripheral 1-receptors). Stimulation o 1-receptors on renal

492 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY Compromis e d ca rdia c function

Compromis e d ca rdia c function

De cre a s e d a rte ria l blood pre s s ure

De cre a s e d a rte ria l blood pre s s ure

Ba rore ce ptor re fle x

Incre a s e d re nin

β

Incre a s e d s ympa the tic outflow

α Incre a s e d a ldos te rone

Incre a s e d ATII

Va s ocons triction

Ba rore ce ptor re flex

-Antagonists

Incre a s e d re nin

Incre a s e d s ympa the tic outflow

Incre a s e d ATII

Va s ocons triction

ACE inhibitors Incre a s e d a ldos te rone

Vasodilators Incre a s e d a fte rloa d

Na + re te ntion

Intra va s cula r volume e xpa ns ion

Incre a s e d pre loa d

Incre a s e d myoca rdia l O 2 de ma nd Wors e ning he a rt fa ilure

FIGURE 26-13. Neurohumoral effects of heart failure. Compromised car-

Spironolactone Diuretics Na + re te ntion

Intrava s cula r volume expa ns ion

Incre a s e d pre loa d

Venodilators

Incre a s e d a fte rloa d

Incre a s e d myoca rdia l O 2 de ma nd Wors e ning he a rt fa ilure

FIGURE 26-14. Pharmacologic modulation of the neurohumoral effects of

diac unction leads to decreased arterial blood pressure, which activates baroreceptors that increase sympathetic outf ow. -Adrenergic sympathetic outf ow ( ) causes vasoconstriction, which increases a terload. The increased a terload creates a greater pressure against which the heart must contract and thereby increases myocardial O2 demand. -Adrenergic sympathetic outf ow ( ) increases juxtaglomerular cell release o renin. Renin cleaves angiotensinogen to angiotensin I, and angiotensin I is then converted to the active hormone angiotensin II (AT II). AT II has a direct vasoconstrictor action; it also increases aldosterone synthesis and secretion. Aldosterone increases collecting duct Na reabsorption, leading to intravascular volume expansion and increased preload. Together, the increased a terload and preload increase myocardial O2 demand. In the already compromised heart, these increased stresses can lead to worsening heart ailure.

heart failure. Many therapeutic agents used in the management o heart ailure modulate the neurohumoral systems that are activated by compromised cardiac unction. The renin-angiotensin-aldosterone system can be inhibited by (1) adrenergic antagonists, which inhibit renin release by the juxtaglomerular cells o the kidney; (2) ACE inhibitors, which prevent the conversion o angiotensin I to the active hormone angiotensin II; and (3) spironolactone, which competitively antagonizes aldosterone binding to the mineralocorticoid receptor. Diuretics promote Na excretion and thereby counteract the Na retention stimulated by activation o the renin-angiotensin-aldosterone system. Venodilators counteract the e ect o intravascular volume expansion by increasing peripheral venous capacitance and thereby decreasing preload. Direct arterial vasodilators alleviate the -adrenergic receptor-mediated and angiotensin II receptormediated vasoconstriction induced by increased sympathetic outf ow. Cardiac glycosides, -adrenergic agonists, and cardiac phosphodiesterase inhibitors are also used in HF to increase myocardial contractility (not shown).

juxtaglomerular (JG) cells promotes the release o renin. JG cells also release renin in response to the decreased renal per usion that accompanies decreased cardiac output. Renin cleaves circulating angiotensinogen to angiotensin I, which is subsequently converted by angiotensin converting enzyme (ACE) to angiotensin II (AT II). AT II acts through AT1 receptors to increase arterial vasomotor tone. AT II also activates several physiologic mechanisms that increase intravascular volume, including aldosterone release rom the adrenal glands (thus promoting salt and water retention), vasopressin (ADH) release rom the posterior pituitary gland, and thirst center activation in the hypothalamus. In addition, AT II appears to be an important mediator o vascular and myocardial hypertrophy. The tachycardia and increased intravascular volume that accompany activation o these neurohumoral mechanisms help to maintain orward cardiac output, and the systemic vasoconstriction provides a mechanism by which central regulatory centers can override local autoregulation o blood

f ow. Together, these mechanisms allow the cardiovascular system to maintain per usion o critical organs in the setting o reduced cardiac output. However, sympathetic stimulation o the heart also increases myocardial oxygen demand by increasing both a terload (arteriolar constriction) and preload (retention o sodium and water). Continued sympathetic stimulation eventually results in down-regulation o -adrenergic receptors, urther impairing the ability o the system to maintain orward output. The central aim of the current pharmacologic management of HF is to modulate the action of these neurohumoral effectors (Fig. 26-14).

CLINICAL MANAGEMENT OF HEART FAILURE The pharmacologic treatment o HF has expanded dramatically over the past three decades. Numerous large-scale clinical trials have demonstrated that the new, “load-active”

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

therapies are associated with statistically signif cant reductions in morbidity and mortality in patients with HF. In addition, improvements in the detection and treatment o hypertension and the management o complex multivessel CAD have dramatically altered the clinical course o patients with contractile dys unction. It is help ul to organize the treatment strategies or contractile dys unction in patients who exhibit or are at risk to develop symptomatic heart ailure according to the ollowing physiologic goals: preload reduction, a terload reduction, and contractility enhancement (increased inotropy). Table 26-5 provides a summary o the hemodynamic e ects and mechanisms o action o the drug classes that are commonly used to treat heart ailure.

Preload Reduction Diuretics

Diuretics have long been cornerstones o the pharmacologic management o patients with le t ventricular ailure and remain integral components o the treatment o patients with congestive symptoms and/or intravascular volume overload.

493

However, despite the e f cacy o these agents at reducing congestive symptoms, there is no evidence o a mortality benef t rom treatment with either loop diuretics or thiazide diuretics. The natriuretic agents most commonly used in HF are the loop diuretics urosemide and bumetanide. These drugs inhibit the Na -K -2Cl co-transporter (NKCC2) in the thick ascending limb o Henle, resulting in increased excretion o sodium, potassium, and water. Thiazide diuretics such as hydrochlorothiazide are also used to treat congestive symptoms, particularly in patients with hypertensive heart disease and LV systolic dys unction. Thiazides inhibit sodium and chloride reabsorption via the Na -Cl co-transporter (NCC) in the distal convoluted tubule. Thiazides are less e f cacious natriuretic agents than loop diuretics and are o ten ine ective as monotherapy or congestive symptoms in patients with chronic kidney disease. Thiazides are sometimes coadministered with loop diuretics in patients with reduced GFR and re ractory volume overload and in selected patients with HF in whom treatment with loop diuretics alone does not achieve adequate diuresis. (Re er to Chapter 21 or an

TABLE 26-5 Pharmacologic Agents Used in the Treatment of Heart Failure DRUG OR DRUG CLASS

MECHANISM OF ACTION

HEMODYNAMIC EFFECT

CLINICAL NOTES

Inhibit AT II generation → ↓ AT1 receptor activation

Decreased a terload Decreased preload

May cause hyperkalemia

Competitive antagonists at -adrenergic receptor → ↓ renin release

Decreased a terload Decreased preload

May be relatively contraindicated in severely decompensated heart ailure

Competitive antagonist at aldosterone receptor

Decreased preload

Mortality benef t may be independent o hemodynamic e ects; may cause hyperkalemia

Drugs with Proven Mortality Reduction ACE inhibitors -Antagonists

Spironolactone

Drugs or Treatments Used to Improve Symptoms Na /H2O restriction

Decreases intravascular volume

Decreased preload

May help limit edema ormation

Diuretics

Inhibit renal Na reabsorption

Decreased preload

Furosemide most e ective or treating congestive symptoms

Aquaretics

Competitive antagonists at vasopressin V2 receptor → ↓ renal aquaporin expression and membrane tra f cking → ↓ ree water reabsorption

Decreased preload

Increased output o soluteree urine; increased serum sodium

Digoxin

Inhibits Na /K ATPase → ↑ intracellular Ca 2 → ↑ contractility

Increased contractility

Delays atrioventricular nodal conduction

Organic nitrates

Increase NO → venous smooth muscle relaxation → ↑ venous capacitance

Decreased preload

Reduces myocardial O2 demand

Dobutamine

Stimulates -adrenergic receptors

Increased contractility ( 1 e ect) Decreased a terload ( 2 e ect)

Used in the acute setting only

Inamrinone, milrinone

Inhibit phosphodiesterase → ↑ -adrenergic e ect

Increased contractility Decreased a terload Decreased preload

Used in the acute setting only

494 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

extended discussion o diuretics.) In the introductory case, the decrease in intravascular volume achieved with urosemide signif cantly improves Mr. N’s congestive symptoms, and he may require long-term administration o oral urosemide to stabilize these symptoms.

Aquaretics

Patients with heart ailure have increased circulating levels o vasopressin, and the extent o vasopressin elevation correlates with the severity o heart ailure. Selective antagonism o the vasopressin V2 receptor results in increased output o solute- ree urine and increased serum sodium levels in patients with heart ailure. The clinical application o vasopressin antagonists (so-called aquaretics) in heart ailure remains controversial, but both conivaptan and tolvaptan are approved or use in patients with heart ailure. Conivaptan is available as an intravenous in usion or the treatment o hypervolemic hyponatremia. In patients with acute decompensated heart ailure requiring hospitalization, addition o oral tolvaptan to a standard therapy regimen increases weight loss and decreases edema over the f rst seven days but has no signif cant long-term e ect on recurrent hospitalizations or mortality (EVEREST trial).

Aldosterone Receptor Antagonists

Spironolactone is a potassium-sparing diuretic that acts as a competitive antagonist at the aldosterone receptor, thus decreasing sodium–potassium exchange in the distal tubule and collecting duct o the nephron. A clinical trial o this agent in patients with systolic HF has received much attention (RALES trial). In this study, patients with severe HF were treated with low-dose spironolactone (25–50 mg daily); enrolled patients were ree o signif cant renal impairment and were concomitantly receiving standard therapy or heart ailure (ACE inhibitor, -blocker, loop diuretic digoxin). In patients treated with spironolactone, all-cause mortality (including sudden cardiac death and death rom progressive heart ailure) was reduced by approximately 30%, as were hospital admissions or exacerbations o HF. A subsequent trial with a similar agent, eplerenone, conf rmed these f ndings in patients with HF a ter MI (EPHESUS trial). Spironolactone is o ten administered in combination with an ACE inhibitor and/or angiotensin receptor blocker (see below). Because spironolactone, ACE inhibitors, and angiotensin receptor blockers all decrease K excretion, plasma K levels must be monitored care ully, and potassium supplementation must be undertaken with caution.

Venodilators

Venodilator agents are o ten co-administered with diuretics in patients with congestive symptoms. The prototypical venodilator is nitroglycerin (NTG). This drug increases venous capacitance and thereby decreases venous return to the heart. The decrease in venous return results in reduced LV chamber volume and reduced LV diastolic pressure. These e ects o the nitrates decrease myocardial oxygen demand, which may be especially benef cial in patients with coexisting angina and LV dys unction. Nitrates may also be particularly e ective in cases where le t HF results rom acute myocardial ischemia. In this condition, LV relaxation is impaired, LV compliance is decreased, and LV diastolic pressure is typically elevated. By increasing venous capacitance, nitrates reduce venous return to the

heart and decrease LV diastolic volume. In turn, the decrease in diastolic volume leads to a decrease in myocardial oxygen consumption. In addition, nitrates may alleviate ischemia, thereby improving diastolic relaxation. Thus, the benef cial e ects o nitrate administration in this setting include both preload reduction and improvement in LV compliance.

Afterload Reduction ACE Inhibitors

ACE inhibitors reversibly inhibit angiotensin converting enzyme (ACE). The resulting decrease in angiotensin II (AT II) leads to several potential benef ts. AT II is an important component o the neurohumoral regulation o the ailing circulation. In response to renal hypoper usion, the kidney increases renin secretion, which results in increased production o AT II, as noted above (also see Chapter 21). In turn, AT II stimulates the adrenal gland to secrete aldosterone. Overall, activation o the renin-angiotensin-aldosterone system increases vasomotor tone as well as sodium and water retention. These hemodynamic alterations result in increased intravascular volume (leading, ultimately, to increased LV diastolic f lling and increased LV stroke volume) and peripheral redistribution o the cardiac output (mediated by the vasoconstrictor e ects o AT II). Administration o an ACE inhibitor reverses the vasoconstriction and volume retention that characterize reninangiotensin-aldosterone system activation. The reduction in a terload decreases the impedance to LV ejection and thereby increases LV stroke volume. The reversal o aldosteronerelated volume retention decreases preload. These e ects are synergistic in patients with HF: as stroke volume increases, GFR is also increased, leading to increased delivery o sodium and water to the distal nephron, where (in the absence o renin-stimulated elevation o aldosterone levels) natriuresis and diuresis occur. ACE inhibition can also increase venous capacitance (and thereby reduce preload) by decreasing degradation o the endogenous vasodilator bradykinin. By altering the myocardial remodeling that occurs a ter ST elevation myocardial in arction, ACE inhibitors can provide urther benef t in patients with concomitant HF and CAD. ACE inhibitors have a statistically signif cant impact on survival in patients with heart ailure. This mortality benef t was f rst demonstrated in patients with severe heart ailure in the CONSENSUS trial: the mortality reduction approximated 40% at 6 months and 31% at 1 year. The mortality benef t o the ACE inhibitors was conf rmed in a broader spectrum o patients in the SOLVD Treatment trial (16% reduction in mortality) and the V-He t II trial (28% reduction in mortality), as well as in patients in the convalescent phase ollowing MI (SAVE trial, 19% reduction in mortality). AT1 antagonists (sometimes called angiotensin receptor blockers or ARBs) are a class o agents that inhibit the renin-angiotensin-aldosterone axis at the level o the angiotensin II receptor. The hemodynamic prof le o these agents is similar to that o the converting enzyme inhibitors. Recent clinical trials have demonstrated a mortality benef t or AT1 antagonists in patients with severe systolic HF (LV ejection raction, 40%) who are unable to take ACE inhibitors. In patients with HF who are already taking an ACE inhibitor, the addition o an AT1 antagonist reduces hospital readmissions or HF but does not reduce mortality (CHARMAdded trial).

C h a p t e r 2 6 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure

Recent data suggest that combining blockade o the reninangiotensin system with up-regulation o the natriuretic peptides bradykinin and adrenomedullin may improve clinical outcomes. Up-regulation o the natriuretic peptides can be achieved through inhibition o neprilysin, an endopeptidase that degrades bradykinin and adrenomedullin. In a recent clinical trial, combination therapy with an ACE inhibitor and a neprilysin inhibitor resulted in a 20% reduction in cardiovascular death and a 21% reduction in heart ailure hospitalization compared to therapy with an ACE inhibitor alone in patients with severe systolic HF. These data, published in the PARADIGM-HF trial, may represent a new therapeutic modality or patients with heart ailure and reduced systolic unction.

-Adrenoceptor Antagonists

Much recent attention has been directed at the use o adrenoceptor antagonists in the treatment o patients with HF. Although the use o -antagonists might seem counterintuitive, clinical trials have now established that these agents increase survival in heart ailure patients. The benef ts o antagonists in patients with heart ailure have been variably attributed to (1) inhibition o renin release, (2) attenuation o the cytotoxic and signaling e ects o elevated circulating catecholamines, and, more generally, (3) prevention o myocardial ischemia. Thus, -antagonists, like ACE inhibitors, may attenuate the adverse e ects o neurohumoral regulators in patients with heart ailure. Furthermore, because -antagonists and ACE inhibitors act via distinct mechanisms and have non-overlapping toxicities, it is reasonable to co-administer these classes o drugs to HF patients.

Vasodilators

Hydralazine is a direct-acting vasodilator that decreases systemic vascular resistance and thereby reduces a terload. The mechanism o action o hydralazine remains to be determined. The arterial vasodilation produced by hydralazine is particularly pronounced when the drug is administered intravenously. The clinical use o hydralazine has been limited by several actors, including the induction o re ex tachycardia during intravenous administration, the development o tachyphylaxis, and the occurrence o a drug-induced lupus syndrome during chronic administration. This agent has demonstrated a mortality benef t in HF when co-administered with organic nitrates (AHEFT Trial). The nitrate–hydralazine combination is typically reserved or patients who cannot tolerate therapy with an ACE inhibitor, although in certain populations, particularly A rican Americans, the addition o nitrates and hydralazine to standard therapy may be superior.

Inotropic Agents Cardiac Glycosides

Digitalis glycosides inhibit the sarcolemmal Na /K ATPase in cardiac myocytes. This action increases intracellular Na , activates the Na /Ca2 exchanger, and increases intracellular Ca2 , including the Ca2 stores in the sarcoplasmic reticulum. This, in turn, leads to increased calcium release upon myocyte stimulation, resulting in increased myocardial contractility (i.e., upward/le tward shi t o the ESPVR). Although patients with HF o ten experience relie o congestive symptoms during treatment with the cardiac glycosides, these drugs have not been shown to decrease mortality.

495

Sympathomimetic Amines

Dobutamine is the parenteral sympathomimetic amine used most commonly in the treatment o decompensated systolic HF (pulmonary congestion accompanied by reduced orward cardiac output). This agent is a synthetic congener o epinephrine that stimulates 1-receptors and, to a lesser extent, 2-receptors and 1-receptors. The stimulation o 1receptors predominates at therapeutic in usion rates, leading ultimately to an increase in the contractility o cardiac myocytes. Stimulation o vascular 2-receptors causes arterial vasodilation and a reduction in a terload. The combined e ects o increased contractility and decreased a terload lead to improvement in overall cardiac per ormance. Dobutamine is typically used in the acute setting (i.e., intensive care unit). In the introductory case, i Mr. N were to become hypotensive due to decreased cardiac output or to develop evidence o decreased end-organ per usion such as a rise in serum creatinine, dobutamine could be administered acutely to stabilize his hemodynamic status.

Phosphodiesterase Inhibitors

Phosphodiesterase inhibitors (such as inamrinone and milrinone ) inhibit the degradation o cAMP in cardiac myocytes and thereby increase intracellular calcium and enhance contractility (inotropy). In the systemic vasculature, these agents cause dilation o both arteriolar resistance vessels and venous capacitance vessels, thereby decreasing a terload and preload. As a result o these aggregate e ects, phosphodiesterase inhibitors have been re erred to as ino-dilators. Despite these positive actions, both phosphodiesterase inhibitors and sympathomimetic amines are reserved or shortterm treatment o patients with acute decompensation o heart ailure. Indeed, long-term treatment with oral phosphodiesterase inhibitors has been shown to increase mortality.

Combination Therapy The drugs described in this chapter o er a number o approaches to the pharmacotherapy o heart ailure. Some agents, most notably ACE inhibitors and -antagonists, have demonstrated signif cant mortality benef t in randomized clinical trials and should probably be viewed as the new cornerstones o therapy. Other drugs, such as digoxin and diuretics, have been mainstays o symptomatic relie despite a lack o mortality benef t. Use o combination therapies must be approached cautiously in HF patients to avoid adverse e ects such as hypotension, arrhythmias, electrolyte imbalances, and renal insu f ciency. Nonetheless, it is typical or these patients to require multidrug regimens to optimize their unctional status.

CONCLUSION AND FUTURE DIRECTIONS Hypertension, ischemic heart disease, and HF are common cardiovascular diseases that occur singly and in combination. Therapeutic strategies target the cellular and molecular pathways that are dys unctional in these disease states. Combination therapy with drugs rom multiple classes is o ten required to address the complex pathophysiology o these conditions and achieve the desired therapeutic result. Current research in cardiovascular genomics and neurohumoral pathways promises to provide new understanding

496 PRINCIPLES OF CARDIOVASCULAR PHARMACOLOGY

o the pathophysiology o cardiovascular disease. For example, the pathophysiology o essential hypertension may, in many cases, involve mutations or polymorphisms in the genes that code or angiotensinogen, renin, the angiotensin II receptor (AT1), endothelin, the glucocorticoid receptor, the insulin receptor, endothelial nitric oxide synthase, and the epithelial Na channel (ENaC). As the genetic determinants o cardiovascular regulation are clarif ed, it may be possible to identi y high-risk patients prospectively and to develop targeted therapies that exert their therapeutic e ects on the molecular and cellular mechanisms predicted to drive the disease in these patients. In recent years, agents targeting neurohumoral pathways—such as ACE inhibitors and -antagonists—have become cornerstones o therapy or all cardiovascular diseases. Large clinical trials have consistently demonstrated that these drugs reduce adverse cardiovascular events, including mortality, in patients with hypertension, patients with coronary artery disease and prior MI, and patients with systolic HF. Over the past 25 years, increased understanding o basic disease mechanisms has improved the physician’s ability to alter both the clinical expression and progression o cardiovascular diseases: examples include recent advances in the primary prevention o coronary artery disease and the positive impact o neurohumoral modulation on the progression o HF. Current research aims to identi y and characterize new drug targets, including a host o signaling molecules that are abnormal in the ailing heart. Elevated levels o in ammatory mediators—such as tumor necrosis actor- (TNF- ), interleukin-6 (IL-6), and endothelin-1—and enzymes—such as inducible nitric oxide synthase, collagenases, and matrix metalloproteinases—have all been reported to contribute in some way to the detrimental structural and unctional changes that occur in the ailing heart.

Acknowledgment We thank April W. Armstrong and Thomas P. Rocco or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Hypertension

ALLHAT O f cers and Coordinators or the ALLHAT Collaborative Research Group. Major outcomes in high-risk hypertensive patients randomized to angiotensin-converting enzyme inhibitor or calcium channel blocker

vs. diuretic: the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT). JAMA 2002;288:2981–2997. (Results o a major trial comparing agents or initial treatment o hypertension.) Jamerson K, Weber MA, Bakris GL, et al. Benazepril plus amlodipine or hydrochlorothiazide or hypertension in high-risk patients. N Engl J Med 2008;359:2417–2428. (Clinical trial suggesting benef t o combination therapy with ACE inhibitor and calcium channel blocker.) James PA, Oparil S, Carter BL, et al. 2014 evidence-based guidelines or the management o high blood pressure in adults. JAMA 2014;311:507–520. (Current guidelines or classi ying and treating hypertension.)

Ischemic Heart Disease

Abrams J. Chronic stable angina. N Engl J Med 2005;352:2524–2533. (Clinical pharmacology o chronic coronary artery disease treatments.) Anderson JL, Adams CD, Antman EM, et al. ACC/AHA 2007 guidelines or the management o patients with unstable angina and non-ST elevation myocardial in arction. Summary article: a report o the American College o Cardiology/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol 2007;50:652–726. (Current guidelines or evaluating and treating patients with unstable angina and non-ST elevation myocardial in arction.) Armstrong EJ, Morrow DA, Sabatine MS. In ammatory biomarkers in acute coronary syndromes. Part I: introduction and cytokines. Part II: acute-phase reactants and biomarkers o endothelial cell activation. Part III: biomarkers o oxidative stress and angiogenic growth actors. Part IV: matrix metalloproteinases and biomarkers o platelet activation. Circulation 2006;113:72–75, 152–155, 289–292, 382–385. (Four-part series reviewing pathophysiology and clinical evidence concerning the role o in ammatory mediators in acute coronary syndromes.) Cannon CP, Braunwald E, McCabe CH, et al. Intensive versus moderate lipid lowering with statins a ter acute coronary syndromes. N Engl J Med 2004;350:1495–1504. (Trial demonstrating clinical benef t or aggressive statin therapy a ter acute coronary syndrome.) Libby P. The molecular mechanisms o the thrombotic complications o atherosclerosis. J Intern Med 2008;263:517–527. (Molecular basis o coronary artery atherosclerosis.)

Heart Failure

ACCF/AHA 2009 ocused update: guidelines or the diagnosis and management o heart ailure in adults. J Am Coll Cardiol 2009;53:e1–e90. (Consensus guidelines or management o heart ailure.) Jessup M, Brozena S. Heart ailure. N Engl J Med 2003;348:2007–2018. (Clinical approach to heart ailure.) McMurray JJV, Packer M, Desai AS, et al. Angiotensin-neprilysin inhibition versus enalapril in heart ailure. N Engl J Med 2014;371:993–1004. (Trial showing mortality benef t to inhibition o neprilysin in addition to ACE inhibition.) Opie LH. Cellular basis or therapeutic choices in heart ailure. Circulation 2004;110:2559–2561. (Molecular basis o heart ailure therapeutics.) Taylor AL, Ziesche S, Yancy C, et al. Combination o isosorbide dinitrate and hydralazine in blacks with heart ailure. N Engl J Med 2004;351:2049–2057. (Trial showing mortality benef t in sel -identif ed black patients.)

S ERM

Es trogee n

Coffa ctor Y

Es troo ge n re ce p tor

Coo fa ctoo r X Nuu cle u s

D NA X Ge nnee 1 Y G e ne 2 X Y G e ne 3

Es trogg e n re ce ptor

IV

G e nee s 1 , 2, a n d 3 e xp re s s e d : F u ll a gonn is t

Principles of Bre a s t: only y Y cofa ctto r e xpre s s e d Endocrine Pharmacology

S ERM

Es trogee n

C offa ctor Y

Es troo ge n re ce p tor

Nuu cle u s

D NA X Ge ne 1 Y G e ne 2 X Y Ge nee 3

G e ne 2 e xp re s s e d : F u ll a gonn is t

Es trog ge n re ce ptor

Hypo Hy poth th a la la m mus

27

C H CR

Pharmacology of the Hypothalamus and Pituitary Gland P ituita ry gla nd

An a n d Va id ya a n d Urs u la B. Ka is e r

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498–499 INT HYPOTHALAMIC AND PITUITARY PHY HYSI SIO OLOGY . . . . . . . . . . . . 498 Relationship Between the Hypothalamus Re t a nd Pitui uita itary a ry Gla land n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498 Feedback Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 5011 PHYSIOLOGY, PATHOPHYSIOLOGY, AND PHARM MAC ACOL OLOG OGY Y OF INDIVIDUAL AXES S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501 Ante An teri rior Pituitary i Gland. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501 Hypothalamic-Pituitary-Growth Hormone Axiss . . . . . . . . . 501

INTRODUCTION The hypothalamus and pituitary gland unction cooperatively as master regulators o the endocrine system. Together, hormones secreted by the hypothalamus and pituitary gland control important homeostatic and metabolic unctions, including reproduction, growth, lactation, thyroid and adrenal gland physiology, and water homeostasis. This chapter introduces the physiology and regulation o hypothalamic and pituitary hormones through a discussion o eedback regulation and the various axes o hormonal regulation. The pharmacologic utility o hypothalamic and pituitary actors is then discussed, with emphasis on the regulation o specif c endocrine pathways. Three concepts are o special importance in this chapter: (1) hypothalamic control o pituitary hormone release, (2) negative eedback inhibition, and (3) endocrine axes. A thorough understanding o these pathways and their mechanisms provides the oundation or understanding the use o pharmacotherapy to modulate the hypothalamic-pituitary axes.

HYPOTHALAMIC AND PITUITARY PHYSIOLOGY Relationship Between the Hypothalamus and Pituitary Gland From a developmental perspective, the pituitary gland consists o two closely associated organs. The anterior pituitary (adenohypophysis) is derived rom ectodermal tissue. 498

ACTH H

Cortis rtis o l

Hypothalamic-Pituiitary-Pro Pro Pr rola lact ctin tin Axis . . . . . . . . . . . . . . . . . 504 Hypothalamic-Pituiitary-T Thyroid Axi xiss . . . . . . . . . . . . . . . . . . 50 5055 Hypothalamic-Pituiitary y-A -Adr d enall Axi xiss . . . . . . . . . . . . . . . . . . 50 5055 Adre dre na lamic-Pitu Hypothalamic-P Pit itui uiitary ry-G ry -Gon -G onad on a all Axi ad xiss . . . . . . . . . . . . . . . . . 506 gla nd dor Pi Pittuiitary Glaa nd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507 Antidiuretic Hormoone n (AD A H H)) . . . . . . . . . . . . . . . . . . . . . . . . . 507 Oxytocin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 CONCLUSION AND FUTU URE DIRECTIONS . . . . . . . . . . . . . . . . . . 509 Sugg Su gges este tedd Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .509

The posterior pituitary (neurohypophysis) is a neural structure derived rom the ventral sur ace o the diencephalon. The pref xes adeno- and neuro- denote the oral ectodermal and neural ectodermal origin o the anterior and posterior pituitary gland components, respectively. An intermediate lobe also exists in most mammals but is vestigial in humans. Although the anterior and posterior pituitary glands derive rom di erent embryologic origins, the hypothalamus controls the activity o both lobes. The connection between hypothalamus and pituitary gland is one o the most important points o interaction between the nervous and endocrine systems. The hypothalamus acts as a neuroendocrine transducer by integrating neural signals rom the brain and converting those signals into chemical messages (largely peptides) that regulate the secretion o pituitary hormones. In turn, the pituitary hormones alter the activities o peripheral endocrine organs. Hypothalamic control o the anterior pituitary gland occurs via hypothalamic secretion o hormones into the hypothalamic-pituitary portal vascular system (Fig. 27-1). The initial capillary bed o this portal system is ormed rom branches o the superior hypophyseal artery that an around the axon terminals o hypothalamic neurons. Endothelial enestrations in this capillary bed allow hypothalamic actors to be released into the bloodstream. These capillaries then coalesce into short veins that extend to the anterior pituitary gland. Upon arriving at the anterior pituitary, the veins branch into a second capillary bed and bathe the endocrine cells o the anterior pituitary gland with hormones secreted by the hypothalamus.

C h a p t e r 2 7 Pharmacology of the Hypothalamus and Pituitary Gland

GR is a 42-ye a r-o ld s a le s e xe cu tive . S h e tra ve ls co n s ta n tly a n d p rid e s h e rs e l o n b e in g e n e rg e tic a n d s u rp a s s in g s a le s p ro je ctio n s e a ch q u a rte r. Th re e ye a rs a g o , s h e d e ve lo p e d irre g u la r m e n s e s , a n d s h e th e n co m p le te ly s to p p e d m e n s tru a tin g . Ove r th e p a s t 2 ye a rs , s h e h a s b e g u n to e e l in cre a s in g ly a tig u e d , h a s d i f cu lty ru s h in g th e le n g th o a irp o rt te rm in a ls , a n d is b o th e re d b y re q u e n t h e a d a ch e s . S h e h a s a lwa ys h a d a f rm h a n d s h a ke , b u t la te ly, s h e h a s a ls o n o tice d th a t h e r w e d d in g rin g is e xce s s ive ly tig h t. GR is a ls o ru s tra te d th a t s h e h a s re ce n tly h a d to re p la ce h e r e n tire s h o e co lle ctio n b e ca u s e h e r s h o e s ize h a s in cre a s e d ro m 7½ to 9, w ith a n e e d o r in cre a s e d w id th a s w e ll. Ad d itio n a lly, s h e h a s n o tice d in cre a s e d p e rs p ira tio n , e ve n w h e n s h e is n o t e xe rtin g h e rs e l , a n d in cre a s e d s p a cin g b e tw e e n h e r te e th . Co n ce rn e d a b o u t h e r p ro g re s s ive co s m e tic ch a n g e s a n d h e r la ck o m e n s e s , GR tu rn s to In te rn e t s e a rch e s o r m o re in o rm a tio n a n d co m e s a cro s s a co n d itio n ca lle d a cro m e ga ly. S tru ck b y th e u n ca n n y re s e m b la n ce s b e tw e e n h e r co m p la in ts a n d th o s e s h e h a s re a d a b o u t o n th e In te rn e t, GR a rra n g e s to s e e h e r d o cto r o r u rth e r e va lu a tio n . A s e ru m in s u lin -like g ro w th a cto r (IGF-1) le ve l is s ig n if ca n tly e le va te d a te r co rre ctio n o r GR’s a g e a n d g e n d e r, a n d h e r s e ru m g ro w th h o rm o n e le ve l is 10 n g /m L (n o rm a l, 1 n g /m L) a te r a n o ra l g lu co s e lo a d o 75 m g . A m a g n e tic re s o n a n ce im a g in g (MRI) s tu d y o h e r h e a d re ve a ls a p itu ita ry a d e n o m a w ith m a xim a l d ia m e te r o 1.5 cm . Th e s e f n d in g s a re co n s is te n t w ith a d ia g n o s is o a cro m e g a ly d u e to a g ro w th h o rm o n e -s e cre tin g a d e n o m a . A te r re e rra l to a n e n d o crin o lo g is t a n d n e u ro s u rg e o n , GR e le cts to u n d e rg o tra n s s p h e n o id a l p itu ita ry s u rg e ry. GR to le ra te s th e s u rg e ry w e ll, b u t h e r p o s to p e ra tive g ro w th h o rm o n e a n d IGF-1 le ve ls re m a in e le va te d .

In contrast to the indirect vascular connection between the hypothalamus and the anterior pituitary gland, a direct neural connection exists between the hypothalamus and the posterior pituitary gland. Neurons synthesize hormones, destined or storage in the posterior pituitary gland, in cell bodies o the hypothalamic supraoptic and paraventricular nuclei. These hormones are then transported down axons to the posterior pituitary gland, where they are stored in neuronal terminals until a release stimulus occurs. The posterior pituitary gland can, there ore, be thought o as an extension o the hypothalamus. As with the anterior pituitary gland, the enestrated endothelial cells in a capillary bed surrounding the posterior pituitary gland, in this case arising rom the in erior hypophyseal artery, acilitate release o hormones into the systemic circulation. During development and proli eration, the ate o anterior pituitary gland cells is determined by a network o transcription actors that shepherd the terminal di erentiation o these cells into thyrotrophs, corticotrophs, lactotrophs,

499

Ba s e d o n th e co n tin u e d e le va tio n in s e ru m g ro w th h o rm o n e a n d IGF-1 le ve ls , GR’s e n d o crin o lo g is t re co m m e n d s m e d ica l tre a tm e n t w ith o ctre o tid e . GR to le ra te s th e th rice -d a ily in je ctio n s w e ll, e xce p t o r o cca s io n a l m ild n a u s e a . A te r 2 w e e ks o re q u e n t in je ctio n s , GR s w itch e s to a lo n g -a ctin g , d e p o t o rm o o ctre o tid e th a t is in je cte d o n ce a m o n th . GR is m u ch h a p p ie r w ith th e re d u ce d re q u e n cy o d ru g a d m in is tra tio n , a lth o u g h s h e co n tin u e s to e xp e rie n ce m ild n a u s e a a n d b lo a tin g a s a d ve rs e e e cts o th is m e d ica tio n . A te r 6 m o n th s o d e p o t o ctre o tid e in je ctio n s , GR’s g ro w th h o rm o n e a n d IGF-1 le ve ls re m a in e le va te d . GR is ru s tra te d a t th e la ck o im p rove m e n t o h e r b io ch e m ica l a s s a ys b u t d o e s e e l th a t s h e h a s m o re e n e rg y th a n b e o re tre a tm e n t, a n d h e r m e n s e s h a ve re s u m e d . GR’s e n d o crin o lo g is t re co m m e n d s tre a tm e n t w ith p e g vis o m a n t a s a n a lte rn a tive m e d ica l a p p ro a ch to tre a tin g th e e e cts o h e r e le va te d g ro w th h o rm o n e le ve ls . GR b e g in s d a ily in je ctio n s w ith p e g vis o m a n t. S ix m o n th s la te r, GR’s IGF-1 le ve l is in th e n o rm a l ra n g e . GR is a ga in yin g a ro u n d th e n a tio n in p u rs u it o in cre a s e d s a le s , a n d s h e s to p s in to w n ju s t lo n g e n o u g h to co m p le te h e r ye a rly h e a d MRI a n d live r u n ctio n te s ts .

Questions 1 . Why was it necessary for GR to receive injections of octreotide and pegvisomant rather than taking the drugs orally? 2 . Why are serum levels of IGF-1 a more appropriate screening test for acromegaly than growth hormone levels? 3 . What anatomical and hormonal considerations are raised by the abrupt cessation of normal menstruation? 4 . How do octreotide and pegvisomant act to lower IGF-1 levels?

somatotrophs, and gonadotrophs. Three examples o transcription actors that are instrumental in anterior pituitary cell development are Pit-1, T-Pit, and Prophet o Pit-1 (Prop-1). The anterior pituitary gland is a heterogeneous collection o numerous cell types, each o which has the capacity to respond to specif c stimuli and to release specif c hormones into the systemic circulation. Each o the several hypothalamic releasing or inhibiting actors alters the hormone secretion pattern o one or more anterior pituitary gland cell types (Table 27-1). Releasing actors also modi y other cellular processes in the anterior pituitary gland, including hormone synthesis and pituitary cell growth. Interestingly, the relationship between hypothalamic releasing factors and pituitary gland hormones is not always 1:1, nor is the interaction always stimulatory. Somatostatin, or example, primarily inhibits the release o growth hormone (GH), but it can also inhibit release o thyroid-stimulating hormone (TSH) and prolactin. Conversely, thyrotropin-releasing hormone (TRH)

500 PRINCIPLES OF ENDOCRINE PHARMACOLOGY Hypotha la mic a re a

Optic chia s m

Pa rave ntricula r a nd s upra optic nucle i

S upe rior hypophys e a l a rte ry Hypotha la micpituita ry porta l s ys te m

Infe rior hypophys e a l a rte ry

To s ys te mic circula tion

P ituita ry gla nd: Ante rior lobe Pos te rior lobe

To s ys te mic circula tion

FIGURE 27-1. The hypothalamic-pituitary portal system. Neurons in the hypothalamus release regulatory actors that are carried by the hypothalamicpituitary portal system to the anterior pituitary gland, where they control the release o anterior pituitary hormones. Posterior pituitary hormones are synthesized in cell bodies o the supraoptic and paraventricular neurons in the hypothalamus and then transported down axonal pathways to terminals in the posterior pituitary gland. These hormones are stored in the posterior pituitary gland rom which they are released into the systemic circulation. Note the separate vascular supplies to the anterior and posterior lobes o the pituitary gland.

primarily stimulates the release o TSH, but it can also cause release o prolactin. The overlapping activities o some releasing actors and release-inhibiting actors, together with the antagonistic actions o some stimulatory and inhibitory hypothalamic actors, provide a mechanism or the precise regulation o secretory pathways. With the exception of dopamine, all known hypothalamic releasing factors are peptides. The anterior pituitary gland hormones are proteins and glycoproteins. Anterior pituitary gland hormones all into three groups. Somatotropic hormones—growth hormone (GH) and prolactin—are 191 and 198 amino acids long, respectively, and exist as monomeric proteins that share signif cant structural homology. Glycoprotein hormones—luteinizing hormone (LH), folliclestimulating hormone (FSH), and thyroid-stimulating hormone (TSH)—are heterodimeric proteins with carbohydrates attached to certain residues. These three hormones share the

same homologous subunit, which is also shared by the human chorionic gonadotropin (hCG) hormone, but each has a unique subunit that con ers biological specif city. Adrenocorticotropic hormone (ACTH) belongs to a separate class, as it is processed by proteolysis rom a larger precursor protein. O importance, intact peptides and proteins are not absorbed across the intestinal lumen; local proteases digest them into their constituent amino acids. For this reason, therapeutic administration o a peptide hormone or hormone antagonist must be accomplished by a non-oral route—this is why, in the introductory case, it was necessary or GR to take octreotide and pegvisomant by injection. The response o an anterior pituitary gland cell to a hypothalamic actor is initiated when the hypothalamic actor binds to specif c G protein-coupled receptors located on the plasma membrane o the appropriate anterior pituitary cell type. Most o these receptors alter the levels o either intracellular

TABLE 27-1 Anterior Pituitary Gland Cell Types, Hypothalamic Control Factors, and Hormonal Targets ANTERIOR PITUITARY GLAND CELL TYPE

STIMULATORY HYPOTHALAMIC FACTORS

INHIBITORY HYPOTHALAMIC FACTORS

PITUITARY HORMONES RELEASED

MAJ OR TARGET ORGAN OF HORMONE

HORMONES PRODUCED BY TARGET ORGAN

Somatotroph

GHRH, ghrelin

Somatostatin

GH

Liver, cartilage

Insulin-like growth actors

Lactotroph

TRH

Dopamine, somatostatin

Prolactin

Mammary gland

None

Thyrotroph

TRH

Somatostatin

TSH

Thyroid gland

Thyroxine, triiodothyronine

Corticotroph

CRH

None known

ACTH

Adrenal cortex

Cortisol, aldosterone, adrenal androgens

Gonadotroph

GnRH

None known

LH and FSH

Gonads

Estrogen, progesterone, testosterone, inhibin

Each anterior pituitary gland cell type responds to multiple hypothalamic stimulatory and inhibitory actors. Integration o these signals determines the relative extent o hormone release by the anterior pituitary gland. Each hormone has one or more specif c target organs, which are, in turn, stimulated to release their own hormones. These target hormones cause eedback inhibition at the hypothalamus and anterior pituitary gland. ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; FSH, ollicle-stimulating hormone; GH, growth hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone.

C h a p t e r 2 7 Pharmacology of the Hypothalamus and Pituitary Gland

cyclic adenosine monophosphate (cAMP), or inositol 1,4,5trisphosphate (IP3) and calcium (Ca2 ) (see Chapter 1, Drug–Receptor Interactions). The molecular details o receptor signaling provide a basis or understanding hypothalamic actor action. For example, growth hormone-releasing hormone (GHRH) binding to its receptors on somatotrophs increases intracellular cAMP and Ca2 levels, whereas somatostatin binding to its receptors on somatotrophs decreases intracellular cAMP and Ca2 . These signaling pathways provide a biochemical explanation or the opposing activities o GHRH and somatostatin on somatotroph release o GH. The timing and pattern o hypothalamic actor release are important determinants o anterior pituitary cell response. Most hypothalamic releasing actors are secreted in a cyclical or pulsatile, rather than continuous, manner. For example, the hypothalamus releases pulses o gonadotropinreleasing hormone (GnRH) with a periodicity o a ew hours. The requency and magnitude o GnRH release determine the extent o pituitary gonadotropin release as well as the ratio o LH secretion to FSH secretion. Interestingly, continuous administration o GnRH suppresses rather than stimulates pituitary gonadotroph activity. These di erent pharmacologic e ects o GnRH—depending on the requency and pattern o administration—have important clinical consequences, as discussed below. Although not studied in as much detail, the majority o the other hypothalamic releasing actors are also thought to be secreted in a pulsatile manner.

Feedback Inhibition End-product inhibition tightly controls hypothalamic and pituitary gland hormone release. For each hypothalamicpituitary-target organ system, an integrated picture can be constructed o how each set o hormones a ects the system. Each pathway, including one or more hypothalamic actors, its pituitary gland target cell type, and the ultimate target gland(s), is re erred to as an endocrine axis ; the term axis is used to connote one o multiple homeostatic systems that the hypothalamus and pituitary gland control. A simplif ed model consists o f ve endocrine axes, with a single type o anterior pituitary gland cell at the center o each axis (see Table 27–1 or anterior pituitary gland cell types). Each axis regulates an important aspect o endocrine homeostasis and is, there ore, subject to close regulation. Feedback inhibition is usually discussed in terms o loops, because the regulatory connection between a given hormone and its target creates a “loop” that alters the subsequent extent o hormone release. These eedback loops closely regulate the hypothalamicpituitary axes by providing levels o control at each stage o action (Fig. 27-2). In general, systemic hormones produced by target organs negatively regulate the pituitary and hypothalamus to maintain an equilibrium level o hormone release. Just as regulatory loops are re erred to based on a hormone’s relationship to its target organ, many endocrine diseases are described based on whether the disease etiology is a disorder o the hypothalamus, pituitary gland, or target organ. The disease is re erred to as primary, secondary, or tertiary, depending on whether the underlying abnormality is in the target organ, pituitary gland, or hypothalamus, respectively. Thereore, a primary endocrine disorder is caused by target organ pathology, a secondary disorder ref ects pituitary disease, and a tertiary endocrine disorder results rom hypothalamic pathology. Whether the underlying disease cause is primary, secondary, or tertiary can have important consequences or disease diagnosis and treatment, as discussed below.

Hypotha la mus

P ituita ry gla nd

501

CRH

ACTH

Cortis ol

Adre na l gla nd

FIGURE 27-2. Hypothalamic-pituitary-target organ feedback. The general mechanism of hypothalamic-pituitary-target organ feedback is depicted here, using the hypothalamic-pituitary-adrenal axis as an example. Stimulatory hypothalamic factors (CRH in this case) stimulate the release of pituitary hormones (ACTH in this case). In response to pituitary hormone signals, the target organ (the adrenal gland in this case) produces a hormone (cortisol in this case). In addition to its systemic physiologic actions (not shown), cortisol negatively regulates the hypothalamic-pituitary-adrenal axis by inhibiting CRH and ACTH. ACTH also negatively regulates CRH, providing more sensitive control of the axis.

PHYSIOLOGY, PATHOPHYSIOLOGY, AND PHARMACOLOGY OF INDIVIDUAL AXES Anterior Pituitary Gland Hypothalamic-Pituitary-Growth Hormone Axis

The hypothalamic-pituitary-growth hormone axis regulates general processes that promote growth. Somatotrophs o the anterior pituitary gland produce and secrete growth hormone. GH is f rst expressed at high concentrations during puberty; it is secreted in a striking pulsatile manner, with the largest pulses usually occurring at night during sleep. Most o the anabolic e ects o GH are mediated by insulin-like growth actors, especially insulin-like growth factor 1 (IGF-1), a hormone released into the circulation by hepatocytes in response to stimulation by GH. Although several cell types are capable o producing IGF-1, hepatocytes contribute the overwhelming majority o detectable IGF-1 in the circulation. Unlike GH, which has a short circulating hal -li e and a pulsatile pattern o secretion, IGF-1 is protein-bound and stable in the circulation or longer periods o time at steady concentrations. Thus, IGF-1 measurements represent an integrated surrogate or GH activity that is stable throughout the day, and IGF-1 levels are a more appropriate tool than GH levels in screening or acromegaly (as in the introductory case). Several environmental and biological stimuli regulate GH secretion. Environmental actors such as hypoglycemia, sleep, exercise, and adequate nutritional status can all increase GH secretion. Endogenous biological inputs that promote GH release include hypothalamic GHRH, sex steroids (most notably during puberty), dopamine, and ghrelin. Ghrelin is an important endogenous growth hormone-releasing peptide that has been

502 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

identif ed and well characterized in the last decade. Ghrelin acts synergistically with GHRH to promote GH release, acting on a receptor that is distinct rom the GHRH receptor. The majority o ghrelin is secreted by gastric undal cells during the asting state, linking growth with nutritional status and energy balance. Nonpeptide, orally active ghrelin mimetics are currently under clinical investigation as GH secretagogues, and antagonists are being studied or appetite control. Several environmental actors inhibit GH release, including A Norma l a xis

hyperglycemia, sleep deprivation, and poor nutritional status. The most signif cant endogenous biological actors that inhibit GH secretion are somatostatin, IGF-1, and GH. Pathophysiology and Pharmacology o Growth Hormone Def ciency

Failure to secrete growth hormone or to enhance IGF-1 secretion during puberty results in growth retardation (Fig. 27-3A–D). GH def ciency most commonly results B Growth hormone ins e ns itivity

Ghre lin GHRH S oma tos ta tin

Ghre lin GHRH S oma tos ta tin

GH

GH

IGF-1

C S e conda ry de ficie ncy Ghre lin GHRH S oma tos ta tin

GH

IGF-1

IGF-1

D Te rtia ry de ficie ncy Ghre lin GHRH S oma tos ta tin

GH

IGF-1

E Growth hormone e xce s s Ghre lin GHRH S oma tos ta tin

GH

IGF-1

FIGURE 27-3. Hypothalamic-pituitary-growth hormone axis in health and disease. A. In the normal hypothalamic-pituitary-growth hormone axis, hypothalamic secretion o growth hormone-releasing hormone (GHRH) or ghrelin stimulates release o growth hormone (GH), while somatostatin inhibits release o GH. Secreted GH then stimulates the liver to synthesize and secrete insulin-like growth actor 1 (IGF-1), which promotes systemic growth. IGF-1 also inhibits GH release rom the anterior pituitary gland. B. In GH insensitivity, the anterior pituitary gland secretes GH, but the liver is unresponsive to stimulation by GH. As a result, IGF-1 secretion is reduced (indicated by dashed lines). The decreased eedback inhibition o GH release results in higher plasma levels o GH (thick line). C. In secondary def ciency, the pathology lies in an unresponsive anterior pituitary gland, which secretes reduced amounts o GH. Because GH levels are low, the liver is not stimulated to produce IGF-1. D. In tertiary def ciency, the hypothalamus ails to secrete GHRH appropriately (dashed line); the role o ghrelin in this condition is unknown. Lack o su f cient GHRH results in lack o adequate stimulation o GH secretion by the anterior pituitary gland and, there ore, diminished production o IGF-1. E. In GH excess, GH is most commonly hypersecreted rom an anterior pituitary adenoma. Elevated, and unregulated, GH levels result in increased hepatic production o IGF-1 and thus in systemic trophic e ects. Because GH secretion occurs via an autonomous adenoma in the pituitary, negative eedback by IGF-1 is usually less e ective.

C h a p t e r 2 7 Pharmacology of the Hypothalamus and Pituitary Gland

rom de ective hypothalamic release o GHRH (tertiary def ciency, Fig. 27-3D) or rom pituitary insu f ciency (secondary def ciency, Fig. 27-3C). Importantly, however, ailure o IGF-1 secretion in response to GH (Laron dwarf sm or primary def ciency, Fig. 27-3B) is one etiology o short stature that is not amenable to treatment with GH. Sermorelin (synthetic GHRH) can be administered parenterally to help determine the disease etiology. Tesamorelin, a novel GHRH analogue, has also been shown to augment basal and pulsatile GH secretion and has been used in the treatment o HIVassociated lipodystrophy. I a patient possesses de ective hypothalamic release o GHRH but has normally unctioning anterior pituitary gland somatotrophs, administration o exogenous GHRH results in increased GH release. As o 2008, sermorelin has become unavailable in the United States due to discontinuation o industrial manu acturing. Alternative exogenous agents currently used to stimulate GH release include glucagon, arginine , clonidine , and insulin-induced hypoglycemia. Most cases o growth hormone-dependent growth retardation are treated with replacement recombinant human growth hormone , re erred to by the generic name somatropin. Typical dosing schedules involve daily subcutaneous or intramuscular injection. Newer GH analogues with longer hal -lives, which would allow less requent dosing, are currently in development. Somatropin therapy is costly and thus approved or use in the United States only or specif c indications. In adults, either conf rmed GH def ciency or panhypopituitarism (at least three hormonal axes a ected) is required or approval, although unapproved use in competitive sports is prevalent. Some pediatric indications or GH use include idiopathic short stature, chronic kidney disease, Turner’s syndrome, and Prader-Willi syndrome. The use o somatropin in AIDS cachexia and in critical illness is an area o active study, although not yet approved or use. Orally bioavailable peptidomimetics o growth hormone are an active area o research. Recombinant IGF-1, known by the generic name mecasermin, is an e ective treatment or patients with GH insensitivity (socalled Laron dwarf sm). Mecasermin is also approved or use in patients with GH def ciency and antibodies against growth hormone. Mecasermin administration can be associated with adverse e ects, including hypoglycemia and rare intracranial hypertension. Pathophysiology and Pharmacology of Growth Hormone Excess

GH excess usually results rom a somatotroph adenoma (Fig. 27-3E). Rarer syndromes o GH excess include ectopic production o GH or GHRH but are beyond the scope o this chapter. This entity has two di ering disease presentations, depending on whether the GH excess occurs be ore or a ter closure o the bone epiphyses. Gigantism occurs i GH is secreted at abnormally high levels in children be ore closure o the epiphyses because increased IGF-1 levels promote excessive longitudinal bone growth. A ter the epiphyses close, abnormally high levels o GH result in acromegaly, as illustrated in the introductory case. This condition occurs because IGF-1, although it can no longer stimulate long bone growth, can still promote growth o organs and cartilaginous tissue. Typical mani estations include the nonspecif c symptoms that GR initially experienced, such as increased hand thickness, enlarging shoe size, hyperhidrosis, and atigue. Other requent f ndings include large acial structures,

503

macroglossia, and organomegaly. Consequences o a pituitary mass lesion (adenoma) may also be evident, including headache, loss o other pituitary hormone unctions (as mani ested by cessation o menstruation, in the case o GR), and visual f eld loss. Available management options or a somatotroph adenoma are surgical resection, medical therapy, and radiation therapy. Transsphenoidal surgical resection o the adenoma is the current standard o care. As seen in the case o GR, surgical treatment has variable success, especially when the adenoma exceeds 1 centimeter in size, and adjuvant medical therapy is requently required. Medical options include somatostatin receptor agonists (also known as somatostatin receptor ligands [SRLs] or somatostatin analogues), dopamine analogues, and GH receptor antagonists. Somatostatin receptor ligands (SRLs) are the mainstay o medical therapy. Somatostatin physiologically inhibits growth hormone secretion, making it a logical treatment or somatotroph adenomas. Somatostatin itsel is rarely used clinically because it has a hal -li e o only a ew minutes. Octreotide and lanreotide are synthetic, longer acting peptide analogues o somatostatin that have been used with extensive experience. Pasireotide is a somatostatin analogue f rst approved or the treatment o Cushing’s disease; it has also shown clinical e f cacy in the treatment o acromegaly and is now approved or this indication. In addition, trials are underway to evaluate the e f cacy o an orally active octreotide in acromegaly. Somatostatin receptors are distributed widely, and somatostatin and its analogues a ect many secretory processes. There ore, octreotide can be used or several indications, including treatment o esophageal varices and certain hormone-secreting tumors. However, systemic administration o SRLs can lead to diverse adverse e ects, including nausea, diarrhea, gallstones, and glucose dysregulation. Sustainedrelease ormulations o SRLs, as exemplif ed in the introductory case, allow less requent dosing but do not appear to alter the adverse e ect prof le. The e f cacy o SRLs lies in their ability to normalize GH and IGF-1 levels in approximately 60–80% o acromegalic patients and to decrease pituitary adenoma size in 40–50% o a ected patients. Dopamine is a hypothalamic actor that acts mainly on lactotrophs to physiologically inhibit prolactin release. Dopamine also stimulates somatotrophs to release GH under physiologic conditions, but patients with acromegaly can have a paradoxical decrease in growth hormone secretion in response to dopamine. This e ect may be due, in part, to the shared embryonic lineage o lactotrophs and somatotrophs; in act, 20–30% o somatotroph adenomas also secrete excess prolactin. Based on this observation, the dopamine analogues bromocriptine and cabergoline are sometimes used as adjunctive agents in the treatment o acromegaly. Although these agents are much less expensive than SRLs and can be administered orally, dopamine receptor agonists are generally much less e ective than SRLs and are thus typically used as second-line agents in the medical management o acromegaly. These agents are discussed below in relation to the hypothalamic-pituitary-prolactin axis. The GH molecule has two binding sites, each o which is capable o binding one GH receptor monomer. GH action requires dimerization o the receptor a ter GH binding in order to initiate receptor activation and intracellular signaling. Pegvisomant is a GH analogue that has been modif ed such that one o the sites binds to the GH receptor with higher

504 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

a f nity than the native molecule, but the other binding site is inactive. There ore, although pegvisomant binds tightly to the monomeric GH receptor, it prevents the receptor dimerization required or subsequent receptor activation and intracellular signaling. In e ect, there ore, the drug acts as a competitive antagonist o GH activity. Pegvisomant also contains multiple polyethylene glycol (PEG) residues, which prolong the hal -li e o the drug and thereby allow once-daily dosing. O the available medical therapies, pegvisomant has the most potent IGF-1 reducing potential, but it also increases GH levels by decreasing IGF-1-mediated eedback inhibition o GH secretion. Concerns have arisen regarding increased tumorigenesis and accelerated somatotroph adenoma growth in the setting o the elevated GH levels induced by pegvisomant use; however, to date, no convincing data have emerged to support these concerns. Current practice suggests the per ormance o an annual pituitary MRI scan to monitor pituitary adenoma growth in patients taking pegvisomant. Despite its e ectiveness at achieving biochemical control o IGF-1 excess, pegvisomant has several major limitations, including limited experience with its use, expensive cost, and liver unction abnormalities. As seen in the case o GR, pegvisomant is currently used as a second- or third-line medical agent a ter SRL therapy has been attempted. In the uture, the use o pegvisomant may become more prevalent i its overall sa ety prof le remains avorable.

Hypothalamic-Pituitary-Prolactin Axis

Lactotrophs o the anterior pituitary gland produce and secrete prolactin. Their activity is inhibited by hypothalamic secretion o dopamine . TRH can enhance prolactin release, in addition to stimulating anterior pituitary thyrotrophs. Estrogen and breast eeding also enhance prolactin release, as described below. Unlike other cells o the anterior pituitary gland, lactotrophs are under tonic inhibition by the hypothalamus, presumably mediated by hypothalamic release o dopamine (Fig. 27-4). Therefore, a disease condition that interrupts the hypothalamic-pituitary portal system results in decreased secretion of most anterior pituitary gland hormones but causes increased prolactin release. In patients taking phenothiazine antipsychotics or metoclopramide (see Chapter 14, Pharmacology o Dopaminergic Neurotransmission), increased levels o prolactin are requently observed because these agents are dopamine receptor antagonists. Prolactin secretion does not appear to be regulated by any identif ed negative eedback system. The physiologic actions o prolactin involve regulation o mammary gland development and milk protein biosynthesis and secretion. Prolactin levels are normally low in men and nonpregnant women. Increased estrogen levels during pregnancy stimulate lactotrophs to secrete increasing quantities o prolactin. During pregnancy, however, estrogen antagonizes prolactin action in the breast; this prevents lactation until a ter parturition. Suckling provides a power ul neural stimulus or prolactin release; prolactin levels increase as much as 100- old within 30 minutes a ter the initiation o breast eeding. The positive eedback o breast eeding on prolactin secretion ensures continued replenishment o milk reserves. I a mother does not breast eed, prolactin levels decrease over the course o several weeks. Interestingly, increased prolactin levels suppress estrogen synthesis, both by antagonizing hypothalamic release o

TRH

Dopa mine

P RL

Bre a s ts / Ma mma ry tis s ue

Es troge n

La cta tion

FIGURE 27-4. Regulation of the hypothalamic-pituitary-prolactin axis. Secretion of prolactin by anterior pituitary gland lactotrophs is tonically inhibited by hypothalamic dopamine. Hypothalamic TRH and circulating estrogens stimulate prolactin release. These stimulatory and inhibitory inputs on lactotrophs result in a baseline equilibrium of prolactin production. Disruption of this equilibrium results in an imbalance of prolactin production; for example, interruption of the pituitary stalk diminishes hypothalamic dopamine delivery to lactotrophs, resulting in increased prolactin secretion.

GnRH and by decreasing gonadotroph sensitivity to GnRH. The resulting decrease in LH and FSH release decreases end-organ stimulation o the hypothalamic-pituitary-gonadal axis, resulting in both decreased estrogen synthesis and suppression o ovulation while a woman is breast eeding. Chronically high secretion of prolactin, such as by a prolactinoma, also suppresses the hypothalamic-pituitary-gonadal axis. For this reason, prolactinomas are a common cause o in ertility, especially in women, who may present with oligomenorrhea or amenorrhea. Bromocriptine is a synthetic dopamine receptor agonist that inhibits lactotroph cell growth and prolactin secretion and is an established medical therapy or prolactinoma. Bromocriptine is orally bioavailable. As with octreotide, many o the adverse e ects o bromocriptine result rom systemic actions o the drug. The adverse e ects o bromocriptine include nausea and vomiting, presumably because the area postrema in the medulla, which stimulates nausea and lies outside the blood–brain barrier, possesses dopamine receptors. The adverse e ect prof le o dopamine receptor agonists is dependent on their relative specif city or the various dopamine receptor subtypes (see Chapter 14). Cabergoline and quinagolide are two other dopamine receptor agonists used to treat prolactinoma; the ormer is commonly used in the United States and the latter is available only in Europe. The advantages o cabergoline include a weekly or bi-weekly dosing interval and less requent gastrointestinal adverse e ects. Although both cabergoline and

C h a p t e r 2 7 Pharmacology of the Hypothalamus and Pituitary Gland

bromocriptine are considered category B or pregnancy (i.e., inadequate well-controlled sa ety data in pregnancy or adverse e ects in animal studies), most practitioners typically use bromocriptine over cabergoline in pregnancy because o its signif cantly longer track record o experience and sa ety. Recent reports have shown a link between the use o cabergoline and valvular heart disease. Comparative studies have correlated this risk with the higher dose cabergoline therapy used in Parkinson’s disease, while the smaller doses requently used to treat prolactinoma have not to date shown a signif cant link to valvular heart disease.

Hypothalamic-Pituitary-Thyroid Axis

The hypothalamus secretes thyrotropin-releasing hormone (TRH), which stimulates thyrotrophs in the anterior pituitary gland to produce and secrete TSH. In turn, TSH promotes biosynthesis and secretion o thyroid hormone by the thyroid gland. Thyroid hormone regulates overall body energy homeostasis. Thyroid hormone negatively controls hypothalamic and pituitary release o TRH and TSH, respectively (see Fig. 28-4). Because thyroid hormone replacement is an e ective therapy or hypothyroidism, TRH and TSH are used mainly or diagnosis o disease etiology. I hypothyroidism is caused by an unresponsive thyroid gland (primary def ciency), serum TSH levels will be high because o decreased negative eedback rom thyroid hormone. For this reason, serum TSH is the main test used in screening for primary thyroid disease. TRH administration would produce an exaggerated increase in TSH, although this test is no longer used regularly in clinical practice. Conversely, i hypothyroidism is caused by a de ect in pituitary TSH production (secondary def ciency), the TSH level will not be high despite the presence o low thyroid hormone levels. In this scenario, i TRH were to be administered, the normally expected rise in TSH would be absent or signif cantly reduced. Most cases o hyperthyroidism are primary, resulting rom excessive thyroid hormone production or release rom the thyroid gland; in this setting, thyroid hormone levels are high and TSH levels are low (see Chapter 28, Pharmacology o the Thyroid Gland). Rarely, hyperthyroidism can result rom TSH-secreting pituitary adenomas (secondary hyperthyroidism), with inappropriately nonsuppressed (high or normal) TSH levels in the ace o high thyroid hormone levels. Surgical resection is the recommended therapy or TSH-secreting pituitary tumors. I surgery is not curative or is contraindicated, medical therapy with octreotide or lanreotide can be e ective in inhibiting TSH secretion, controlling hyperthyroidism, and reducing tumor volume. Recombinant TSH (thyrotropin) is commonly used in conjunction with radioactive iodine treatment o thyroid cancer. Thyrotropin is administered be ore radioactive iodine therapy to maximize uptake o radiolabeled 131I isotope into thyroid tissue in patients with thyroid cancer. This approach enables the administration o smaller quantities o radioisotope, maintaining maximum radiation exposure specif cally to thyroid tissue with less radiation exposure to other tissues. Other aspects o thyroid gland pharmacology are discussed in Chapter 28.

Hypothalamic-Pituitary-Adrenal Axis

Neurons rom the paraventricular nucleus o the hypothalamus synthesize and secrete corticotropin-releasing hormone (CRH). CRH binds to cell sur ace receptors on corticotrophs o the anterior pituitary gland and stimulates corticotrophs to

505

synthesize and release adrenocorticotropic hormone (ACTH; also called corticotropin). ACTH is synthesized as part o proopiomelanocortin (POMC), a precursor polypeptide that is cleaved into multiple e ector molecules. In addition to ACTH, cleavage o POMC yields melanocyte-stimulating hormone (MSH), lipotropin, and -endorphin. MSH has e ects on skin pigmentation. ACTH is structurally similar to MSH, and high concentrations o ACTH can bind to and activate MSH receptors. In primary hypoadrenalism, increased ACTH levels result in enhanced skin pigmentation. ACTH stimulates the synthesis and secretion o adrenocortical steroid hormones, including glucocorticoids, androgens, and mineralocorticoids (Fig. 27-5A). ACTH is required or secretion o glucocorticoids and adrenal androgens. Mineralocorticoid production is also regulated by potassium balance and volume status, and ACTH has a relatively minor role in regulating mineralocorticoids. ACTH also has a trophic e ect on the zona asciculata and zona reticularis o the adrenal cortex (see Fig. 29-1); excessive ACTH secretion causes adrenal hyperplasia, while ACTH def ciency ultimately causes adrenal atrophy. Among the several steroid hormone products o adrenal biosynthesis, cortisol is arguably the most crucial. In addition to serving as the main eedback inhibitor o pituitary ACTH release, cortisol unctions as a “stress hormone” and is involved in vascular tone, electrolyte balance, and glucose homeostasis. Def ciency o cortisol can rapidly lead to critical illness or death, while cortisol excess results in Cushing’s syndrome (Fig. 27-5B). A synthetic orm o ACTH, known as cosyntropin, can be used to diagnose suspected cases o adrenal insu f ciency and also to assist in ascertaining whether the insu f ciency is primary or secondary. Administration o cosyntropin to a patient with primary adrenal insu f ciency will ail to increase plasma cortisol concentration due to the inherent dys unction o adrenal biosynthesis. Conversely, administration o cosyntropin to a patient with new-onset secondary adrenal insu f ciency will result in a robust increase in plasma cortisol. However, patients with long-standing secondary adrenal insu f ciency may have a blunted cortisol response to cosyntropin, owing mainly to progressive adrenal cortical atrophy in the absence o the trophic e ects o ACTH. Conditions requiring physiologic replacement o glucocorticoids are usually treated with synthetic analogues o cortisol, rather than ACTH, because use o the target hormone generally allows or more precise physiologic control. Cortisol physiology and pharmacology are discussed in greater detail in Chapter 29, Pharmacology o the Adrenal Cortex. CRH is used as a diagnostic tool in petrosal sinus sampling or ACTH. CRH can be used to distinguish whether excessive cortisol secretion results rom an ACTH-secreting pituitary adenoma or rom an ectopic ACTH-secreting tumor (Fig. 27-5). I the hypercortisolism derives rom a pituitary corticotroph adenoma (Cushing’s disease), administration o CRH will usually increase blood ACTH levels (Fig. 27-5C). This response is not seen in the case o an ectopic ACTHsecreting tumor, which secretes ACTH at a constant autonomous rate (Fig. 27-5D). Cushing’s syndrome resulting rom a primary adrenal tumor is o ten treated with surgical resection; however, several medical therapies also exist. Metyrapone , ketoconazole , and mitotane all have potent inhibitory e ects on adrenal steroidogenesis and can be used to reduce cortisol production, while mifepristone antagonizes peripheral cortisol receptor

506 PRINCIPLES OF ENDOCRINE PHARMACOLOGY A Norma l a xis

somatostatin receptors, with the highest a f nity or somatostatin receptor subtype 5. In clinical trials, increased cortisol levels in patients with Cushing’s disease were signif cantly reduced during treatment with pasireotide. Hyperglycemia was a common adverse e ect even though cortisol levels were reduced.

B P rima ry a dre na l tumor/ Cus hing's s yndrome

CRH

Hypothalamic-Pituitary-Gonadal Axis ACTH

Gonadotrophs are unique among anterior pituitary gland cells because they secrete two glycoprotein hormones—LH and FSH. Together, these hormones are re erred to as gonadotropins. LH and FSH are both heterodimers composed o and subunits. LH and FSH share the same subunit with TSH and hCG but possess unique subunits. Gonadotrophs regulate the secretion o LH and FSH independently. This axis is diagrammed in Figure 27-6.

ACTH

Cortis ol

Cortis ol

C P ituita ry a de noma / Cus hing's dis e a s e

GnRH (puls a tile )

D Ectopic ACTH-s e cre ting tumor

GnRH (continuous ) ACTH

Activin LH

ACTH

a nd

FS H Cortis ol

Cortis ol

Tumor

ACTH

Es troge n (+/-) Inhibin (-)

Te s tos te rone (-) Inhibin (-)

Ova rie s or te s te s

FIGURE 27-5. Hypothalamic-pituitary-adrenal axis in health and disease. A. In the normal hypothalamic-pituitary-adrenal axis, hypothalamic secretion of corticotropin-releasing hormone (CRH) stimulates release of adrenocorticotropic hormone (ACTH). ACTH, in turn, stimulates synthesis and secretion of cortisol by the adrenal cortex. Cortisol inhibits further release of CRH and ACTH. B. A primary adrenal tumor causes Cushing’s syndrome by autonomously producing cortisol (thick line), independent of regulation by ACTH. The excessive cortisol production suppresses ACTH production (dashed line). C. An ACTH-producing pituitary adenoma causes Cushing’s disease by autonomously secreting excessive levels of ACTH (thick line), which stimulate the adrenal gland to produce increased levels of cortisol (thick line). ACTH secretion by the tumor has a blunted sensitivity to feedback inhibition by cortisol. D. An ectopic ACTH-secreting tumor (such as a small cell carcinoma of the lung) also stimulates the adrenal gland to produce increased levels of cortisol, which suppress pituitary ACTH production. However, circulating ACTH levels remain elevated due to the ectopic-source production of the hormone.

binding (see Chapter 29). These therapies can also be used in patients with Cushing’s disease, although they are not targeted at the pituitary tumor itsel . To date, there have been ew e ective treatments targeted at the level o the pituitary to reduce ACTH secretion in Cushing’s disease. Pasireotide was recently approved or therapy o Cushing’s disease. Pasireotide is a somatostatin analogue that targets

FIGURE 27-6. Hypothalamic-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) is secreted by the hypothalamus in a pulsatile fashion, stimulating gonadotroph cells of the anterior pituitary gland to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH stimulate the ovaries or testes to produce the sex hormones estrogen or testosterone, respectively, which inhibit further release of LH and FSH. Paradoxically, however, the increasing estrogen levels that are secreted from developing follicles during the follicular phase of the menstrual cycle induce a positive feedback, midcycle ovulatory surge of LH and FSH secretion. Inhibin is also produced by the gonads in response to FSH and exerts negative feedback on gonadotrophs to inhibit further release of FSH. Locally produced pituitary activin acts in a paracrine fashion to stimulate FSH secretion. Exogenous pulsatile GnRH can be used to induce ovulation in women with infertility of hypothalamic origin. However, continuous administration of GnRH suppresses the gonadotroph response to endogenous GnRH and thereby causes decreased production of sex hormones. Analogues of GnRH with increased metabolic stability and prolonged half-lives take advantage of this effect and are used to suppress sex hormone production in clinical conditions such as precocious puberty and prostate cancer.

C h a p t e r 2 7 Pharmacology of the Hypothalamus and Pituitary Gland

Gonadotropins control hormone production by the gonads, promoting the synthesis o androgens and estrogens. The e ects o estrogen and other reproductive hormones on the anterior pituitary gland are complex. In males, gonadotropins are inhibited via negative eedback by testosterone. In contrast, in emales, depending on the rate o change and absolute concentration o estrogen, as well as the stage o the menstrual cycle, estrogen can exert both inhibitory and excitatory e ects on gonadotropins. Inhibin is a hormone produced in the gonads that has inhibitory e ects primarily on FSH secretion, with little e ect on LH secretion. Activin is a paracrine actor that is produced and acts locally both in the pituitary and in the gonads and unctions in the pituitary gland to stimulate primarily FSH secretion (Fig. 27-6). Endocrine control o the reproductive process is discussed in greater detail in Chapter 30, Pharmacology o Reproduction. Native GnRH, which has a short half-life, can be administered in a pulsatile fashion to stimulate patterned gonadotropin release, while GnRH analogues with longer half-lives are used to suppress production of sex hormones by desensitizing the pituitary gland to the stimulating activity of the native releasing factor (Fig. 27-6). The main pharmacologic di erence among the currently approved GnRH agonists is the route o administration. Leuprolide is the most commonly used GnRH agonist and can be administered as a daily subcutaneous injection or as a monthly depot injection. Osmotic pump implants (see Chapter 55, Drug Delivery Modalities) are also available that deliver leuprolide acetate at a controlled rate or up to 12 months. Long-acting agonists are utilized therapeutically to suppress gonadotropins in several clinical conditions, including in vitro ertilization, endometriosis, uterine f broids, precocious puberty, and androgen-dependent prostate cancer. Their main drawback is that gonadotroph suppression does not occur immediately; instead, there is a transient (several days) increase (“ are”) in sex hormone levels, ollowed by a lasting suppression o hormone synthesis and secretion. FSH is used to stimulate ovulation or in vitro ertilization. Two US Food and Drug Administration (FDA)-approved ormulations are available. Urofollitropin is purif ed FSH isolated rom the urine o postmenopausal women, and follitropin is a recombinant orm o FSH. Both agents e ectively stimulate ovulation but may cause ovarian hyperstimulation syndrome . Interestingly, a rare orm o ovarian hyperstimulation syndrome that occurs during pregnancy ( amilial gestational ovarian hyperstimulation syndrome) is caused by an inherited mutation in the FSH receptor. This mutation allows human chorionic gonadotropin (hCG), a hormone present in high concentrations during the early stages o pregnancy, to stimulate the FSH receptor. The resulting overstimulation o the FSH receptor is thought to cause the ollicular enlargement and other sequelae characteristic o this syndrome. Whether similar mutations in the FSH receptor could be associated with cases o drug-induced ovarian hyperstimulation syndrome is an area o active investigation. The GnRH receptor antagonists cetrorelix and ganirelix are sometimes used in assisted reproduction; they suppress premature surges in LH in the early to mid- ollicular phase o the menstrual cycle, resulting in improved rates o implantation and pregnancy (see Chapter 30). GnRH antagonists also have applications or palliation o metastatic prostate cancer. In this situation, a direct GnRH antagonist has the advantage o avoiding the initial surge in testosterone caused by treatment with GnRH agonists.

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Posterior Pituitary Gland The anterior lobe o the pituitary gland secretes numerous hormones, while the posterior lobe o the pituitary gland (neurohypophysis) secretes only two hormones: antidiuretic hormone (ADH) and oxytocin. ADH is an important regulator o plasma volume and osmolality, while oxytocin has physiologic e ects on uterine contraction and lactation.

Antidiuretic Hormone (ADH)

ADH is a peptide hormone produced by magnocellular cells o the hypothalamus. Cells in this region possess osmoreceptors that sense changes in extracellular osmolality. Increased osmolality stimulates ADH secretion rom nerve terminals in the posterior pituitary gland. ADH binds to two types o receptors: V1 and V2. V1 receptors, located in systemic arterioles, mediate vasoconstriction. This property gives ADH its alternative name, vasopressin. V2 receptors, located in the nephron, stimulate the cell sur ace expression o water channels in order to increase water reabsorption in the collecting duct, as discussed in Chapter 21, Pharmacology o Volume Regulation. These two actions o ADH combine to maintain vascular tone by (1) increasing blood pressure and (2) increasing water reabsorption. Disruption o ADH homeostasis results in two important pathophysiologic conditions. Excessive secretion o ADH causes the syndrome of inappropriate ADH (SIADH); def cient secretion o ADH or decreased responsiveness to ADH causes diabetes insipidus . In SIADH, ADH secretion occurs irrespective o plasma volume status or osmolality. One o the most common causes o SIADH is the ectopic secretion o ADH by small cell carcinoma o the lung, but SIADH may also be caused by a medication e ect or result rom almost any pulmonary process, central nervous system insult, or pituitary surgery. Excessive ADH secretion results in persistent stimulation o V1 and V2 receptors, causing hypertension and excessive water retention. The inappropriate water retention can result in low extracellular sodium concentration. Until recently, i the source o excess ADH could not be removed, the only e ective therapy or SIADH was restriction o uid intake or administration o hypertonic saline. Over the past decade, the discovery and clinical use o vasopressin receptor antagonists has provided more therapeutic options or the treatment o SIADH. Conivaptan and tolvaptan are vasopressin receptor antagonists that have recently been approved by the FDA or SIADH-induced hyponatremia. Tolvaptan is a specif c V2 receptor antagonist approved or use in heart ailure, while conivaptan is a mixed V1a and V2 receptor antagonist approved or use in euvolemic and hypervolemic hyponatremia. Both are available as oral agents. Demeclocycline (a tetracycline antibiotic; see Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation) and lithium (see Chapter 15, Pharmacology o Serotonergic and Central Adrenergic Neurotransmission) are two other pharmacologic treatments that can also be used to treat SIADH. Both diabetes insipidus and diabetes mellitus are characterized by symptoms o thirst, polydipsia, and polyuria. Despite their phenotypic similarities, however, the etiologies o diabetes mellitus and diabetes insipidus are unrelated. Diabetes insipidus is a disorder o vasopressin def ciency or resistance, whereas diabetes mellitus is caused by def cient

508 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

or secrete ADH. In this condition, administration of the exogenous ADH analogue, desmopressin, results in stimulation of V2 receptors and a robust concentration of urine and decrease in thirst (Fig. 27-7). Nephrogenic diabetes insipidus results from an inability of renal collecting duct cells to respond to ADH (or, in other words, resistance to ADH). Nephrogenic diabetes insipidus can be caused by a mutation in the V2 receptor, such that ADH is unable to bind the receptor

production of insulin or target tissue insensitivity to insulin (see Chapter 31, Pharmacology of the Endocrine Pancreas and Glucose Homeostasis). Diabetes insipidus is characterized by polyuria and polydipsia secondary to an inability to concentrate urine and retain free water at the level of the renal collecting duct. A distinction is made between two types of diabetes insipidus. Neurogenic diabetes insipidus results from an inability of hypothalamic neurons to synthesize

B

ADH

A

Wa te r

V2 -re ce ptor

D

P ituita ry gla nd No cha nge in wa te r cha nne l e xpre s s ion Colle cting duct ce ll

ADH

ADH

Wa te r

C Wa te r

Wa te r

V2 -re ce ptor

P ituita ry gla nd

Wa te r

Incre a s e d wa te r cha nne l e xpre s s ion

Mis s ing or unre s pons ive V2 -re ce ptor

No cha nge in wa te r cha nne l e xpre s s ion

Colle cting duct ce ll

Desmopressin

Wa te r Wa te r

Wa te r

V2 -re ce ptor

Incre a s e d wa te r cha nne l e xpre s s ion

FIGURE 27-7. Comparison of neurogenic and nephrogenic diabetes insipidus. A. Antidiuretic hormone (ADH), released by nerve terminals in the posterior pituitary gland, stimulates V2 receptors on renal collecting duct cells and thereby increases expression o water channels in the apical membrane o these cells. Increased water channel expression increases water ux through the cell. B. In neurogenic diabetes insipidus, the posterior pituitary gland is unable to secrete ADH. Consequently, there is no stimulation o renal V2 receptors by ADH, and the collecting duct cells do not increase water channel expression. C. Exogenous administration o desmopressin, an ADH analogue, can replace the def ciency o posterior pituitary gland-derived ADH and thereby treat neurogenic diabetes insipidus. D. In nephrogenic diabetes insipidus, the V2 receptor is either missing or unresponsive to stimulation by ADH. The lack o unctional V2 receptors prevents the cell rom responding to ADH with an increase in water channel expression.

C h a p t e r 2 7 Pharmacology of the Hypothalamus and Pituitary Gland

or stimulate receptor signaling, or by medication-induced resistance; lithium is one such medication. In nephrogenic diabetes insipidus, administration o desmopressin results in little or no change in urine concentration or thirst because the V2 receptor is insensitive to ADH and its analogues. Patients with nephrogenic diabetes insipidus can be treated with diuretics such as amiloride or hydrochlorothiazide . The proposed mechanism by which these diuretics prevent excessive loss o ree water is paradoxical: they induce a volume-contracted state, which promotes enhanced absorption o water in the proximal tubule and thereby decreases delivery o water to the site o ADH resistance, the collecting ducts.

Oxytocin

Oxytocin is a peptide hormone produced by paraventricular cells o the hypothalamus. Many o the known physiologic roles o oxytocin involve muscle contraction; two such e ects are milk release during lactation and uterine contraction. In the milk letdown response, stimuli to the hypothalamus cause oxytocin release into the blood rom nerve terminals in the posterior pituitary gland. Oxytocin causes contraction o myoepithelial cells surrounding the mammary gland alveoli. This is an important physiologic action during breast eeding. In addition, it has long been known that administration o oxytocin causes uterine contraction. Oxytocin release is probably not the physiologic stimulus or initiation o labor during pregnancy; however, oxytocin is used pharmacologically to induce labor exogenously.

CONCLUSION AND FUTURE DIRECTIONS Hormones o the hypothalamus and pituitary gland can be used as pharmacologic agents to modi y the respective endocrine axes o each hormone. Recognizing the relationships and e ects o primary, secondary, and tertiary disorders o any hypothalamic-pituitary axis is o paramount importance in understanding the appropriate diagnostic and treatment

509

choices. Hypothalamic hormones can be used as diagnostics to determine the causes o underlying endocrine pathology (CRH, GHRH, TRH) or as therapeutics to suppress an axis (GnRH, somatostatin, dopamine). Hormones o the anterior pituitary gland can be given as replacement therapy in cases o def ciency (GH) or used diagnostically (ACTH). The posterior pituitary gland produces two hormones, ADH and oxytocin, which can be used to treat neurogenic diabetes insipidus and to induce labor, respectively. Recent advances have led to new medical therapies that extend the ability to treat pituitary hormone hypersecretion due to Cushing’s disease. Future directions in hypothalamic and pituitary gland pharmacology will include design o new drug delivery systems; synthesis o orally active, nonpeptide analogues o hormones; and investigations to better understand hormone receptor mechanisms and signaling to assist in the design o new pharmacotherapies.

Acknowledgment We thank Ehrin J. Armstrong and the late Armen H. Tashjian, Jr. or their valuable contributions to this chapter in the First and Second Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Colao A, Petersenn S, Newell-Price J, et al. A 12-month phase 3 study o pasireotide in Cushing’s disease. N Engl J Med 2012;366:914–924. (Recent clinical trial of pasireotide.) Gadelha MR, Bronstein MD, Brue T, et al. Pasireotide C2402 Study Group. Pasireotide versus continued treatment with octreotide or lanreotide in patients with inadequately controlled acromegaly (PAOLA): a randomised, phase 3 trial. Lancet Diabetes Endocrinol 2014;2:875–884. (Clinical trial of pasireotide for acromegaly.) Hays R. Vasopressin antagonists—progress and promise. N Engl J Med 2006;355:2146–2148. (Perspective on SIADH and the future of vasopressin antagonists.) Melmed S. Acromegaly. N Engl J Med 2006;355:2558–2273. (Review of growth hormone pathophysiology and treatment for acromegaly.) Verhelst J, Abs R. Hyperprolactinemia. Treat Endocrinol 2003;2:23–32. (Review of the pathophysiology and management of hyperprolactinemia.)

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C h a p t e r 2 7 Pharmacology o the Hypothalamus and Pituitary Gland 513

Na +/ I- s ym ym portee r (NIS (N )

28

Extr t a c e llu ll la r s p a cce

I-

2Naa +

I-

2Na +

I-

Cl-

Pe rcc hl hloo rate Thio c yan anat n at a tee Pe rtee c h hne tatee Na +-inde pe nde ntt C l-/I- excha nge r (pe ndrin) n)

Pharmacology of the Thyroid Gland Th Thio T h amine s Io dide s (hig (h ig h)

An t h o ny Ho lle n b e rg a n d W illia m W. Ch in

C lITG Thyroid h pe ro roxida id a s e (orga nifica tion) (or TG-MIT, T TG-DIT Thyroid hy pee ro roxid xida da s e Colloid (coupling)) s ppaa c e T3 T T44 TG G

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514 514–515 14 Trrea eatm tment nt oof Hy nt Hyper yyp rthyro rthyro rt yroidism . . . . . . . . . . . . . . . . . . . . . . . . . . 519 Inhibi bitors bit bi iitors to t o Iodide Iodide od odi ddide i Uptak Upta Uptake Uptake tak ak . . . . . . . . . . . . . . . . . . . . . . . . . . . 519 THYROID GLAND PHYS YSIO IOLO LOG GY. . . . . . . . . . . . . . . . . . . . . . . . . . . 5144 I and did dide ideHormone s (hi (hig h)Relea Inhib nhhibitors hibi biito torrs o Organif Orrgganif niff ca ccation atio ationIo Hormo Rele eleas elease ele leass e . . . . . . 520 Synthesis andd Se Secc retion of Thyroid Hormones. . . . . . 131 . . . .-. . 51 514 Inhibitors In nhhibitors bitorrs o Periph bitors Peri her Pe era ral a Thy hyrro rroid id Hor Hormone ne Met Metabolism Meta eta t . . . 521 Meta Me tabo bolism of Thyroid Hormones . . . . . . . . . . . . . . . . . . . .I. . . 5166 Other Drugs Other Ot ug A Affecting Thyroid hy hyyroid oidd Hormone Ho Horm e Home Hom Ho meos m me osta tasi siss . . . . . 521 T3,T4 T3 , T4 Th Effects of Thyroid Hormones on Target Tisss ue uess . . . . . . . . . . . 516 Lithium m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 Hypothalamic-Pituitary-Thyyroid id Axi xiss . . . . . . . . . . . . . . . . . . . . 517 Ami Amiod miodarone od e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 PATHOP OPHY HYSI SIOL OLO OGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518 Corticoste osteroids stero teer e rroid eroids oids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 Follicula licu r ce ll Co PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 519 Tyrosinee Kinase Ty K Inhibitors ib s . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 Treaa tm tmen entt of Hyp ypot othy hyro roid idis ism m . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5199 T3 AND FUTURE T3 T4DIRECTIONS . . . . . . . . . . . . . . . . . . 521 CONCLUSION riphe iph con conv Suggested Reading . . . . . . . . .Pe. .riphe . . .e.raa. l. conve . . .v.e.rss. ion .o. . . . . . . .522 Pro pylthio urac il

INTRODUCTION The thyroid gland has diverse and important e ects on many aspects o metabolic homeostasis. Follicular thyroid cells constitute the majority o thyroid tissue; these cells produce and secrete the classical thyroid hormones: thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3). Thyroid hormones regulate growth, metabolism, and energy expenditure, rom oxygen consumption to cardiac contractility. Parafollicular C cells o the thyroid gland secrete calcitonin, a regulator o bone mineral homeostasis. Calcitonin is discussed in Chapter 32, Pharmacology o Bone Mineral Homeostasis. The major diseases o the thyroid gland involve disruption o the normal hypothalamic-pituitary-thyroid axis (see Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland). Replacement o def cient thyroid hormone is an e ective and established therapy or hypothyroidism. Treatment o hyperthyroidism is more complex, with options including antithyroid drugs, radioactive iodine, and surgical excision o abnormal tissue. Understanding the pathways and mechanisms o eedback regulation o thyroid hormone synthesis and thyroid hormone actions serves to explain the rationale or e ective drug treatment o thyroid diseases.

THYROID GLAND PHYSIOLOGY Synthesis and Secretion of Thyroid Hormones The thyroid is an endocrine gland located in the neck inerior to the larynx and spanning the ventral sur ace o the trachea. The main unction o the thyroid gland is to produce 514

T3

the thyroid hormones, T3 and T4. Structurally, the thyroid hormones are built on a backbone o two tyrosine molecules that are iodinated and connected by an ether linkage (Fig. 28-1). An important structural eature o thyroid hormones is the placement o iodines on this backbone. The position and relative orientation o iodines attached to the tyrosine residues determine the specif c orm o thyroid hormone. 3,5,3 ,5 -Tetraiodothyronine (thyroxine, T4) has our iodines attached to the tyrosine backbones and is the major orm o thyroid hormone secreted by the thyroid gland. 3,5,3 -Triiodothyronine (T3) has three iodines. Most T3 is produced by peripheral or extra-thyroidal 5 deiodination o T4 (see below). A biologically inactive orm o thyroid hormone is 3,3 ,5 -triiodothyronine, also re erred to as reverse triiodothyronine (rT3) because the single iodine is on the opposite tyrosine in the backbone relative to T3. In a normal individual, circulating thyroid hormone consists o about 90% T4, 9% T3, and 1% rT3, and most o the hormone is bound to plasma proteins (both specif c binding proteins and albumin). Iodine is a trace element that is a crucial component o thyroid hormone structure. Thyroid ollicular cells, which synthesize and secrete thyroid hormones, selectively concentrate iodide (I ) via a Na /I symporter (NIS) located on the basolateral membrane o the cell (Fig. 28-2). This active transport mechanism has the ability to concentrate iodide to intracellular concentrations up to 500 times that o plasma; most individuals have thyroid gland to plasma iodide ratios o approximately 30. Once inside thyroid gland ollicular cells, iodide is transported across the apical membrane o the cell via a Na independent Cl /I exchanger (one is known as pendrin) into the colloid space (Fig. 28-2). Iodide is then oxidized

C h a p t e r 2 8 Pharmacology o the Thyroid Gland

Over the course o a ew m onths, Diana L, 45 ye a rs o ld , n o tice s a n u m b e r o d is co n ce rtin g ch a n g e s in th e wa y s h e e e ls a n d in h e r g e n e ra l a p p e a ra n ce . Ms . L e e ls n e rvo u s a ll th e tim e ; s m a ll e ve n ts m a ke h e r ju m p y. S h e a ls o ke e p s th e te m p e ra tu re u n u s u a lly co ld in h e r h o u s e , to th e p o in t w h e re h e r h u s b a n d a n d ch ild re n b e g in to co m p la in . Be ca u s e o th e s e s ym p to m s a n d th e o cca s io n a l e e lin g th a t h e r h e a rt “ s kip s a b e a t,” Ms . L g o e s to s e e h e r d o cto r. A te r s o m e q u e s tio n in g , h e p a lp a te s h e r n e ck a n d n o te s th a t h e r thyro id g la n d is d i u s e ly e n la rg e d . He a ls o n o te s th a t Ms . L’s e ye s a re m o re p ro m in e n t th a n n o rm a l. Te s ts o r th yro id h o rm o n e le ve ls re ve a l h ig h s e ru m re e triio d o thyro n in e (T3) a n d lo w thyro tro p in (TS H). In a d d itio n , a te s t o r TS H re ce p to r a n tib o d y is p o s itive . Ms . L is d ia g n o s e d w ith Gra ve s ’ d is e a s e , a o rm o h yp e rth yro id is m , a n d tre a te d w ith m e th im a zo le . Alth o u g h in itia lly co m o rte d b y th e a ct th a t h e r d o cto r ca n e xp la in h e r s ym p to m s , s h e s o o n b e co m e s d is co u ra g e d b e ca u s e s h e d o e s n o t n o tice a ny im p rove m e n t o r a co u p le o w e e ks . A te r a m o n th , h o w e ve r, h e r s ym p to m s b e g in to s u b s id e . Re p e a t te s ts co n f rm th a t h e r thyro id h o rm o n e le ve ls a re n o rm a lize d . On e ye a r a te r s ta rtin g tre a tm e n t w ith m e th im a zo le , h o w e ve r, s h e b e g in s to re e xp e rie n ce p a lp ita tio n s a n d e e ls

by the enzyme thyroid peroxidase (Fig. 28-2). This oxidation reaction creates a reactive iodide intermediate that couples to specif c tyrosine residues on thyroglobulin. Thyroglobulin is a protein synthesized by thyroid ollicular cells and secreted at the apical sur ace into the colloid space. Thyroid peroxidase is also concentrated at the apical sur ace, O H2 N

OH

I 3

3'

I

5'

O

5

OH

I

515

a n xio u s . He r d o cto r co n f rm s th a t h e r th yro id h o rm o n e le ve ls a re a ga in e le va te d , d e s p ite m e th im a zo le th e ra p y. A te r d is cu s s io n w ith h e r d o cto r, Ms . L e le cts to u n d e rg o tre a tm e n t w ith ra d io a ctive io d in e . S h e to le ra te s th e tre a tm e n t w e ll, a n d te s tin g ove r th e n e xt 3 ye a rs s h o w s th a t s h e h a s n o rm a l thyro id h o rm o n e le ve ls . Ho w e ve r, 4 ye a rs a te r ra d io a ctive io d in e tre a tm e n t, s h e d e ve lo p s s ym p to m s th a t a re th e o p p o s ite o h e r o rig in a l p ro b le m s : s h e e e ls tire d a n d co ld a ll th e tim e , a n d s h e ga in s 30 p o u n d s ove r th e co u rs e o 6 m o n th s . He r d o cto r co n f rm s th a t Ms . L h a s d e ve lo p e d hyp o thyro id is m . He p re s crib e s thyroxin e (T4), w h ich s h e n o w ta ke s o n ce a d a y, a n d s h e e e ls w e ll a ga in .

Questions 1 . Why was Ms. L’s serum thyrotropin level low but her triiodothyronine level high? 2 . Which eatures o the thyroid gland make radioactive iodine a generally sa e and specif c therapy or hyperthyroidism? 3 . Why did Ms. L develop hypothyroidism a ter treatment with radioactive iodine? 4 . What is the mechanism o action o methimazole? Why did methimazole eventually stop working?

and it is thought that generation o oxidized iodide at this sur ace allows the iodide to react with tyrosine residues in the newly secreted thyroglobulin molecules. The process o thyroglobulin iodination is known as organif cation. Organif cation results in thyroglobulin molecules containing monoiodotyrosine (MIT) and diiodotyrosine (DIT) residues; these tyrosine residues have one or two covalently attached iodines, respectively. A ter MITs and DITs are generated within thyroglobulin, thyroid peroxidase also catalyzes coupling between these residues. An MIT joined to DIT generates T3, while the joining o two DITs creates T4. Note again that the majority o plasma T3 is produced by metabolism o T4 in the circulation (also see the ollowing section, “Metabolism o Thyroid Hormones”)

I

Thyroxine (T4) Outer ring deiodination

FIGURE 28-1. Structure and peripheral metabolism o thyroid hormones. Inner ring deiodination

O H2 N

O

I

OH

3

I

3'

OH

H2 N

I

OH

3

I

3'

OH

O

5 I

3,5,3'-Triiodothyronine (T3) (biologica lly a ctive )

O

5' I

3,3',5'-Triiodothyronine (rT3) (biologica lly ina ctive )

Thyroid hormones are synthesized rom two derivatized tyrosine molecules that are attached by an ether linkage. The outer ring is hydroxylated, whereas the inner ring is linked covalently to thyroglobulin during thyroid hormone synthesis. Iodine is attached to three or our positions o the tyrosine backbone, creating several di erent substitution patterns. Thyroxine (T4) has our iodines attached, two on each ring. Thyroxine is the predominant thyroid hormone produced by the thyroid gland. Triiodothyronine (T3) has two iodines attached to the inner ring but only one iodine attached to the outer ring. In contrast, reverse triiodothyronine (rT3) has two iodines on the outer ring but only one iodine on the inner ring. During peripheral metabolism, thyroxine is deiodinated by deiodinases present in target tissues and in the liver. The pattern o deiodination produces either T3 or rT3. I the iodine is removed rom the outer ring by 5 -deiodinase, the biologically active T3 is produced. I the iodine is removed rom the inner ring, the biologically inactive rT3 is produced.

516 PRINCIPLES OF ENDOCRINE PHARMACOLOGY Na +/ I- s ymporte r (NIS )

Extra ce llula r s pa ce

2Na +

I-

I-

2Na +

I-

Cl-

Na +-inde pe nde nt Cl-/I- e xcha nge r (pe ndrin)

ClITG Thyroid peroxidase (orga nifica tion) Colloid TG-MIT, s pa ce TG-DIT Thyroid peroxidase (coupling) T3 T4 TG

T3,T4

Follicula r ce ll

T3

T4 P e riphe ra l conve rs ion T3

FIGURE 28-2. Thyroid hormone synthesis, storage, and release. Follicular cells of the thyroid gland concentrate iodide (I ) from plasma via a basolateral membrane Na /I symporter (NIS). Iodide is further transported to the colloid space via the sodium-independent Cl /I exchanger (pendrin). In a reaction (called organif cation) catalyzed by thyroid peroxidase, iodide reacts covalently with tyrosine residues on thyroglobulin (TG) molecules at the apical membrane of the follicular cell. Addition of one I to tyrosine results in the formation of monoiodinated tyrosine (MIT); addition of two I to tyrosine results in the formation of diiodinated tyrosine (DIT). MIT and DIT associate covalently on thyroglobulin via a mechanism known as coupling, which is also catalyzed by thyroid peroxidase. The derivatized thyroglobulin is stored as colloid within follicles in the thyroid gland. Upon stimulation by TSH, thyroid follicular cells endocytose colloid into lysosomal compartments, where the thyroglobulin is degraded to yield free T4, free T3, and uncoupled MIT and DIT. T3 and T4 are secreted into the plasma, and MIT and DIT are deiodinated intracellularly to yield free iodide for use in new thyroid hormone synthesis (not shown). The thyroid gland secretes more T4 than T3, although T4 is converted to T3 in peripheral tissues. The monocarboxylate 8 transporter (MCT8) is localized at the basolateral membrane of thyroid follicular cells and is one of the transporters involved in secretion of thyroid hormones from the thyroid gland (not shown).

and that nascent T3 and T4 are covalently part o the thyroglobulin protein at this point. These thyroglobulin molecules are then stored in the lumen o the ollicle as colloid. When thyroid-stimulating hormone (discussed below) stimulates the thyroid gland to secrete thyroid hormone, the ollicular cells endocytose colloid. The ingested thyroglobulin enters lysosomes, where proteases digest the thyroglobulin. Proteolytic digestion releases ree T3, T4, MIT, and DIT. T3 and T4 are transported across the ollicular cell basolateral membrane and into the blood. Free MIT and DIT are rapidly deiodinated within the cell, allowing the iodide to be recycled or new thyroid hormone synthesis.

Most endocrine organs concurrently synthesize and release new hormone when activated rather than storing large quantities o precursor hormone. The thyroid gland is unusual among endocrine glands in that it stores large quantities of thyroid prohormone in the form of thyroglobulin. It is not understood why the thyroid gland maintains this elaborate pathway or hormone synthesis and release, but doing so makes it possible to maintain plasma thyroid hormone at a constant level despite uctuations in the availability o dietary iodide.

Metabolism of Thyroid Hormones Thyroid hormone circulates mostly bound to plasma proteins, notably thyroid-binding globulin (TBG) and transthyretin. Although T4 is the predominant thyroid hormone ound in the blood, the physiologic activity o T3 is our old higher than that o T4 on target tissues. Some serum T4 is inactivated by deamination, decarboxylation, or conjugation and excretion by the liver. Most T4, however, is deiodinated to the more active T3 orm in several locations in the body. This deiodination reaction is catalyzed by the enzyme iodothyronine 5 -deiodinase (Fig. 28-1). There are three subtypes o deiodinase. Type I 5 -deiodinase , expressed in the liver and kidneys, is important or converting T4 to the majority o serum T3. Type II 5 -deiodinase is expressed primarily in the pituitary gland, brain, and brown at. This enzyme is located intracellularly and converts T4 to T3 locally. Type III 5-deiodinase is responsible largely or conversion o T4 to the biologically inactive rT3. The presence o T4 in the blood provides a bu er, or reservoir, or thyroid hormone e ects. Most T4 to T3 conversion occurs in the liver, and many pharmacologic agents that increase hepatic cytochrome P450 enzyme activity also increase T4 to T3 conversion. In addition, T4 has a hal -li e in the plasma o approximately 6 days, whereas plasma T3 has a hal -li e o only 1 day. Because T4 has a long plasma half-life, changes in thyroid hormone-regulated functions caused by pharmacologic intervention are generally not observed for a period of 1 to 2 weeks, as seen with Ms. L in the introductory case.

Effects of Thyroid Hormones on Target Tissues Thyroid hormones have e ects on virtually every cell o the body. While the majority o the e ects o thyroid hormones likely occur at the level o gene transcription, there is growing evidence that these hormones also act at the plasma membrane and/or in the cytoplasm. Both modes o action are mediated by hormone binding to thyroid hormone receptors (TRs). Free hormone enters the cell by both passive di usion and active transport, the latter mediated by hormone-specif c and nonspecif c carriers such as organic anion and monocarboxylate transporters that are located in the plasma membrane. The best described o these is the monocarboxylate 8 transporter (MCT8), although other transporters likely also contribute to thyroid hormone transport. Once inside the cell, thyroid hormone binds to thyroid hormone receptors. These TRs are proteins containing thyroid hormone-binding, DNAbinding, and dimerization domains. There are two classes o thyroid hormone receptor, termed TR and TR . In addition, both TR and TR can be expressed as multiple iso orms. TR monomers can interact in a dimerization reaction to orm homodimers or with another transcription actor, retinoid

C h a p t e r 2 8 Pharmacology o the Thyroid Gland

X receptor (RXR), to orm heterodimers. These TR dimers

bind to gene regulatory regions and are activated by binding o thyroid hormones. Together, the multiple di erent combinations o TRs and the variability in their tissue distributions create tissue specif city or thyroid hormone e ects. In the absence o hormone, thyroid hormone receptor dimers associate with corepressor molecules and constitutively bind to (and thereby inactivate) thyroid hormone-stimulated genes. Binding o thyroid hormone to TR:RXR or TR:TR dimers promotes dissociation o the corepressors and recruitment o coactivators to the DNA. Thus, thyroid hormone binding to TR dimers serves as a molecular switch rom inhibition to activation o gene transcription (Fig. 28-3). Thyroid hormone also acts to down-regulate gene expression by a TRdependent mechanism, the exact nature o which is not ully understood. For example, thyroid hormone is able to downregulate TSH subunit gene expression, causing negative eedback o thyroid hormone on the hypothalamic-pituitarythyroid axis (see Chapter 27). Increasing evidence suggests that thyroid hormone also has nongenomic e ects on mitochondrial metabolism and that it interacts with plasma membrane receptors to stimulate intracellular signal transduction. Thyroid hormone is important in in ancy or growth and development o the nervous system. Congenital def ciency o thyroid hormone results in cretinism, a severe but preventable orm o mental retardation. Genetic mutations in the MCT8 transporter are associated with Allan-Herndon-Dudley syndrome , a neurologic disease that presents in male children with a constellation o motor, cognitive, and developmental problems. These children also have abnormalities in thyroid

RXR

TR

De cre a s e d tra ns cription

Core pre s s or

5'

3'

No thyroid hormone

T3 Coa ctiva tor RXR

Core pre s s or

T3 TR

DNA tra ns cription

5'

3'

With thyroid hormone

FIGURE 28-3. Thyroid hormone receptor actions. In the absence o thyroid hormone, the thyroid hormone receptor (TR):retinoid X receptor (RXR) heterodimer associates with a corepressor complex, which binds to promoter regions o DNA and inhibits gene expression. In the presence o thyroid hormone (T3), the corepressor complex dissociates rom the TR:RXR heterodimer, coactivators are recruited, and gene transcription occurs. This example demonstrates the action o T3 on a TR:RXR heterodimer, but similar mechanisms are probable or TR:TR homodimers. A use ul therapeutic strategy in the uture may involve pharmacologic targeting o tissue-specif c corepressors or coactivators.

517

unction, with normal TSH levels in the setting o elevated circulating T3 levels and low T4 levels. Thus, MCT8 mutations appear to impair central eedback o thyroid hormones (see below), leading to elevated levels o T3 in the presence o a normal level o TSH. The low T4 level appears to be multi actorial; one actor is that MCT8 is involved in secretion o T4 rom the thyroid gland. MCT8 mutations might lead to severe neurologic disease because o the lack o transport o thyroid hormone into key areas o the brain during development. It is also possible, however, that MCT8 transports other actors that are key to normal neurologic development. In the adult, thyroid hormone regulates general body metabolism and energy expenditure. Enzymes regulated by thyroid hormone include the Na /K ATPase and many o the enzymes o intermediary metabolism, both anabolic and catabolic. At high levels o thyroid hormone, this e ect can result in utile cycling and a consequent increase in body temperature—this is why Ms. L started turning down the heat in her home. Many o the e ects o thyroid hormone resemble the e ects o sympathetic neural stimulation, including increased cardiac contractility and heart rate, excitability, nervousness, and diaphoresis (sweating). These symptoms were also seen in Ms. L—she was nervous all the time and was startled by slight provocations. Conversely, low levels o thyroid hormone result in myxedema , a hypometabolic state characterized by lethargy, dry skin, coarse voice, and cold intolerance.

Hypothalamic-Pituitary-Thyroid Axis Thyroid hormone secretion ollows a negative regulatory eedback scheme similar to that o the other hypothalamicpituitary-target organ axes (Fig. 28-4). Thyrotropin-releasing hormone (TRH) is a tripeptide secreted by the hypothalamus that travels via the hypothalamic-pituitary portal circulation to the anterior pituitary gland (see Chapter 27). TRH binds to a G protein-coupled receptor located in the plasma membrane o anterior pituitary gland thyrotropes, or TSHproducing cells. This stimulates a signal transduction cascade that ultimately promotes the synthesis and release o thyroid-stimulating hormone (TSH). TSH is the most important direct regulator o thyroid gland unction. TSH stimulates every known aspect o thyroid hormone production, including iodide uptake, organif cation, coupling, thyroglobulin internalization, and secretion o thyroid hormone. In addition, TSH promotes increased vascularization and growth o the thyroid gland. In pathologic conditions where TSH or a TSH mimic (see below) is secreted at high levels, the thyroid gland can enlarge to several times its normal size, resulting in the characteristic di usely hypertrophied thyroid gland re erred to as a goiter, which Ms. L’s doctor noted when he palpated her neck. Negative eedback o the hypothalamic-pituitary-thyroid axis occurs through regulatory actions o thyroid hormone on both the hypothalamus and pituitary gland. Secreted thyroid hormone di uses and is transported into the thyrotropes o the anterior pituitary gland, where it binds and activates nuclear thyroid hormone receptors. These bound receptors inhibit TSH gene transcription and, hence, TSH synthesis. Thyroid hormone also has important regulatory e ects on the hypothalamus; thyroid hormone binding to receptors in hypothalamic cells inhibits transcription o the gene that codes or the TRH precursor protein.

518 PRINCIPLES OF ENDOCRINE PHARMACOLOGY A Normal axis

B Graves' disease

TRH

TS H

Thyroid gla nd

C Hashimoto's thyroiditis

TRH

TS H

Thyroid hormone

Thyroid hormone

Ta rge t tis s ue s

Stimulatory autoantibody

TRH

TS H

Thyroid hormone

Thyroid hormone Ta rge t tis s ue s

Destructive autoantibody

Thyroid hormone

Thyroid hormone Ta rge t tis s ue s

FIGURE 28-4. The hypothalamic-pituitary-thyroid axis in health and disease. A. In the normal axis, thyrotropin-releasing hormone (TRH) stimulates thyrotropes o the anterior pituitary gland to release thyroid-stimulating hormone (TSH). TSH stimulates synthesis and release o thyroid hormone by the thyroid gland. Thyroid hormone, in addition to its e ects on target tissues, inhibits urther release o TRH and TSH by the hypothalamus and anterior pituitary gland, respectively. B. In Graves’ disease, a stimulatory autoantibody autonomously activates the TSH receptor in the thyroid gland, resulting in sustained stimulation o the thyroid gland, increased plasma thyroid hormone (thick lines), and suppression o TRH and TSH release (dashed lines). C. In Hashimoto’s thyroiditis, a destructive autoantibody attacks the thyroid gland, causing thyroid insu f ciency and decreased synthesis and secretion o thyroid hormone (dashed lines). Consequently, eedback inhibition on the hypothalamus and anterior pituitary gland does not occur, and plasma TSH levels rise (thick lines).

PATHOPHYSIOLOGY The pathophysiology o thyroid diseases can be understood as a disturbance o the physiologic hypothalamic-pituitarythyroid axis. For example, a physiologic decrease in thyroid hormones normally activates TSH synthesis and release, which leads to increased release o thyroid hormones by the thyroid gland and to restoration o normal thyroid hormone levels. Thyroid gland pathology can also cause thyroid hormone insu f ciency, which also reduces the negative eedback o thyroid hormone on TSH release. Although TSH levels are elevated as a consequence, there is no increase in thyroid hormone release because the diseased thyroid gland cannot respond appropriately. Most common thyroid diseases are best categorized as conditions that result in increased (hyperthyroid) or decreased (hypothyroid) thyroid hormone secretion. Two common thyroid diseases are Graves’disease and Hashimoto’s thyroiditis (Fig. 28-4). Each is believed to be autoimmune in origin, but Graves’ disease causes hyperthyroidism, whereas Hashimoto’s thyroiditis ultimately results in hypothyroidism. Graves’ disease demonstrates the importance o plasma thyroid hormone in regulating homeostasis o the hypothalamicpituitary-thyroid axis. In this syndrome, an IgG autoantibody specif c or the TSH receptor is produced. This autoantibody is known as thyroid-stimulating immunoglobulin (TSIg). The antibody acts as an agonist, activating the TSH receptor and thereby stimulating thyroid ollicular cells to synthesize and release thyroid hormone. Unlike TSH, however, TSIg is not subject to negative feedback control; it continues to stimulate thyroid function even when plasma thyroid hormone levels

rise into the pathologic range. Because the autoantibody in Graves’ disease acts independently o the hypothalamicpituitary-thyroid axis, thyroid hormone homeostasis is disrupted. Clinical symptoms o hyperthyroidism result, and laboratory studies show high plasma thyroid hormone levels, low or undetectable TSH levels, and high TSIg levels. In the introductory case, Ms. L’s TSH levels were low because her excess plasma thyroid hormone suppressed release o TSH by her anterior pituitary gland. Hashimoto’s thyroiditis, in contrast, results in selective destruction o the thyroid gland. Antibodies specif c or many thyroid gland proteins, including thyroglobulin and thyroid peroxidase, can be ound in the plasma o patients with Hashimoto’s thyroiditis. As with Graves’ disease, the underlying etiology o this disease is thought to be autoimmune. The clinical course o Hashimoto’s thyroiditis involves a gradual in ammatory destruction o the thyroid gland with resultant hypothyroidism. Early in the course o the disease, destruction o thyroid ollicular cells can release excessive quantities o stored colloid, resulting in transiently increased levels o thyroid hormone. Eventually, the gland is almost completely destroyed, and clinical symptoms o hypothyroidism develop (e.g., lethargy and decreased metabolic rate). Therapy or Hashimoto’s thyroiditis involves pharmacologic replacement with oral synthetic thyroid hormone. Other causes o hypothyroidism and hyperthyroidism include developmental anomalies, subacute (de Quervain’s) thyroiditis, and thyroid adenomas and carcinomas. Details o the underlying pathophysiologies di er, but pharmacologic intervention in each case rests on determining whether the patient is hypothyroid, euthyroid, or hyperthyroid.

C h a p t e r 2 8 Pharmacology o the Thyroid Gland

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic treatment o thyroid gland diseases involves either replacement o def cient thyroid hormone or antagonism o excessive thyroid hormone. Replacement is sel evident, while the antagonists work at multiple steps in thyroid hormone synthesis and action (Fig. 28-5). In addition, a number o pharmacologic agents used or nonthyroid disease indications have important e ects on peripheral thyroid hormone metabolism. The mechanisms o their action are discussed at the end o this section.

Treatment of Hypothyroidism Thyroid hormone is a well-established and sa e therapy or long-term treatment o hypothyroidism. Therapy aims to replace missing endogenous thyroid hormone with regularly administered exogenous thyroid hormone. The exogenous thyroid hormone is structurally identical to endogenous thyroid hormone (generally T4) and is produced by chemical synthesis.

Extra ce llula r s pa ce

Thionamides Iodides (high)

2Na +

I-

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Perchlorate Thiocyanate Pertechnetate Na +-inde pe nde nt Cl-/I- e xcha nge r (pe ndrin)

ClITG Thyroid peroxidase (orga nifica tion) TG-MIT, TG-DIT Thyroid peroxidase Colloid (coupling) s pa ce T3 T4 TG

Iodides (high)

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T4

519

Many trials with replacement hormone debated whether it would be more e f cacious to provide replacement o T3 or T4. T3 is the metabolically more active orm o thyroid hormone, and one might have anticipated that replacement o def cient thyroid hormone with T3 would more e ectively normalize thyroid homeostasis. However, a number o f ndings argue against this. First, most thyroid hormone in the blood is in the orm o T4, although T4 has lower activity than T3 and is eventually metabolized to T3. Having a large reservoir o thyroid “prodrug” (T4) in the plasma may be important, perhaps as an e ective bu er to normalize metabolic rates over a wide range o conditions. Second, the hal -li e o T4 is 6 days, as compared to the 1-day hal li e o T3. The extended hal -li e o T4 allows a patient to take just one thyroid hormone replacement pill per day. For these reasons, levothyroxine , the L-isomer o T4, is still the treatment o choice or hypothyroidism. (One possible exception is myxedema coma, where the aster onset o T3 may provide enhanced recovery rom li e-threatening hypothyroidism.) The e f cacy o thyroid hormone replacement is monitored by assays o plasma TSH and thyroid hormone levels. TSH is an accurate marker o thyroid hormone activity because anterior pituitary gland release o TSH is exquisitely sensitive to eedback control by thyroid hormone in the blood. Once a patient is taking a stable dose o levothyroxine, monitoring o TSH levels can generally be per ormed less requently, such as every 6 months to a year. Sudden alterations in TSH levels despite constant dosing o levothyroxine may be due to drug–drug interactions a ecting absorption and metabolism. For example, resins such as sodium polystyrene sulfonate (Kayexalate®) and cholestyramine may decrease absorption o T4. Adequate gastric acidity is also required or absorption o exogenous levothyroxine; the dose o levothyroxine may there ore need to be increased in patients who become in ected with Helicobacter pylori or who start taking a proton pump inhibitor. Drugs that increase the activity o certain hepatic P450 enzymes, including rifampin and phenytoin, increase the hepatic clearance o T4. In such cases, it may be necessary to increase the dose o T4 in order to maintain a euthyroid state.

Treatment of Hyperthyroidism There are pharmacologic agents that target each step o thyroid hormone synthesis, rom initial uptake o iodide, to organif cation, coupling, and peripheral conversion o T4 to T3. Clinically, both radioactive iodine and thionamides are available or the treatment o hyperthyroidism. -Adrenergic antagonists are also sometimes used to ameliorate some o the symptoms o hyperthyroidism.

P e riphe ra l conve rs ion

Propylthiouracil

Inhibitors of Iodide Uptake T3

FIGURE 28-5. Pharmacologic interventions affecting thyroid hormone synthesis. Anions with a molecular radius approximately equal to that o the iodide anion (I ), such as perchlorate, thiocyanate, and pertechnetate, compete with iodide or uptake by the Na /I symporter. Radioactive 131I , when concentrated within thyroid cells, causes selective destruction o the thyroid gland. High levels o iodide transiently depress thyroid unction by inhibiting organif cation, coupling, and proteolysis o thyroglobulin. Thionamides, such as propylthiouracil and methimazole, inhibit organif cation and coupling; propylthiouracil also inhibits peripheral conversion o T4 to T3. TG-MIT, thyroglobulin-monoiodotyrosine; TG-DIT, thyroglobulin-diiodotyrosine.

Iodide is taken up by the thyroid ollicular cell via a Na / I symporter. Certain anions with the approximate atomic radius o iodide, such as perchlorate , thiocyanate , and pertechnetate , compete with iodide or uptake into the thyroid gland ollicular cell (Fig. 28-5). This results in a decreased amount o iodide available or thyroid hormone synthesis. The e ects o anion uptake inhibitors are usually not immediately apparent because o the large store o preormed thyroid hormone in the colloid. Anion uptake inhibitors can be used in the treatment o hyperthyroidism; these agents reduce the intrathyroidal

520 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

supply o iodide available or thyroid hormone synthesis. However, their use is uncommon because o the potential or causing aplastic anemia, and the thionamides (see below) are generally more e ective. Because many o these uptake inhibitors are also used as radiopaque contrast materials, it is important to keep this physiologic antagonism in mind whenever a patient has symptoms o hypothyroidism a ter extensive radiographic studies employing contrast material.

Inhibitors o Organif cation and Hormone Release Iodides

Two distinct types o iodide are used in clinical practice. Both take advantage o the thyroid gland’s selective uptake and concentration o iodide to levels much higher than that in the blood. The f rst agent, 131I , is a radioactive iodine isotope that strongly emits -particles toxic to cells. The Na /I symporter expressed on thyroid ollicular cell membranes cannot distinguish 131I rom normal stable iodide (127I ). There ore, 131 I becomes sequestered within the thyroid gland. This makes radioactive 131I a specif c and e ective therapy or hyperthyroidism. The concentrated intracellular radioactive iodine continues to emit -particles, resulting in selective local destruction o the thyroid gland. Radioactive iodine is used to treat thyrotoxicosis, and this agent serves as an alternative to surgery in the treatment o hyperthyroidism. There is a concern that patients may eventually develop hypothyroidism a ter treatment with radioactive iodine, because it is di f cult to ascertain or a given patient the extent to which radioactive 131I will kill all or most o his or her thyroid ollicular cells. The goal is to administer enough 131I to result in a euthyroid state, without precipitating hypothyroidism. This desired result is not always obtained; or example, in the introductory case, Ms. L eventually developed hypothyroidism a ter treatment with 131I . Regardless, the development o hypothyroidism is easier to manage clinically than hyperthyroidism. Based on epidemiologic studies, it is unlikely that therapeutic doses o radioactive iodine have any e ect on the incidence o thyroid cancer. The second clinically important pharmacologic agent is, paradoxically, stable inorganic iodide. High levels o iodide inhibit thyroid hormone synthesis and release, a phenomenon known as the Wolff-Chaikoff effect. This phenomenon is likely mediated by down-regulation o the Na /I symporter in the thyroid gland. The negative eedback e ect o high intrathyroidal iodide concentrations is reversible and transient; thyroid hormone synthesis and release returns to normal a ew days a ter the plasma iodide concentration is increased. There ore, inorganic iodide is not a use ul long-term therapy or hyperthyroidism. This phenomenon does, however, have other important uses. For example, high iodide dosing reduces the size and vascularity o the thyroid gland. Because o this, iodide is o ten administered be ore thyroid gland surgery, resulting in technically easier excision o the gland. Iodide can also have important preventative e ects. When the nuclear accident at Chernobyl occurred, there was concern that radioactive iodine released into the air over Poland could cause population-wide thyroid gland destruction. As a preventative measure, millions o Polish children were given large doses o iodide or a number o days to suppress thyroid gland unction temporarily and thereby to avoid uptake o environmental radioactive iodine.

Thionamides

The thionamides propylthiouracil and methimazole are important and use ul inhibitors o thyroid hormone production. Thionamides compete with thyroglobulin or oxidized iodide in a process that is catalyzed by the enzyme thyroid peroxidase (Fig. 28-5). By competing or oxidized iodide, thionamide treatment causes a selective decrease in the organif cation and coupling o thyroid hormone precursors and thereby inhibits thyroid hormone production. Iodinated thionamides may also be capable o binding to thyroglobulin, urther antagonizing any coupling reactions. Recall that thyroid ollicular cells store a large quantity o nascent thyroid hormone in the orm o colloid. This colloid can provide a su f cient amount o thyroid hormone or more than a week in the absence o any new synthesis. Because thionamides a ect the synthesis but not the secretion o thyroid hormone, the e ects o these drugs are not mani ested until several weeks a ter the initiation o treatment (as in the introductory case). Thionamide treatment o ten results in goiter ormation. For this reason, the drugs are commonly re erred to as goitrogens . Inhibition o thyroid hormone production by thionamides results in up-regulation o TSH release by the anterior pituitary gland in an attempt to reestablish homeostasis. The increased plasma TSH cannot raise thyroid hormone levels because o the action o the thionamide, however. In response to stimulation by the elevated TSH, the thyroid gland hypertrophies in an attempt to increase thyroid hormone synthesis. This results in the eventual ormation o a goiter. Propylthiouracil is considered the prototype thionamide; methimazole is another requently used drug in this class. Propylthiouracil inhibits thyroid peroxidase as well as peripheral T4 to T3 conversion, whereas methimazole has only been shown to inhibit thyroid peroxidase. Propylthiouracil has a short hal -li e that necessitates dosing three times a day, while methimazole can be administered once daily. Both propylthiouracil and methimazole are generally well tolerated. The most requent adverse e ect o these agents is a pruritic rash early in the course o treatment, which may remit spontaneously. Arthralgias are also a common reason or stopping these agents. Propylthiouracil and methimazole can inter ere with the vitamin K-dependent synthesis o prothrombin, leading to hypoprothrombinemia and an increased bleeding tendency. Three rare but serious complications o propylthiouracil and methimazole are agranulocytosis, hepatotoxicity, and vasculitis. Agranulocytosis occurs in 0.1% o cases, usually within the f rst 90 days o treatment with these agents. Because o this risk, all patients taking thionamides should have a baseline measurement o white blood cell count and should be advised to discontinue the drug immediately i they develop ever or a sore throat. Hepatotoxicity is also a rare adverse e ect o the thionamides. The hepatitis is typically cholestatic in pattern and may represent an allergic reaction to the drugs. Severe hepatitis leading to liver ailure and death has been associated with propylthiouracil treatment. Vasculitis rom these agents can mani est as druginduced lupus or an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Because the incidence o serious adverse e ects appears to be less requent with methimazole than with propylthiouracil, methimazole is generally the pre erred agent in clinical practice. Two exceptions to this rule are thyroid storm and pregnancy. In the acute management o severe

C h a p t e r 2 8 Pharmacology of the Thyroid Gland

hyperthyroidism (thyroid storm), the additional ability o propylthiouracil to block peripheral conversion o T4 to T3 makes this drug the more attractive agent. In pregnancy, propylthiouracil is the pre erred agent because it has a more extensive sa ety record and because methimazole use during pregnancy has been associated with the development o aplasia cutis. The thionamides are generally e ective at controlling hyperthyroidism. A high percentage o patients taking these agents will go into remission over the course o 6 months to a year and may be able to maintain a euthyroid state a ter discontinuation o these medications. Some patients, however, will develop persistent hyperthyroidism despite treatment, as in the introductory case. Such patients require more def nitive treatment o their hyperthyroidism by either radioactive iodine therapy or surgical removal o the thyroid gland.

Inhibitors of Peripheral Thyroid Hormone Metabolism Although the majority o thyroid hormone is synthesized in the thyroid gland as T4, thyroid hormone acts at peripheral sites principally as T3. Conversion o T4 to T3 is dependent on a peripheral 5 -deiodinase, and inhibitors o this enzyme are e ective adjuncts in treating the symptoms o hyperthyroidism. As mentioned above, propylthiouracil inhibits both organif cation and coupling in the thyroid gland and peripheral conversion o T4 to T3. Two other agents, -adrenergic blockers and ipodate, are discussed below. -Adrenergic Blockers

-Adrenergic antagonists are use ul therapies or the symptoms o hyperthyroidism. Many o the e ects o high plasma thyroid hormone levels resemble nonspecif c -adrenergic stimulation (e.g., sweating, tremor, tachycardia), although circulating catecholamine levels are not elevated. It is hypothesized that thyroid hormones may increase the responsiveness o tissues such as the heart to -adrenergic stimulation, perhaps by up-regulating -adrenergic receptor expression or altering G protein-mediated signaling. It has also been demonstrated that -blockers can reduce peripheral conversion o T4 to T3, but this e ect is not thought to be clinically relevant. Because o its rapid onset o action and short elimination hal -li e (9 minutes), esmolol is a pre erred parenteral -adrenergic antagonist or the treatment o thyroid storm. Ipodate

Ipodate is a radiocontrast agent ormerly used or visualization o the biliary ducts in endoscopic retrograde cholangiopancreatography (ERCP) procedures. In addition to its use ulness as a radiocontrast agent, ipodate signif cantly inhibits conversion o T4 to T3 by inhibiting the enzyme 5 -deiodinase. Although ipodate was sometimes used in the past to treat hyperthyroidism, it is no longer commercially available.

Other Drugs Affecting Thyroid Hormone Homeostasis Lithium

Lithium, a drug used in the treatment o bipolar disorder (see Chapter 15, Pharmacology o Serotonergic and Central Adrenergic Neurotransmission), can cause hypothyroidism. Lithium is actively concentrated in the thyroid gland, and high levels o lithium inhibit thyroid hormone release rom thyroid ollicular cells. There is some evidence that lithium may inhibit thyroid hormone synthesis as well. The mechanism(s) responsible or these actions is unknown.

521

Amiodarone

Amiodarone is an antiarrhythmic drug (see Chapter 24, Pharmacology o Cardiac Rhythm) that has both positive and negative e ects on thyroid hormone unction. Amiodarone structurally resembles thyroid hormone and it contains a high concentration o iodine (each 200-mg tablet o amiodarone contains 75 mg o iodine). Metabolism o amiodarone releases this iodine as iodide, resulting in increased plasma concentrations o iodide. The increased plasma iodide is concentrated in the thyroid gland; this can result in hypothyroidism by the Wol -Chaiko e ect. Amiodarone can also cause hyperthyroidism by two mechanisms. In type I thyrotoxicosis, the excess iodide load provided by amiodarone leads to increased thyroid hormone synthesis and release. In type II thyroiditis, an autoimmune thyroiditis is induced that leads to release o excess thyroid hormone rom the colloid. Because o its close structural similarity to thyroid hormone, amiodarone may also act as an analogue o thyroid hormone at the level o the receptor. In addition, amiodarone competitively inhibits type I 5 -deiodinase. This results in decreased peripheral conversion o T4 to T3 and increased plasma concentrations o rT3.

Corticosteroids

Corticosteroids, such as cortisol and glucocorticoid analogues, also inhibit the 5 -deiodinase that converts T4 to T3. Because T4 has less physiologic activity than T3, treatment with corticosteroids reduces net thyroid hormone activity. In addition, corticosteroids suppress TSH levels, likely by inhibiting transcription o TRH in the hypothalamus. Despite these e ects on the hypothalamic-pituitary-thyroid axis, chronic use o high-dose corticosteroids does not appear to cause clinically signif cant hypothyroidism.

Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors (TKIs) are e ective therapeutics or the treatment o many malignancies (see Chapter 40, Pharmacology o Cancer: Signal Transduction). Many o these drugs can also alter thyroid hormone levels. The f rst TKI ound to cause hypothyroidism was sunitinib, but subsequently, this same adverse e ect was identif ed in patients treated with other TKIs as well. It has also been recognized that TKIs can induce hyperthyroidism. This apparent paradoxical e ect suggests that targeting o receptor tyrosine kinases, such as the vascular endothelial growth actor receptor, may lead to alterations in angiogenesis and/or autoimmunity that a ect the hypothalamic-pituitary-thyroid axis. It is likely that the mechanisms o thyroid dys unction in patients treated with TKIs are multi actorial, and thyroid unction must be monitored in such patients.

CONCLUSION AND FUTURE DIRECTIONS Thyroid hormone synthesis consists o a complex set o synthesis and degradation steps. This pathway creates numerous points or pharmacologic intervention, rom iodide uptake to peripheral conversion o T4 to T3. Thyroid hormone replacement is a sa e and e ective long-term therapy or thyroid hormone def ciencies. Several e ective therapies exist or the management o thyrotoxicosis. Radioactive iodine and thionamides are commonly used or this purpose, leading to selective destruction o the thyroid gland and antagonism o organif cation and coupling, respectively.

522 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Future potential therapies or diseases o the thyroid gland may ocus on treating the etiology o autoimmune thyroid diseases, such as Graves’ disease and Hashimoto’s thyroiditis, and better def ning the molecular targets o thyroid hormone action.

Acknowledgment We thank Ehrin J. Armstrong and the late Armen H. Tashjian, Jr. or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Biondi B, Warto sky L. Treatment with thyroid hormone. Endocr Rev 2014;35:433–512. (Extensive discussion of the uses of thyroid hormone as a replacement hormone and a therapeutic agent.)

Brent GA. Clinical practice. Graves’ disease. N Engl J Med 2008;358: 2594–2605. (Reviews the clinical approach to Graves’ disease and discusses other causes of hyperthyroidism.) Cheng SY, Leonard JL, David PJ. Molecular aspects o thyroid hormone actions. Endocr Rev 2010;31:139–170. (A comprehensive treatise on the action of thyroid hormones in the cell.) Cooper DS. Antithyroid drugs. N Engl J Med 2005;352:905–917. (An excellent, detailed summary of the clinical uses and adverse effects of methimazole and propylthiouracil.) Fekete C, Lechan RM. Central regulation o hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions. Endocr Rev 2014;35:159–194. (Review of the regulatory aspects of thyroid physiology.) Medical Letter, Inc. Drugs or hypothyroidism and hyperthyroidism. Treat Guidel Med Lett 2006;4:17–24. (Review of therapeutic considerations, including important drug interactions.) Portulano C, Paroder-Belenitsky M, Carrasco N. The Na /I– symporter (NIS): mechanism and medical impact. Endocr Rev 2014;35:106–149. (Excellent review of this critical transporter of iodide into the thyroid follicular cell.)

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C h a p t e r 2 8 Pharmacology o the Thyroid Gland 523

29

Fe e dba c k inhibi biti tio on

ACTH Ante An t rio te iorr pi pitu tuit ita it ta ry glla nd d

Pharmacology of the Adrenal Cortex Ra je s h Ga rg a n d Ga il K. Ad le r

Adree na l gl glaa n d

INTRODUCTION O & CASE . . . . . . . . Amino g lute thimid 524–525 Meta tabo bolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535 e th h im imid ide Physiologic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535 Ph OVERVIEW V OF THE H AD DRE RENA NAL L CORT TeEX K tto o c o nazo le (hig h). . 5244 Ch Ke ole lees te telati rol C ol oRegula ro l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535 tion GLUCOCOR O OR RTI TICO COIIIDS CO DS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 Pathophysiology Pat o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535 Physi Ph s iology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 S CC CC Aldosterone A ld Hypo unction . . . . . . . . . . . . . . . . . . . . . . . . . . . 535 Sy Synthesis . . . . . . . . . . . . . . . . . Tr . .rilo . . . s. .tan .an .n. e. . . . . . . . . . . . . . . 525 t c o nazo le Ke to Aldosterone d Metabolism M m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 P ree gne ne nolone Hyper unctioon . . . . . . . . . . . . . . . . . . . . . . . . . . 535 Pharmacologic m Class se sess and nd Agents . . . . . . . . . . . . . . . . . . . . . 536 Ph hys ysio iolo logi lo gicc Ac gi Acti tions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. . . . 526 52 Mineralo Mi cort rtic rt icoi ic oidd Re Rece cept ce e ptor Agonistss . . . . . . . . . . . . . . . . . 536 17 1 7 oco R Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5227 Mineralocorticoid Receptor Mi Min ece Antagonistss . . . . . . . . . . . . . . 536 Pathop h phyysiolog oogyy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5288 P rog rogee s tte ro ronee 117 17-h 7 hyd ydr d roxy xypr xypre pree ggn pr gne nee nolone n n ollone S . . . . . . .De h. .yd y. . roe p. ia ndros te rone e536 . . . . . . . . . . . . . . . . . . . . . Adrenal Insu A s f ciencyy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528 ADRENAL ANDROGENS 20 Physiology si i o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 5366 Glucocc or G orti tico ccoid id Exc xcesss . . . . . . . . . .21 . . . . . . . . . . . .17 . . . . . . . . 529 3 3 Trrilo ril iillo.s. tane rilo t536 anee Pathophysiology op . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .T. rri . il.lo . .s. tan Pharmacologic r Classes and Agentss . . . . . . . . . . . . . . . . . . . . . 529 Pharmacologic m Classes and Agents . . . . . . . . . . . . . . . . . . . . . 536 Cortisol andd Glucocorticoid Analogues C l . . . . . . . . . 529 11-de 11 de d e ooxyco xyco xycor o r ticoss te t rone 17-h ydr 17 ydr t rone Andros A te ne dione d oxyproge s te And Inhibitors n o Adrenocortical Hormone m Synthesis . . . . . . . . 534 CO CONC NCLU LUSI USION ON AND FUT UTUR URE E20 DIRE RECT CTIO IONS NS . . . . . . . . . . . . . . . . . . 53 5377 Glucocorticoid Gl G i id Receptor R Antagonists A i s . . . . . . . . . . . . . . . . . 534 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .538 MINERALOCORTICOIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534 Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534

INTRODUCTION Like the pituitary gland, the adrenal gland consists o two organs used together during embryologic development. The outer adrenal cortex originates rom mesoderm, and the inner adrenal medulla is derived rom neural crest cells. The adrenal cortex synthesizes and secretes steroid hormones essential or salt balance, intermediary metabolism, and, in emales, androgenic actions. The adrenal medulla synthesizes and secretes the catecholamine epinephrine, which is important, although not essential, or maintaining sympathetic tone. This chapter ocuses on the adrenal cortex; because o its importance in neuropharmacology, the adrenal medulla is discussed in Chapter 11, Adrenergic Pharmacology. The therapeutic utility o adrenocortical hormones spans almost every area o medicine. This is largely because o the use ulness o glucocorticoid analogues as e f cacious and potent anti-in ammatory agents. Un ortunately, long-term systemic glucocorticoid therapy also produces a substantial number o predictable but undesirable adverse e ects. The physiology o mineralocorticoids has been studied in the etiology o hypertension, cardiovascular disease, and renal disease, and there is considerable interest in the use o mineralocorticoid receptor antagonists as therapies or these disorders. Adrenal androgens, although lacking a def nitive 524

therapeutic indication at present, are the ocus o investigation or use in emale sexual dys unction. Both def ciency and excess o adrenocortical hormones can cause human disease. Def ciency states are treated by replacing the hormones in the orm o therapeutic agents, while inhibitors o adrenocortical biosynthetic enzymes and antagonists at adrenocortical hormone receptors can be used to treat hormone excess.

OVERVIEW OF THE ADRENAL CORTEX The adrenal cortex synthesizes three classes o hormones: mineralocorticoids , glucocorticoids , and androgens . Histologically, the adrenal cortex is divided into three zones. Moving rom the capsule toward the medulla, these regions are the zona glomerulosa, zona asciculata, and zona reticularis (Fig. 29-1). The glomerulosa is responsible or mineralocorticoid production and is under the control o angiotensin II, blood potassium concentration, and, to a lesser extent, adrenocorticotropic hormone (ACTH). The asciculata and reticularis synthesize glucocorticoids and androgens, respectively. Both the asciculata and reticularis are mainly under the control o ACTH, which, in turn, is regulated by corticotropin-releasing hormone (CRH), vasopressin, and cortisol (see Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland).

C h a p t e r 2 9 Pharmacology o the Adrenal Cortex 525

Eight-year-old J ohnny f nds that he ca n b a re ly ca tch h is b re a th a t tim e s , e s p e cia lly w h ile e xe rcis in g . His a s th m a com es an d go es, bu t no th erapy seem s to s to p th e a s th m a a tta cks co m p le te ly. Alth o u g h h is d o cto r is co n ce rn e d th a t it co u ld s tu n t J oh nny’s g rowth , sh e even tu ally pres crib es oral pred nis one (a glucocorticoid analogue) and tells J ohnny’s p are nts to m ake s ure he takes th e m edication e very day. A ter a ew weeks, J ohnny’s asthm a attacks s ubsid e, and he is able to have a airly n orm al ch ildh oo d. Du rin g th is tim e , th e d o ctor p a ys clos e a tte n tio n to J ohnny’s linear gro wth . Two years later, J ohnny’s d octo r d e cid e s th a t a n e w in h a le d g lu co co rtico id co u ld be a sa er m edication or him . J ohnny s witches to the in h aled glu cocortico id a nd d is co n tin u es oral pred n isone. Three days later, he develops a respiratory in ection and is brought to the em ergency departm ent with low b lood p res sure an d a tem p erature o 103°F. Based

Through its mineralocorticoid, glucocorticoid, and adrenal androgen products, the adrenal cortex plays a role in diverse aspects o homeostasis. The ollowing discussion considers the physiology, pathophysiology, and pharmacology o each class o adrenal hormones. The glucocorticoids are discussed f rst, ollowed by the mineralocorticoids and the adrenal androgens.

Zona glome rulos a Angiote ns in II K+

Aldosterone synthase

Aldos te rone

11β-hydroxylase 17α-hydroxylase

Cortis ol, a ndroge ns

ACTH

Zona fa s cicula ta / re ticula ris

FIGURE 29-1. Regions of the adrenal cortex. The adrenal cortex is divided into three regions. The outermost region, the zona glomerulosa, synthesizes aldosterone and is regulated by circulating levels o angiotensin II, potassium, and, to a lesser extent, adrenocorticotropic hormone (ACTH). The zona asciculata and zona reticularis synthesize cortisol and adrenal androgens. ACTH released rom the anterior pituitary gland stimulates production o both cortisol and adrenal androgens. Tissue-specif c expression o enzymes in each zone o the adrenal cortex—aldosterone synthase in the glomerulosa, steroid 11 -hydroxylase and steroid 17 -hydroxylase in the asciculata/ reticularis—determines the specif city o hormone production in that zone.

on his history o prednis one us e, J ohnny is im m edia te ly g ive n hyd ro co rtis o n e (co rtis o l) in tra ve n o u s ly, as well as a salin e in u sio n. J o hnny recovers and or the next 6 m onths s lowly tapers his oral prednisone dose with continued use o the inhaled glucocorticoid. Eve ntu ally, h e is a ble to ta ke th e in ha led glu co co rtico id alone as an e ective therap y o r h is as th m a.

Questions 1 . Why are cortisol analogues such as prednisone used or treating asthma? 2 . Why did abrupt cessation o oral prednisone precipitate J ohnny’s clinical presentation in the emergency department? 3 . Why are inhaled glucocorticoids sa er than oral glucocorticoids or long-term treatment o asthma? 4 . Why did the doctor monitor J ohnny’s linear growth?

GLUCOCORTICOIDS Physiology Synthesis

Cortisol, the endogenous glucocorticoid, is synthesized rom

cholesterol. Its synthesis begins with the rate-limiting conversion o cholesterol to pregnenolone, a reaction catalyzed by side-chain cleavage enzyme (Fig. 29-2). This f rst step converts the 27-carbon cholesterol into a 21-carbon precursor common to all adrenocortical hormones. From this precursor, steroid metabolism can proceed down three di erent pathways to generate mineralocorticoids, glucocorticoids, or adrenal androgens. An oxidase enzyme catalyzes each step in the pathway o adrenocortical hormone synthesis. The oxidase enzymes are mitochondrial cytochromes , similar to the liver cytochrome P450 oxidase system. Tissue-specif c expression o particular oxidase enzymes in each zone o the adrenal cortex provides the biochemical basis or the di erences among the hormonal end products o the di erent zones o the cortex. For example, the zona asciculata synthesizes cortisol but not aldosterone or androgens (Fig. 29-1). This is because enzymes required uniquely or cortisol synthesis, such as steroid 11 -hydroxylase, are expressed in the zona asciculata, whereas enzymes required or aldosterone and androgen synthesis are not. The human zona glomerulosa does not express steroid 17 -hydroxylase, which is required or production o cortisol and androgens but is not required or production o aldosterone (Fig. 29-2).

Metabolism

Approximately 90% o circulating cortisol is bound to plasma proteins, the most important o which are corticosteroidbinding globulin (CBG, also re erred to as transcortin) and albumin. CBG has high a f nity or cortisol but low overall capacity, whereas albumin has low cortisol a f nity but high overall capacity. Only molecules o cortisol that are unbound to protein (the so-called ree raction) are bioavailable, that is, available to di use through plasma membranes

526 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Fe e dba c k inhibition

ACTH Ante rior pituita ry gla nd

Adre na l gla nd

Aminoglutethimide Ketoconazole (high)

Chole s te rol S CC

Trilostane

P re gne nolone

3

Ketoconazole

17 P roge s te rone

17-hydroxypre gne nolone 17

21 11-de oxyco r ticos te rone Metyrapone

3

17-hydroxyproge s te rone

11

Co r ticos te rone AS

20 Trilostane 20

De hydroe pia ndros te rone 3

Trilostane

Andros te ne dione

21 11-de oxyco r tis ol Metyrapone

11

Aldos te rone

Co r tis ol

Te s tos te rone

Mine ra loco r ticoids

Glucoco r ticoids

S e x s te roids

FIGURE 29-2. Hormone synthesis in the adrenal cortex. The hormones o the adrenal cortex are steroids derived rom cholesterol. The rate-limiting step in adrenal hormone biosynthesis is the modif cation o cholesterol to pregnenolone by side-chain cleavage enzyme. From this step, pregnenolone metabolism can be directed toward the ormation o aldosterone, cortisol, or androstenedione. The ux o metabolites through each o these pathways depends on the tissue-specif c expression o enzymes in the di erent cell types o the cortex and on the relative activity o the di erent synthetic enzymes. Note that several enzymes are involved in more than one pathway and that de ects in these enzymes can a ect the synthesis o more than one hormone. For example, a de ect in steroid 21-hydroxylase prevents the synthesis o both aldosterone and cortisol. This overlap o synthetic activities also contributes to the nonselective action o glucocorticoid synthesis inhibitors such as trilostane. Enzymes are shown as numbers or letters—SCC, side-chain cleavage enzyme; 3, 3 -hydroxysteroid dehydrogenase; 17, steroid 17 -hydroxylase; 20, 17, 20-lyase; 21, steroid 21-hydroxylase; 11, steroid 11 -hydroxylase; AS, aldosterone synthase. Aminoglutethimide and high levels o ketoconazole inhibit side-chain cleavage enzyme. Ketoconazole also inhibits 17, 20-lyase. Trilostane inhibits 3 -hydroxysteroid dehydrogenase. Metyrapone inhibits steroid 11 -hydroxylase.

into cells. Thus, the a f nity and capacity o plasma binding proteins regulate the availability o active hormone and, consequently, hormone activity. The liver and kidneys are the primary sites o peripheral cortisol metabolism. Through reduction and subsequent conjugation to glucuronic acid, the liver is responsible or inactivating cortisol in the plasma. The conjugation reaction makes cortisol more water soluble, thus enabling renal excretion. Importantly, the liver and kidneys express di erent iso orms o the enzyme 11 -hydroxysteroid dehydrogenase , a regulator o cortisol activity. The two iso orms catalyze opposing reactions. In distal collecting duct cells o the kidney, 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD 2) converts cortisol to the biologically inactive compound cortisone , which (unlike cortisol) does not bind to the mineralocorticoid receptor (see below; Fig. 29-3B). Expression o 11 -HSD 2 protects the mineralocorticoid receptor rom activation by cortisol in a variety o cell types, including endothelial cells and vascular smooth

muscle cells. In contrast, cortisone can be converted back to cortisol (also re erred to as hydrocortisone) in the liver by 11 -hydroxysteroid dehydrogenase type 1 (11 -HSD 1, Fig. 29-3A). The interplay between these opposing reactions determines overall glucocorticoid activity. In addition, as discussed below, the activity o these enzymes is important in glucocorticoid pharmacology.

Physiologic Actions

Like other steroid hormones, unbound cortisol di uses across the plasma membrane into the cytosol o target cells, where the hormone binds to a cytosolic receptor. There are two types o glucocorticoid receptors: the Type I (mineralocorticoid) and Type II glucocorticoid receptors . The Type I receptor is expressed in the organs o excretion (kidney, colon, salivary glands, sweat glands) and other tissues including the hippocampus, vasculature, heart, adipose tissue, and peripheral blood cells. The Type II receptor has a broader tissue distribution. The Type I glucocorticoid receptor is synonymous

C h a p t e r 2 9 Pharmacology of the Adrenal Cortex 527 A

OH

O OH

O H H

OH

OH

HO H

11β-HSD 1 (live r)

H

O

H

H

O

O Cortis one

B

Cortis ol

OH

O

HO H H

OH

OH

O

(kidne y)

O

OH

H

11β-HSD 2

H

O

H

H

O Cortis ol

Cortis one

(a gonis t a t mine ra locorticoid re ce ptor)

(ina ctive a t mine ra locorticoid re ce ptor)

FIGURE 29-3. 11 -Hydroxysteroid dehydrogenase. The enzyme 11 -hydroxysteroid dehydrogenase (11 -HSD) exists in two isoforms, which catalyze opposing reactions. A. In the liver, 11 -hydroxysteroid dehydrogenase type 1 (11 -HSD 1) converts 11-keto glucocorticoids such as cortisone to 11-hydroxy glucocorticoids such as cortisol. B. In vitro, cortisol is a potent agonist at the mineralocorticoid receptor (MR). In the kidney, however, MRs are “shielded” from cortisol by the action of the enzyme 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD 2), which converts cortisol to inactive cortisone. This mechanism ensures that, at physiologic levels, cortisol does not exert mineralocorticoid effects. At high concentrations, however, cortisol can overwhelm the capacity of 11 -HSD 2, leading to stimulation of renal MRs.

with the mineralocorticoid receptor. The nomenclature is un ortunate, and this chapter herea ter re ers to the Type I receptor as the “mineralocorticoid receptor.” Once cortisol binds to its cytosolic receptor and orms a hormone–receptor complex, the complex is transported into the nucleus. In the case o cortisol, a homodimerized hormone–receptor complex binds to gene promoter elements re erred to as glucocorticoid response elements (GREs) or negative GREs , which enhance or inhibit the expression o specif c genes, respectively. The glucocorticoid receptor also regulates transcription through direct and indirect interaction with co-activator molecules. Cortisol has pro ound e ects on mRNA expression; about 10% o all human genes are estimated to contain GREs. Because the expression o such a large number o genes is a ected by activation o GREs, cortisol has physiologic actions in most tissues. These actions can be divided generally into metabolic e ects and anti-in ammatory e ects. Metabolic effects of cortisol increase nutrient availability by raising blood glucose, amino acid, and triglyceride levels. Cortisol increases blood glucose by antagonizing insulin action and by promoting gluconeogenesis in the asting state. Cortisol also increases muscle protein catabolism, leading to release o amino acids that can be utilized by the liver as uels or gluconeogenesis. By potentiating growth hormone action on adipocytes, cortisol increases the activity o hormone-sensitive lipase and the subsequent release o ree atty acids (lipolysis). Free atty acids urther increase insulin resistance. Cortisol levels increase as a component o stress responses induced by a wide range o stimuli, such as vigorous exercise, psychological stress, acute trauma, surgery, ear, severe in ection, hypoglycemia, and pain. By

increasing blood glucose, the physiologic e ects o glucocorticoids maintain energy homeostasis during the stress response, thus ensuring that critical organs such as the brain continue to receive nutrients. Cortisol also has multiple anti-in ammatory actions. Cortisol negatively regulates cytokine release rom cells o the immune system by inhibiting nuclear actor B (NF- B); this action may be an important mechanism to limit the extent o immune responses and to regulate the in ammatory response. In turn, certain cytokines, including IL-1, IL-2, IL-6, and TNF- , can stimulate hypothalamic release o CRH, which stimulates ACTH and cortisol release. This series o stimulatory and inhibitory e ects creates a eedback loop in which in ammatory cytokines and cortisol are coordinately regulated to control immune and in ammatory responses (Fig. 29-4). Glucocorticoid-mediated suppression o the in ammatory response also has important pharmacologic implications or clinical conditions such as organ transplantation, rheumatoid arthritis, and asthma. Indeed, the introductory case demonstrates that glucocorticoids are an e ective therapy or asthma. The exact mechanisms by which glucocorticoids act to ameliorate the symptoms o asthma are unknown but are thought to be related to the ability o glucocorticoids to reduce in ammation in the airways (see below and Chapter 48, Integrative In ammation Pharmacology: Asthma).

Regulation

The hypothalamic-pituitary unit coordinates the production o cortisol (re er to Chapter 27 or an overview). In response to central circadian rhythms and to stress, neurons in the paraventricular nucleus o the hypothalamus synthesize and

528 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Hypotha la mus

The rmore gula tory ce nte rs

CRH

Immune s timulus

Fe ve r Ma cropha ge s

P ituita ry gla nd

Infla mma tory cytokine s (IL-1α, IL-1β, IL-6, TNF-α)

ACTH

Cortis ol Anti-infla mma tory fa ctors (a nne xin-1, IκB-α, MKP -1, GILZ, S LP I) Adre na l gla nd

Me dia tors of infla mma tion (e icos a noids , s e rotonin, PAF, bra dykinin)

FIGURE 29-4. The immune-adrenal axis. Cortisol has pro ound immunosuppressive e ects. Cortisol inhibits the action o several mediators o inf ammation (eicosanoids, serotonin, platelet activating actor [PAF], bradykinin) and stimulates anti-inf ammatory actors (annexin-1, inhibitor o nuclear actor B [I B- ], mitogen-activated kinase phosphatase-1 [MKP-1], glucocorticoid-induced leucine zipper protein [GILZ], secretory leukoprotease inhibitor [SLPI]). Cortisol also inhibits the release o a number o cytokines rom macrophages, including IL-1 , IL-1 , IL-6, and TNF- . Because these cytokines in turn promote the hypothalamic release o corticotropin-releasing hormone (CRH) and thereby increase serum cortisol levels, it is hypothesized that the stressinduced increase in cortisol limits the extent o the inf ammatory response.

secrete corticotropin-releasing hormone (CRH), a peptide hormone. CRH then travels through the hypothalamic-pituitary portal system and binds to G protein-coupled receptors on the sur ace o corticotroph cells in the anterior pituitary gland. CRH binding stimulates the corticotrophs to synthesize proopiomelanocortin (POMC), a precursor polypeptide that is cleaved into multiple peptide hormones including ACTH. Neurons in the paraventricular nucleus can also respond to stress by synthesizing and secreting arginine vasopressin. This vasopressin is released into the hypothalamic-pituitary portal system together with CRH, and it synergizes with CRH to increase the release o ACTH by the anterior pituitary gland. Interestingly, the stress-responsive parvocellular neurons that secrete CRH and vasopressin into the hypothalamic-pituitary portal system are di erent rom the osmolality-responsive magnocellular neurons that synthesize vasopressin and transport this hormone to the posterior pituitary gland (see Chapter 27), even though both types o neurons are located in the paraventricular nucleus o the hypothalamus. Proteolytic cleavage o POMC yields not only ACTH but also -melanocyte-stimulating hormone (MSH), lipotropin, and -endorphin. MSH binds to receptors on skin melanocytes, promoting melanogenesis and thereby increasing skin pigmentation. Because o the similarities between the ACTH and MSH peptide sequences, high concentrations o ACTH can also bind to and activate MSH receptors. This action becomes apparent in primary hypoadrenalism (see below), in which increased ACTH levels result in increased skin pigmentation. The role o lipotropin in human physiology is uncertain but is thought to involve control o lipolysis. -Endorphin is an endogenous opioid that is important or pain modulation and or regulation o reproductive physiology. Because steroid hormones are able to di use reely across cell membranes and because the adrenal gland stores little

cortisol, ACTH regulates cortisol production by promoting synthesis o the hormone. ACTH also has a trophic e ect on the zona asciculata and zona reticularis o the adrenal cortex, and hypertrophy o the cortex can occur in response to chronically elevated levels o ACTH. As in other endocrine axes, the hormone (cortisol) produced by the target organ (adrenal cortex) exerts negative eedback regulation at the level o both the hypothalamus and the anterior pituitary gland. High cortisol levels decrease both synthesis and release o CRH and ACTH. Because ACTH has important trophic e ects on the adrenal cortex, the absence o ACTH leads to atrophy o the cortisolproducing zona asciculata and the androgen-producing zona reticularis. However, the aldosterone-producing zona glomerulosa cells continue to unction in the absence o ACTH because angiotensin II and blood potassium continue to stimulate the production o aldosterone.

Pathophysiology Diseases a ecting glucocorticoid physiology can be divided into disorders o hormone def ciency and disorders o hormone excess. Addison’s disease is the classic example o adrenocortical insu f ciency, while Cushing’s syndrome exemplif es cortisol excess.

Adrenal Insu f ciency

Addison’s disease is an example o a primary adrenal insu -

f ciency in which the adrenal cortex is selectively destroyed, most commonly due to a T cell-mediated autoimmune reaction but alternatively due to in ection, inf ltration, cancer, or hemorrhage. Destruction o the cortex results in decreased synthesis o all classes o adrenocortical hormones. By comparison, secondary adrenal insu f ciency is caused by hypothalamic or pituitary disorders or by prolonged administration o exogenous glucocorticoids. In secondary

C h a p t e r 2 9 Pharmacology o the Adrenal Cortex 529

adrenal insu f ciency, the decrease in ACTH levels causes decreased synthesis o sex hormones and cortisol but does not alter aldosterone synthesis (see earlier discussion). Regardless o the underlying cause, adrenal insu f ciency has serious consequences and can be li e threatening i le t untreated in the setting o stress. Patients with adrenal insu f ciency requently experience atigue, loss o appetite, weight loss, dizziness on standing, and nausea. Hyperkalemia is common in primary adrenal insu f ciency due to the lack o aldosterone. I adrenal insu f ciency is the result o high-dose, prolonged therapy with exogenous glucocorticoids, then the dose o glucocorticoid should be tapered slowly to allow the hypothalamic-pituitary-adrenal (HPA) axis time to regain ull activity. Importantly, it can take up to 1 year or the HPA axis to recover unction a ter discontinuation o exogenous glucocorticoid treatment. In the introductory case, Johnny was switched rom an oral glucocorticoid to an inhaled glucocorticoid that delivered a much lower systemic concentration o glucocorticoid. His adrenal cortex had atrophied because he had been maintained or 2 years on chronically high doses o prednisone; there ore, he was unable to produce a su f cient amount o cortisol in response to the stress o a respiratory in ection. As a result, he arrived in the emergency department with acute adrenal insu f ciency and required intravenous therapy with saline and hydrocortisone.

Glucocorticoid Excess

Cushing’s syndrome re ers to a number o underlying patho-

physiologies, all o which increase cortisol production. The term Cushing’s disease is reserved or ACTH-secreting pituitary adenomas that lead to increased cortisol production (Fig. 27-5C). Other causes o Cushing’s syndrome include ectopic secretion o ACTH, most commonly by small cell carcinomas o the lung (Fig. 27-5D), and (rarely) ectopic CRH production. Cushing’s syndrome can also result rom cortisol-secreting tumors (adenoma or carcinoma) o the adrenal cortex (Fig. 27-5B). However, iatrogenic Cushing’s syndrome—secondary to pharmacologic treatment with exogenous glucocorticoids—is by ar the most common cause o Cushing’s syndrome. The clinical eatures o Cushing’s syndrome result rom chronic overstimulation o target organs by endogenous or exogenous glucocorticoids. These eatures—which can include centripetal adipose redistribution, hypertension, proximal limb myopathy, osteoporosis, immunosuppression, and diabetes mellitus—re ect amplif cation o the normal physiologic actions o glucocorticoids in a variety o target tissues. In cases o endogenous Cushing’s syndrome, cortisol-mediated activation o mineralocorticoid receptors leads to volume expansion, hypertension, and hypokalemia.

Pharmacologic Classes and Agents Cortisol and Glucocorticoid Analogues

Drug therapy with glucocorticoids is indicated or two main purposes. First, exogenous glucocorticoids can be used as replacement therapy in cases o adrenal insu f ciency. The goal o this therapy is to administer physiologic doses o glucocorticoids to ameliorate the e ects o the adrenal insu f ciency. Second, and more commonly, glucocorticoids are administered at pharmacologic doses to suppress in ammation and immune responses associated with disorders such as asthma, rheumatoid arthritis, and organ rejection a ter transplantation.

Because pharmacologic levels o systemic glucocorticoids invariably result in severe adverse e ects, strategies to minimize these untoward responses to glucocorticoids have ocused on local delivery o glucocorticoids to the area(s) requiring treatment. By limiting systemic exposure to the drug, HPA axis suppression and other eatures o iatrogenic Cushing’s syndrome can be minimized or avoided. Examples o local glucocorticoid delivery include inhaled glucocorticoids or asthma, topical glucocorticoids or inammatory skin conditions, and intra-articular glucocorticoids or arthritis. A large number o glucocorticoid analogues have been synthesized. The ollowing discussion highlights the di erences among some commonly used cortisol analogues— including prednisone , prednisolone , f udrocortisone , and dexamethasone —by comparing the structures, potencies, and durations o action o these compounds to those o cortisol. Structure and Potency

Glucocorticoids can be divided into two classes based on the structural moiety present at the 11-carbon position. Compounds with a hydroxyl ( OH) group at the 11-carbon position, such as cortisol, possess intrinsic glucocorticoid activity. In contrast, compounds with a carbonyl ( O) group at the 11 carbon, such as cortisone, are inactive. The liver enzyme 11 -HSD 1 must reduce the 11-carbonyl compound to its 11-hydroxyl congener in order or the compound to be active (Fig. 29-3). In other words, cortisone is an inactive prodrug until it is converted by the liver to the active drug cortisol. The native activity o a glucocorticoid is especially important or topically administered drugs because the skin does not possess appreciable amounts o 11 -HSD 1 (see below). Also, whenever possible, the active drug orm is preerred over the inactive prodrug orm or patients with liver dys unction because such patients may not be able to convert the prodrug to its active orm. The basic cortisol “backbone” is essential or glucocorticoid activity, and all synthetic glucocorticoids are analogues of the endogenous glucocorticoid cortisol (Fig. 29-5).

OH HO 1

9

H

O OH

16

2

F O

H

6

FIGURE 29-5. Synthetic modi cations to the cortisol backbone. Four modif cations to the cortisol backbone are common in synthetic glucocorticoids. Addition o a 1–2 double bond (far left box), a methyl group at carbon 6, or a methyl group at carbon 16 increases the glucocorticoid activity o the compound relative to that o cortisol. Addition o uorine to carbon 9 increases glucocorticoid activity and markedly increases mineralocorticoid activity; the mineralocorticoid e ect is blunted i 9- uorination is combined with 16-methylation. Simultaneous addition o the 1–2 double bond, methyl at carbon 16, and uorine at carbon 9 creates dexamethasone, which has very potent glucocorticoid activity and essentially no mineralocorticoid activity.

530 PRINCIPLES OF ENDOCRINE PHARMACOLOGY A

OH

O OH

HO 11

OH

OH

O

11

H

HO

OH

HO

11

H

H

O

OH

H

H H

H

H

O

H

O

O Cortis ol

Methylprednisolone

Prednisolone

OH

O OH

HO 11

OH

OH

HO 11

H

F

O

H

F

H

H

O

O

Fludrocortisone

Dexamethasone

B

OH O 11

H

O

OH

OH

O 11

H H

O

H

O OH

H H

O Cortis one

Prednisone

FIGURE 29-6. Glucocorticoid analogues. Panel A shows a number o 11-hydroxy glucocorticoids, while panel B shows two 11-keto congeners. Note that the drugs in panel A are physiologically active, while the drugs in panel B are prodrugs that must be activated by 11 -HSD 1 to become active compounds. The structural class to which a glucocorticoid analogue belongs can be an important consideration in therapeutic decision making. For example, because the skin lacks signif cant 11 -HSD 1 activity, only 11-hydroxy glucocorticoids can be used in topical glucocorticoid creams. HSD, hydroxysteroid dehydrogenase.

For example, addition o a double bond between carbons 1 and 2 o cortisol creates prednisolone (Fig. 29-6), which has 4–5 times the anti-in ammatory potency o cortisol. Further addition o an -methyl group (where is def ned as the side-group orientation axial to the compound, while is the equatorial orientation) to carbon 6 o prednisolone creates methylprednisolone , which has an anti-in ammatory potency 5–6 times that o cortisol. Although prednisolone and methylprednisolone have signif cantly higher glucocorticoid potency than cortisol, their potency at the mineralocorticoid receptor is lower than that o cortisol. In contrast, addition o an - uorine (F) to carbon 9 o cortisol increases both the glucocorticoid and mineralocorticoid potencies o the resulting compound, known as f udrocortisone (Fig. 29-6). Because o its remarkably enhanced mineralocorticoid activity, udrocortisone is use ul in the treatment o mineralocorticoid def ciency states (see below). Dexamethasone incorporates two o the above changes to the cortisol backbone (1–2 double bond, 9 - uorine) as well as the addition o an -methyl group at the 16-carbon position (Fig. 29-6). This compound has more than 18 times the glucocorticoid potency o cortisol but virtually no mineralocorticoid activity.

A number o other permutations have been made to the cortisol backbone in other synthetic glucocorticoids, but the earlier discussion highlights the pertinent structural di erences among the most common synthetic glucocorticoids. Clinically, it is most important to be aware of the potency of each agent relative to cortisol, especially when considering a change from one analogue to another that has different relative glucocorticoid and mineralocorticoid activities. In general, glucocorticoids used at pharmacologic doses should have minimal mineralocorticoid activity to avoid the consequences o mineralocorticoid excess (i.e., hypokalemia, volume expansion, and hypertension). Table 29-1 summarizes the relative glucocorticoid potencies and mineralocorticoid activities o several common glucocorticoid analogues. Duration o Action

The duration o glucocorticoid action is a complex pharmacokinetic variable that depends on: 1. Fraction o the drug bound to plasma proteins. More than 90% o circulating cortisol is protein-bound, primarily to CBG and, to a lesser degree, to albumin. In contrast, glucocorticoid analogues generally bind to CBG with low a f nity. As a result, approximately

C h a p t e r 2 9 Pharmacology of the Adrenal Cortex 531

TABLE 29-1 Relative Potencies and Durations of Action of Representative Glucocorticoid Analogues PHARMACOLOGIC AGENT

RELATIVE GLUCOCORTICOID POTENCY

Hydrocortisone (cortisol)

RELATIVE MINERALOCORTICOID ACTIVITY

DURATION OF ACTION

1

1

Short

Prednisolone

4–5

0.25

Short

Methylprednisolone

5–6

0.25

Short

Dexamethasone

18

Short-acting agents have a tissue half-life of

12 hours, and long-acting agents have a half-life of

two-thirds o a typical glucocorticoid analogue circulates in the plasma bound to albumin, while the rest is present as ree steroid. Because only the ree steroid is metabolized, the extent o binding to plasma proteins is a determinant o the drug’s duration o action. 2. A f nity o the drug or 11 -HSD 2. Glucocorticoids that have a lower a f nity or 11 -HSD 2 have a longer plasma hal -li e because such drugs are not trans ormed into inactive metabolites as rapidly. 3. Lipophilicity o the drug. Increased lipophilicity promotes partitioning o the drug into adipose stores; the resulting decrease in the drug’s metabolism and excretion extends its plasma hal -li e. 4. A f nity o the drug or the glucocorticoid receptor. Increased a f nity o a glucocorticoid analogue or the glucocorticoid receptor increases the duration o action o the drug, because drug bound to the receptor continues to exert its e ect until the drug–receptor complex dissociates. Together, these our variables result in a characteristic duration o action prof le or each glucocorticoid analogue. Table 29-1 summarizes the duration o action o representative analogues as “short” or “long.” In general, glucocorticoid agents with higher anti-inf ammatory (glucocorticoid) potency have a longer duration o action. Replacement Therapy

Treatment o primary adrenal insu f ciency is aimed at physiologically replacing both glucocorticoids and mineralocorticoids. Oral hydrocortisone is the glucocorticoid o choice. Because glucocorticoid replacement therapy must continue or li e, the therapeutic goal is to administer the smallest possible e ective dose o cortisol so as to minimize the adverse e ects o chronic glucocorticoid excess. Patients with primary adrenal insu f ciency also require mineralocorticoid replacement, as described below. Patients with secondary adrenal insu f ciency require only glucocorticoid replacement because mineralocorticoid production is preserved by the renin-angiotensin system (see Chapter 21, Pharmacology o Volume Regulation). Pharmacologic Dosing

Glucocorticoids are important mediators o the stress response, regulating both glucose homeostasis and the immune system. Glucocorticoids have ound wide clinical use as anti-in ammatory agents because o their pro ound e ects on immune and in ammatory processes. Pharmacologic levels o glucocorticoids inhibit

Effects at Pharmacologic Levels.

0.01

Long

48 hours.

cytokine release and thereby decrease IL-1, IL-2, IL-6, and

TNF- action (Fig. 29-4). Local regulation o cytokine release is crucial or leukocyte recruitment and activation, and disruption o this signaling process pro oundly inhibits immune unction. Glucocorticoids also block the synthesis o arachidonic acid metabolites by inhibiting the action o phospholipase A2. As discussed in Chapter 43, Pharmacology o Eicosanoids, arachidonic acid metabolites such as thromboxanes, prostaglandins, and leukotrienes mediate many o the early steps o in ammation, including vascular permeability, platelet aggregation, and vasoconstriction. By blocking the production o these metabolites, glucocorticoids signif cantly down-regulate the in ammatory response. The multiple e ects described above make glucocorticoids use ul drugs in the treatment o many in ammatory and autoimmune diseases, such as asthma, rheumatoid arthritis, Crohn’s disease, polyarteritis nodosa, temporal arteritis, and immune rejection a ter organ transplantation. It is important to note, however, that pharmacologic glucocorticoid therapy does not correct the underlying disease etiology but only limits the e ects o inf ammation. Thus, discontinuing chronic glucocorticoid therapy o ten results in the resumption o in ammatory symptoms, unless the disorder has gone into spontaneous remission or been treated by other means. Endogenous glucocorticoids a ect many metabolic processes, and pharmacologic dosing with exogenous glucocorticoids amplif es those actions. Because o this, adverse e ects typically accompany prolonged pharmacologic dosing with glucocorticoids. Increased susceptibility to inection is a potential adverse e ect o long-term immunosuppression by exogenous glucocorticoids. Glucocorticoids raise plasma glucose levels, as described above, and pharmacologic doses o glucocorticoids ampli y these e ects. Insulin resistance and increased plasma glucose concentrations necessitate increased pancreatic -cell production o insulin to normalize blood glucose levels. As a result, diabetes mellitus is a common complication o long-term glucocorticoid administration, especially in patients with decreased pancreatic -cell reserve. Pharmacologic dosing o glucocorticoids inhibits the vitamin D-mediated absorption o calcium. This results in secondary hyperparathyroidism and, there ore, increases bone resorption. Glucocorticoids also directly suppress osteoblast and osteocyte unction. These mechanisms contribute to bone loss, and long-term glucocorticoid therapy o ten results in osteoporosis. Steroid-induced bone resorption can be prevented with bisphosphonates, which

532 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

inhibit osteoclast unction and thus slow the progression o bone loss (see Chapter 32, Pharmacology o Bone Mineral Homeostasis). Chronic administration o glucocorticoids also slows linear bone growth in children, and glucocorticoid administration can cause growth retardation. Short stature can be the result in children who take glucocorticoids throughout adolescence. For this reason, Johnny’s physician monitored his growth closely while he was treated with oral prednisone. Pharmacologic doses o glucocorticoids can cause selective atrophy o ast-twitch muscle f bers, resulting in catabolism and weakness o (primarily) the proximal muscles. Glucocorticoids also cause a characteristic redistribution o at, with peripheral wasting o adipose stores and central obesity. Excessive at deposition also occurs on the back o the neck (bu alo hump) and ace (moon acies). In considering the potential or adverse e ects o glucocorticoids, it is important to understand the concept o a population at risk. Not all individuals treated with glucocorticoids develop the same adverse e ects because genetic and environmental variability place di erent individuals at risk or di erent sequelae o therapy. For example, a patient with borderline diabetes who is treated with glucocorticoids is likely to develop overt diabetes, whereas a patient with su f cient pancreatic -cell reserve may not experience this adverse e ect. By care ully def ning a patient’s risk actors, it is o ten possible to predict the patient’s predisposition or adverse e ects o glucocorticoids. Problems can be associated with the discontinuation o chronic glucocorticoid therapy. During long-term therapy with pharmacologic doses o glucocorticoids, high plasma glucocorticoid levels suppress the release o CRH and ACTH, resulting in atrophy o the adrenal cortex. Abrupt cessation o glucocorticoid therapy can precipitate acute adrenal insu ciency because a substantial period o time (months) is required to reactivate the hypothalamic-pituitary-adrenal axis. Even a ter ACTH secretion recovers, additional months may be required or the adrenal cortex to begin secreting physiologic levels o cortisol. Thus, it is axiomatic that chronic glucocorticoid treatment should, whenever possible, be tapered slowly with gradually decreasing doses. This taper allows the hypothalamus, anterior pituitary gland, and adrenal cortex to resume normal unction gradually, thus avoiding adrenal insu f ciency and, it is hoped, avoiding exacerbation o the underlying in ammatory condition.

Withdrawal from Glucocorticoid Treatment.

Routes o Administration

Several di erent drug delivery methods allow selective targeting o glucocorticoid to a particular tissue. The relevant concept is that glucocorticoids can be administered locally at many times the normal plasma concentration while minimizing systemic adverse e ects. Examples o these methods include inhaled, topical, and depot preparations o glucocorticoids. The administration o glucocorticoids during pregnancy is also an example o selective targeting because the placenta can metabolically partition glucocorticoids between mother and etus. Inhaled glucocorticoids are the ormulation o choice in the chronic treatment o asthma. Glucocorticoids reduce asthma symptoms by inhibiting

Inhaled Glucocorticoids.

airway in ammatory responses, especially eosinophilmediated in ammation. The exact mechanism(s) is unknown but is thought to involve inhibition o cytokine release and subsequent inhibition o the in ammatory cascade (see Chapter 48). Because systemic therapy with glucocorticoids can lead to serious adverse e ects, e orts have been made to develop inhaled glucocorticoids with low oral bioavailability, thereby allowing high-dose delivery directly to the airway mucosa while minimizing systemic dosing. The goal o inhaled glucocorticoid therapy is to maximize the local-to-systemic ratio o glucocorticoid concentration. Because the inhaled glucocorticoid is delivered directly to the in amed organ, rather than via the systemic circulation, less inhaled glucocorticoid than oral glucocorticoid is required to control airway in ammation. The inhaled route o administration makes glucocorticoids sa er or long-term dosing, especially in children. Microcrystalline powders and metered-dose inhalers o potent glucocorticoids such as beclomethasone , ciclesonide , f unisolide , f uticasone , mometasone , and triamcinolone (Fig. 29-7) are currently available as inhaled ormulations. I a patient treated chronically with systemic glucocorticoids is switched to inhaled glucocorticoids, care must be taken not to stop the systemic dosing abruptly. In the introductory case, acute adrenal insu f ciency occurred because Johnny was switched acutely rom oral prednisone to an inhaled glucocorticoid. On average, inhaled preparations deliver approximately 20% o the dose to the lung, while the other 80% is swallowed. However, the glucocorticoids available as inhaled ormulations (see above) have signif cant f rst-pass hepatic metabolism, and the swallowed portion is converted to inactive metabolites by the liver. For example, less than 1% o swallowed uticasone is systemically bioavailable. Thus, Johnny’s abrupt switch rom an orally available glucocorticoid to an inhaled ormulation caused acute adrenal insu f ciency. Acute adrenal insu f ciency can be li e threatening and should be treated immediately with a large dose o intravenous glucocorticoid; or this reason, Johnny was given an intravenous in usion o hydrocortisone. Eventually, a ter Johnny was placed back on oral prednisone, he was able to taper the dose slowly and, once his hypothalamic-pituitary-adrenal axis had been reactivated, to use the inhaled glucocorticoid alone. Oropharyngeal candidiasis is a potential local complication o inhaled glucocorticoid therapy, because some glucocorticoid is delivered directly to the oral and pharyngeal mucosa. This results in local immunosuppression and permits in ection with opportunistic organisms. Oropharyngeal candidiasis can be avoided by rinsing the mouth with water a ter each administration o aerosolized glucocorticoid or by using anti ungal mouthwash. Intranasal administration o a glucocorticoid analogue is an e ective therapy or allergic rhinitis. Glucocorticoids pro oundly suppress the eosinophilic response and are o ten superior to antihistamines in the treatment o this disorder. Topical preparations o glucocorticoids are available or multiple dermatologic disorders, including psoriasis, lichen planus, and atopic dermatitis. Cutaneous administration delivers an extremely low percentage o the glucocorticoid systemically, allowing topical dosing at many- old higher local concentrations than could be achieved sa ely with systemic administration. The

Cutaneous Glucocorticoids.

C h a p t e r 2 9 Pharmacology o the Adrenal Cortex 533

O O

O O

HO H Cl

O

O

O

H

O

HO

O

S H

Beclomethasone dipropionate

OH

F O O

H

H

O F

HO O

H

F

Fluticasone propionate

H

O

CH2 Cl Budesonide

O

O HO

O

O O

O O HO

O

Cl

O

H

Mometasone furoate

O

O

OH

Ciclesonide

H O O

F

HO

H

OH

HO

OH

O

H

O

OH H

O Triamcinolone

H

O F Flunisolide

FIGURE 29-7. Structures of common inhaled glucocorticoids. Most o the inhaled glucocorticoids are halogenated analogues o cortisol that are highly potent glucocorticoid agonists with little mineralocorticoid activity (halogen atoms are shown in blue). Their high potency allows low doses o the inhaled glucocorticoids to inhibit the local in ammatory response that is a critical component o asthma pathophysiology. In addition, because several o these compounds are subject to almost complete f rst-pass metabolism in the liver, the raction o inhaled glucocorticoid that is inadvertently swallowed (80% o the inhaled dose) becomes inactivated so that it is not systemically bioavailable. The raction o inhaled glucocorticoid delivered to the lung is eventually absorbed into the systemic circulation.

glucocorticoid that is administered must be biologically active because the skin has little, i any, o the 11 -HSD 1 enzyme needed to convert glucocorticoid prodrugs to active compounds. Hydrocortisone, methylprednisolone, and dexamethasone are e ective steroids or cutaneous use. Depot intramuscular preparations o glucocorticoid analogues last or days to weeks and can be

Depot Glucocorticoids.

an alternative to daily or alternate-day oral glucocorticoids in the treatment o inf ammatory diseases. Although depot ormulations reduce the necessity or daily oral administration, these preparations are seldom used because the dose cannot be titrated on a requent basis. Depot preparations o methylprednisolone suspended in polyethylene glycol are commonly used, however, or intra-articular administration. This approach can be indicated or inf ammatory processes

534 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

restricted to the joints, such as rheumatoid arthritis or gout. Intra-articular glucocorticoid injection is use ul in acute attacks o gout that are unresponsive to colchicine or indomethacin. Intra-articular and bursa injections require the use o active glucocorticoid, because joint tissue lacks 11 -HSD 1. Use in Pregnancy

The placental–maternal barrier provides another example o selective glucocorticoid targeting. During pregnancy, the placenta metabolically separates the etus rom the mother. Because o this, prednisone can be administered to the mother during pregnancy without etal side e ects. The maternal liver activates the prednisone to prednisolone, but placental 11 -HSD 2 converts the prednisolone back to inactive prednisone. Because the liver does not unction during etal li e, the etus does not, in turn, activate prednisone. Therefore, use of prednisone in pregnancy does not result in delivery of an active glucocorticoid to the fetus. Glucocorticoids promote lung development in the etus. I glucocorticoid therapy is indicated to promote etal lung maturation, dexamethasone is commonly administered to the mother. Dexamethasone is a poor substrate or placental 11 -HSD 2 and there ore crosses the placenta in active orm rom the maternal circulation to the etal circulation, where it stimulates lung maturation. The dose o dexamethasone must be titrated care ully because exposure to excessive glucocorticoid can have deleterious e ects on etal development.

Inhibitors of Adrenocortical Hormone Synthesis

Several compounds are available to inhibit hormone biosynthesis by the adrenal cortex. Although these drugs have some specif city or individual adrenal enzymes (Table 29-2), it is not generally possible to alter the production o a single adrenal hormone independent o other hormones. The enzymes necessary or adrenal hormone synthesis are P450 enzymes, and use o these inhibitors is also associated with potential toxicity to hepatic P450 enzymes. In general, these agents can be divided into drugs that a ect earlier versus later steps in adrenal hormone synthesis. The agents that inhibit early TABLE 29-2 Sites of Action and Pathways Affected by Inhibitors of Adrenal Hormone Synthesis

INHIBITOR

SITE OF ACTION

ADRENAL STEROIDOGENIC PATHWAYS AFFECTED

Mitotane

Mitochondria

All

Aminoglutethimide

Side-chain cleavage enzyme

All (aromatase also inhibited in ovary)

Ketoconazole

Primarily 17, 20-lyase

Low concentrations: ↓ Androgen synthesis High concentrations: ↓ Synthesis of all adrenal and gonadal steroid hormones

Metyrapone

Steroid 11 -hydroxylase

Cortisol and aldosterone synthesis

Trilostane

3 -Hydroxysteroid dehydrogenase

All (primarily cortisol and aldosterone synthesis)

steps have broad e ects, while those a ecting later steps have more selective actions. Mitotane is a structural analogue o DDT (a potent insecticide) that is toxic to adrenocortical mitochondria and thus has broad e ects on adrenal steroidogenesis. Although used in requently, mitotane may be indicated or medical adrenalectomy in cases o severe Cushing’s disease or adrenocortical carcinoma. Patients taking mitotane commonly develop hypercholesterolemia because o the drug’s concomitant inhibition o cholesterol oxidase. Aminoglutethimide inhibits side-chain cleavage enzyme (Fig. 29-2). Aminoglutethimide also inhibits the enzyme aromatase, which is important or conversion o androgens to estrogens. Consistent with its ability to inhibit aromatase, aminoglutethimide has been ound to be e ective as a therapy or breast cancer. However, it is not used or this purpose because o the availability o more selective aromatase-inhibiting drugs (see Chapter 30, Pharmacology o Reproduction). Ketoconazole is an anti ungal agent that acts by inhibiting the ungal P450 enzymes (see Chapter 36, Pharmacology o Fungal In ections). The enzymes that mediate adrenal and gonadal hormone synthesis are also members o the P450 enzyme amily, and high doses o ketoconazole also suppress steroid synthesis in these organs. This agent inhibits primarily 17, 20-lyase, which is important or adrenal androgen synthesis. High doses o ketoconazole also inhibit side-chain cleavage enzyme, the enzyme that converts cholesterol to pregnenolone. Because pregnenolone generation is required or the synthesis o all adrenal hormones, highdose ketoconazole has broadly inhibitory e ects on adrenocortical hormone synthesis. Metyrapone and trilostane have more selective e ects on adrenal hormone synthesis. Metyrapone inhibits steroid 11 -hydroxylase, resulting in impaired cortisol and aldosterone synthesis (Fig. 29-2). Metyrapone has been used as a diagnostic drug to test the hypothalamic and pituitary response to decreased circulating cortisol levels. Trilostane is a reversible inhibitor o 3 -hydroxysteroid dehydrogenase. This agent reduces cortisol production in the adrenal cortex and is used to treat Cushing’s disease in dogs. Trilostane is not approved or use in humans.

Glucocorticoid Receptor Antagonists

Mifepristone (RU-486) is a progesterone receptor antagonist

used to induce abortion early in pregnancy (see Chapter 30). At higher concentrations, mi epristone also blocks the glucocorticoid receptor. Mi epristone has been approved or treatment o hyperglycemia in Cushing’s syndrome. Mi epristone potentially could be use ul or the treatment o li ethreatening elevated glucocorticoid levels, such as in ectopic ACTH syndrome, although its clinical use ulness or this purpose has not been evaluated ully.

MINERALOCORTICOIDS Physiology Synthesis

Like cortisol, aldosterone is a 21-carbon steroid hormone derived rom cholesterol. Enzymes unique to aldosterone synthesis are expressed only in the zona glomerulosa. Aldosterone secretion is stimulated by angiotensin II, blood potassium concentration, and ACTH (Fig. 29-1).

C h a p t e r 2 9 Pharmacology of the Adrenal Cortex 535

Metabolism

Circulating aldosterone binds with low a f nity to transcortin, albumin, and a specif c aldosterone binding protein. Only 50% to 60% o circulating aldosterone is bound to transport proteins, and aldosterone has a short elimination hal -li e (20 minutes). Orally administered aldosterone also has high f rst-pass hepatic metabolism; approximately 75% o the hormone is metabolized to an inactive orm during each pass through the liver. As a result, orally administered aldosterone is not an e ective replacement therapy or adrenal insu f ciency states.

Physiologic Actions

Mineralocorticoids play important roles in regulating sodium reabsorption in the kidney, the colon, and sweat and salivary glands. Circulating aldosterone di uses across the plasma membrane and binds to a cytosolic mineralocorticoid receptor (synonymous with the Type I glucocorticoid receptor). The aldosterone:mineralocorticoid receptor complex is then transported into the nucleus, where it increases or decreases transcription o specif c genes through interactions with transcriptional complexes and binding to hormone-responsive-element DNA-binding domains on specif c gene promoters. In addition to these transcriptional e ects, aldosterone has rapid e ects on intracellular signaling pathways. These nongenomic actions appear to be mediated by hormone binding to mineralocorticoid receptors located on the cell sur ace. The physiologic and pathophysiologic roles o this second signaling mechanism are an active area o investigation. In the kidney, aldosterone increases transcription o serum and glucocorticoid-induced kinase 1 (SGK1). SGK1 increases Na /K ATPase activity in the basolateral membrane and induces the apical Na channel o distal nephron cells, resulting in increased sodium reabsorption and potassium secretion across the luminal epithelium o the nephron (see Chapter 21). As a result, sodium retention, potassium excretion, and H excretion are all enhanced by aldosterone. Increased sodium retention is accompanied by increased water retention and, thus, extracellular volume expansion. Excess aldosterone can cause hypokalemic alkalosis and hypertension, while hypoaldosteronism can cause hyperkalemic acidosis and hypotension. The mineralocorticoid receptor is also expressed in cells not involved in sodium reabsorption, including endothelial cells, vascular smooth muscle cells, cardiomyocytes, adipocytes, neurons, and in ammatory cells. Preclinical studies demonstrate a role or the mineralocorticoid receptor in the pathophysiology o vascular injury, atherosclerosis, heart disease, renal disease, and stroke. Activation o the mineralocorticoid receptor increases oxidative stress, promotes inammation, regulates adipocyte di erentiation, and reduces insulin sensitivity. In humans, antagonists o aldosterone action at the mineralocorticoid receptor, such as spironolactone and eplerenone, reduce morbidity and mortality in heart ailure, improve vascular unction, reduce cardiac hypertrophy, and reduce albuminuria. These benef cial e ects o mineralocorticoid receptor blockade appear to be independent o changes in blood pressure.

Regulation

Three systems regulate aldosterone synthesis: the reninangiotensin system, blood potassium levels, and ACTH.

The renin-angiotensin-aldosterone system is a central regulator o extracellular uid volume. Decreases in extracellular uid volume decrease per usion pressure at the a erent arteriole o the renal glomerulus, which acts as a baroreceptor. This stimulates the juxtaglomerular cells to secrete renin, a protease that cleaves the prohormone angiotensinogen to angiotensin I. Angiotensin I is then converted to angiotensin II by angiotensin converting enzyme, which is expressed at high concentrations by the capillary endothelium o the lungs. Angiotensin II has direct arteriolar pressor e ects, and it stimulates aldosterone synthesis by binding to and activating a G protein-coupled receptor in zona glomerulosa cells o the adrenal cortex. In a eedback loop, angiotensin II also inhibits renin secretion. Potassium loading increases aldosterone synthesis independent o renin activity. Because aldosterone activity at the distal nephron promotes potassium excretion, this control mechanism serves a homeostatic role in regulating potassium balance. Finally, ACTH acutely stimulates aldosterone synthesis in the zona glomerulosa. Changes in ACTH levels contribute to the circadian regulation o aldosterone and to the aldosterone increases associated with acute stress, such as hypoglycemia. Unlike cortisol, aldosterone does not negatively regulate ACTH secretion.

Pathophysiology Aldosterone Hypofunction

Aldosterone hypo unction (hypoaldosteronism) can result rom a primary decrease in aldosterone synthesis or action or rom a secondary decrease in aldosterone regulators such as angiotensin II. Most cases o hypoaldosteronism result rom decreased aldosterone synthesis. De ects in the gene coding or steroid 21-hydroxylase, an enzyme necessary or both aldosterone and glucocorticoid synthesis, lead to congenital adrenal hyperplasia (discussed below under adrenal androgen pathophysiology) and cause salt wasting as a result o aldosterone def ciency. Addison’s disease , or primary adrenal insu f ciency, results in hypoaldosteronism secondary to destruction o the zona glomerulosa. Most cases o Addison’s disease are caused by autoimmune adrenalitis; other causes o adrenal cortex destruction include tuberculosis, metastatic cancer, and hemorrhage. In each case, aldosterone hypo unction can lead to salt wasting, volume depletion, hyperkalemia, and acidosis. Hypoaldosteronism can also result rom states o decreased renin production (so-called hyporeninemic hypoaldosteronism, which is common in diabetic renal insu f ciency). Both resistance to the action o aldosterone at the level o the mineralocorticoid receptor and inactivating mutations o the aldosterone-regulated epithelial sodium channel in the cortical collecting duct o the nephron result in clinical hypoaldosteronism, despite normal to elevated aldosterone levels in the blood.

Aldosterone Hyperfunction

Primary hyperaldosteronism results rom excess aldoste-

rone production by the adrenal cortex. Bilateral zona glomerulosa adrenal hyperplasia and aldosterone-producing adenomas are the two most common causes. Increased aldosterone synthesis leads to positive sodium balance, with consequent extracellular volume expansion, suppression

536 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

o plasma renin activity, potassium wasting and hypokalemia, and hypertension. Independent o its e ect on blood pressure, primary hyperaldosteronism also has adverse cardiovascular e ects, including endothelial dys unction, increased intima-media thickness, vascular sti ness, and increased le t ventricular wall thickness. Primary hyperaldosteronism is also a cause o insulin resistance. The prevalence o primary hyperaldosteronism is much higher than initially thought, occurring in 5% to 10% o all patients with hypertension and up to 25% o patients with resistant hypertension.

Pharmacologic Classes and Agents Mineralocorticoid Receptor Agonists

Pathophysiologic conditions leading to hypoaldosteronism necessitate replacement with physiologic doses o a mineralocorticoid. It is not possible to administer aldosterone itsel as a therapeutic agent, because the liver converts more than 75% o oral aldosterone to an inactive metabolite during f rst-pass metabolism. Instead, the cortisol analogue f udrocortisone , which has minimal f rstpass hepatic metabolism and a high mineralocorticoid-toglucocorticoid potency ratio, is used. The adverse e ects o udrocortisone therapy are all related to the ability o this agent to mimic a state o mineralocorticoid excess, including hypertension, hypokalemia, and even heart ailure. To ensure that an appropriate dose o drug is being administered, it is important to monitor serum potassium and blood pressure levels closely in all patients receiving udrocortisone.

Mineralocorticoid Receptor Antagonists

Spironolactone (also discussed in Chapters 21 and 30) is a

competitive antagonist at the mineralocorticoid receptor, but the drug also binds to and inhibits the androgen and progesterone receptors to a lesser extent. The latter actions, which result in adverse e ects such as gynecomastia in males, limit the use ulness o this agent in some patient subsets. Eplerenone is a mineralocorticoid receptor antagonist that binds selectively to the mineralocorticoid receptor, thereby reducing the incidence o gynecomastia relative to spironolactone. Both spironolactone and eplerenone can be used as antihypertensive agents, and both are approved or use in patients with heart ailure. Antagonism o the mineralocorticoid receptor can result in signif cant hyperkalemia. Because many patients with heart ailure are prescribed both spironolactone or eplerenone and an angiotensin converting enzyme inhibitor (which also raises blood potassium levels), it is important to monitor potassium levels closely in these patients.

ADRENAL ANDROGENS Physiology Sex steroids produced by the adrenal cortex, primarily dehydroepiandrosterone (DHEA), have an uncertain role in human physiology. DHEA seems to be a prohormone that is converted to more potent androgens, primarily testosterone, in the periphery. Adrenocortical androgens are an important source o testosterone in emales; these hormones are necessary or the development o emale axillary and pubic hair

at the time o puberty, when adrenal androgen secretion is activated (adrenarche).

Pathophysiology Congenital adrenal hyperplasia (CAH) and polycystic ovarian syndrome are two important diseases related to adrenocorti-

cal androgen production. Congenital adrenal hyperplasia is a clinical term related to several di erent inherited enzyme def ciencies in the adrenal cortex. Enzyme de ects leading to increased adrenocortical androgen production cause hirsutism and virilization in emales. Polycystic ovarian syndrome, discussed in Chapter 30, may be caused by congenital adrenal hyperplasia in a small subset o patients. The most common orm o congenital adrenal hyperplasia results rom a def ciency o steroid 21-hydroxylase . Def ciency o 21-hydroxylase results in the inability o adrenocortical cells to synthesize both aldosterone and cortisol (Fig. 29-8). Because cortisol is the main negative eedback regulator o pituitary ACTH release, the decreased cortisol synthesis that results rom 21-hydroxylase def ciency disinhibits ACTH release. Increased ACTH restores the cortisol level in patients with partial enzyme de ects, but it also induces shunting o precursor compounds into the “unblocked” androgen pathway, resulting in greater production o DHEA and androstenedione. The liver subsequently converts these compounds into testosterone. In severe 21-hydroxylase def ciency, there may be a virilizing e ect on the developing emale etus. As a result, emale neonates with severe 21-hydroxylase def ciency typically have masculinized or ambiguous external genitalia. In the male neonate, however, increased adrenal androgens may have little or no noticeable phenotypic e ect. In ants with severe 21-hydroxylase def ciency are commonly diagnosed in in ancy during an acute salt-wasting crisis, which results rom the inability to synthesize aldosterone and cortisol. Mild 21-hydroxylase def ciency may mani est later in li e as hirsutism, acne, and oligomenorrhea in young women a ter menarche. Treatment o congenital adrenal hyperplasia due to severe enzyme de ects requires physiologic replacement doses o glucocorticoids and mineralocorticoids. Treatment o congenital adrenal hyperplasia in patients with mild enzyme de ects may include therapy with exogenous glucocorticoid to suppress excessive hypothalamic and pituitary release o CRH and ACTH, thus decreasing the production o adrenal androgens.

Pharmacologic Classes and Agents Androgens synthesized by the adrenal gland can be viewed as prohormones. Because no specif c receptors or either DHEA or androstenedione have been described, the activity o these hormones depends on their conversion to testosterone, and subsequently to dihydrotestosterone, in peripheral target tissues. As discussed above, adrenal androgen excess can cause a variety o syndromes in women; the pharmacologic interruption o excessive androgenic activity is discussed in Chapter 30. DHEA is not regulated by the US Food and Drug Administration (FDA) and is commonly used as an over-thecounter drug. Population cross-sectional studies have shown a reciprocal relationship between an age-related decline in DHEA levels and the risk o cardiovascular disease and

C h a p t e r 2 9 Pharmacology o the Adrenal Cortex 537

Fe e dba ck inhibition

ACTH Ante rior pituita ry gla nd

Hype rpla s tic a dre na l corte x

Chole s te rol

P re gne nolone

P roge s te rone

17-hydroxypre gne nolone

De hydroe pia ndros te rone

17-hydroxyproge s te rone

Andros te ne dione

21 11-de oxycorticos te rone

21 Corticos te rone

11-de oxycortis ol

Aldos te rone

Cortis ol

Mine ra locorticoids

Glucocorticoids

Te s tos te rone S e x s te roids

FIGURE 29-8. Congenital adrenal hyperplasia. Steroid 21-hydroxylase def ciency, the most common cause o congenital adrenal hyperplasia, results in impaired biosynthesis o aldosterone and cortisol (dashed lines). There ore, steroid hormone synthesis in the adrenal cortex is shunted toward increased production o sex steroids (thick lines). The lack o cortisol production decreases the negative eedback on corticotroph cells o the anterior pituitary gland (dashed line), causing increased ACTH release (thick blue arrow). Increased levels o ACTH induce adrenal hyperplasia and urther stimulate the synthesis o sex steroids. This pathway can be interrupted by administering exogenous cortisol. The def cient enzyme is shown as a number: 21, steroid 21-hydroxylase.

cancer. Replacement therapy with DHEA may be indicated or cases o Addison’s disease in which there is bona f de DHEA def ciency. Exogenous DHEA can be converted to testosterone by the liver. As a result, DHEA is commonly abused or its anabolic e ects.

CONCLUSION AND FUTURE DIRECTIONS Aldosterone, cortisol, and adrenal androgens regulate many aspects o basic homeostasis. Aldosterone regulates extracellular uid volume by promoting sodium reabsorption and uid retention. Cortisol regulates diverse physiologic processes, including energy homeostasis and in ammatory responses. The physiologic role o adrenal androgens is unknown, but pathophysiologic states causing increased adrenal androgen production have signif cant masculinizing e ects in women. Antagonists o aldosterone are currently used to control high blood pressure and to improve clinical

outcomes in heart ailure. Accumulating evidence suggests that antagonists selective or the aldosterone receptor may become important therapies or a range o cardiovascular and renovascular diseases. E orts are ongoing to develop aldosterone receptor antagonists that are tissue-specif c and lack e ects on potassium homeostasis. Also, aldosterone synthase inhibitors are being developed and may be used in the uture to reduce aldosterone production. Glucocorticoid pharmacology is an immense f eld, primarily because glucocorticoids are used to suppress in ammation in many di erent disease states. Chronic glucocorticoid use is associated with a multitude o predictable adverse e ects, and uture research in this area will attempt to minimize the adverse e ects o glucocorticoid therapy while maintaining the anti-in ammatory actions. Such e orts could include the development o tissue-selective glucocorticoid agonists and antagonists (analogous to the selective estrogen receptor modulators), as well as urther ref nement o drug delivery methods. Attempts are also being made to design sustained-release preparations o hydrocortisone to better

538 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

mimic the circadian rhythm o cortisol or treatment o Addison’s disease. The pharmacology o adrenal androgens needs to be studied more extensively to determine the indications, i any, or DHEA therapy.

Acknowledgment We thank Ehrin J. Armstrong and Robert G. Dluhy or their valuable contributions to this chapter in the First and Second Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Barnes PJ. Glucocorticosteroids: current and uture directions. Br J Pharmacol 2011;163:29–43. (Reviews mechanisms mediating the anti-in ammatory e ects o glucocorticoids, mechanisms o glucocorticoid resistance, and therapeutic implications.) Charmandari E, Nicolaides NC, Chrousos GP. Adrenal insu f ciency. Lancet 2014;383:2152–2167. (Discusses the pathophysiology, clinical presentation, diagnosis, and management o adrenal insu f ciency.) Kolkho P, Borden SA. Molecular pharmacology o the mineralocorticoid receptor: prospects or novel therapeutics. Mol Cell Endocrinol 2012;350: 310–317. (Reviews the pharmacology o steroidal and non-steroidal mineralocorticoid receptor antagonists and potential or tissue-specif c drugs.)

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540 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

30 Pharmacology of Reproduction Eh rin J . Arm s t ro n g a n d Ro b e rt L. Ba rb ie ri

INTROD DUC CTIION ON & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55441– 41– 1 54 542 PHYSIOLO LOGY LO GY Y OF RE REPR PR PROD ROD O U UC CTI TIV VE HORM VE MONE NE ES . . . . . . . . . . . . . 54 5411 Synthesi ssiis is of Progee st stin inss , Anddrogee ns ns, s , an nd Estroggen e s . . . . . . . 54 5411 Horm mon onee A Acc ti tion on a nd Metabbolliss m . . . . . . . . . . . . . . . . . . . . . . . 54 5422 Hypo Hy p th po t al alam mic ic-P -Pittui -P u taa ry ry-R -Rep e roodu d cct ctio tioon Ax Axiis . . . . . . . . . . . . . . . 544 In nte tegr e grat attio io off Enddoc ion ocrine Con o tr trol ol:: Th The Mens Me nss tr t ua u l Cy Cycl clle . . . . 545 P TH PA T OPHYSI S OLOG SI GY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5466 Disrruption of thee Hy Hypo p th hal alam am mic-P -P Pit itui uita t ry-Rep eproduction ep Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5477 Inappropriate Grow wth of Ho orm r onne-Dependent eeTiissuee s . . . . 54 5477 Decr crea ease sedd Es Estr t og gen or An Andr drog rogen Sec ecre r tion . . . . . . . . . . . . . 548 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 548 B Inhibitors of Gonadal Hormones . . . . . . . . . . . . . . . . . . . . . . . . 548 Synthesis Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548 Receptor Antagonistss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549

INTRODUCTION This chapter presents endocrine pharmacology relevant to both the male and emale reproductive tracts. Although men and women di er in their hormonal prof les, androgens and estrogens are both under the control o the anterior pituitary gland gonadotropins, luteinizing hormone (LH) and ollicle-stimulating hormone (FSH), and ultimately regulated by hypothalamic release o gonadotropinreleasing hormone (GnRH). Female hormone patterns are temporally more complex and cyclic than male patterns: hormonal control o the menstrual cycle is an illustrative example o how sex hormones are integrated into a complex physiologic system. Understanding the menstrual cycle also provides a basis or understanding the pharmacology o contraception. A number o diseases are treated pharmacologically via modif cation o reproductive hormone activity; these range rom in ertility and endometriosis to breast and prostate cancer. Key concepts in this chapter include (1) the interactions between estrogen and the pituitary gland, (2) the e ects o GnRH release requency on gonadotropin release, (3) the tissue selectivity o estrogen receptor agonists and antagonists, and (4) the various strategies used to antagonize the e ects o endogenous sex hormones, rom suppression o the hypothalamic-pituitary-reproduction axis to antagonism at the target tissue receptor.

Horm Ho rmon ones e andd Hor ormo mone Analogues: Contraception . . . . . . . 55 mo 552 Comb mbin inat atiion Estr trog oggenn–P Prrogge stin Contraception . . . . . . . . 552 Progestin-Only Conntraception . . . . . . . . . . . . . . . . . . . . . . . . 553 Emergency (Mornin ing-After) Contraceptio ionn . . . . . . . . . . . . 554 Male Ma le Contraception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 5544 Hormones and nd Hor ormo mone Analogues: Replacement . . . . . . . . 554 Estrogens and Proggesti tinss . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554 Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 555 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .555

PHYSIOLOGY OF REPRODUCTIVE HORMONES Synthesis of Progestins, Androgens, and Estrogens The synthesis o progestins, androgens, and estrogens is closely intertwined. All three groups are steroid hormones derived rom the metabolism o cholesterol. The synthesis o these hormones is similar to that o adrenal sex hormones, which is discussed in Chapter 29, Pharmacology o the Adrenal Cortex. The terminology progestins, androgens, and estrogens denotes a number o related hormones rather than a single molecule in each group (Fig. 30-1). The progestins consist o progesterone , a common precursor to testosterone and estrogen synthesis (see also Fig. 29-2), and a number o synthetically altered progesterone derivatives used or therapeutic purposes. Progestins generally exert antiproli erative e ects on the emale endometrium by promoting the endometrial lining to secrete rather than proli erate (see below). Progesterone is also required or the maintenance o pregnancy. Androgens , all o which have masculinizing properties, include dehydroepiandrosterone (DHEA), androstenedione, testosterone , and dihydrotestosterone (DHT); among the androgens, testosterone is considered the classic circulating androgen and DHT the classic intracellular 541

542 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Am y J f rs t n o tice s th a t h e r h a ir is th in n in g d u rin g h e r te e n a g e ye a rs . Eve n th o u g h s h e lo s e s s o m e h a ir o n h e r s ca lp , Ms . J n o tice s e xce s s ive h a ir g ro w th o n h e r a ce ; s h e s o m e tim e s h a s to s h a ve to re m o ve in a p p ro p ria te h a ir g ro w th . At a g e 24, s h e g o e s to h e r d o cto r co m p la in in g o b o th h e r h a ir p ro b le m a n d th e a ct th a t h e r p e rio d s a re irre g u la r. On u rth e r q u e s tio n in g , th e d o cto r d is cove rs th a t th e lo n g e s t in te rva l b e tw e e n h e r m e n s tru a l cycle s h a s b e e n 6 m o n th s a n d th e s h o rte s t 22 d a ys . Wh e n Ms . J d o e s h a ve p e rio d s , th e y a re h e a vy a n d la s t o r m o re th a n h e r p re vio u s a ve ra g e o 5 d a ys . Th e in cre a s e d h a ir g ro w th o n h e r a ce , e xtre m itie s , a b d o m e n , a n d b re a s ts h a d b e g u n a ro u n d a g e 15. Ms . J a ls o re p o rts a p ro b le m w ith b e in g o ve rw e ig h t s in ce h ig h s ch o o l, a lth o u g h in m id d le s ch o o l, s h e h a d b e e n e xtre m e ly a ctive in s o cce r, f e ld h o cke y, a n d s w im m in g . Th e d o cto r o rd e rs s e ve ra l te s ts a n d f n d s th a t Ms . J h a s m ild ly e le va te d re e a n d to ta l

androgen. Androgens are required or conversion to a male phenotype during development and or male sexual maturation. Estrogens re er to a number o substances that share a common eminizing activity. 17 -Estradiol is the most potent naturally occurring estrogen, while estrone and estriol are less potent. Note that all estrogens are derived rom the aromatization o precursor androgens (Fig. 30-1). The ovary and placenta most actively synthesize the aromatase enzyme that converts androgens to estrogens, but other nonreproductive tissues such as adipose tissue, hypothalamic neurons, and muscle can also aromatize androgens to estrogen. A ter menopause, the majority o circulating estrogen is derived rom adipose tissue. This is also the main source o circulating estrogens in men.

Hormone Action and Metabolism Progestins, androgens, and estrogens are all hormones that bind to a related super amily o nuclear hormone receptors; glucocorticoids, mineralocorticoids, vitamin D, and thyroid hormone also bind to the same super amily o receptors. Once synthesized, these hormones di use into the plasma, where they bind tightly to carrier proteins such as sex hormone-binding globulin (SHBG) and albumin. Only the unbound raction o hormone is able to di use into cells and bind to an intracellular receptor. Interestingly, testosterone is essentially a prohormone. Testosterone binds to the androgen receptor but with only modest a f nity. As a result, testosterone has only modest androgenic activity. Instead, testosterone is converted in target tissues to the more active dihydrotestosterone (Fig. 30-2), which binds to the androgen receptor with an a f nity ten old higher than that o testosterone. The ormation o dihydrotestosterone rom testosterone is catalyzed by the enzyme 5 -reductase . There are at least two subtypes o 5 -reductase. Di erential tissue expression o these enzymes provides some pharmacologic specif city or the 5 -reductase inhibitors. The importance

te s to s te ro n e le ve ls a n d a n in cre a s e d ra tio o p la s m a LH to FS H. Ba s e d o n th e s e f n d in g s , th e d o cto r te lls Ms . J th a t s h e p ro b a b ly h a s a d is o rd e r ca lle d p o lycys tic o va ria n s yn d ro m e (PCOS ). He re co m m e n d s co m b in a tio n o ra l co n tra ce p tive s to re g u la rize h e r m e n s tru a l cycle s . He a ls o p re s crib e s s p iro n o la cto n e to a m e lio ra te h e r p ro b le m s w ith h a ir g ro w th a n d b a ld in g .

Questions 1 . What is the pathophysiologic link between excessive hair growth and infertility in polycystic ovarian syndrome? 2 . Why was spironolactone prescribed to treat Ms. J ’s hair problem? 3 . How do oral contraceptives act, and how would they help regulate Ms. J ’s menstrual cycles?

o dihydrotestosterone as the most active androgen is highlighted in individuals with inherited def ciencies o 5 reductase. Males lacking this enzyme are phenotypically emale because they are unable to convert testosterone to dihydrotestosterone and are thus unable to activate a program o male di erentiation during development. The estrogen receptor (ER) is the best studied o the sex hormone receptors and serves as an example or all three receptor types (i.e., estrogen receptor, androgen receptor, and progesterone receptor). Because progestins, androgens, and estrogens are lipophilic steroid hormones, the raction o hormone that remains unbound to plasma proteins can reely di use across the plasma membrane into the cytosol o cells. Once inside the cell, the hormone ligand binds to its specif c intracellular receptor, which subsequently dimerizes. For example, association o estrogen with the estrogen receptor causes dimerization o two estrogen–estrogen receptor complexes, and the dimer then binds to estrogen response elements (EREs) in promoter regions o DNA. This binding to EREs, together with the recruitment o coactivators or corepressors, enhances or inhibits the transcription o specif c genes and thereby causes the physiologic e ects o the hormone. There are two subtypes o estrogen receptors—ER and ER . In addition, it is now recognized that many estrogen receptor actions involve association o the receptor with other transcription co actors. In other words, dimerization o the estrogen receptor and subsequent binding o the dimer to EREs are insu f cient to explain the complex and varied actions o estrogen in di erent tissues. The specif c transcription actors that are recruited by the estrogen receptor appear to be tissue-dependent and ligand-dependent and probably account or some o the target specif city o estrogen action. Although the subtypes and molecular associations o the androgen and progesterone receptors have not been studied as thoroughly as those o the estrogen receptor, it is likely that the same complexities exist or these receptors. The recognition that di erential binding o modular transcription actors

C h a p t e r 3 0 Pharmacology of Reproduction

543

HO

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FIGURE 30-1. Synthesis of progestins, androgens, and estrogens. Progestins, androgens, and estrogens are steroid hormones derived from cholesterol. The major progestins include progesterone and 17 -hydroxyprogesterone. The androgens include dehydroepiandrosterone (DHEA), androstenedione, and testosterone. Estrogens include estrone and estradiol. Estrogens are aromatized forms of their conjugate androgens: androstenedione is aromatized to estrone, and testosterone is aromatized to estradiol. Estradiol and estrone are both metabolized to estriol, a weak estrogen (not shown). Some of the precursor–product relationships among the hormones are omitted for clarity (see Fig. 29-2). HSD, hydroxysteroid dehydrogenase.

544 PRINCIPLES OF ENDOCRINE PHARMACOLOGY GnRH (puls a tile )

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FIGURE 30-2. Intracellular conversion of testosterone to dihydrotestosterone. Testosterone circulates in the plasma bound to sex hormone-binding globulin (SHBG) and albumin (not shown). Free testosterone di uses through the plasma membrane o cells into the cytosol. In target tissues, the enzyme 5 -reductase converts testosterone to dihydrotestosterone, which has increased androgenic activity relative to testosterone. Dihydrotestosterone binds with high a f nity to the androgen receptor, orming a complex that is transported into the nucleus. Homodimers o dihydrotestosterone and androgen receptor initiate transcription o androgen-dependent genes. Finasteride and dutasteride, drugs used in the treatment o benign prostatic hyperplasia and male pattern hair loss, inhibit the enzyme 5 -reductase.

to ERs could alter estrogenic e ects will likely prove a burgeoning area o pharmacologic research in the near uture, as pharmaceutical researchers continue to develop receptor agonists and antagonists with selective actions in specif c tissues. Selective estrogen receptor modulators (SERMs, see below) are the f rst drugs to take advantage o the tissue selectivity o sex hormone receptor unction. In some cells, a membrane-bound G protein-coupled estrogen receptor (GPER) is also activated by estradiol, resulting in activation o adenylyl cyclase (G s e ect), mobilization o intracellular calcium (G e ect), and transactivation o the epidermal growth actor receptor (G e ect). The role o GPER activation in the physiology and pathophysiology o estrogen action is an area o active investigation.

Hypothalamic-Pituitary-Reproduction Axis The hypothalamic-pituitary-reproduction axis regulates sex hormone synthesis. Gonadotropin-releasing hormone (GnRH) resides at the top o this three-tiered hierarchy. The hypothalamus secretes GnRH in pulses (Fig. 30-3). GnRH travels via the hypothalamic–pituitary portal system to stimulate gonadotroph cells o the anterior pituitary gland. Stimulation o gonadotroph cells via a G protein-coupled cell sur ace receptor increases the synthesis and secretion o LH and FSH, which are jointly re erred to as the gonadotropins. Although one cell type produces both LH and FSH, the synthesis and release o these two hormones are controlled independently. Current research suggests that the

FIGURE 30-3. The hypothalamic-pituitary-reproduction axis. The hypothalamus secretes gonadotropin-releasing hormone (GnRH) into the hypothalamic–pituitary portal system in a pulsatile pattern. GnRH stimulates gonadotroph cells in the anterior pituitary gland to synthesize and release luteinizing hormone (LH) and ollicle-stimulating hormone (FSH). These two hormones, re erred to as gonadotropins , promote ovarian and testicular synthesis o estrogen and testosterone, respectively. Estrogen and testosterone inhibit release o GnRH, LH, and FSH. Depending on the time in the menstrual cycle, the concentration o estrogen in the plasma, and the rate at which estrogen concentration increases in the plasma, estrogen can also stimulate pituitary gonadotropin release (e.g., at ovulation). Both the ovaries and testes secrete inhibin, which selectively inhibits FSH secretion, and activin, which selectively promotes FSH secretion.

rate o GnRH secretion may pre erentially alter the secretion patterns o LH and FSH. Pulsatile secretion o GnRH is critical or the proper unctioning o the hypothalamicpituitary-reproduction axis. When GnRH is administered continuously, gonadotroph release of LH and FSH is suppressed rather than stimulated. This e ect has the important pharmacologic consequence that pulsatile administration o exogenous GnRH stimulates gonadotropin release, whereas continuous GnRH administration inhibits LH and FSH release and thereby blocks target cell unction. LH and FSH have analogous but somewhat di erent e ects in males and emales. The pertinent target cells in the male are the Leydig and Sertoli cells o the testis, while the thecal and granulosa cells o the ovary mediate gonadotropin unction in the emale (Fig. 30-4). In each case, a two-cell system is coordinated to mediate sex hormone actions. In the male, LH stimulates testicular Leydig cells to increase the synthesis o testosterone, which then di uses into neighboring Sertoli cells. In the Sertoli cell, FSH stimulation increases the production o androgen binding protein (ABP), which is important or maintaining the high testicular concentrations

C h a p t e r 3 0 Pharmacology of Reproduction LH

FS H

Male

LH-R

FS H-R

ABP Te s tos te rone s ynthe s is

Te s tos te rone Te s tos te rone -ABP

Le ydig ce ll

S e rtoli ce ll

LH

FS H

Female

LH-R

FS H-R

Aromatase Androge n s ynthe s is

Androge n Es troge n

The ca l ce ll

Gra nulos a ce ll

FIGURE 30-4. Two-cell systems for gonadal hormone action. In the male , the binding of luteinizing hormone (LH) to the LH receptor (LH-R) activates testosterone synthesis in Leydig cells. Testosterone then diffuses into nearby Sertoli cells, where the binding of follicle-stimulating hormone (FSH) to its receptor (FSH-R) increases levels of androgen binding protein (ABP). ABP stabilizes the high concentrations of testosterone that, together with other FSH-induced proteins synthesized in Sertoli cells, promote spermatogenesis in the nearby germinal epithelium (not shown). In the female , LH acts in an analogous manner to promote androgen (androstenedione) synthesis in thecal cells. Androgen then diffuses into nearby granulosa cells, where aromatase converts androstenedione to estrone, which is then reduced to the biologically active estrogen, estradiol. FSH increases aromatase activity in granulosa cells, promoting the conversion of androgen to estrogen. Note that dihydrotestosterone is not a substrate for aromatase.

o testosterone necessary or spermatogenesis. In addition, FSH stimulates the Sertoli cell to produce other proteins necessary or sperm maturation. In the emale, LH stimulates the thecal cells to synthesize the androgen androstenedione, which is then aromatized to estrone and estradiol in the granulosa cells under the in uence o FSH. Both Sertoli cells and granulosa cells synthesize and secrete the regulatory proteins inhibin A, inhibin B, and activin. Inhibins secreted by the gonad act on the anterior pituitary gland to inhibit the release o FSH, while activin stimulates FSH release. Neither the inhibins nor activin has an e ect on anterior pituitary gland LH release (Fig. 30-3). The role o these regulatory proteins in controlling hormone action is still

545

not completely understood. In the male, testosterone is also an important negative regulator o pituitary gland and hypothalamic hormone release. The role o estrogen in the emale is more complex and can involve either positive or negative eedback depending on the prevailing hormonal milieu; this topic is addressed below as part o the menstrual cycle discussion. In the emale, the combination o estradiol and progesterone synergistically suppresses GnRH, LH, and FSH secretion by actions at both the hypothalamus and pituitary gland.

Integration of Endocrine Control: The Menstrual Cycle The emale menstrual cycle is governed by the cycling o hormones with an approximate periodicity o 28 days (normal range, 24–35 days). This cycle begins at the onset o puberty and continues uninterrupted (with the exception o pregnancy) until menopause (Fig. 30-5). The start o the cycle, cycle day 1, is arbitrarily def ned as the f rst day o menstruation. Ovulation occurs at the midportion (about day 14) o each cycle. The portion o the menstrual cycle be ore ovulation is o ten re erred to as the follicular or proliferative phase; during this time, the developing ovarian follicle produces most o the gonadal hormones, which stimulate cellular proliferation o the endometrium. Subsequent to ovulation, the corpus luteum produces progesterone, and the endometrium becomes secretory rather than proli erative. The second hal o the menstrual cycle is thus o ten re erred to as the luteal or secretory phase, depending on whether the ovary or the endometrium is considered as the rame o re erence. At the start o the menstrual cycle, there is low production o estrogen and inhibin A. As a result, the anterior pituitary gland secretes increasing amounts o FSH and LH. These hormones stimulate the maturation o our to six ollicles, each o which contains an ovum arrested in the f rst stage o meiosis. Maturing ollicles secrete increasing concentrations o estrogen, inhibin A, and inhibin B. Estrogen causes the ollicles to increase the expression o LH and FSH receptors on thecal and granulosa cells, respectively. Receptor upregulation increases the ollicular response to pituitary gland gonadotropins and allows one ollicle to secrete increasing quantities o estrogen. The increased plasma estrogen and inhibin levels partially suppress pituitary gland LH and FSH release. In turn, the decreased gonadotropin levels cause other ollicles to become atretic, so that usually, only one ollicle matures. At the same time, increased estrogen levels stimulate the uterine endometrium to proli erate rapidly. As the dominant ollicle continues to grow, it secretes high, sustained levels o estrogen. Although the mechanism is still not completely understood, the combination o high estrogen levels and the rapid rate o increase o estrogen levels causes a brie positive eedback e ect on gonadotroph release o gonadotropins, stimulating rather than inhibiting release o LH and FSH. The resulting midcycle surge o LH and FSH stimulates the dominant ollicle to swell and to increase the activity o its proteolytic enzymes. Approximately 40 hours a ter the onset o the LH surge , the ollicle ruptures and ovulation occurs. The ovum is released into the peritoneal cavity and is then taken up by a allopian tube, where it begins its route toward the uterus. I the oocyte becomes ertilized in the allopian tube, it reaches the uterus approximately 4 days a ter ovulation and implants into the endometrium approximately 5–6 days a ter ovulation.

546 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Follicula r pha s e

The cellular remains o the ruptured ovarian ollicle become the corpus luteum. Cells o the corpus luteum secrete estrogen and progesterone, not just estrogen. The presence of progesterone in the second half of the menstrual cycle causes the endometrium to switch from a proliferative to a secretory state. The endometrium begins synthesizing proteins necessary or implantation o a ertilized egg. The blood supply to the endometrium also increases to provide increased nutrients i pregnancy ensues. The corpus luteum has a li espan o approximately 14 days. I ertilization and implantation o a viable blastocyst do not occur within 14 days o ovulation, the corpus luteum becomes atretic and ceases its production o estrogen and progesterone. Without the trophic e ects o estrogen and progesterone, the endometrial lining sheds and menstruation begins. In the absence o estrogen and progesterone, the inhibition o gonadotrophs is removed, and production o FSH and LH increases. This stimulates the development o new ovarian ollicles and the beginning o another menstrual cycle. I ertilization does occur, implantation within the uterine lining causes the blastocyst to secrete human chorionic gonadotropin (hCG). The presence o hCG stimulates the corpus luteum to remain viable and continue secreting progesterone. Because hCG is one o the f rst proteins produced by the embryo that is unique to pregnancy, pregnancy tests assay or the presence o hCG. hCG production decreases a ter 10–12 weeks o pregnancy, when the placenta begins to secrete progesterone autonomously. Special considerations attending the use o drugs in pregnancy are discussed in Box 6-1.

Lute a l pha s e

Ovula tion

Ova ria n follicle

Growing

Ma ture

Corpus lute um

Es troge n

P roge s te rone (+ e s troge n)

m

l

)

60

(

m

l

L

U

/

H

40 20 0

10

(

m

l

F

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(

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PATHOPHYSIOLOGY

10 5

P

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o

(

g

n

e

g

s

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Me ns e s

S e cre tory

E

n

d

o

m

e

t

r

i

u

m

0

2

6

10

14

18

22

26

Day

FIGURE 30-5. The menstrual cycle. The menstrual cycle is divided into the ollicular phase and the luteal phase. Ovulation def nes the transition between these two phases. During the ollicular phase, gonadotroph cells o the anterior pituitary gland secrete LH and FSH in response to pulsatile GnRH stimulation. Circulating LH and FSH promote growth and maturation o ovarian ollicles. Developing ollicles secrete increasing amounts o estrogen. At f rst, the estrogen has an inhibitory e ect on gonadotropin release. J ust be ore the midpoint in the menstrual cycle, however, estrogen exerts a brie positive eedback e ect on LH and FSH release. This is ollowed by ollicular rupture and release o an egg into the allopian tube. During the second hal o the cycle, the corpus luteum secretes both estrogen and progesterone. Progesterone induces a change in the endometrium rom a proli erative to a secretory type. I ertilization and implantation o a blastocyst do not occur within 14 days a ter ovulation, the corpus luteum involutes, secretion o estrogen and progesterone declines, menses occurs, and a new cycle begins.

Pathophysiologic processes in the reproductive tract reect one o three general mechanisms o dysregulation (Table 30-1). The f rst is disruption o the hypothalamicpituitary-reproduction axis, which causes a number o underlying disorders that can lead to in ertility. The second is inappropriate growth o estrogen-dependent or testosteronedependent tissue. This can lead to breast cancer or prostate cancer, as well as to benign but clinically important conditions such as endometriosis or endometrial hyperplasia. Finally, decreased estrogen secretion, as in menopause, or decreased androgen secretion, as in some aging men, is associated with a number o undesirable health consequences.

TABLE 30-1 General Mechanisms of Reproductive Tract Disorders for Which Pharmacologic Agents Are Currently Used MECHANISM

EXAMPLES

Disruption o the hypothalamic-pituitaryreproduction axis

Polycystic ovarian syndrome Prolactinoma

Inappropriate growth o hormone-dependent tissue

Breast cancer Prostatic hyperplasia, prostate cancer Endometriosis, endometrial hyperplasia Leiomyomas (uterine f broids)

Decreased estrogen or androgen secretion

Hypogonadism Menopause

C h a p t e r 3 0 Pharmacology of Reproduction

Disruption o the Hypothalamic-PituitaryReproduction Axis The hypothalamic-pituitary-reproduction axis is normally tightly regulated via eedback inhibition or stimulation o hormone activity, with the goal o producing a success ul menstrual cycle every month. When this axis is disrupted, in ertility can result. Common causes o in ertility due to disruption o sex hormone production include polycystic ovarian syndrome and prolactinomas. Polycystic ovarian syndrome (PCOS) is a complex syndrome characterized by anovulation or oligo-ovulation and by increased levels o plasma androgen. PCOS is a common problem a ecting between 3% and 5% o women o reproductive age. The diagnosis is typically clinical, as in the case o Ms. J, and based on the concurrent f ndings o oligoovulation and hirsutism (excessive hair growth). Although multiple etiologies are likely to be responsible or PCOS, all o the etiologies result in increased androgen secretion and suppression o normal ovulatory cycles. The increased androgen secretion results in masculinization; as seen in Ms. J’s case, male pattern baldness and inappropriate acial hair growth are common. Many women with PCOS are treated with both an estrogen–progestin contraceptive to suppress ovarian production o testosterone and an antiandrogen, such as spironolactone (see below), to abrogate the masculinizing e ects o increased circulating testosterone. Three primary hypotheses have been advanced to explain the development o PCOS. The f rst, re erred to as the LH hypothesis , is based on the observation that many women with PCOS have an increased requency and amplitude o pituitary LH pulses. In act, 90% o women with PCOS have increased circulating LH. Increased LH activity stimulates thecal cells o the ovary to synthesize increased amounts o androgens, including androstenedione and testosterone. In addition, the increased LH and androgen levels prevent normal ollicle growth, in turn preventing ollicle secretion o large amounts o estrogen. The absence o an estrogen “trigger” prevents the LH surge and ovulation. As seen in the introductory case, patients with PCOS menstruate irregularly, and the menstrual periods that they do have tend to have heavy ow. The second hypothesis, re erred to as the insulin theory, is based on the observation that many women with PCOS are obese and insulin resistant and secrete increased insulin. Increased insulin decreases the production o sex hormonebinding globulin (SHBG), which results in a higher concentration o ree testosterone and there ore greater androgenic e ects on peripheral tissues. It has also been observed that insulin can directly synergize with LH to increase androgen production by thecal cells. Interestingly, in women with PCOS, medications that specif cally treat insulin resistance, such as met ormin, may result in regular ovulatory menses and normalization o testosterone levels. The third hypothesis is the ovarian hypothesis . This explanation posits dysregulation o sex steroid synthesis at the level o the thecal cell. For example, an abnormal increase in the activity o the oxidative enzymes responsible or androgen synthesis could lead to greater thecal cell production o androgens in response to any given stimulus. It is important to note that these hypotheses are not mutually exclusive and that PCOS could result rom a combination o two or three mechanisms. When the cellular mechanisms underlying this disease are better elucidated, new pharmacologic therapies

547

can be developed to treat the etiology o the disease rather than its e ects. Prolactinomas are another common cause o in ertility among women o reproductive age. These clonal, benign tumors o lactotrophs in the anterior pituitary gland can cause in ertility through two parallel pathways. First, increased prolactin levels suppress estrogen synthesis, both by antagonizing the hypothalamic release o GnRH and by decreasing gonadotroph sensitivity to GnRH. This antagonism decreases LH and FSH release and thereby decreases end-organ stimulation by the hypothalamic-pituitary-reproduction axis. The second mechanism, common to all pituitary gland tumors, is a crowding-out or mass e ect. Because the pituitary gland is enclosed in the bony sella turcica, lactotroph proli eration in the anterior pituitary gland leads to crowding o other cell types and thereby inhibits the unction o nearby gonadotroph cells. Prolactin-secreting tumors typically remain responsive to the inhibitory e ect o dopamine agonists. In most cases, chronic administration o dopamine agonists such as cabergoline or bromocriptine suppresses prolactin secretion and causes the tumor cells to shrink, thereby decreasing the size o the tumor and restoring normal gonadotroph unction and ovulation.

Inappropriate Growth o Hormone-Dependent Tissues The growth o breast tissues is dependent on many hormones, including estrogen, progesterone, androgens, prolactin, and insulin-like growth actors. Many (but not all) breast cancers express the estrogen receptor (ER), and the growth o such cancers is o ten stimulated by endogenous levels o estrogen and inhibited by antiestrogens. When a breast carcinoma is ound to express the ER, an estrogen receptor antagonist (either a pure antagonist such as ulvestrant or a selective estrogen receptor modulator such as tamoxi en; see below) or an estrogen synthesis inhibitor (an aromatase inhibitor such as anastrozole , letrozole , exemestane , or ormestane ) is commonly administered to slow tumor growth. Prostate growth is androgen-dependent and requires the local conversion o testosterone to dihydrotestosterone by type II 5 -reductase in stromal cells o the prostate. Both enzyme inhibition (f nasteride or dutasteride ) and receptor antagonist ( utamide , bicalutamide , nilutamide , or enzalutamide ) strategies are used to treat conditions in which the growth o prostate tissue is dysregulated, such as benign prostatic hyperplasia and metastatic prostate cancer (see below). Endometriosis is the growth o endometrial tissue outside the uterus. The act that endometriosis is usually ound in areas surrounding the allopian tube (ovaries, rectovaginal pouch, and uterine ligaments) has led to the hypothesis that endometriosis could result rom retrograde migration o endometrial tissue via the allopian tubes during menstruation. Other etiologies are possible, however, including metaplastic tissue growth rom the peritoneum or spread o endometrial cells to extrauterine sites via lymphatic ducts. There is also evidence o increased aromatase activity in endometrial tissue rom such patients. Because oci o endometriosis respond to estrogen stimulation, endometriosis grows and regresses with the menstrual cycle. This can lead to severe pain, abnormal bleeding, and the ormation o adhesions in the peritoneal cavity. In turn, adhesion ormation can lead to in ertility. Because endometriosis is usually estrogendependent, treatment with long hal -li e GnRH agonists o ten achieves regression o the disease.

548 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Decreased Estrogen or Androgen Secretion The e ects o decreased sex hormone production vary depending on the age o the patient at the onset o symptoms. Hypogonadism results i sex hormone production is impaired be ore adolescence. Patients with hypogonadism do not undergo sexual maturation, but proper hormone replacement can, in many cases, allow the development o secondary sexual characteristics. Menopause is a normal physiologic response to exhaustion o the ovarian ollicles. Throughout a woman’s li etime, ollicles are arrested in meiosis. Only a small percentage o ollicles mature during the menstrual cycle; the rest eventually become atretic. Menstrual cycles cease when all o the ollicles are depleted rom the ovaries. Follicle depletion leads to a decrease in estrogen and inhibins (because developing ollicles are the main estrogen and inhibin source in premenopausal women) and an increase in LH and FSH (because estrogen and inhibins suppress gonadotropin release). After menopause, androstenedione continues to be converted to estrone by aromatase in peripheral (mainly adipose) tissues. However, estrone is a less potent estrogen than estradiol. Because o the relative lack o estrogen a ter menopause, many women experience hot ashes, vaginal dryness, and decreased libido. Postmenopausal women are also at risk or osteoporosis. The role o estrogen in the maintenance o bone mass is discussed in urther detail in Chapter 32, Pharmacology o Bone Mineral Homeostasis.

Men do not experience a sudden decrease in sex hormones in a manner analogous to the emale menopause, but androgen secretion does decline gradually with age. Although controversy currently exists over the role o androgen therapy in normal elderly men, androgen replacement is indicated in cases o adult hypogonadism where both low testosterone levels and symptoms o hypogonadism are present.

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic agents have been developed to target most o the steps in gonadal physiology and pathophysiology. The relevant drug classes include modulators o anterior pituitary gland gonadotroph activity and specif c antagonists o peripheral hormone action. In addition, sex hormones are o ten used as replacement therapy or to modi y gonadotropin release (Fig. 30-6).

Inhibitors of Gonadal Hormones Synthesis Inhibitors GnRH Agonists and Antagonists

Under physiologic conditions, the hypothalamus releases GnRH in a pulsatile ashion. The requency o GnRH pulses controls the relative release o LH and FSH by the anterior pituitary gland. In contrast, continuous administration o GnRH

GnRH (puls a tile ; e ndoge nous )

Ante rior pituita ry gla nd

GnRH agonists (continuous; leuprolide, goserelin, nafarelin, buserelin, triptorelin)

LH/FS H

GnRH receptor antagonists (cetrorelix, ganirelix, degarelix)

Ova rie s / te s te s

Aromatase inhibitors Andros te ne dione (exemestane, Aro m a ta s e formestane, anastrozole, letrozole) Es troge n SERMs (+/-) Ge ne tra ns cription

Te s tos te rone

P roge s te rone

5α -re d u c ta s e

5α -reductase inhibitors (finasteride, dutasteride)

Dihydrote s tos te rone Mifepristone, ulipristal Ge ne tra ns cription

Ge ne tra ns cription

Androgen receptor antagonists (flutamide, bicalutamide, nilutamide, enzalutamide, spironolactone)

FIGURE 30-6. Pharmacologic modulation of gonadal hormone action. Pharmacologic modulation o gonadal hormone action can be divided into inhibitors o hormone synthesis and hormone receptor antagonists. Continuous administration o GnRH suppresses LH and FSH release rom the anterior pituitary gland, thus preventing gonadal hormone synthesis. GnRH receptor antagonists (cetrorelix, ganirelix, degarelix) are also used or this purpose. Finasteride and dutasteride inhibit the enzyme 5 -reductase, thus preventing conversion o testosterone to the more active dihydrotestosterone. Aromatase inhibitors (exemestane, ormestane, anastrozole, letrozole) inhibit production o estrogens rom androgens. A number o hormone receptor antagonists and modulators prevent the action o endogenous estrogens (some SERMs), androgens (f utamide, bicalutamide, nilutamide, enzalutamide, spironolactone), and progesterone (mi epristone, ulipristal).

C h a p t e r 3 0 Pharmacology o Reproduction

suppresses, rather than stimulates, pituitary gonadotroph activity. It is possible to suppress the hypothalamic-pituitaryreproduction axis either by continuous administration o a GnRH agonist (leuprolide , goserelin, na arelin, buserelin, or triptorelin) or by administration o a GnRH receptor antagonist (cetrorelix, ganirelix, or degarelix). Continuous administration o a GnRH agonist is used to treat hormone-dependent tumors such as prostate cancer and, in some cases, breast cancer. Individual agents are discussed in detail in Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland. Currently available GnRH analogues are peptides and are administered by non-oral routes such as injection or nasal spray. A nonpeptide oral GnRH antagonist, elagolix, is in late clinical trials or the treatment o pelvic pain caused by endometriosis. 5 -Reductase Inhibitors Finasteride and dutasteride are inhibitors o 5 -reductase,

the enzyme that converts testosterone to dihydrotestosterone. Finasteride is a selective inhibitor o the type II reductase, which is highly expressed in the prostate. Dutasteride is an inhibitor o both type II reductase and type I reductase (which is expressed in skin and prostate). Recall that dihydrotestosterone binds to the androgen receptor with higher a f nity than testosterone. Blocking the local conversion of testosterone to dihydrotestosterone effectively abrogates the local action of testosterone. Prostate cells are dependent on androgen stimulation or survival, and administration o a reductase inhibitor slows the growth o prostate tissue. Finasteride and dutasteride are approved or treatment o symptoms resulting rom benign prostatic hyperplasia, such as decreased urine ow and di f culty initiating urination. These drugs are potential alternatives to transurethral resection o the prostate (TURP), which is a common surgical treatment or symptomatic prostatic hyperplasia. One year o therapy can result in up to 25% reduction in prostate size. These drugs are most e ective or patients with larger prostates because the greatest clinical changes are observed in prostates that are already signif cantly hypertrophied. Adverse e ects include decreased libido and erectile dys unction.

Aromatase Inhibitors

Because estrogens are synthesized rom androgen precursors via the action o aromatase, blocking the aromatase enzyme can e ectively inhibit estrogen ormation. This approach is used to inhibit the growth o estrogen-dependent tumors such as estrogen receptor (ER)-positive breast cancer. A number o highly selective aromatase inhibitors have recently been developed. Anastrozole and letrozole are competitive inhibitors o aromatase, while exemestane and ormestane bind covalently to aromatase. All o these agents are currently used in the treatment o metastatic breast cancer and in the prevention o recurrences in cancers primarily treated with surgery and radiation. Recent trials suggest that aromatase inhibitors are more e ective than estrogen receptor antagonists, such as tamoxi en, or the treatment o breast cancer. However, aromatase inhibitors produce pro ound suppression o estrogen action, and estrogen is a major regulator o bone density. There ore, women taking aromatase inhibitors have an increased risk o osteoporotic ractures. Approximately 20% o breast cancer occurs in premenopausal women. In these patients, the combination o a GnRH agonist (to suppress ovarian hormone production) and an aromatase inhibitor (to suppress estrogen ormation) has been shown to signif cantly reduce the risk o distal tumor recurrence.

549

TABLE 30-2 Tissue-Specif c Agonist and Antagonist Activity o Selective Estrogen Receptor Modulators BREAST

ENDOMETRIUM

BONE

Estrogen Tamoxi en Raloxi ene Bazedoxi ene Estrogen, the physiologic hormone, has stimulatory e ects in breast, endometrium, and bone. Tamoxi en is an antagonist in breast tissue and is there ore used in the treatment o estrogen receptor-positive breast cancer. Raloxi ene is an agonist in bone but an antagonist in breast and endometrium. Raloxi ene is approved or prevention and treatment o osteoporosis in postmenopausal women and prevention o breast cancer. Bazedoxi ene is an antagonist in breast and endometrium but an agonist in bone. Bazedoxi ene is approved or treatment o hot f ashes and prevention o postmenopausal osteoporosis. Clomiphene (not shown in the table) is a SERM that acts as an estrogen receptor antagonist in hypothalamus and anterior pituitary gland; it is used clinically to increase FSH secretion, thereby inducing ovulation.

Receptor Antagonists Selective Estrogen Receptor Modulators

The term selective estrogen receptor modulator (SERM) is based on the observation that certain so-called antiestrogen drugs are not pure antagonists but rather mixed agonists/antagonists (Table 30-2). These pharmacologic agents inhibit estrogenic e ects in some tissues, while promoting estrogenic e ects in other tissues. The basis or tissue selectivity may include several mechanisms. First, there are two estrogen receptor subtypes, ER and ER , and the expression o these receptor subtypes is tissuespecif c. Second, the ability o the estrogen receptor to interact with other transcription co actors (coactivators and corepressors) depends on the structure o the ligand that is bound to the receptor. Figure 30-7 provides an example. Assume that the binding o 17 -estradiol (called Estrogen in the f gure) to the estrogen receptor causes a con ormational change in the receptor, so that two transcriptional co actors, X and Y, can also bind to the receptor. This complex can then activate three genes: an X-dependent gene, a Y-dependent gene, and a gene that depends on both X and Y. In contrast, the binding o a SERM to the estrogen receptor causes a di erent con ormational change in the receptor, so that transcription actor X is able to bind, but transcription actor Y is not. As a result, the SERM-receptor-X complex can activate the X-dependent gene but not the Y-dependent gene or the (X Y)-dependent gene. In addition, assume that transcription actors X and Y are expressed in bone cells but that breast cells express only transcription actor Y. In the breast, this SERM acts as an antagonist because (1) the inability o Y to associate with the SERM-estrogen receptor complex prevents the SERM rom activating any estrogen-dependent e ects and (2) binding o the SERM to the estrogen receptor competitively inhibits the binding o endogenous estrogen to the receptor. In bone, however, this SERM acts as a partial agonist because it can activate X-dependent but not Y-dependent genes.

550 PRINCIPLES OF ENDOCRINE PHARMACOLOGY A Bone : both X a nd Y cofa ctors e xpre s s e d S ERM

Es troge n

Cofa ctor Y Cofa ctor Y Es troge n re ce ptor

Cofa ctor X Nucle us

Es troge n re ce ptor

Cofa ctor X Nucle us

DNA

DNA

X Ge ne 1 Y Ge ne 2 X Y Ge ne 3

X Ge ne 1 Y Ge ne 2 X Y Ge ne 3

Only Ge ne 1 e xpre s s e d: P a rtia l a gonis t

Ge ne s 1, 2, a nd 3 e xpre s s e d: Full a gonis t

B Bre a s t: only Y cofa ctor e xpre s s e d S ERM

Es troge n

Cofa ctor Y Cofa ctor Y Es troge n re ce ptor

Nucle us

Es troge n re ce ptor

Nucle us

DNA

DNA

X Ge ne 1 Y Ge ne 2 X Y Ge ne 3

X Ge ne 1 Y Ge ne 2 X Y Ge ne 3

Ge ne 2 e xpre s s e d: Full a gonis t

No ge ne s e xpre s s e d: Anta gonis t

FIGURE 30-7. A model or the tissue specif city o action o SERMs. Selective estrogen receptor modulators (SERMs) exhibit tissue-specif c estrogen receptor antagonist or partial agonist activity. This tissue specif city o action seems to be explained by the ollowing observations: (1) transcriptional coactivators and/or corepressors are expressed in a tissue-specif c manner, (2) a SERM–estrogen receptor (ER) complex can associate with some coactivators or corepressors but not others, and (3) genes can be activated or inhibited by di erent combinations o SERM–ER complexes and coactivators or corepressors. In the example shown, assume that bone cells express coactivators (co actors) X and Y, whereas breast cells express only coactivator Y. The estrogen–ER complex can associate with X and Y, whereas the SERM–ER complex can associate with only X. A. In bone cells, estrogen binding to ER and recruitment o coactivators X and Y induce expression o Genes 1, 2, and 3. The SERM–ER complex cannot bind coactivator Y, and the SERM–ER–co actor X complex induces expression o only Gene 1. In bone, then, estrogen is a ull agonist, whereas the SERM is a partial agonist. B. In breast cells, estrogen binding to ER and recruitment o coactivator Yinduce expression o Gene 2, but the SERM is unable to promote expression o any gene. In breast, then, the SERM acts as an antagonist. For simplicity, this model shows only coactivators, although corepressors are also involved in SERM action.

These tissue-specif c actions o SERMs have important implications or both the desired e ects and the adverse e ects o pharmacologic agents. I it were possible to design a SERM that inhibits estrogen-dependent growth o breast carcinoma without causing estrogen-induced endometrial hyperplasia, then the undesirable adverse e ects o tamoxi en (discussed below) could be reduced. It is likely that SERMs with ref ned specif city will have important implications or the treatment o osteoporosis, breast cancer, and perhaps even cardiovascular disease. The six SERMs in current clinical use are tamoxi en, raloxi ene, toremi ene, bazedoxi ene, ospemi ene, and clomiphene. Tamoxi en is the only SERM currently approved or use in the treatment and prevention o breast cancer. Tamoxi en has been employed in the palliative treatment o metastatic breast cancer and as adjuvant therapy a ter lumpectomy. Tamoxifen is an estrogen receptor antagonist in breast tissue

but a partial agonist in endometrium and bone. These pharmacodynamic e ects result in inhibition o the estrogendependent growth o breast cancer but also stimulation o endometrial growth. Because o the latter e ect, tamoxien administration is associated with a our old to six old increase in the incidence o endometrial cancer. There ore, in order to minimize the risk o iatrogenic endometrial cancer, tamoxi en is typically administered or no more than 5 years. Raloxi ene is a newer SERM that possesses estrogen receptor agonist activity in bone but antagonist activity in both breast and endometrial tissue. Its mechanism o action is illustrated in Figure 30-7 and its molecular structure is shown in Figure 30-8. Consistent with this prof le o tissue-specif c actions, raloxi ene does not appear to increase the incidence o endometrial cancer. The agonist activity o raloxi ene in bone decreases bone resorption and thus delays or prevents

C h a p t e r 3 0 Pharmacology o Reproduction

A

551

B

FIGURE 30-8. Structural comparison o estrogen (natural ligand) and raloxi ene (SERM) bound to the estrogen receptor. The ligand-binding domain o the human estrogen receptor-alpha is displayed in ribbon ormat rom the yellow-brown N-terminus to the dark blue C-terminus. The natural ligand 17 -estradiol (estrogen) and the selective estrogen receptor modulator (SERM) raloxi ene are displayed in space-f lling ormat. A. In the estrogen-bound structure, the position o the orange helix (H12) def nes the agonist con ormation o the receptor that recruits coactivators and thereby regulates transcription o estrogenregulated genes (see Fig. 30-7). B. In the raloxi ene-bound structure, the bulky side chain o raloxi ene disrupts the agonist con ormation o the receptor (note that helix H12 is substantially displaced); in this con ormation, the receptor is capable o recruiting some coactivators but not others (see Fig. 30-7).

the progression o osteoporosis in postmenopausal women (discussed in more detail in Chapter 32). Raloxi ene is approved or use in prevention o breast cancer and prevention and treatment o osteoporosis. In a large clinical trial comparing raloxi ene and tamoxi en or prevention o breast cancer in women at high risk, both agents resulted in a 50% reduction in the development o invasive breast cancer. Tamoxi en treatment was associated with more cases o endometrial hyperplasia, endometrial cancer, cataracts, and deep vein thrombosis than raloxi ene. However, tamoxi en also prevented more cases o noninvasive breast cancer than raloxi ene. Toremi ene is an estrogen receptor antagonist in breast tissue. It is approved or treatment o metastatic breast cancer in postmenopausal women. Bazedoxi ene is a unique SERM that blocks estrogen-induced proli eration o the endometrium. In menopausal women with a uterus, the combination o estrogen and bazedoxi ene is approved or treatment o hot ashes. Bazedoxi ene is also approved or prevention o postmenopausal osteoporosis. Ospemi ene is an estrogen receptor agonist in vaginal tissue and it improves vulvovaginal atrophy in postmenopausal women. It is approved or treatment o moderate to severe pain with sexual relations in postmenopausal women. Clomiphene is a SERM used to induce ovulation. The drug acts as an estrogen receptor antagonist in the hypothalamus and anterior pituitary gland and as a partial agonist in the ovaries. In women with PCOS, the antagonist activity o clomiphene in the hypothalamus and anterior pituitary gland results in relie o the negative eedback inhibition imposed by endogenous estrogen and, there ore, in the increased release

o GnRH and gonadotropins, respectively. The increased levels o FSH stimulate ollicle growth, resulting in an estrogen trigger signal, an LH surge, and ovulation. The main adverse e ect is that clomiphene can cause multiple ollicles to grow, resulting in increased ovarian size. Unlike the administration o exogenous FSH (see Chapter 27), however, clomiphene use is seldom associated with the ovarian hyperstimulation syndrome. The aromatase inhibitor letrozole can also reduce the negative eedback o estrogen on the hypothalamus and pituitary and induce ovulation in women with PCOS. In obese women with PCOS, letrozole is more e ective than clomiphene in inducing ovulation and pregnancy. Androgen Receptor Antagonists

Androgen receptor antagonists competitively inhibit the binding o endogenous androgens to the androgen receptor. By this mechanism, receptor antagonists block the action o testosterone and dihydrotestosterone on their target tissues. The androgen receptor antagonists include f utamide , bicalutamide , nilutamide , and enzalutamide . These agents are approved only or the treatment o metastatic prostate cancer, but utamide is also used therapeutically in the treatment o benign prostatic hyperplasia. Spironolactone , originally approved as an aldosterone receptor antagonist (see Chapter 21, Pharmacology o Volume Regulation), also has signif cant antagonist activity at the androgen receptor. Like other androgen receptor antagonists, spironolactone can be used as a competitive inhibitor o testosterone action. Ms. J was treated with spironolactone to antagonize the excessive androgen stimulation o her hair ollicles and thus to ameliorate her hirsutism. A compound derived

552 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

rom spironolactone, drospirenone , has both progestational and antiandrogen e ects. It is used as a progestin in some estrogen–progestin contraceptives. Selective Progesterone Receptor Modulators Mifepristone (also re erred to as RU-486) is a selective proges-

terone receptor modulator (SPRM) used to induce abortion at up to 70 days o pregnancy. As noted above, progesterone is crucial or maintenance o the endometrium during pregnancy; the hormone stabilizes the uterine lining and promotes vessel growth and secretory activities o the decidua. Acting as an antagonist, mi epristone inhibits progesterone action by binding competitively to the progesterone receptor. Blockade o progesterone action results in decay and death o the decidua, and lack o nourishment rom the decidua causes the blastocyst to die and detach rom the uterus. Because the blastocyst is no longer secreting hCG, the corpus luteum involutes, and involution o the corpus luteum causes progesterone synthesis and secretion to decrease. Mi epristone is commonly administered in sequence with misoprostol, a prostaglandin analogue (see Chapter 43, Pharmacology o Eicosanoids). Misoprostol stimulates uterine contractions, and the combined e ects o progesterone antagonism and uterine contractions are more than 95% e ective in terminating f rst-trimester pregnancy. Because mi epristone is administered as a single dose, adverse e ects related to progesterone antagonism are rare. Instead, the main potential or complication lies in the subsequent abortion, which can result in excessive vaginal bleeding. In addition, co-administration o misoprostol can cause nausea and vomiting. Ulipristal, a second SPRM, is approved or use in emergency contraception (see below). Asoprisnil is an investigational progesterone receptor modulator that does not cause abortion but inhibits the growth o tissues derived rom the endometrium and myometrium. Preliminary studies indicate that ulipristal and asoprisnil may be e ective in the treatment o endometriosis and uterine leiomyomata (f broids). The di erences in the tissue specif cities o mi epristone, ulipristal, and asoprisnil are probably due to their di erences in in uencing the binding o transcription co actors to the progesterone receptor complex.

OH C

HO

C

CH

CH3 O Mestranol

FIGURE 30-9. Structure of synthetic estrogens. Ethinyl estradiol and mestranol are used in combination estrogen–progestin contraceptives.

transport o both egg and sperm, even i ovulation were to occur. In combination, these mechanisms explain the 95% e f cacy o combination oral contraception. The estrogen used in combination estrogen–progestin contraceptives is either ethinyl estradiol or mestranol (Fig. 30-9). Use o “unopposed” estrogens promotes endometrial growth, and early studies o estrogen-dominant contraceptives determined that these agents increase the risk o endometrial cancer. Because o this f nding, or a woman with a uterus, estrogen is always co-administered with a progestin to limit the extent o endometrial growth. Numerous progestins (Figs. 30-10 and 30-11) are used in estrogen–progestin contraceptives, and all are potent progesterone receptor agonists. Ideally, the progestin would possess activity only at progesterone receptors, but almost all currently available progestins also have some androgenic crossreactivity. Progestins vary in their androgenic activity. On a molar basis, norgestrel and levonorgestrel have the highest androgenic activity, while norethindrone and norethindrone acetate (Fig. 30-10) have lower androgenic activity. The so-called

OH

O OCCH3

C

CH

O

O

O CH3

Medroxyprogesterone acetate

The development o sa e, e f cacious contraceptives or women has revolutionized sexual practices. The two classes o widely used oral contraceptives are estrogen–progestin combinations and progestin-only contraception. The development o male contraception is an active area o research; current approaches to this therapy are discussed brie y at the end o the section. Combination estrogen–progestin contraception suppresses GnRH, LH, and FSH secretion and ollicular development, thereby inhibiting ovulation. The combination o an estrogen and a progestin is the most potent known method to suppress GnRH, LH, and FSH secretion. Co-administration o estrogen and progestin may also inhibit pregnancy by a number o secondary mechanisms, including alterations in tubal peristalsis, endometrial receptivity, and cervical mucus secretions. The latter actions could together inhibit the proper

CH

Ethinyl estradiol

Hormones and Hormone Analogues: Contraception

Combination Estrogen–Progestin Contraception

OH

Norethindrone O OCCH3

O OCCH3

C

CH

O

O

O CH3

Megestrol acetate

Norethindrone acetate

FIGURE 30-10. Structure of synthetic progestins. Medroxyprogesterone acetate is commonly combined with estrogen or hormone therapy in postmenopausal women. Megestrol acetate is o ten used as therapy or endometrial cancer. Norethindrone was the f rst progestin to be synthesized in quantities su f cient to mass-produce combination estrogen–progestin contraceptives. Norethindrone acetate is commonly used in contraceptives; it is metabolized to the parent compound, norethindrone.

C h a p t e r 3 0 Pharmacology of Reproduction OH

OH H2 C

C CH

C CH

O

Desogestrel

Levonorgestrel

OCCH3

OH C

O

O

C CH

CH

HON

Gestodene

Norgestimate

FIGURE 30-11. Structure of progestins commonly used in oral contraceptives. Levonorgestrel is the most androgenic of the commonly used progestins. Desogestrel, gestodene, and norgestimate are less androgenic than levonorgestrel.

third-generation progestins—ethynodiol, norgestimate , gestodene , and desogestrel (Fig. 30-11)—have even lower androgen receptor cross-reactivity. Drospirenone is a unique synthetic progestin that also has antiandrogenic activity. Combination estrogen–progestin contraceptives are available in three delivery systems: a vaginal ring, transdermal patches, and oral tablets. The vaginal ring consists o a silastic cylinder packed with ethinyl estradiol and a progestin, etonogestrel. The steroids are released with zero-order kinetics (see Chapter 3, Pharmacokinetics). The ring is placed in the vagina and remains there or 21 days. It is then removed, and 7 days later, a new ring is placed. During the 7 days ollowing removal o the ring, menses may ensue (see below). The contraceptive transdermal patch consists o a matrix that continually releases ethinyl estradiol and a synthetic progestin, norelgestromin. The patch is changed weekly or 3 weeks. During the ourth week, no patch is utilized and menses may occur. Classical regimens o combination oral contraceptive tablets consist o 21 days o drugs ollowed by 7 days o a placebo pill. The 7-day placebo period removes exogenous hormone stimulation, causing the endometrium to slough and resulting in withdrawal bleeding. Because the administration o progestin throughout the cycle inhibits the proli erative growth o the endometrium, most women experience lighter menstrual periods when taking combination oral contraceptives, and a woman’s menstrual cycle o ten becomes more regular. The 21-7 cycle ormulation was meant to simulate a 28-day cycle but is relatively arbitrary. By combining pill packs, “long cycles” o 42 active hormone pills ollowed by 7 days o hormone pills, or 63 active hormone pills ollowed by 7 days o hormone pills, can easily be prescribed. “Long cycle” regimens reduce the requency o menstrual bleeding but may increase the requency o irregular, unscheduled bleeding—so-called breakthrough bleeding. An even longer cycle ormulation o ethinyl estradiol and levonorgestrel is available, in which the drug combination is administered or 84 days ollowed by 7 days o placebo. This ormulation has contraceptive e f cacy equal to that o the classical regimen and reduces to our the total number o menstrual cycles

553

each year. Formulations containing 24 daily hormone pills and 4 days o placebo are also available. An advantage o this ormulation is that ovulation is not as likely to occur i a woman orgets to start her new cycle o pills or 3 or 4 days. Combination oral contraceptive ormulations include monophasic and triphasic hormone schedules. The standard ormulation, used by the majority o women, is a constant (monophasic) dose o estrogen and progestin or 21 days. Triphasic ormulations incorporate a constant dose o estrogen with a dose o progestin that increases each week during the 21 days o the cycle. The main advantage o triphasic administration is that the total amount o progestin administered over each month is reduced. Indeed, the general trend in recent years has been to decrease the quantities o administered estrogen and progestin to the smallest amount necessary or inhibition o ovulation. However, there are no clearly established di erences in either the adverse e ects or the clinical e f cacy o monophasic compared to triphasic therapy. In general, the lowest e ective dose o ethinyl estradiol is pre erred because low-dose estrogen is thought to minimize the risk o deep vein thrombosis (see below). A number o studies have been per ormed to assess the adverse e ects o long-term contraceptive use. These studies have shown that the incidence o deep vein thrombosis and the incidence o pulmonary embolism are increased with combination oral contraception. These complications occur in requently, and the absolute number o adverse events is low. Interestingly, pregnancy is associated with a greater risk o deep vein thrombosis and pulmonary embolism than treatment with estrogen-containing contraceptives. Studies have ailed to demonstrate any increase (or decrease) in breast cancer. Use o oral contraceptives is associated with an increase in gallbladder disease because estrogens increase the biliary concentration o cholesterol relative to that o bile salts, and the resulting decrease in cholesterol solubility promotes the ormation o gallstones. Oral contraceptives should not be administered to women over 35 who smoke, because the administration o contraceptives to this population is associated with an increase in thrombotic cardiovascular events. Recent studies have ocused on the benef ts rather than the adverse e ects o oral contraception. Modern combination oral contraceptives reduce the risk o endometrial cancer, probably because constant administration o a progestin inhibits endometrial growth. In addition, exogenous administration o an estrogen–progestin combination reduces the risk o ovarian cancer, probably by lowering circulating levels o gonadotropins. Overall, the consensus is that oral contraceptives have more benef cial than harm ul medical e ects.

Progestin-Only Contraception

In situations where estrogen may be contraindicated, the use o continuous low-dose oral progestins may be warranted. The two progestin-only oral contraceptives available in the United States, commonly re erred to as the mini-pill, are norethindrone and desogestrel. Progestin-only oral contraception prevents ovulation 70–80% o the time, probably because progestins alter the requency o GnRH pulsing and decrease anterior pituitary gland responsiveness to GnRH. Despite the relatively high requency o ovulation, this orm o contraception is 96–98% e ective, suggesting that secondary mechanisms—such as alterations in cervical mucus, endometrial receptivity, and tubal peristalsis—are also at work. Because progesterone inhibits

554 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

endometrial proli eration and promotes endometrial secretion, it may also be the case that an egg is unable to implant in an endometrium that is continually exposed to progestin. Patients taking these drugs do not typically menstruate, but breakthrough spotting and irregular, light menstrual periods commonly occur during the f rst year o administration. Progestin-only contraceptives are also available as injectables and implants. Medroxyprogesterone acetate ( ormulated as 104 mg or subcutaneous injection or 150 mg or intramuscular injection) can be given parenterally every 3 months (Fig. 30-10). This dosage orm is especially e ective or women who have di f culty remembering to take a daily (pill) or weekly (patch) agent. A silastic implant is also available that releases etonogestrel and is e ective or 3 years. The implant is typically inserted into the dorsal side o the orearm.

Emergency (Morning-After) Contraception

Emergency contraception re ers to the administration o medications to prevent pregnancy a ter ailure o a barrier contraceptive (condom breakage) or recent unprotected intercourse (including sexual assault). Both levonorgestrel, a potent progestin, and ulipristal, an SPRM, are approved as emergency contraceptives. Emergency hormonal contraception is most e ective when administered within 120 hours o the exposure. Both levonorgestrel and ulipristal prevent pregnancy by inter ering with ovulation.

Male Contraception

The goal o hormonal male contraception is to suppress endogenous production o sperm reversibly, generating a state o azoospermia (absence o sperm in the ejaculate) without suppressing libido or erectile unction. Reliable inhibition o spermatogenesis is a di f cult task, because even a 99% reduction in spermatogenesis could result in a su f cient number o viable sperm or ertilization. Initial studies o male contraception centered on the parenteral administration o testosterone esters such as testosterone enanthate or testosterone undecanoate. As an end product o the hypothalamicpituitary-reproduction axis, testosterone signif cantly suppresses gonadotropin release. The reduced circulating levels o LH and FSH are unable to stimulate Sertoli cell unction, and decreased spermatogenesis results. In a largescale clinical trial, this approach produced a contraceptive ailure rate o 1 per 100 man-years. Recent clinical trials indicate that the administration o both an androgen and a progestin may be superior to an androgen alone in suppressing spermatogenesis because the combination more completely suppresses GnRH secretion and gonadotropin release. The ollowing combinations have been demonstrated to be e ective, reversible male contraceptives: parenteral testosterone enanthate plus daily oral levonorgestrel; parenteral testosterone undecanoate plus injectable medroxyprogesterone acetate; and transdermal administration o testosterone and a synthetic progestin, Nestorone ® . The main di f culties with this approach have been the large population variability in the degree o spermatogenesis inhibition (on average, only 60% o men become azoospermic) and the signif cant adverse e ects o acne, weight gain, polycythemia, and a potential increase in prostate size.

Hormones and Hormone Analogues: Replacement Estrogens, progestins, and androgens are used as replacement therapies in cases o hormone def ciency.

Estrogens and Progestins

The realization that estrogen loss at menopause has deleterious e ects has led to the development o perimenopausal and postmenopausal hormone replacement therapy ( or additional detail, see Chapter 32). The principal indication or such therapy is to suppress hot ashes and treat atrophy o the urogenital tissues, which may mani est as dry vagina. For women with a uterus, estrogen therapy must be combined with progestin therapy to prevent the induction o endometrial cancer. For women without a uterus, estrogen alone is typically given or hormone therapy. The Women’s Health Initiative (WHI) is a large clinical trial that evaluated the health benef ts and risks o hormone therapy in postmenopausal women. Separate clinical trials tested estrogen alone against a placebo in women without a uterus and continuous estrogen–progestin against a placebo in women with a uterus. The results o the study, expressed as the relative risk or various endpoints o hormone treatment versus placebo, are presented in Table 30-3. Estrogen treatment did not increase the risk o coronary heart disease or breast cancer, but it did increase the risk o stroke and it decreased the risk o osteoporotic racture. Continuous estrogen–progestin treatment increased the risk o stroke, pulmonary embolism, and breast cancer, and it decreased the risk o osteoporotic racture (see Chapter 32). Given the balance o risks and benef ts, the current recommendation or postmenopausal women is to use hormone therapy only to treat bothersome symptoms such as vasomotor symptoms or vaginal dryness and to use the lowest possible dose o hormone therapy or the shortest period o time. A ter the results o the WHI trial were published in 2002, there was a marked decrease in the number o menopausal women using estrogen–progestin therapy and a parallel reduction in the number o cases o diagnosed breast cancer. Many synthetic progestins are available or use in hormone therapy or menopausal women. Recent epidemiologic studies TABLE 30-3 Summary of Findings from the Women’s Health Initiative ESTROGEN ALONE VS. PLACEBO

CONTINUOUS ESTROGEN– PROGESTIN VS. PLACEBO

Sample size

10,739

16,608

Mean age o subjects

63 years old

63 years old

Mean duration o hormone use

6.8 years

5.2 years

Coronary heart disease

0.91 (0.75–1.12)

1.29 (1.02–1.63)

Breast cancer

0.77 (0.59–1.01)

1.26 (1.00–1.59)

Stroke

1.39 (1.10–1.77)

1.41 (1.07–1.85)

Pulmonary embolism

1.34 (0.87–2.06)

2.13 (1.39–3.25)

Osteoporotic hip racture

0.61 (0.41–0.91)

0.67 (0.47–0.96)

Osteoporotic vertebral racture

0.62 (0.42–0.93)

0.65 (0.46–0.92)

Data represent hazard ratios (95% conf dence intervals) o various events during treatment with hormone therapy or placebo. Conf dence intervals that cross the value o 1.00 are not statistically signif cant (p .05).

C h a p t e r 3 0 Pharmacology of Reproduction

555

report that micronized progesterone may be associated with a lower risk o breast cancer than other commonly used synthetic progestins such as medroxyprogesterone acetate. Micronization is a process in which the progesterone crystals are synthesized with a diameter in the nanometer range, thus acilitating their absorption when administered orally. Like contraceptives, hormone therapy is available as oral tablets, transdermal patches, and vaginal rings and tablets. A vaginal ring that elutes estradiol at a controlled dose rate (see Chapter 55, Drug Delivery Modalities) provides local administration o estrogen and minimal systemic absorption o the drug. The vaginal ring is an e ective therapy or postmenopausal vaginal dryness and atrophy.

resulting in gynecomastia. In addition, high plasma levels o androgens are associated with erythrocytosis, severe acne, and derangements in lipid metabolism (increased low-density lipoprotein [LDL] and decreased high-density lipoprotein [HDL]). Some athletes have recently started to use injections o hCG to stimulate endogenous Leydig cell testosterone production, hoping to avoid detection by sports authorities. SERMs and aromatase inhibitors have also been used by athletes in an attempt to increase endogenous LH secretion and Leydig cell testosterone production.

Androgens

The male and emale hormones o reproduction share signif cant mechanistic overlap with one another. Androgens, estrogens, and progestins are all steroid hormones that exert their physiologic action by binding to intracellular receptors, translocating to the nucleus, and altering gene transcription. Recent evidence suggests that estrogens may also act on membrane receptors to mediate nongenomic e ects. Derangements in the physiologic e ects o reproductive hormones can involve disruption o the hypothalamic-pituitary-reproduction axis, inappropriate growth o hormone-dependent tissue, or decreased activity o gonadal hormones at target tissues. Currently available pharmacologic agents can modi y the endocrine axis (e.g., GnRH agonists), inhibit synthesis o active hormones (e.g., 5 -reductase inhibitors, aromatase inhibitors), or inhibit endorgan e ects at the receptor level (e.g., SERMs, SPRMs, antiandrogens). Oral contraceptives, such as estrogen–progestin combinations and progestin-only contraception, disrupt the exquisite cyclicity o the menstrual cycle and thus suppress ovulation. The development o an e ective male contraceptive has met a number o obstacles but should represent a major pharmacologic advance in the uture. Exciting progress is also being made in the design o new SERMs that possess a variety o tissue-specif c activities; such research may result in new agents e ective or both prevention o breast cancer and treatment o postmenopausal osteoporosis.

Androgen replacement is an e ective therapy or hypogonadism. Oral testosterone is ine ective because o its high f rst-pass metabolism by the liver. Two esters o testosterone, testosterone enanthate and testosterone cypionate , can be administered intramuscularly. A preparation o either o these agents, injected every 2–4 weeks, increases plasma testosterone to physiologic concentrations in hypogonadal men. Transdermal testosterone patches have also been developed; this drug delivery system has the advantages that plasma testosterone levels remain relatively constant and f rst-pass hepatic metabolism is bypassed. Testosterone is also available in a topical gel ormulation; using this preparation on a once-a-day application schedule, plasma testosterone levels gradually increase until they reach physiologic replacement levels a ter 1 month o application. Testosterone can also be administered as a tablet that adheres to the buccal mucosa, resulting in rapid systemic absorption o the drug. Aging men sometimes develop symptoms and signs o hypogonadism, such as decreased energy, decreased libido, gynecomastia, decreased muscle mass, and acial hair growth. Recent guidelines recommend that androgen replacement therapy be o ered to men only with consistent symptoms and signs o hypogonadism and low plasma testosterone levels ( 3.0 ng/mL). Testosterone should not be administered to men with prostate cancer because it may stimulate growth o the tumor. A novel approach is to combine parenteral testosterone replacement with androgen receptor blockade using utamide. The testosterone replacement stimulates an increase in muscle mass, even in the presence o androgen receptor blockade. However, utamide does block prostate enlargement caused by testosterone replacement. Some athletes abuse androgens by sel -administration at supratherapeutic levels. Androgens have been demonstrated to increase muscle mass and at- ree mass. In one survey, approximately 5% o high school athletes reported that they had used androgen supplements. Almost every type o androgen has been abused in an attempt to enhance athletic per ormance, including the adrenal hormone precursors androstenedione and dehydroepiandrosterone. Covert laboratories are continuously inventing new synthetic androgens that have not yet been recognized by standard drug testing programs. These “designer” androgens are meant to enhance athletic per ormance and to be undetectable by sports regulatory authorities. Pharmacologic doses o androgens suppress the hypothalamic-pituitary-reproduction axis, resulting in suppression o testicular unction, decreased sperm production, and impaired ertility. Because many androgens can be converted to estrogens by aromatase, pharmacologic doses o androgens can also cause an increase in plasma estrogen,

CONCLUSION AND FUTURE DIRECTIONS

Suggested Reading Borst SE, Yarrow JG, Conover CF, et al. Musculoskeletal and prostate e ects o combined testosterone and f nasteride administration in older hypogonadal men: a randomized controlled trial. Am J Physiol Endocrinol Metab 2014;306:E433–E442. (Example o combining testosterone and f nasteride.) Legro RS, Arslanian SA, Ehrmann DA, et al. Diagnosis and treatment o polycystic ovary syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2013;98:4565–4592. (Recent clinical recommendations regarding treatment o polycystic ovarian syndrome.) Legro RS, Brzyski RG, Diamond MP, et al. Letrozole versus clomiphene or in ertility in the polycystic ovary syndrome. N Engl J Med 2014;371:119– 129. (Clinical trial that demonstrated improved ertility rates using letrozole among patients with polycystic ovarian syndrome.) Manson JE, Chlebowski RT, Ste anick ML, et al. Menopausal hormone therapy and health outcomes during the intervention and extended poststopping phases o the Women’s Health Initiative randomized trials. JAMA 2013;310:1353–1368. (Integrated overview o long-term health benef ts and risks with menopausal hormone therapy.) Pagani O, Regan MM, Walley BA, et al. Adjuvant exemestane with ovarian suppression in premenopausal breast cancer. N Engl J Med 2014;371: 107–118. (Clinical trial that demonstrated reduced rates o recurrent breast cancer with exemestane.) Winiko B, Dzuba IG, Chong E, et al. Extending outpatient medical abortion services through 70 days o gestational age. Obstet Gynecol 2012; 120:1070–1076. (Clinical trial o medication pregnancy termination through day 70 o pregnancy.)

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558 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

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560 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

r tis s uee s

B lo o d

β c e llll E n do doge ge n ous ins n ulin (fro (f ro m β cee ll) ll)) o r Exxo g e no no u us ins ulin

Glu ccos e Glu ( rom GI tra (f ra ctt a nd live r)

G luc Gluc u c ooss e ucos

Gluc Gl ucoo s e

Me ta bolis m

31 Ins ulilinn s e cr cree tio tionn

Me ta ta bboo liss m

To tis is s ue s

To tis s u e s

ATP

Diazoxide

I s ulin In cle c e ll

Pharmacology of the Endocrine Pancreas and Glucose Homeostasis Ins uli linn

S ulf u lffo ny n ylur yl g linid in ide s , GLP-1 ana DPPP 4 in inh h

IIn nss uli u linn

Adipo s e c e lll

Live r c e ll ll

Giu lio R. Ro m e o a n d S t e ve n E. S h o e ls o n Glucos e

Glycog ge n

Gluc Gl ucos e

Triglyce ride

Gluc Gl ucos os e

Gl G yc y og oge n

Glucos Gluc o e Gluc Gl uc INTRODUC INTRODU INTRO ODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561–562 62 PHARMACO CO CO OLO LOGI L GIC CLASSES AND A AG GEN G NTS . . . . . . . . . . . . . . . . 56 5699 rapy apy py for Diab abet etes es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569 BIOC BI OCHE HEM MISTRY AND PHYSIOLOG GY . . . . . . . . . . . . . . . . . . . . . .P. 561 P A γ Therap PAR Gl ge ne n s is Glucone oge Insulin Replacement: Exogee no In nouus us Insul IInsulin n. . . . . . . . . . . . . . . 5770 Panc Pa ncre reat atic Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5 561 Metforminn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 Energy Homeostasis i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 Bigg u uan anid ide s id Insulin Secretaggog In ogue ueBi y e reas and Glinidess . . . . . GL 571P--1 analo g ue , GLPne s Energy Repletion and the Fed State . . . . . .Thiazo . . . . . . .lidine . . . . . .dio 562n GLP GL P-11-B Based “Increti tin”” Therapi pies es:: GL GLPP 1 Re Recept p or DPPDP P -4 in inhi hibi bito to r, Fasting and Starvationn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5633 Agoni nist stss (G (GLP LP-11Ra R ’s)) and DPP-4 Inhibitors. . . . . . . . . . . am 572 lin anallo g ue amyl Insu uli linn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563 Inhibitors of Glucose Reabsorpti tion in th thee Ki Kidn dney ey:: Biochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563 SGLT-2 Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564 Thiazolidinediones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 Action at Target Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Amylin Analogue: Pramlintide . . . . . . . . . . . . . . . . . . . . . . . . 573 Glucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Bile Acid Sequestrants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573 Amylin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Inhibitors of Intestinal Glucose Absorption. . . . . . . . . . . . . 573 Somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Dopamine Receptor Agonistss . . . . . . . . . . . . . . . . . . . . . . . . 573 Incretins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Pharmacologic Management Strategies. . . . . . . . . . . . . . . 573 PATHOPHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Therapy for Hyperinsulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . 573 Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Glucagon as a Therapeutic Agent. . . . . . . . . . . . . . . . . . . . . . . 574 Type 1 Diabetess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Type 2 Diabetess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 574 Morbidity and Mortalityy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 Hypoglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569

INTRODUCTION This chapter reviews the physiology and pharmacology o insulin, glucagon, and the other major hormones that regulate glucose homeostasis. Diabetes mellitus is clinically the most common disease o these endocrine axes, and the majority o the chapter is devoted to the physiology and pharmacology o insulin. Type 1 diabetes mellitus is caused by an absolute def ciency o insulin secretion, and type 2 diabetes mellitus is caused by insu f cient (or dys unctional) insulin secretion to overcome insulin resistance in target tissues. Medical students may be interested to note that Charles Best, a ourth-year medical student in Canada, had a signif cant role in the identif cation o insulin. Along with his mentor, Frederick Banting, Best isolated a pancreatic extract rom dogs that could reduce blood glucose in diabetic dogs and humans. Although the 1923 Nobel Prize in Medicine or Physiology was jointly awarded to surgeon Frederick Banting and physiologist J. J. R. MacLeod, Banting shared his award with Best.

BIOCHEMISTRY AND PHYSIOLOGY Pancreatic Anatomy The pancreas is a glandular organ that contains both exocrine and endocrine tissue. The exocrine portion—which constitutes 99% o the pancreatic mass—secretes bicarbonate and digestive enzymes into the gastrointestinal (GI) tract. Scattered within the exocrine tissue are nearly one million small islands o endocrine tissue that secrete hormones directly into the blood. These tiny endocrine glands, collectively called islets of Langerhans , include several di erent cell types that secrete di erent hormones: -cells release glucagon; -cells release insulin and amylin; -cells release somatostatin and gastrin; and PP cells release pancreatic polypeptide.

Energy Homeostasis The storage and subsequent release o nutrients provides a homeostatic mechanism or sustained cellular nutrition in the absence o continuous eeding. Multiple hormones are 561

562 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

At h e r a n n u a l ch e ck-u p , 55-ye a r-o ld Mrs . S co m p la in s o a tig u e a n d re q u e n t u rin a tio n (p o lyu ria ), e ve n a t n ig h t. S h e a ls o re p o rts d rin kin g la rg e vo lu m e s o lu id s (p o lyd ip s ia ) to q u e n ch h e r th irs t. Th e s e s ym p to m s h a ve w o rs e n e d d u rin g th e p a s t 2 ye a rs a n d co rre la te w ith a 15-lb w e ig h t ga in ove r th e s a m e p e rio d . He r cu rre n t b o d y m a s s in d e x (BMI; th e w e ig h t in kilo g ra m s d ivid e d b y th e s q u a re o th e h e ig h t in m e te rs ) is 32, th u s m e e tin g crite ria o r cla s s I o b e s ity. S h e is “ ru s tra te d ” b y h e r in e e ctive n e s s to cu rb p o rtio n s ize s a n d m a in ta in a ro u tin e o p hys ica l a ctivity. S h e d e n ie s o th e r u rin a ry s ym p to m s ; h e r kid n e y u n ctio n is e s s e n tia lly n o rm a l. He r p a s t m e d ica l h is to ry is re m a rka b le o r hyp e rlip id e m ia o r th e p a s t 10 ye a rs . Bo th o h e r p a re n ts d ie d o co ro n a ry h e a rt d is e a s e in th e ir e a rly 60s . On p h ys ica l e xa m in a tio n , Mrs . S is m o d e ra te ly o b e s e b u t o th e rw is e a p p e a rs n o rm a l. Glu co s e is d e te cte d in h e r u rin e , b u t p ro te in s a n d ke to n e s a re n o t. He r b lo o d te s ts a re s ig n if ca n t o r e le va te d g lu co s e (210 m g /d L), to ta l ch o le s te ro l (340 m g /d L), a n d Hb A1c (a m e a s u re o g lu co s e co va le n tly b o u n d to h e m o g lo b in ; 8.2%) le ve ls . Th e p h ys icia n e xp la in s to Mrs . S th a t s h e m o s t like ly h a s typ e 2 d ia b e te s m e llitu s . In th is d is e a s e , th e b o d y a ils to re s p o n d n o rm a lly to in s u lin (in s u lin re s is ta n ce ) a n d ca n n o t

involved in controlling the uptake, utilization, storage, and release o nutrients. Insulin promotes the uptake and storage o glucose and other small, energy-containing molecules. The “counterregulatory” hormones oppose the actions o insulin and promote glucose release (Table 31-1). These hormones include (1) glucagon rom islet -cells, (2) the catecholamines norepinephrine and epinephrine rom the sympathetic nervous system and adrenal medulla, (3) the glucocorticoid cortisol rom the adrenal cortex, and (4) growth hormone rom the pituitary gland. Glucagon-like peptide-1 (GLP-1) rom the GI tract enhances insulin release in response to an ingested meal, while amylin suppresses endogenous production o glucose in the liver. Blood glucose is easily measured and provides an accurate guide to the current balance o insulin and the counterregulatory hormones. This balance normally keeps blood glucose levels within a narrow range (70–120 mg/dL) regardless o recent ood intake. Blood glucose excursions are much greater in patients with diabetes, potentially rising higher than 400 mg/dL in instances o poor control. The drugs used to treat diabetes, particularly insulin and the insulin secretagogues (sul onylureas and glinides), may reduce blood glucose values below the normal range o 70 mg/dL. Severe hypoglycemia, which is def ned by the patient requiring assistance to administer glucose or glucagon to restore normal blood glucose levels, is dangerous because organs— particularly the brain—depend on a constant supply o glucose or proper unctioning. Conversely, chronic hyperglycemia is toxic to many cells and tissues.

p ro d u ce a s u f cie n t a m o u n t o in s u lin to ove rco m e th is re s is ta n ce . Th e p h ys icia n d is cu s s e s th a t li e s tyle in te rve n tio n s (a d h e rin g to a b a la n ce d d ie t w ith co n tro lle d to ta l ca lo rie in ta ke a s w e ll a s in cre a s in g h e r e xe rcis e ) a re th e co rn e rs to n e o d ia b e te s tre a tm e n t. Th e p h ys icia n a ls o d is cu s s e s th e b e n e f ts a n d ris ks o a n tid ia b e tic m e d ica tio n s a n d p re s crib e s m e t o rm in (a b ig u a n id e ) o r h e r d ia b e te s .

Questions 1 . What are the cellular and molecular actions of insulin? 2 . What is the etiology of diabetes mellitus, and how is type 1 diabetes mellitus different from type 2 diabetes mellitus? 3 . In addition to alleviating her polyuria and polydipsia, why is it important to control Mrs. S’s diabetes (i.e., what acute and chronic complications could arise)? 4 . What do the blood glucose and HbA1c levels indicate about Mrs. S’s diabetes? Are there circumstances under which one parameter could be elevated and the other could be normal? 5 . What are the effects of antidiabetic medications on body weight? Why might Mrs. S’s physician have chosen metformin to treat her diabetes?

Energy Repletion and the Fed State

A ter a meal, complex carbohydrates are broken down to monosaccharides (e.g., glucose, galactose, and ructose) in the lumen o the GI tract and transported into GI epithelial cells by a combination o active and passive apical membrane transporters. Sugars are then transported by basal membrane transporters rom the epithelial cell cytosol to intercellular spaces, rom which the sugars continue into the bloodstream. Elevated glucose in the blood is the signal or pancreatic -cells to release insulin, which enters the portal vein. The liver, there ore, receives the highest concentrations o insulin concurrently with the nutrients absorbed rom the digestive tract. The liver and the other energy-storing tissues, such as skeletal muscle and adipose tissue, are the primary target tissues for insulin (Fig. 31-1). Insulin also acts on pancreatic -cells to suppress the secretion o glucagon. The hormone leptin is not directly involved in nutrient regulation, but it plays an important role in the neuroendocrine response to energy storage and long-term energy balance. Leptin is secreted by adipocytes and detected by receptors in the hypothalamus. Its concentration in the plasma is proportional to total at mass. Thus, leptin signals from the periphery to the central nervous system that energy stores (in the form of adipose tissue) are replete. Leptin also suppresses appetite, which switches the body rom an energy-accumulating state to a state o energy utilization. This allows growth and reproduction to be switched on, whereas the lack o leptin, as occurs in prolonged starvation, results in persistently increased appetite and suppression o energy-utilizing unctions.

C h a p t e r 3 1 Pharmacology of the Endocrine Pancreas and Glucose Homeostasis

563

TABLE 31-1 Effects of Selected Hormones on Energy Homeostasis HORMONE

SOURCE

TARGET TISSUES

ACTION

Glucagon

-Cell (pancreas)

Liver (adipose, skeletal muscle)

Promotes glycogenolysis and gluconeogenesis in liver

Insulin

-Cell (pancreas)

Liver (adipose, skeletal muscle)

Promotes uptake of glucose, amino acids, and fatty acids from blood into cells for storage as glycogen, protein, and triglyceride

Amylin

-Cell (pancreas)

Central nervous system

Suppresses glucagon release Slows gastric emptying Decreases food intake

Somatostatin

-Cell (pancreas) GI tract Hypothalamus

Other islet cells, GI tract, brain, and pituitary gland

Decreases release of insulin and glucagon Decreases GI tract motility and hormone release Decreases growth hormone secretion

Epinephrine

Adrenal medulla

Many

Promotes glycogenolysis in liver Lipolytic via activation of hormone-sensitive lipase

Cortisol

Adrenal cortex

Many

Antagonizes insulin action at target tissues Promotes gluconeogenesis in liver, protein breakdown in muscle

GLP-1

Ileum

Endocrine pancreas, stomach, brain, heart

Increases -cell mass and insulin secretion Delays gastric emptying Decreases food intake and glucagon secretion

Leptin

Adipocytes

CNS (basomedial hypothalamus)

Signals adequacy of body energy stores Decreases food intake Permits energy-intensive neuroendocrine functions

Physiologically, insulin and glucagon are the two most important hormones controlling glucose homeostasis. Insulin promotes energy storage in target tissues. Glucagon, epinephrine, cortisol, and growth hormone—the “counterregulatory” hormones—act to raise blood glucose and thereby to counteract the effects of insulin. By acting as a “fat sensor,” leptin signals total-body energy storage and regulates long-term energy balance. GI, gastrointestinal. GLP-1, glucagon-like peptide-1.

A key intracellular mediator o energy storage is the nuclear receptor peroxisome proliferator-activated receptor(PPAR ). PPAR is a transcription actor that has roles in both adipose cell di erentiation and lipid metabolism. PPAR activation decreases serum ree atty acid levels and increases lipogenesis in adipose tissue, which increases the storage o atty acids in adipose tissue. This allows other tissues—such as the liver—to lower their at content, lower their glucose production, and increase their insulin sensitivity. PPAR is the target or the thiazolidinedione (TZD) class o diabetes drugs. Like many small hydrophilic molecules, glucose is reely f ltered at the glomerulus but is normally reabsorbed almost completely by the sodium-glucose co-transporter 2 (SGLT-2) in the proximal convoluted tubule. There ore, the glucose content o the urine is normally negligible. However, the transport maximum (Tmax) or SGLT-2 is reached at glucose concentrations o 180–200 mg/dL, so, in diabetes, when blood glucose levels are over 200 mg/dL and SGLT-2 is saturated, glucose does appear in the urine. (Urinary glucose “dipsticks” were used by patients with diabetes to monitor glycemic control long be ore “f nger stick” measures o blood glucose came into use.) The principle o losing glucose in the urine has been capitalized on with the development o selective inhibitors o SGLT-2, which cause enough glucose to remain in the urine that blood glucose levels, HbA1c levels, and even body weight are lowered.

Fasting and Starvation

As the blood glucose concentration decreases, pancreatic cells release increasing amounts o glucagon and pancreatic

-cells secrete decreasing amounts o insulin. In contrast to insulin, which promotes the cellular uptake o glucose in the ed state, glucagon mobilizes glucose rom the liver by stimulating gluconeogenesis and glycogenolysis. As asting continues, catecholamine and glucocorticoid levels also increase, promoting the release o atty acids rom adipose tissue and the breakdown o protein to amino acids in muscle. In low-energy (low adenosine triphosphate [ATP]) states, the enzyme adenosine 5 -monophosphate-activated protein kinase (AMPK) also triggers a shi t rom anabolic to catabolic activities. AMPK is present in tissues throughout the body, and it helps to regulate energy metabolism at both the cellular and organismal levels. Exercise activates AMPK, which increases muscle uptake o glucose. Activated AMPK also decreases glucose production and the synthesis o lipids and proteins by the liver. The pharmacologic e ects o metormin and other biguanides are incompletely understood despite intense investigation but are contributed to by both AMPK-dependent and AMPK-independent mechanisms.

Insulin Biochemistry

Insulin is a 51-amino acid protein composed o two peptide chains that are linked by two disulf de bridges. Its name comes rom the Latin insula (meaning “island,” a ter the islets o Langerhans). The human pancreas contains approximately 8 mg o insulin, o which 0.5 to 1.0 mg is secreted and replenished daily. Insulin is initially synthesized in pancreatic -cells as preproinsulin, which is processed f rst to proinsulin and then insulin and ree connecting (C) peptide (Fig. 31-2).

564 PRINCIPLES OF ENDOCRINE PHARMACOLOGY Ga s tric e mptying

GI tract

GLP-1 analogue, amylin analogue

Die ta ry comple x ca rbohydra te s

α -glucosidase inhibitors

Other tissues

Glucosidases

Blood

β cell Endoge nous ins ulin (from β ce ll) or Exogenous insulin

Glucos e (from GI tra ct a nd live r)

Glucos e

Glucos e

Me ta bolis m

Ins ulin s e cre tion Me ta bolis m

To tis s ue s

To tis s ue s Diazoxide

Ins ulin

Ins ulin

Muscle cell

Glucos e

Sulfonylureas, glinides, GLP-1 analogue, DPP-4 inhibitor

Ins ulin

Adipose cell

Glycoge n

ATP

Liver cell

Glucos e

Glucos e

Triglyce ride

Glycoge n

Glucos e P PARγ

Gluca gon

Glucone oge ne s is

Thiazolidinediones

Biguanides

GLP-1 analogue, DPP-4 inhibitor, amylin analogue

FIGURE 31-1. Physiologic and pharmacologic regulation of glucose homeostasis. Dietary complex carbohydrates are broken down to simple sugars in the GI tract by the action of glucosidases; simple sugars are then absorbed by GI epithelial cells and transported into the blood. Glucose in the blood is taken up by metabolically active tissues throughout the body. Glucose metabolism in pancreatic -cells increases cytosolic ATP, which stimulates insulin secretion. Released insulin acts on plasma membrane insulin receptors in target tissues (muscle, adipose, liver) to increase glucose uptake and storage as glycogen or triglyceride. Glucose is also taken up by other cells and tissues to fuel metabolism. In muscle and liver, insulin promotes glucose storage as glycogen. In adipose cells, insulin promotes glucose conversion to triglycerides. Peroxisome proliferator-activated receptor (PPAR ) also promotes the conversion of glucose to triglycerides in adipocytes. Glucagon promotes both hepatic gluconeogenesis and glycogen breakdown; the newly generated glucose is transported out of the liver cell into the blood. Note that glucose from dietary complex carbohydrates and insulin secreted by pancreatic -cells both enter the liver in high concentrations through the portal circulation (not shown). Pharmacologic interventions that decrease blood glucose levels include delaying gastric emptying with a GLP-1 analogue or an amylin analogue; inhibiting intestinal -glucosidases with an -glucosidase inhibitor; administering exogenous insulin; augmenting -cell insulin secretion with sulfonylureas (SFUs), glinides, or incretins; suppressing glucagon and gluconeogenesis with incretins, an amylin analogue, or biguanides; and enhancing the action of insulin in adipose cells with thiazolidinediones. To treat hyperinsulinemic hypoglycemia, diazoxide inhibits pancreatic -cell insulin secretion.

Secretion

Resting pancreatic -cells are poised to secrete insulin, which is pre ormed and stored in secretory vesicles at the plasma membrane. The low basal rate o insulin secretion is increased dramatically upon exposure o -cells to glucose. Glucose enters -cells via GLUT2, a specif c plasma membrane transporter. As blood glucose rises (e.g., a ter eeding), more enters the cell, where it is phosphorylated to glucose-6-phosphate and enters the glycolytic pathway and the citric acid cycle. ATP is produced and ADP consumed by these processes, and the ATP/ADP ratio in the -cell rises.

This modulates the activity o a membrane-spanning ATPsensitive K channel (K /ATP channel). When open, the K / ATP channel hyperpolarizes the cell by allowing an outward f ux o K , and insulin release is inhibited. When closed, the cell depolarizes and insulin is released. Because ATP inhibits the channel and ADP activates the channel, a high intracellular ATP/ADP ratio closes the K /ATP channel. Depolarization o the -cell activates voltage-gated Ca2 channels to promote the in ux o extracellular Ca2 and the usion o insulin-containing vesicles with the plasma membrane, which increases insulin release into the circulation (Fig. 31-3).

C h a p t e r 3 1 Pharmacology o the Endocrine Pancreas and Glucose Homeostasis Dipe ptide cle a va ge s ite

NH2

S UR1

Kir6.2

Inhibitors

Arg Lys

565

Activators

1 2

Mg 2+-ADP

Sulfonylurea/ glinide

3

Diazoxide Cys

Cys

ATP

Cys Cys

K+/ATP channe l

GLUT2 tra ns porte r Arg Arg

Proinsulin

Cys

COOH As n

Lys P ro

Cys

K+ conducta nce

Glucos e

Dipe ptide cle a va ge s ite

Me mbra ne de pola riza tion

ATP Me ta bolis m ADP

B-cha in NH2

Ca 2+

P a ncre a tic β ce ll

1 2

2

3

As n

C-pe ptide

4

Cha nge d to lys ine in insulin glulisine

Ins ulin ve s icle s

COOH

A-cha in NH2

1

Ca 2+ influx

Ins ulin s e cre tion

FIGURE 31-3. Physiologic and pharmacologic regulation of insulin reCys Cys

Cha nge d to gluta mic a cid in insulin glulisine

Cys Cys

Myris toyla te d in insulin detemir Two a dditiona l a rginine re s idue s in insulin glargine

Cha nge d to glycine in insulin glargine

COOH 19 Cys

Insulin

Cys

COOH As n

NH2

Lys P ro

27

Re ve rs e d in insulin lispro

26

Cha nge d to a s pa rtic a cid in insulin aspart

FIGURE 31-2. Processing of human insulin. Preproinsulin is synthesized and exported into the endoplasmic reticulum, where the signal peptide (not shown) is cleaved to generate proinsulin (top panel). Intramolecular disulf de bonds (cys–cys) aid in the proper olding o proinsulin. Proinsulin is transported to secretory vesicles, where prohormone convertases act on dipeptide cleavage sites in proinsulin (boxes) to generate insulin and connecting (C) peptide. Two disulf de bonds aid in holding the A chain and B chain o insulin together. Insulin and C peptide are secreted together rom the pancreatic -cell (bottom panel). Modif cations to insulin’s amino acid sequence result in the altered pharmacokinetics o the various insulin analogues; lispro, aspart, and glulisine are rapid-acting insulins, whereas glargine and detemir have slower absorption. The substitutions are: in lispro, the positions o ProB28 and LysB29 are reversed; in aspart, ProB28 is replaced by aspartic acid; in glulisine, AsnB3 and LysB29 are replaced by lysine and glutamic acid, respectively; in glargine, AsnA21 is replaced by glycine, and two additional arginines are added to the carboxyl terminus o the B-chain; and in detemir, a atty acid (myristic acid) is esterif ed to the -amino group o LysB29.

lease from pancreatic -cells. When the K /ATP channel is open in its basal state, less insulin is released; when the K /ATP channel is closed, more insulin is released. In the basal state, the plasma membrane o the -cell is hyperpolarized, and the rate o insulin secretion rom the cell is low. However, when glucose is available, it enters the cell via GLUT2 transporters in the plasma membrane and is metabolized to generate intracellular ATP. ATP binds to and inhibits the plasma membrane K /ATP channel. Inhibition o the K /ATP channel decreases plasma membrane K conductance; the resulting depolarization o the membrane activates voltage-gated Ca 2 channels and thereby stimulates an in ux o Ca 2 . Ca 2 mediates usion o insulincontaining secretory vesicles with the plasma membrane, leading to insulin secretion. The K /ATP channel, an octamer composed o Kir6.2 and SUR1 subunits, is the target o several physiologic and pharmacologic regulators. ATP binds to and inhibits Kir6.2, while sul onylureas (SFUs) and glinides bind to and inhibit SUR1; all three o these substances promote insulin secretion. The GLP-1 mimetic exenatide, acting as an agonist at G protein-coupled GLP-1 receptors in the plasma membrane o the pancreatic -cell, also stimulates glucose-dependent insulin secretion. This action o exenatide appears to be mediated by an increase in intracellular cyclic AMP and may involve an indirect e ect on the K /ATP channel (not shown). Mg2 -ADP and diazoxide bind to and activate SUR1, thereby inhibiting insulin secretion. (For clarity, only our o the eight K /ATP channel subunits are shown.)

-cell K /ATP channels are octameric structures containing four subunits of Kir6.2 and four subunits of the sulfonylurea receptor, SUR1. The Kir6.2 tetramer forms the pore of the K /ATP channel, while the associated SUR1 proteins regulate the channel’s sensitivity to ADP and pharmacologic agents, including sulfonylurea and related insulin secretagogue drugs. Mutations in Kir6.2 or SUR1 can result in hyperinsulinemic hypoglycemia because the channel remains closed and the -cell remains continually depolarized even when the extracellular glucose concentration and the intracellular ATP/ADP ratio are low.

566 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

In addition to blood glucose, G protein-mediated pathways, parasympathetic activity, and the GI hormones GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) also inhibit K /ATP channel activity and stimulate insulin secretion. -Cell exposure to nutrients promotes insulin transcription, translation, processing, and packaging in addition to insulin secretion.

Action at Target Tissues

Insulin binds receptors on the sur ace o target cells. Although virtually all tissues express insulin receptors , the energy-storing tissues (liver, muscle, and adipose) express higher levels o the receptor and have greater responses to insulin and thus constitute the main insulin target tissues. The insulin receptor (Fig. 31-4) is a glycoprotein consisting o our disulf de-linked subunits and two intracellular tyrosine kinase domains. The binding o insulin to the extracellular portion o the insulin receptor activates the intracellular tyrosine kinase, which phosphorylates itsel and the intracellular insulin receptor substrate proteins (IRS). Tyrosine-phosphorylated IRS-1 recruits second messenger proteins that contain phosphotyrosine-binding src homology 2 (SH2) domains. Type IA phosphatidylinositol 3-kinase (PI3-kinase) is one such SH2 domain-containing protein that generates second messenger products important or many aspects o insulin action. Although the details linking the insulin receptor second messengers to the metabolic e ects o insulin remain open to investigation, the metabolic e ects o insulin action are well understood: insulin is the classic anabolic (energy storing) hormone (Fig. 31-1). In the liver, insulin increases Ins ulin Glucos e

Ins ulin re ce ptor GLUT4 P

P

GLUT4 P

S hc

Glucos e Hexokinase

IRS prote ins

Grb-2

Grb-2

S OS

S OS

S HP -2

P I3-kina s e

? Mitoge ne s is

P rote in s ynthe s is

Glucos e -6phos pha te

p85 p110 Glucos e tra ns port

Glucagon Glucagon—a single-chain polypeptide o 29 amino acids— is a catabolic (energy releasing) hormone secreted by pancreatic -cells. When plasma glucose levels are low, glucagon mobilizes glucose, at, and protein rom storage as energy sources. Besides low glucose and high insulin levels, stimuli or glucagon secretion include sympathetic nervous system activity, stress, exercise, and high plasma levels o amino acids (because the latter indicates a state o starvation). Glucagon binding to its G protein-coupled receptor on the plasma membrane o hepatocytes increases intracellular cAMP and activates protein kinase A, a serine/threonine kinase. Glucagon promotes hepatic glycogenolysis and gluconeogenesis (Fig. 31-1). Glucagon also promotes lipolysis in adipose tissue. The liver and kidneys degrade glucagon; like insulin, its circulating hal -li e is about 6 minutes.

Amylin

Tra ns loca tion

P

glucokinase activity, thereby mediating the phosphorylation and trapping o glucose in hepatocytes and ueling glycogen synthesis, glycolysis, and atty acid synthesis. In skeletal muscle and adipose tissue, insulin stimulates translocation o the insulin-responsive glucose transporter, GLUT4, rom intracellular vesicles to the cell sur ace, which acilitates the transport o glucose into the cell (Fig. 31-4). In muscle, insulin also increases amino acid uptake, stimulates the ribosomal protein synthesis machinery, and promotes glycogen synthase activity and subsequent glycogen storage. In adipose tissue, insulin promotes the expression o lipoprotein lipase, which hydrolyzes triglycerides rom circulating lipoproteins or uptake into at cells. Once inside the at cell, glucose and atty acids are stored predominantly as triglycerides. This process is enhanced by the deactivation o hormone-sensitive lipase, which degrades intracellular triglyceride. Insulin is rapidly degraded in the liver and kidney, with a circulating hal -li e o 6 minutes.

Me ta bolis m/ s tora ge

Glycoge n s ynthe s is

FIGURE 31-4. Downstream effects of insulin receptor activation. Insulin binding to the extracellular portion of the receptor activates tyrosine kinase domains inside the cell, leading to receptor “autophosphorylation” and tyrosine phosphorylation of cytoplasmic substrate proteins including Shc and insulin receptor substrate (IRS) proteins. Phosphorylated Shc promotes mitogenesis. Phosphorylated IRS proteins interact with many other signaling proteins (Grb-2, SHP-2, p85, and p110) to effect changes in cellular function. The IRS interaction with p85 and p110 recruits phosphatidylinositol 3-kinase (PI3-kinase). PI3-kinase activates signaling cascades that control many aspects of cellular insulin action, including glucose transport (via the translocation of GLUT4 glucose transporters to the cell surface), protein synthesis, and glycogen synthesis. Glucose that enters the cell is rapidly phosphorylated by hexokinase and subsequently used for metabolism or stored in the cell as glycogen or triglyceride.

Amylin is a 37-amino acid protein that is packaged together with insulin in secretory granules o the -cell. Insulin and amylin are co-secreted ollowing a meal. Amylin binds receptors within the central nervous system, and its glucoseregulatory actions complement those o insulin. Specif cally, amylin suppresses glucagon release, slows gastric emptying, and decreases ood intake. Together, these actions avor gradual entry o glucose into the circulation ollowing a meal. Amylin is cleared by the kidney; its hal -li e is approximately 10 minutes.

Somatostatin Somatostatin has 14- and 28-amino acid orms that are selectively produced in pancreatic -cells, the gastrointestinal tract, and the hypothalamus. Primary unctions o somatostatins are to inhibit (1) the release o pituitary growth hormone and thyroid-stimulating hormone (see Chapter 27, Pharmacology o the Hypothalamus and Pituitary Gland), (2) the secretion o pancreatic insulin and glucagon, and (3) GI motility and the release o various GI hormones. The stimuli or pancreatic somatostatin release are similar to those or insulin (i.e., high plasma levels o glucose, amino acids, and atty acids). Local somatostatin release allows the hormone to act in a paracrine ashion. The circulating hal li e o somatostatin is only 2 minutes.

C h a p t e r 3 1 Pharmacology o the Endocrine Pancreas and Glucose Homeostasis

Incretins Glucagon-like peptide-1 (GLP-1) and glucagon are both encoded by the glucagon gene, but their sequences are di erent due to di erential mRNA splicing in the enteroendocrine L cells o the distal small intestine and in pancreatic -cells, respectively. Bioactive orms o GLP-1 are 29 or 30 amino acids in length. Blood levels o GLP-1 are low during asting and rise a ter a meal. GLP-1 acts on G protein-coupled receptors located on islet - and -cells and in the central and peripheral nervous systems, heart, kidney, lung, and GI tract. At the pancreatic -cell, GLP-1 augments insulin secretion in response to an oral glucose load (hence GLP-1’s “incretin” effect). At the pancreatic -cell, GLP-1 suppresses glucagon secretion. GLP-1 acts in the stomach to delay gastric emptying and at the hypothalamus to decrease appetite. GLP-1 has a short hal -li e in the circulation (1–2 minutes) due to enzymatic degradation by dipeptidyl peptidase-4 (DPP-4).

PATHOPHYSIOLOGY Diabetes Mellitus As early as AD 200, the Greek physician Aretaeus observed patients who had excessive thirst and urination. He named this condition diabetes, which is Greek or “to siphon, or pass through.” Later, physicians added mellitus (Latin or “honeyed, sweet”) to the disease name a ter noticing that diabetic patients produce urine that contains sugar. The designation diabetes mellitus also distinguishes this disorder rom diabetes insipidus (see Chapter 27), in which either the secretion o antidiuretic hormone (ADH) or the response to ADH action in the kidney is dysregulated, such that water

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is not reabsorbed in the collecting duct o the nephron and copious amounts o dilute urine are produced. The syndrome of diabetes mellitus results from a heterogeneous group of metabolic disorders that have hyperglycemia in common (Table 31-2). Hyperglycemia can result rom an absolute lack o insulin (type 1 diabetes mellitus) or rom a relative insu f ciency o insulin production in the ace o insulin resistance (type 2 diabetes mellitus ). The pathophysiology o diabetes that occurs during pregnancy or is induced by other causes (e.g., pancreatitis) is beyond the scope o this chapter.

Type 1 Diabetes

Type 1 diabetes mellitus, which accounts or 5% o cases in the United States, results rom the autoimmune destruction o pancreatic -cells. No insulin is produced in the absence o -cells, so circulating insulin concentrations are near zero. The unavailability of insulin to promote nutrient entry into cells, coupled with the unopposed actions of counterregulatory hormones, induces a starvation-like response by the cells and tissues of the body. Glycogenolysis and gluconeogenesis proceed unchecked in the liver, thereby increasing the concentration o blood glucose even though it is already too high. Skeletal muscle protein breakdown leads to the release o amino acids, which travel to the liver to urther uel gluconeogenesis. In adipose tissue, triglycerides are broken down and glycerol and atty acids are released into the circulation. In addition, the liver breaks down atty acids or use as gluconeogenic uels and or export as ketone bodies that could be used as uel by the brain. These ketones equilibrate into -hydroxybutyrate and acetoacetate. Excessively high concentrations o these “ketoacids” can deplete serum bicarbonate, eventually resulting in a state o high-anion-gap

TABLE 31-2 Type 1 and Type 2 Diabetes Mellitus TYPE 1

TYPE 2

Etiology

Autoimmune destruction o pancreatic -cells

Insulin resistance, with inadequate -cell unction to compensate

Insulin levels

Absent or negligible

Typically higher than normal

Insulin action

Absent or negligible

Decreased

Insulin resistance

Not part o syndrome but may be present (e.g., in obese patients)

Yes

Age of onset

Typically

Typically

Acute complications

Ketoacidosis Wasting

Hyperglycemic hyperosmolar syndrome (can lead to seizures and coma)

Chronic complications

Neuropathy Retinopathy Nephropathy Atherosclerotic cardiovascular disease (ASCVD; includes stroke, peripheral arterial disease, and coronary artery disease)

Same as type 1

Pharmacologic interventions

Insulin

Approximately 10 drug classes are available, including insulin i other therapies ail.

30 years

40 years

Type 1 and type 2 diabetes mellitus are both associated with increased blood glucose levels, but the two diseases result rom distinct pathophysiologic pathways. In type 1 diabetes mellitus, autoimmune destruction o pancreatic -cells leads to an absolute lack o insulin. The etiology o type 2 diabetes is less well understood but seems to involve impaired insulin sensitivity and an inadequate level o compensatory insulin production or an abnormal prof le o secretion by pancreatic -cells. Although type 1 and type 2 diabetes have di erent acute complications (see text), they share similar chronic complications. Insulin is the primary pharmacologic intervention or type 1 diabetes, while type 2 diabetes can be treated with a number o di erent agents.

568 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

metabolic acidosis called diabetic ketoacidosis (DKA). This is a medical emergency that requires immediate, aggressive treatment. DKA is o ten the initial presentation o type 1 diabetes during childhood or adolescence. Some patients with residual -cells can experience a transient “honeymoon” phase, lasting weeks to months, in which adequate endogenous insulin secretion occurs be ore the eventual complete loss o insulin production. Patients in whom the autoimmune -cell destruction proceeds in a “smoldering” ashion are o ten diagnosed in adulthood, a clinical picture re erred to as latent autoimmune diabetes in adults (LADA). The genetic predisposition to type 1 diabetes maps strongly to the human leukocyte antigen (HLA) loci, also known as the major histocompatibility complex (MHC), which encodes proteins involved in antigen presentation. Other genetic loci weakly contribute to type 1 diabetes. In most patients with type 1 diabetes, autoantibodies to -cell proteins can be detected. Environmental and nutritional actors in uence disease development as well; i one member o an identical twin pair is a ected, the incidence o type 1 diabetes in the other twin is about 50%. Because patients with type 1 diabetes produce little or no endogenous insulin, therapy invariably consists o replacement with exogenous insulin.

Type 2 Diabetes

Type 2 diabetes mellitus, which constitutes 90% o cases in the United States, has historically been thought o as “adult-onset diabetes.” The prevalence o type 2 diabetes has dramatically risen over the past three decades and now a ects adolescents as well as adults. Obesity is the single most important risk actor, and more than 80% o type 2 diabetic patients are obese. The disorder typically develops gradually, without obvious symptoms at the onset. It is requently diagnosed either by elevated blood glucose levels in routine screening tests or, as in the introductory case, a ter the disease has become severe enough to be symptomatic. The progression to type 2 diabetes o ten begins with a state o insulin resistance . With increasing age and added weight, tissues that were once normally insulin-responsive become relatively re ractory to insulin action and require increased insulin levels to respond appropriately. Current investigations are ocused on two potential mechanisms or the pathogenesis o insulin resistance: (1) the ectopic accumulation o lipid into liver and muscle and (2) obesity-induced in ammation. Increasing evidence suggests that the immune system plays an important role in insulin resistance, although many o the details remain to be elucidated. In most individuals, the initial insulin resistance is compensated or by increased production o insulin by pancreatic -cells. Indeed, many individuals with obesity and insulin resistance never progress to rank diabetes because their -cells continue to compensate by secreting increased amounts o insulin. In some patients like Mrs. S, however, the -cells eventually ail to keep pace with the increasing demand or insulin. Although patients with type 2 diabetes generally have higher than normal circulating insulin levels, these levels are insu f cient to overcome the insulin resistance in target tissues. The eventual ailure o -cell compensation could result rom the loss o -cells through increased apoptosis (programmed cell death) or rom dys unctional insulin secretion. Type 2 diabetes is a complex, polygenic disorder, meaning that polymorphisms in many genes can contribute to

overall risk, although the degree o risk con erred by each polymorphism is o ten quite small. A great number o such genes have now been identif ed. Most o these genes a ect pancreatic -cells, while a relatively small number o the identif ed type 2 diabetes genes con er risk or either obesity or insulin resistance. There ore, lean, insulin-sensitive patients with type 2 diabetes have a strong predisposition to -cell ailure. Most o the relatively rare amilial, monogenic causes o diabetes are also due to genetic lesions a ecting -cell unction. Mild or early type 2 diabetes can be unmasked in predisposed individuals by transient periods o insulin resistance, or example, as occurs during treatment with glucocorticoids (see Chapter 29, Pharmacology o the Adrenal Cortex) or pregnancy (gestational diabetes). The ability o patients with type 2 diabetes (like Mrs. S) to produce insulin provides the rationale or treating such patients with oral agents that either sensitize target cells to the action o insulin (e.g., met ormin, thiazolidinediones) or increase insulin secretion by pancreatic -cells (e.g., sul onylureas and other insulin secretagogues). Drugs that control blood glucose levels by slowing the absorption o sugars rom the GI tract (e.g., acarbose) are used less requently. Patients with type 2 diabetes who have lost a great deal o -cell unction or are otherwise di f cult to manage with oral agents may benef t rom exogenous insulin therapy.

Morbidity and Mortality

Type 1 and type 2 diabetes are associated with both typespecif c acute morbidities and common chronic complications. In uncontrolled type 1 diabetes, the lack o insulin and unopposed action o counterregulatory hormones leads to a “metabolic storm” that includes the activation o hormone-sensitive lipase in adipose tissue, the breakdown o triglycerides into glycerol and atty acids, and the increased ux o atty acids in the liver mitochondria that uels ketogenesis. Although ketoacidosis is less common in type 2 diabetes because these patients produce insulin, uncontrolled type 2 diabetes can cause a hyperglycemic hyperosmolar syndrome that leads to mental status changes and can progress to seizures, coma, and death. Both type 1 and type 2 diabetes are associated with long-term vascular pathology. These chronic complications include accelerated atherosclerotic cardiovascular disease (ASCVD), retinopathy, nephropathy, and neuropathy. Although the exact mechanisms are unclear, chronic hyperglycemia, chronic hyperlipidemia, and increased in ammatory signaling may all be contributing actors. In treating Mrs. S’s diabetes, the goal is not only to correct her hyperglycemia and associated symptoms (e.g., polydipsia) but also to prevent these serious chronic complications. Roughly 80% o diabetic patients die rom ASCVD. It is there ore critically important to control concomitant cardiovascular risk actors, such as hypertension and atherogenic dyslipidemia, in addition to lowering blood glucose in these patients. Two large, multicenter, randomized clinical trials, the Diabetes Control and Complications Trial/Epidemiology o Diabetes Interventions and Complications (DCCT/EDIC) involving type 1 diabetic patients, and the United Kingdom Prospective Diabetes Study involving newly diagnosed type 2 patients, demonstrated that intensive diabetes management dramatically decreases the onset (primary prevention) and delays the progression (secondary prevention) o the microvascular complications o retinopathy, nephropathy, and neuropathy. These trials also suggested that intensive blood

C h a p t e r 3 1 Pharmacology o the Endocrine Pancreas and Glucose Homeostasis

glucose control initiated early a ter diagnosis may decrease ASCVD in the long-term. However, causal relationships between glycemic control and risk o ASCVD are less clear in patients with established diabetes. Sel -monitoring o blood glucose is key to conf rm “tight” control and to guide changes in diet, exercise activity, and pharmacologic treatment. Blood glucose is measured by f nger stick using a monitor that assesses glucose concentration in small samples o capillary blood. In addition, levels o glycohemoglobin (HbA1c) provide estimates o the average blood glucose level over the previous 8–12 weeks. HbA1c results rom the nonenzymatic glycation o hemoglobin in red blood cells. It is proportional to the average level o glucose in the blood and to the li espan o the red blood cells ( 120 days). HbA1c and blood glucose levels usually rise in parallel, although it is possible or HbA1c to be elevated while blood glucose is normal in a given instance— or example, when the blood glucose level is acutely normal but the glucose levels had been chronically elevated over the previous several months. HbA1c levels are monitored as a marker of average blood glucose control. Mrs. S’s HbA1c level o 8.2% is concerning because the rate o chronic diabetic complications rises dramatically with HbA1c levels greater than 7.5%. HbA1c levels may be misleadingly low in patients with a shortened red blood cell li espan (e.g., patients with hemolytic anemia) or in pregnancy.

Hypoglycemia Hyperinsulinemia is one o several conditions that can result in hypoglycemia, which is a dangerous condition because the brain requires a constant supply o glucose and cannot rely on alternate uels as readily as peripheral tissues can. Hyperinsulinemia has various causes, the most common o which is iatrogenic (i.e., exogenous insulin or insulin secretagogues used to treat type 1 or type 2 diabetes). A central challenge in the therapy o both type 1 and type 2 diabetes is to normalize glucose levels adequately while avoiding hypoglycemia due to overtreatment. Rarer causes o hypoglycemia include insulinomas (insulin-secreting tumors o pancreatic -cells) and mutations in the -cell K /ATP channel (e.g., mutations in Kir6.2 or SUR1 that result in constitutive depolarization; see above).

PHARMACOLOGIC CLASSES AND AGENTS Therapy for Diabetes Therapeutic agents or treating diabetes target various steps in the regulation o normal glucose homeostasis. Currently available classes o agents include (1) exogenous insulin preparations; (2) met ormin, a biguanide; (3) insulin secretagogues (sul onylureas, D-phenylalanine derivatives, and meglitinides); (4) incretins (GLP-1 receptor agonists and DPP-4 inhibitors); (5) inhibitors o glucose reabsorption in the kidney (SGLT-2 inhibitors); (6) PPAR- agonists (thiazolidinediones); (7) analogues o amylin; (8) bile acid sequestrants; (9) inhibitors o intestinal glucose absorption; and (10) dopamine receptor agonists (bromocriptine). The major goals or the pharmacologic therapy o diabetes are to normalize blood glucose levels and other metabolic parameters in order to reduce the risk o long-term

569

complications. Li estyle interventions are a cornerstone o the management o both type 1 and type 2 diabetes, and all patients with diabetes should receive personalized diabetes education ocused on improved diet and exercise. For patients with type 1 diabetes, the strategy is to provide su f cient exogenous insulin to nearly normalize glycemia without substantially increasing the risk o hypoglycemia. Appropriate treatment o type 1 diabetic patients not only achieves normoglycemia but also reverses the metabolic starvation response mediated by the unopposed action o counterregulatory hormones. For example, insulin treatment reverses amino acid breakdown in muscle and ketogenesis in the liver. The treatment o type 2 diabetes is multi aceted but nearly always includes recommendations or a balanced diet rich in f ber and low in high-glycemic-index carbohydrates and saturated ats ( 7%) and a stepwise program o physical activity with the goal o 150 min/week o aerobic, resistance, and exibility training. In the clinical vignette, Mrs. S was diagnosed with type 2 diabetes a ter progressive weight gain, which likely increased her insulin resistance. Exercise and weight loss could dramatically improve her blood glucose control. I a patient is unable or unwilling to make the necessary changes in li estyle, which is o ten the case, then one or more drugs may be used. Currently available drugs o er the potential to individualize diabetes treatment, with decisions about the blood glucose target (i.e., HbA1c level) being guided by age, li e expectancy, comorbidities, and the patient’s expectations. The choice o pharmacologic regimen should take into account the requency o administration, e f cacy, ease o use, adverse e ects, and patient’s ability and pre erence (Table 31-3). TABLE 31-3 Estimated Frequency of Drug Prescribing for Treatment of Diabetes PERCENT OF PATIENTS WITH AT LEAST ONE CLAIM

PERCENT OF TOTAL CLAIMS FOR DIABETES MEDICATIONS

Met ormin

65

32

Sul onylureas

41

21

Insulin

29

22

Thiazolidinediones

20

12

DPP-4 inhibitors

12

4

Incretin mimetic

3

2

Meglitinides

2

1

0.5

0.2

0.4

0.2

DRUG CLASS

-Glucosidase inhibitors Amylin analogue

Estimates are based on the Medco Health Solutions, Inc., 2009 database o 4.5 million adult patients making at least one claim or a diabetes medication, representing a total o 41 million medication claims. The middle column sums to greater than 100% because some patients were prescribed multiple medications. The right column sums to less than 100% because about 5% o claims were or f xed-dose combination therapies, especially sul onylurea/met ormin and DPP-4 inhibitor/met ormin combinations. These results are pooled or all patients with diabetes and do not distinguish between type 1 diabetes and type 2 diabetes. Adapted rom Medco Health Solutions Diabetes TRC. Medco Diabetes Drug Usage: 2009. 2010; Medco Health Solutions, Inc.

570 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

Tight diabetes control (HbA1c 6.5%) might be appropriate in patients with a long li e expectancy and low risk o hypoglycemia but may be harm ul in elderly patients with a history o severe hypoglycemia and signif cant CVD. The biguanide metormin is uni ormly endorsed as the f rst option or patients with type 2 diabetes in the absence o contraindications. Currently, 70% o type 2 diabetic patients in the United States take metormin either alone or in combination with other agents.

Insulin Replacement: Exogenous Insulin

Insulin is not only the oundation or treating patients with type 1 diabetes but also a potentially help ul adjunct or patients with type 2 diabetes when diet and other therapies provide insu f cient control o hyperglycemia. The f rst insulin preparations were derived rom pig and cow sources, but current recombinant human preparations are produced in vitro. The ideal insulin delivery system would mimic normal -cell insulin delivery into the portal circulation, which causes the liver to be exposed to higher concentrations o insulin than peripheral tissues are. Because insulin is a protein, however, it is rapidly degraded by digestive proteases, and it is not generally e ective as an oral agent. Exogenous insulin is there ore administered parenterally, typically by injection with a f ne-gauge needle that creates a small depot at the subcutaneous injection site. This route o administration means that the liver and other target tissues are exposed to similar insulin concentrations, which is unnatural because the liver is exposed to too little insulin and peripheral tissues are exposed to too much. The rate at which the depot o insulin is absorbed depends on a variety o actors, including the solubility o the insulin preparation, the local density o blood and lymphatic vessels, and potential circulatory or subcutaneous actors (e.g., lipoatrophy or lipohypertrophy). All other actors being equal, aster absorption generally translates into aster onset

o action, but high interperson and site-to-site variability in rates o absorption alters the action prof le o the injected insulin. Table 31-4 lists the most commonly used insulin preparations according to two categories: prandial bolus insulins and basal insulins . Patients using insulin usually require both a longer acting basal insulin and a ast-acting prandial bolus insulin or optimal control o hyperglycemia; other therapeutic options are also available (e.g., insulin pumps). Prandial bolus insulins are used to mimic -cell release o insulin in response to a nutrient load and thus act rapidly and or relatively short durations. Regular insulin is the classic prandial bolus insulin. It is structurally similar to endogenous insulin with the addition o zinc ions to promote stability. Regular insulin tends to aggregate into hexamers, and the dissociation o the hexamers into monomers is the rate-limiting step or absorption. Thirty minutes is required or regular insulin to reach the bloodstream a ter subcutaneous administration. Thus, regular insulin should be administered 30 minutes prior to a meal. Several “rapid-acting” engineered insulins are available that enter the circulation more quickly than regular insulin and can be administered just minutes be ore a meal. The analogues are structurally similar to regular insulin but have been modif ed slightly to avor dissociation o the hexamer into monomers (Fig. 31-2). The names o these analogues hint at the modif cations: in insulin lispro, the amino acids proline and lysine at positions B28 and B29 are reversed; in insulin aspart, aspartic acid replaces proline at B28; in insulin glulisine , lysine replaces asparagine at B3 and glutamic acid replaces lysine at B29. In 2014, the US Food and Drug Administration (FDA) approved an inhaled human insulin ormulation (A rezza®) with kinetics similar to “rapid-acting” injectable insulin analogues: onsets o action or both are 10–30 minutes, peaks o action are between 30 and 90 minutes, and overall durations o action are 3–5 hours. Inhaled insulin

TABLE 31-4 Commonly Used Insulin Preparations ACTION PROFILE (HOURS) TYPE AND PREPARATION

CONSTITUENTS

ONSET

PEAK

DURATION

USAGE

Regular

Unmodif ed insulin

0.5–1

2–3

6–8

Meals or acute hyperglycemia

Lispro

Modif ed insulin

0.1–0.25

0.5–3

4

Meals or acute hyperglycemia

Aspart

Modif ed insulin

0.1–0.25

0.5–3

4

Meals or acute hyperglycemia

Glulisine

Modif ed insulin

0.1–0.25

0.5–3

4

Meals or acute hyperglycemia

NPH

Modif ed insulin, protamine

2–4

4–10

12–18

Basal insulin, insulin o choice in pregnancy

Glargine

Modif ed insulin

2–4

None

20–24

Basal insulin

Detemir

Modif ed insulin

2–4

None

20–23

Basal insulin

Prandial bolus

Basal

Modif cations to native human insulin consist o either (1) alterations in the amino acid sequence o the molecule or (2) changes in the physical orm o the molecule. These changes a ect the rate at which insulin is absorbed and the temporal prof le o insulin action. Alterations in the amino acid sequence change the tendency or insulin to aggregate. For example, the modif cation in lispro decreases aggregation, resulting in aster absorption and more rapid action. In contrast, a neutral ormulation like glargine increases aggregation and delays the rate o absorption o insulin rom its subcutaneous injection site, making this preparation a long-acting dosage orm.

C h a p t e r 3 1 Pharmacology of the Endocrine Pancreas and Glucose Homeostasis

should not be used in patients with chronic lung disease, such as asthma or chronic obstructive pulmonary disease (COPD), due to risk o bronchospasm a ter administration o the drug. Basal “long-acting” insulins are administered once or twice daily and provide a more constant low-level release o insulin. NPH (neutral protamine Hagedorn) insulin is the oldest basal insulin that is still in common use. NPH insulin contains regular insulin suspended with zinc and protamine—an arginine-rich protein isolated rom f sh sperm. Protamine prolongs the time required or the absorption o insulin because it remains complexed with insulin until proteolytic enzymes cleave the protamine rom the insulin. NPH insulin must be gently resuspended prior to administration and can exhibit a wide variability in its action prof le. NPH’s peak activity occurs between 4 and 10 hours a ter administration; this variability in peak activity can be associated with an increased risk o hypoglycemia. U-500 insulin is f ve old more concentrated than regular (U-100) insulin in the bottle and initially at the injection site, which promotes hexamerization and retards its absorption. This makes its action prof le a “hybrid” between regular U-100 insulin and NPH insulin, since U-500 insulin peaks at 2–5 hours and lasts or up to 12 hours. U-500 insulin is most o ten used when large daily doses ( 200 units) o insulin are needed and is administered 2–3 times a day be ore meals. Two engineered long-acting insulin preparations are also available. Insulin glargine di ers rom human insulin by the addition o two arginines a ter position B30 and the replacement o asparagine A21 with glycine. These modif cations raise the pKa o insulin glargine rom acidic to neutral, which renders the insulin less soluble and slows its absorption rom the injection site. Insulin detemir di ers rom regular insulin in that myristic acid, a 14-carbon saturated atty acid, is attached to the side chain o lysine B29. The atty acid chain promotes binding o the insulin analogue to serum and tissue albumin, which retards the absorption, action, and clearance o the drug. Compared to NPH, the engineered long-acting insulins provide more constant insulin levels that plateau or many hours to provide basal coverage with lower risk o nocturnal hypoglycemia. A typical basal-bolus regimen consists o a long-acting basal insulin once or twice daily with boluses o ast-acting insulin be ore meals. Advances in insulin preparations and delivery modalities continue to evolve. Premixed insulin preparations consisting o 25–30% ast-acting and 70–75% long-acting analogues are usually administered twice daily. These may be more convenient or some patients due to the reduced number o injections. Insulin pumps providing a continuous, variable-rate insulin in usion are gaining popularity among patients with type 1 diabetes. Insulin pumps allow programmed delivery at both a continuous rate to mimic basal insulin secretion and peaks to match postprandial glucose excursions. This provides greater exibility in dosing while avoiding multiple injections but also requires a high degree o patient understanding and involvement. The major danger with insulin therapy is that administration of insulin in the absence of adequate carbohydrate intake can result in hypoglycemia. While tight glycemic control that aims to maintain near normoglycemia decreases the incidence o diabetic complications, it also increases the requency o hypoglycemic events, especially a ter administration o prandial insulin. Appropriate matching o insulin dose and carbohydrate intake is a major goal or diabetes management. In type 2 diabetic patients such as Mrs. S, insulin resistance is typically more severe in muscle and liver than in at

571

cells. For this reason, insulin pre erentially deposits calories in adipose tissue, and insulin therapy in insulin-resistant patients (especially those who are already obese, like Mrs. S) o ten leads to weight gain.

Metformin

Hepatic glucose production may be abnormally elevated in type 2 diabetes. Metformin acts to decrease glucose production, atty acid synthesis, and cholesterol synthesis in the liver, potentially through AMPK-dependent and AMPKindependent mechanisms. Met ormin also improves glucose uptake in peripheral muscle; the molecular mechanism responsible or this is less well understood. Met ormin increases insulin signaling and is especially e ective at lowering glucose in type 2 diabetics who are obese and insulin resistant. Met ormin is also used or the o -label (not FDAapproved) treatment o other conditions, such as polycystic ovarian syndrome and non-alcoholic atty liver disease, that are associated with insulin resistance and hyperinsulinemia. The most common adverse e ect o met ormin is mild gastrointestinal distress, which is usually transient and can be minimized by slow titration o the dose. A potentially more serious adverse e ect is lactic acidosis . Because biguanides decrease the ux o metabolic acids through gluconeogenic pathways, lactic acid can accumulate to dangerous levels in biguanide-treated patients. This complication is rarely seen with met ormin (as opposed to phen ormin, which is not approved or use in the United States). Lactic acidosis may occur more requently when met ormin is taken by patients who have other conditions predisposing to metabolic acidosis, such as hepatic disease, heart ailure, sepsis, alcohol abuse, or renal disease (because biguanides are excreted by the kidneys). Current guidelines suggest reducing the met ormin dose when the glomerular f ltration rate (GFR) is 45 mL/min and discontinuing met ormin when GFR is 30 mL/min. Biguanides do not directly a ect insulin secretion, and their use is not associated with hypoglycemia. Unlike insulin and insulin secretagogues, biguanides promote a modest decrease in weight or are weight neutral.

Insulin Secretagogues: Sulfonylureas and Glinides

Sulfonylureas (SFUs) have been available in the United States

since the 1950s or the treatment o type 2 diabetes. SFUs stimulate insulin release rom pancreatic -cells, thereby increasing circulating insulin to levels su f cient to overcome the insulin resistance. At the molecular level, SFUs bind the SUR1 subunit of the SFU receptor, which inhibits the -cell K /ATP channel (Fig. 31-3). The SFUs used to treat type 2 diabetes bind with a higher a f nity to SUR1 than to SUR2 iso orms, accounting or their relative -cell specif city. The inhibition o the K /ATP channel by SFUs is unctionally similar to the molecular events occurring a ter a meal, when increased glucose metabolism causes -cell accumulation o intracellular ATP, membrane depolarization, Ca2 in ux, usion o insulin-containing vesicles with the plasma membrane, and insulin secretion (see above). SFUs are orally available and metabolized by the liver. Their major adverse e ect is hypoglycemia resulting rom relative oversecretion o insulin. These medications should be used cautiously in patients who are unable to recognize or respond appropriately to hypoglycemia (e.g., the elderly) and in patients with decreased renal or hepatic unction due to impaired clearance. It is controversial whether glyburide bears

572 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

an especially high risk or hypoglycemia, but the American Geriatric Society recommends that glyburide be avoided in elderly patients. Although SFUs are generally e ective, sa e, and inexpensive generic drugs that, along with met ormin, are mainstays o treatment or type 2 diabetes, their use is waning or two reasons. Unlike some diabetes drugs that maintain e f cacy, the SFUs have been shown to lose e f cacy over time. In addition, due to their mechanism o action, SFUs and glinides are thought to diminish rather than preserve -cell unction over time. SFUs can cause weight gain secondary to increased insulin activity on adipose tissue. This adverse e ect is counterproductive in obese patients such as Mrs. S. There ore, SFUs are better suited or use in non-obese patients. Like SFUs, meglitinides and D-phenylalanine derivatives, known collectively as glinides , stimulate insulin release by binding SUR1 and inhibiting the -cell K /ATP channel. Although they are structurally distinct, SFUs and glinides both bind the SUR1 subunit, albeit at distinct sites on SUR1. The glinides have a rapid onset o action, similar to SFUs, but also a short hal -li e, making them an attractive option or patients with irregular meal schedules or who are prone to late postprandial hypoglycemia. Similar to SFUs, glinides are associated with weight gain.

GLP-1-Based “Incretin” Therapies: GLP-1 Receptor Agonists (GLP-1Ra’s) and DPP-4 Inhibitors Exenatide , a hormone originally isolated rom the salivary

glands o the Gila monster, acts as an agonist at human GLP-1 receptors. The drug is injected subcutaneously either twice a day or weekly in its extended-release ormulation. Liraglutide is a long-acting orm o GLP-1 with palmitic acid attached to the side chain o lysine, which causes it to bind serum and tissue albumin much like insulin detemir. This increases its circulating hal -li e rom 1–2 minutes to 12 hours, making it suitable or daily injection. Albiglutide , a GLP-1 dimer used to human albumin, has a hal -li e o 4–7 days, thereby allowing or weekly injection. These GLP-1 receptor agonists (GLP-1Ra’s) can be used in combination with other antidiabetic drugs but not with DPP-4 inhibitors; the latter combination may enhance the risk o pancreatitis. GLP-1Ra’s have several mechanisms o action, including increased glucose-dependent -cell secretion o insulin, suppressed -cell secretion o glucagon, slowed gastric emptying (which retards ingested nutrient entry into the circulation), and decreased appetite. The most common adverse e ects are nausea and vomiting (20–30%), which o ten improve with prolonged use but are the most common causes or discontinuation o these drugs (5% o patients). Medullary thyroid cancer has been observed in rodents treated with GLP-1Ra’s but not in humans. GLP-1Ra’s carry a “black box” warning or this potential risk. The risk o acute pancreatitis and pancreatic cancer has been debated. The FDA recently issued a press release stating that existing data do not conclusively support a causal link between incretins and pancreatic pathology but also recommending continued monitoring through analysis o ongoing postmarketing clinical trials. Because GLP-1Ra’s augment glucose-dependent insulin secretion, they are not associated with hypoglycemia unless used in conjunction with other antidiabetic agents such as SFUs. GLP-1Ra’s are associated with weight loss in 10–40% o patients. Indeed, liraglutide at doses higher than those routinely used or treatment o diabetes was approved in late 2014 or treatment o obesity.

Dipeptidyl peptidase-4 (DPP-4) is an endogenous enzyme that cleaves and inactivates GLP-1. The DPP-4 inhibitors thus prolong the hal -li e o endogenous GLP-1 by inhibiting DPP-4. These drugs increase circulating GLP-1 and insulin concentrations and decrease glucagon concentration. They are used most commonly in combination with met ormin (see earlier), although they can also be used alone. Currently approved DPP-4 inhibitors include sitagliptin, saxagliptin, linagliptin, and alogliptin. They are taken orally once a day and typically decrease HbA1c levels by 0.5–0.7%. Like metormin, pramlintide, and GLP-1 agonists, DPP-4 inhibitors alone are not associated with hypoglycemia, although an increased risk o hypoglycemia is noted when they are administered with SFUs. They are well tolerated and weight neutral.

Inhibitors of Glucose Reabsorption in the Kidney: SGLT-2 Inhibitors Glucose excretion in the urine represents the net di erence between the amount o glucose f ltered at the glomerulus and the amount reabsorbed by low-a f nity, high-capacity SGLT-2 transporters in the proximal convoluted tubule. The sodium gradient across the luminal membrane o the proximal tubule cells provides the driving orce or glucose reabsorption. Intracellular glucose di uses into the bloodstream via the transporter GLUT2. SGLT-2 capacity is saturated and the transport maximum or glucose is reached at blood glucose levels o 180–200 mg/dL. Above these levels, as o ten occurs in diabetes, glucose is excreted in the urine. Inhibition o SGLT-2 drew interest as a potential therapeutic target when the nonspecif c inhibitor phlorizin was ound to normalize glucose in experimental models o diabetes. While GI adverse e ects and low bioavailability hampered the development o phlorizin or use in humans, the specif c SGLT-2 inhibitors canaglif ozin, empaglif ozin, and dapaglif ozin are now approved or treating patients with type 2 diabetes. As an add-on therapy, SGLT-2 inhibitors decrease HbA1c levels by 0.7–1.0% rom a baseline o 8%. Importantly, the mechanism o action o SGLT-2 inhibitors is independent o both insulin secretion and insulin action, which distinguishes this therapeutic class rom other antidiabetic therapies. The e f cacy o SGLT-2 inhibitors is blunted in patients with reduced GFR (between 30 and 60 mL/min), consistent with their mechanism o action. SGLT-2 inhibitors have a low risk or hypoglycemia, since urine glucose is already negligible at low blood glucose concentrations. The main adverse e ects include urinary tract in ection, vulvovaginitis, and balanitis, which have been reported more o ten in emale patients and are presumably linked to the presence o glucose in the urine. SGLT-2 inhibitors result in weight loss due to the excretion o glucose in the urine. O note, SGLT-2 inhibitors are not approved or treatment o patients with type 1 diabetes and have been linked to cases o euglycemic DKA. This risk is especially high in patients who have rapidly decreased their insulin doses a ter starting SGLT-2 inhibitors, and appears to be the result o increased glucagon secretion.

Thiazolidinediones

The thiazolidinedione (TZD) drugs are insulin “sensitizers” that enhance the action of insulin at target tissues but do not directly a ect insulin secretion. TZDs are synthetic ligands or the transcription actor PPAR , which a ects adipose cell di erentiation and lipid metabolism. By activating PPAR , TZDs promote atty acid uptake and storage in adipose tissue rather than in skeletal muscle or liver. This decrease in

C h a p t e r 3 1 Pharmacology of the Endocrine Pancreas and Glucose Homeostasis

muscle and liver at content enables tissues to be more sensitive to insulin and suppresses glucose production in the liver. Rosiglitazone and pioglitazone are the currently available TZDs. In addition to redistributing lipid stores among adipose, muscle, and liver, the TZDs have anti-in ammatory properties that may contribute to their e f cacy. Adverse e ects o TZDs include weight gain o 2–4 kilograms, uid retention (edema), heart ailure, and risk o bone ractures. Rosiglitazone had been associated with an increased risk o myocardial in arction, leading initially to a special warning, but the FDA recently reevaluated the data and suspended that warning.

Amylin Analogue: Pramlintide

Pramlintide was designed as a stable analogue o human amy-

lin, the -cell hormone that is co-secreted with insulin and may help regulate postprandial glucose levels. Type 1 diabetics lack endogenous amylin, and type 2 diabetics are relatively def cient in amylin. Thus, pramlintide is approved or use in both type 1 diabetics and insulin-requiring type 2 diabetics. Pramlintide’s structure is similar to amylin with the exception o three amino acid substitutions that con er improved solubility and stability (three prolines replace an alanine and two serines). Pramlintide slows gastric emptying, reduces postprandial glucagon and glucose release, and promotes satiety. It is administered as a subcutaneous injection be ore meals. Its most common adverse e ect is nausea, which is o ten limiting but may improve in some patients with continued use. Pramlintide is not associated with hypoglycemia unless it is used in conjunction with other agents that can cause hypoglycemia. Use o pramlintide o ten results in modest weight loss.

Bile Acid Sequestrants

In addition to its well-characterized cholesterol-lowering e ects, colesevelam signif cantly lowers HbA1c levels in patients with type 2 diabetes. Colesevelam binds bile acids in the intestine, leading to an increase in ecal excretion and a decrease in enterohepatic reuptake o these acids. This promotes the conversion o cholesterol to bile acid, leading to hepatic uptake o LDL cholesterol and reduction in serum LDL cholesterol. However, colesevelam also increases triglyceride levels in 50% o patients. The glucose-lowering mechanism o colesevelam is likely related to its binding o bile acids. This could alter the activity o the G protein-coupled receptor TGR5 to increase secretion o GLP-1 or other incretins and inhibit hepatic glycogenolysis. As an add-on therapy, colesevelam reduces HbA1c levels by 0.3–0.5%. It is contraindicated in patients with a history o bowel obstruction or severe hypertriglyceridemia. Its main adverse e ects are constipation, nausea, and dyspepsia.

Inhibitors of Intestinal Glucose Absorption

-Glucosidase inhibitors are carbohydrate analogues that

delay the absorption o dietary carbohydrates by inhibiting intestinal brush border -glucosidase enzymes and thus reduce rates o cleavage o complex carbohydrates into glucose. By retarding the absorption o complex carbohydrates, -glucosidase inhibitors reduce the postprandial peak in blood glucose. -Glucosidase inhibitors are e ective when taken with meals but not at other times. -Glucosidase inhibitors may be used as monotherapy or adjunctive therapy. They pose no risk o hypoglycemia and are most use ul in postprandial hyperglycemia and or newonset patients with mild hyperglycemia. Adverse e ects that limit the use o -glucosidase inhibitors include atulence,

573

bloating, abdominal discom ort, and diarrhea. These adverse e ects are mechanism-based, since delivery o undigested carbohydrate to the distal bowel provides nutrients or colonic bacteria. Thus, -glucosidase inhibitors are contraindicated in patients with in ammatory bowel disease. A dose-dependent increase in hepatic transaminases is reversible a ter drug discontinuation. These agents do not promote weight change.

Dopamine Receptor Agonists

Bromocriptine mesylate is a nonselective dopamine agonist that has been used or decades to treat hyperprolactinemia. The antidiabetic mechanism o action is not completely understood. Growing evidence in animals points to suppression o hepatic glucose production by modulating the hepatic–hypothalamic circuitry. Bromocriptine does not augment insulin secretion or insulin sensitivity in peripheral tissues. When administered daily and within 2 hours a ter awakening, bromocriptine reduces HbA1c levels by 0.5% in poorly controlled type 2 diabetic patients treated with diet alone or with one other drug. As monotherapy, bromocriptine is either weight neutral or results in modest weight gain (1–2 lb over the f rst 6 months).

Pharmacologic Management Strategies

As discussed above, patients with type 1 diabetes require insulin therapy. A subset o patients with type 2 diabetes also receive insulin, and both types o patients benef t rom individual optimization o therapy using combinations o rapid-acting and long-acting insulin preparations. However, patients with type 2 diabetes rarely receive insulin as an initial therapy. Depending on disease severity, patients with type 2 diabetes may f rst receive recommendations or altered li estyle such as diet and exercise. I this is insu f cient, an oral medication is initiated, which is invariably met ormin providing it is tolerated and not contraindicated. In the case o Mrs. S, in addition to counseling her on li estyle, most physicians would prescribe met ormin because it is sa e and e ective and does not lead to weight gain; Mrs. S also does not appear to have renal disease or other contraindications. Starting rom a HbA1c level o 8.2%, behavioral modif cation and met ormin treatment may result in a reduction o HbA1c level to the target o 7%. Advancement to combination therapy should be considered i monotherapy does not achieve the target HbA1c level. Current evidence does not support a “priority list” among the other oral agents and non-insulin injectables as an add-on(s) to met ormin. The decision o which drugs to use in combination with met ormin is based on ease o use, cost, adverse e ect prof le, and patient pre erence. In general, combination therapy with drugs that affect different molecular targets, and that have different mechanisms of actions, has the advantage of improving glycemic control while using a lower dose of each drug and thus reducing adverse effects. See Table 31-5 or a comparison o adverse e ects associated with the long-term use o several di erent therapies or type 2 diabetes.

Therapy for Hyperinsulinemia Although surgical removal is ultimately the treatment o choice or insulinomas, diazoxide , octreotide , and, in some cases, everolimus may stabilize hypoglycemia preoperatively. Diazoxide binds the SUR1 subunit o K /ATP channels in pancreatic -cells and stabilizes the ATP-bound (open) state o the channel so that the -cells remain hyperpolarized and

574 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

TABLE 31-5 Adverse Effects over Ten Years of Use: A Comparison of Several Agents Used as Monotherapy for Type 2 Diabetes Mellitus

AGENT

INCREASE IN WEIGHT (COMPARED TO DIET THERAPY ALONE), KG

Insulin

4.0

2.3

36

Sul onylurea

2.2

0.5

14

0

0

4

Biguanide

SEVERE SYMPTOMATIC HYPOGLYCEMIA,* HYPOGLYCEMIA,** % OF SUBJ ECTS % OF SUBJ ECTS

Because diabetes is a chronic disease, the long-term implications o therapy are an important consideration. Insulin and sul onylureas are both capable o lowering blood glucose to dangerous levels, while biguanides lack this adverse e ect. In addition, biguanide use is not associated with an increase in body weight, while patients taking insulin or a sul onylurea tend to gain weight. *Severe hypoglycemia is def ned as hypoglycemia requiring hospitalization or other third-party intervention. **Symptomatic hypoglycemia is def ned as hypoglycemia not requiring hospitalization. Data rom United Kingdom Prospective Diabetes Study (UKPDS), 1998.

less insulin is released. Diazoxide binds channels containing either SUR1 or SUR2 iso orms and is there ore used not only to decrease insulin secretion by pancreatic -cells but also to hyperpolarize SUR2-expressing cardiac and smooth muscle cells. In a rare orm o genetic hyperinsulinemic hypoglycemia, a mutant SUR1 iso orm is relatively insensitive to Mg2 -ADP but does respond to diazoxide; in most orms o this disease, however, the mutant channel is not transported to the cell sur ace, and diazoxide is ine ective. Octreotide is a somatostatin analogue that is longer acting than endogenous somatostatin. Like somatostatin, this agent blocks hormone release rom endocrine-secreting tumors, such as insulinomas, glucagonomas, and thyrotropinsecreting pituitary adenomas. Octreotide has several other clinical indications as well (see Chapter 27). The mammalian target o rapamycin (mTOR) inhibitor everolimus is FDA-approved or the treatment o neuroendocrine tumors o pancreatic origin. Everolimus has been shown to prolong progression- ree survival in patients with nonresectable locally advanced or metastatic neuroendocrine tumors o the pancreas, including insulinoma.

Glucagon as a Therapeutic Agent Glucagon is used to treat severe hypoglycemia when oral or

intravenous glucose administration is not possible. As with insulin, glucagon is administered by subcutaneous injection. The hyperglycemic action o glucagon is transient, and it requires a su f cient hepatic store o glycogen. Glucagon

is also used as an intestinal relaxant be ore radiographic or magnetic resonance imaging (MRI) o the gastrointestinal tract. The mechanism by which glucagon mediates intestinal relaxation remains uncertain.

CONCLUSION AND FUTURE DIRECTIONS Fuel homeostasis involves the pancreatic hormones insulin, glucagon, amylin, and somatostatin and the GI hormones GLP-1 and GIP. When the levels o these hormones are pathologically altered, an individual can become hyperglycemic (as in diabetes mellitus) or hypoglycemic. Various pharmacologic agents act at several di erent cellular and molecular sites to normalize blood glucose levels. Exogenous insulin, SFUs, glinides, and incretins increase insulin levels, while diazoxide reduces insulin levels. Met ormin and thiazolidinediones increase insulin sensitivity at target tissues. SGLT-2 inhibitors inhibit the reabsorption o glucose in the proximal tubule o the kidney. -Glucosidase inhibitors slow the intestinal absorption o carbohydrates. Amylin analogues slow gastric emptying. Octreotide, a synthetic orm o somatostatin, has wide-ranging inhibitory e ects on hormone secretion. Exogenous glucagon can be used to increase plasma glucose levels. Research on new pharmacologic treatments or early type 1 diabetes include e orts to develop immune modulatory therapies aimed at reversing -cell dys unction. For type 2 diabetes, agents may be developed to inhibit the enzymes o glycogen synthesis and glycogenolysis in order to restrain glucose production (e.g., inhibitors o glycogen synthase kinase 3 to promote glycogen synthesis and inhibitors o hepatic glycogen phosphorylase to suppress glycogenolysis), to modulate the microbiome either directly or indirectly, or to target in ammation using small-molecule anti-in ammatory drugs or selected biologics that block the actions o certain cytokines.

Suggested Reading Drucker DJ. The biology o incretin hormones. Cell Metab 2006;3:153–165. (Reviews basic physiology of GLP-1 and related hormones.) Garber AJ, Abrahamson MJ, Barzilay JI, et al. AACE comprehensive diabetes management algorithm 2013. Endocr Pract 2013;19:327–336. (Reviews principles of management of diabetes, including strategies to escalate pharmacologic treatment through combination therapy.) Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol 2012;13:251–262. (Reviews function and mechanism of action of potential metformin target.) Inzucchi SE, Bergenstal RM, Buse JB, et al. Management o hyperglycemia in type 2 diabetes: a patient-centered approach. Diabetes Care 2012;35:1364–1379. (Clinically oriented approach to treatment of type 2 diabetes, including diet, exercise, insulin and other injectables, oral agents, and combination therapy.) Rena G, Pearson ER, Sakamoto K. Molecular mechanism o action o metormin: old or new insights? Diabetologia 2013;56:1898–1906. (Thorough review of mechanism of action of metformin and its relationship with antidiabetic effects.)

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C h a p t e r 3 1 Pharmacology o the Endocrine Pancreas and Glucose Homeostasis 579

Ca lcium m inta ke 1,000 0 mg g

32

PTH PT H

1,2 25(OH) H)2 D

(exo g e noo us ; o nc e -daily)

300 30 0 mg

3 0 0 mg

(A Abs o rptiio n )

100 0 mg

P la s ma ca lcc iu ium m

Pharmacology of Bone Mineral Homeostasis ((S S e cre tion)

S ma ll inte in te s tine in

800 mg

Fe ca excrestion David M . S lovik a n d Eh rin J .l Arm t ro n g

(Accre tion ion))

300 mgg (Re s orpttio (R ion)

CT (e x o g e no u s )

Bone P TH (e ndogee nous n ; cont co ntin inuo u o us ) uo

2 00 mgg R e na l e xcre tion

P TH

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580–581 INT PHYSIOLOGY OF BONE MINERAL HOM OMEO EOSTASIS . . . . . . . . . . 580 Structure of Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 St Mine nera rall Ba Bala lanc a ncee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 Regulation of Bone Remodellingg . . . . . . . . . . . . . . . . . . . . . . . . 58 5811 Horm Ho rmon onal al Con ontr trol ol of Ca Callciium and Phosphate . . . . . . . . . . . . . 58 5833 Parathyroid Hormonee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 Vita Vi tami minn D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 Fibroblast Growth Factor 23 and Phosphatonins . . . . . . . . 586 Calcitonin, Glucocorticoids, Thyroid Hormone, and Gonadal Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 PATHOPHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 590 Antiresorptive Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590 Hormone Replacement Therapy (HRT)) . . . . . . . . . . . . . . . . 590

INTRODUCTION The 206 bones o the human skeleton are ar rom the li eless structures they are commonly imagined to be. Bones are remodeled continuously and are involved in many unctions besides structural support and protection o internal organs, including hematopoiesis and mineral storage. The ocus o this chapter is on the critical role o bone in mineral homeostasis, the process and regulation o bone remodeling, the diseases that can result when the delicate balances o mineral homeostasis and bone remodeling are perturbed, and the pharmacologic therapies employed to treat these conditions. A key concept regarding the pharmacologic agents discussed in this chapter is the distinction between bone antiresorptive agents, which slow bone loss, and bone anabolic agents, which have the potential to increase overall bone mass.

PHYSIOLOGY OF BONE MINERAL HOMEOSTASIS Specialized cells called osteoblasts and osteoclasts continually remodel the human skeleton in response to mechanical 580

(s timuu la te s Ca 2+ re a bs orption a nnd d e nha nce s P O 4 excre xcre tion) Selective Estrogen Receptor Modu Mo dula du ula lato tors . . . . . . . . . . . . . . 591 Bisphosphonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 5922 RANKL Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 5944 Calcitonin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595 Bone Bo nee Ana nabbolic Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595 Fluoridee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595 Parathyroidd Hormonee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595 Treatment of Secondary Hyperparathyroidism in Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Oral Phosphate Binderss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Calcitriol and Its Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Calcimimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Calcium, Inorganic Phosphate, and Vitamin D . . . . . . . . . . . . 596 Calcium m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596 Inorganic Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597 Vitamin D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 597 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598

orces and endocrine and paracrine actors. Two o the endocrine actors—parathyroid hormone and vitamin D—control bone metabolism or the purpose o maintaining extracellular calcium homeostasis. Other hormones, such as glucocorticoids, thyroid hormone, gonadal steroids, and f broblast growth actor 23 (FGF-23), also have important e ects on bone integrity. This section reviews the cellular and molecular mechanisms that mediate bone ormation and bone resorption and the mechanisms by which hormones (especially parathyroid hormone and vitamin D) maintain plasma calcium levels within a narrow concentration range.

Structure of Bone Bone consists o 25% organic and 75% inorganic components. The organic component includes the cells (osteoblasts, osteoclasts, osteocytes, bone lining cells, bone stromal cells) and osteoid (a matrix consisting primarily o type I collagen f bers and several low-abundance proteins). The inorganic component consists o crystalline calcium phosphate salts, primarily hydroxyapatite . The chemical ormula o hydroxyapatite is (Ca)5(PO4)3OH. Ninety-nine percent o the calcium in the body is stored in the skeleton, mostly

C h a p t e r 3 2 Pharmacology of Bone Mineral Homeostasis

RS is a 60-year-old Caucasian em ale livin g in th e n o rth ea s t wh o co m es to h e r p hys icia n w ith th e re ce n t o ns e t o low b a ck p a in th a t b e ga n w h e n s h e in a d verten tly s tep ped into a p o th ole. S he is otherwise healthy he and has no history o prior ractures. He r m e n s tru a l p e rio d s ce a s e d w h e n s h e w a s 54 ye a rs o ld . S h e h a d little in th e w a y o p o s tm e n o p a u s a l s ym p to m s a n d n e ve r to o k h o rm o n e re p la ce m e n t th e ra p y. Me n a rch e wa s a t a g e 11. S h e h a s o n e ch ild w h o wa s b o rn w h e n RS wa s 38 ye a rs o ld . He r m o th e r d ie d a t a g e 55 w ith b re a s t ca n ce r, a n d h e r s is te r, a g e 58, w a s re ce n tly d ia g n o s e d w ith b re a s t ca n ce r. S h e is la cto s e in to le ra n t a n d a vo id s d a iry. Ad d itio n a lly, s h e d o e s n o t ta ke ca lciu m o r vita m in D s u p p le m e n ts a n d s h e h a s ve ry little s u n lig h t e xp o s u re w ith h e r d a ily a ctivity. Th e re is n o kn o w n a m ily h is to ry o o s te o p o ro s is . He r a th e r a n d m a te rn a l a u n t d ie d in th e ir 60s w ith co ro n a ry a rte ry d is e a s e . He r p hys ica l e xa m in a tio n is u n re m a rka b le e xce p t o r p o in t te n d e rn e s s o ve r lu m b a r ve rte b ra L1. He r w e ig h t is 135 lb , a n d s h e is 64 in ch e s ta ll b u t b e lie ve s s h e h a s lo s t s o m e h e ig h t o ve r th e la s t ye a r. La b o ra to ry s tu d ie s a re a ll w ith in n o rm a l lim its e xce p t o r a lo w 25-OH vita m in D le ve l. La te ra l x-ra y

as hydroxyapatite. Figure 32-1 illustrates the structure o a long bone.

Mineral Balance Calcium is absorbed in the small intestine by two mechanisms: acilitated transport, which occurs throughout the small intestine, and calcitriol-dependent active transport, which occurs mainly in the duodenum. In people ingesting 1,000 mg o dietary calcium per day, approximately 300 mg/ day is normally absorbed by the intestines (Fig. 32-2). At lower calcium intakes, the e f ciency o intestinal calcium absorption is higher, and at higher calcium intakes, the e f ciency o absorption is lower. These adjustments contribute importantly to calcium homeostasis, and intestinal calcium absorption can increase to as much as 600 mg/day in the presence o high levels o calcitriol (the active orm o vitamin D), as discussed below. The absorption o calcium rom the intestine is normally balanced by calcium losses through renal excretion (about 200 mg/day) and salivary and biliary secretion (about 100 mg/day; Fig. 32-2). In contrast to calcium absorption, intestinal absorption o inorganic phosphate is not homeostatically regulated and is typically about two-thirds o the ingested phosphate irrespective o dietary intake.

Regulation of Bone Remodeling Osteoclasts are the cells responsible for bone resorption. Osteoblasts are the cells responsible for bone formation.

Regulation o these two cell types by mechanical, endocrine,

581

o th e s p in e s h o w s a co m p re s s io n ra ctu re o L1 a n d g e n e ra lize d o s te o p e n ia . Me a s u re m e n t o b o n e m in e ra l d e n s ity (BMD) a t th e s p in e a n d h ip re ve a ls va lu e s th a t a re 2.6 s ta n d a rd d e via tio n s b e lo w th e h e a lthy p e a k e m a le va lu e o r b o th s ite s . He r p hys icia n d ia g n o s e s p o s tm e n o p a u s a l o s te o p o ro s is a n d a re ce n t co m p re s s io n ra ctu re o L1. RS a s ks h e r p hys icia n to d is cu s s w ith h e r th e a va ila b le th e ra p e u tic o p tio n s a n d is p a rticu la rly in te re s te d in th e p o te n tia l ris ks a n d b e n e f ts o e a ch o p tio n .

Questions 1 . What medical conditions should be investigated to rule out reversible causes of RS’s osteoporosis? 2 . Why is RS at particularly high risk for osteoporosis? 3 . Given her family history, RS has an increased risk for breast cancer and cardiovascular disease. How does this alter the choice of therapeutic agents that could be prescribed? 4 . What are the therapeutic options available for RS? What are the advantages and disadvantages of each option? 5 . Should RS take calcium and vitamin D in addition to another therapeutic agent?

and paracrine actors determines the balance between bone ormation and bone resorption (see below). The two signaling proteins RANK ligand (RANKL) and macrophage colony-stimulating actor (M-CSF) are together both necessary and su f cient or the maturation o osteoclasts. RANKL, a member o the tumor necrosis actor (TNF) super amily, is synthesized by osteoblasts and by osteoblast precursors, which express RANKL on their cell membranes. RANKL binds to RANK, a receptor expressed on osteoclasts and osteoclast precursor cells in the bone marrow. This binding interaction promotes the di erentiation o osteoclast precursors into mature osteoclasts (Fig. 32-3). Alternatively, RANKL binds with high a f nity to osteoprotegerin (OPG), a soluble extracellular protein synthesized and secreted by osteoblasts. OPG is called a decoy receptor because it prevents RANKL rom interacting with RANK. Inherited def ciency o RANKL or RANK causes a orm o osteopetrosis (def ned as de ective bone resorption and increased bone mass), while inherited def ciency o OPG causes increased bone resorption and osteoporosis. To repair its strength over time and to respond adaptively to mechanical stresses, human bone is continually resorbed and re ormed. This process is called remodeling. Partly because o its large sur ace area on which remodeling can take place, 25% o trabecular bone is remodeled each year in adults. In contrast, only 3% o cortical bone is remodeled each year. This di erence is important because pathologic conditions that disturb bone remodeling preferentially affect bones with a high content of trabecular bone, such as the vertebral bodies.

582 PRINCIPLES OF ENDOCRINE PHARMACOLOGY A

Articula r ca rtila ge

P roxima l e piphys is

Epiphys e a l pla te S pongy tra be cula r bone Compa ct cortica l bone Me dulla ry ca vity

Dia phys is Os te on (Ha ve rs ia n s ys te m)

La me lla e

Remodeling is carried out by the coordinated activity o millions o cellular units—basic multicellular units (BMU)— consisting o osteoblasts and osteoclasts. The process o resorption begins when physical and/or chemical signals (discussed below) recruit osteoclasts to orm a tight ringlike seal with the bone sur ace and extend villus-like projections toward the sur ace within this ring. These villi secrete lactic acid, carbonic acid, and citric acid and use carbonic anhydrase to generate protons and a H -ATPase to pump the protons onto the bone sur ace. (Individuals and experimental animals def cient in this carbonic anhydrase have osteopetrosis.) The tight seal creates a closed, ring-shaped microenvironment beneath the osteoclast, within which the secretion o organic acids and protons consumes hydroxide at the bone sur ace and dissolves hydroxyapatite. The dissolution o hydroxyapatite can be expressed as ollows: (Ca)5(PO4)3OH Æ 5Ca2

Dis ta l e piphys is

Tra be cula r bone Ce ntra l (Ha ve rs ia n) ca na l

Cortica l bone

P e rios te um

B Os te obla s ts

Os te ocla s t

Re s orption la cuna

Os te ocyte

FIGURE 32-1. Structure of bone. A. The upper panel depicts the structure o a long bone (exemplif ed by the humerus). Note that the diaphysis consists o a thick outer layer or cortex o compact cortical bone surrounding the bone marrow. In the epiphysis, the cortex is thinner and surrounds trabecular bone as well as bone marrow; trabecular bone is also ound in the vertebral bodies and much o the pelvis. B. The lower panel shows the detailed structure o bone. Bone remodeling is a dynamic balance between the catabolic activity o osteoclasts and the anabolic activity o osteoblasts. Osteoblasts and osteoclasts are ound on all inner bone sur aces, including the endosteum that lines cortical bone and the many sur aces in trabecular bone. Bone remodeling is most intense in trabecular bone. Consequently, conditions that disrupt bone remodeling and/or bone mineralization a ect trabecular bone pre erentially. For example, osteoporotic ractures occur most commonly in vertebral bodies, which are predominantly trabecular bone.

3PO43

OH

Equation 32-1

According to Le Chatelier’s principle, OH consumption drives this reaction to the right. This is an important mechanism exploited by osteoclasts to resorb the mineral component o bone. Demineralization o the bone matrix exposes it to proteolysis by cathepsin K, collagenases, and other proteases that are concomitantly secreted by the villi. Although this proteolysis totally degrades much o the exposed bone matrix, some o the type I collagen peptide chains escape into the circulation a ter partial proteolysis. The blood or urine level o such type I collagen metabolites (e.g., NTX, CTX) is an index o bone turnover, re ecting breakdown o type I collagen and total body bone resorption. Because o its large hydroxyapatite-covered sur ace area, bone normally adsorbs various nonskeletal proteins and peptides rom its environment, including IGF-I and TGF- . Demineralization exposes these adsorbed growth factors to the proteolytic enzymes secreted by osteoclast villi, but some escape proteolysis and a ect the cellular activity o neighboring osteoclasts, osteoblasts, and osteocytes. A ter about 3 weeks o such bone resorption, cytokines and growth actors liberated rom the matrix, together with hormonal and other actors (see below), begin to stimulate local accumulation o osteoblasts via proli eration, di erentiation, and reduced apoptosis (programmed cell death). These osteoblasts replace the osteoclasts in the resorption cavity (lacuna) and begin to ref ll the cavity with concentric layers, or lamellae , o unmineralized organic matrix (osteoid) (Fig. 32-3). As osteoblasts f ll the cavity with new osteoid, they also secrete alkaline phosphatase , which hydrolyzes phosphate esters including pyrophosphate (a potent inhibitor o bone mineralization). The hydrolysis o pyrophosphate also increases the local concentration o inorganic phosphate. Together, the alkaline phosphatase-catalyzed hydrolysis o pyrophosphate and the liberation o inorganic phosphate promote the crystallization o calcium phosphate salts and mineralization o the bone matrix. As osteoblasts continue to lay down matrix, some eventually become completely surrounded by it and are then called osteocytes (Fig. 32-1). Osteocytes are the most numerous bone cell type, comprising 90–95% o the cells in bone. Osteocytes respond to changes in mechanical strain and help control the balance between bone ormation and resorption via their secretion o sclerostin (a protein that inhibits bone ormation) and other actors. Genetic mutations that delete

C h a p t e r 3 2 Pharmacology o Bone Mineral Homeostasis

583

Ca lcium inta ke 1,000 mg

PTH

1,25(OH)2 D 300 mg

300 mg

(Abs orption)

100 mg

(Accre tion)

P la s ma ca lcium

300 mg

(S e cre tion)

S ma ll inte s tine

(Re s orption)

CT (exogenous)

800 mg

200 mg Re na l excre tion

Fe ca l excre tion P TH

(s timula te s Ca 2+ re a bs orption a nd e nha nce s P O 4 excre tion)

or inactivate sclerostin increase bone ormation without a corresponding increase in bone resorption. Such uncoupling leads to a marked increase in bone mass and bone strength in humans and experimental animals. A monoclonal antibody that inactivates sclerostin, and thereby increases bone mass and bone strength throughout the skeleton, is being evaluated Os te obla s t pre curs or

P TH, s he a r s tre s s , TGF-β

Os te obla s t pre curs or

1 5 RANKL

Ma ture os te obla s ts 2

Os te ocla s t pre curs or Ma ture os te ocla s t RANK

Os te obla s t pre curs or

3

FIGURE 32-2. Daily whole-body calcium balance.

(exogenous; once-daily)

TGF-β, IGF-1, growth fa ctors , cytokine s 4

M-CS F

FIGURE 32-3. Interaction of osteoblasts and osteoclasts in bone remodeling. Bone resorption and bone ormation are coupled by the interactions between osteoblasts and osteoclasts. 1. Factors such as parathyroid hormone (PTH), shear stress, and trans orming growth actor (TGF- ) cause osteoblast precursors to express the osteoclast di erentiation actor RANKligand (RANKL). 2. RANKL binds to RANK, a receptor expressed on osteoclast precursors. 3. The RANKL–RANK binding interaction, together with macrophage colony-stimulating actor (M-CSF), causes osteoclast precursors to di erentiate into mature osteoclasts. 4. As mature osteoclasts resorb bone, matrix-bound actors such as TGF- , insulin-like growth actor 1 (IGF-1), other growth actors, and cytokines are released. 5. These liberated actors stimulate osteoblast precursors to develop into mature osteoblasts, which begin to ref ll the resorption cavities excavated by the osteoclasts.

Bone P TH (e ndoge nous ; continuous )

In a state o whole-body calcium balance, the uxes o calcium include net uptake o 200 mg per day rom the GI tract and excretion o 200 mg per day by the kidneys. Calcitriol [1,25(OH)2D] enhances absorption o Ca 2 rom the GI tract. Continuous secretion o parathyroid hormone (PTH) increases bone ormation and (even more) bone resorption and stimulates renal tubular reabsorption o calcium; both e ects raise plasma Ca 2 . Continuous secretion o PTH also enhances renal clearance o inorganic phosphate (PO4). In contrast, once-daily injection o PTH (in blue) stimulates new bone ormation (accretion) more than it stimulates bone resorption and has only transient (and consequently minor) e ects on renal clearance o Ca 2 and PO4. Exogenous calcitonin (CT; also in blue) inhibits bone resorption.

as a potential treatment or osteoporosis. Mature osteocytes normally alter their secretion o sclerostin in bone regions subjected to mechanical loads and thereby play a critical role in skeletal adaptations to gravity and other mechanical loads by localizing bone remodeling responses to such loads.

Hormonal Control of Calcium and Phosphate Calcium is essential or many important physiologic processes, such as neurotransmitter release, muscle contraction, and blood coagulation, and deviations in extracellular calcium levels can have serious consequences. There ore, the plasma calcium level is tightly regulated. Inorganic phosphate concentrations must also be regulated, in part because changes in plasma inorganic phosphate concentrations a ect plasma calcium levels (see below). Three main hormones— parathyroid hormone (PTH), vitamin D, and FGF-23— mediate calcium and phosphate homeostasis. In addition, calcitonin, glucocorticoids, thyroid hormone, and gonadal steroids have lesser e ects on calcium and phosphate homeostasis. Table 32-1 summarizes the mechanisms and e ects o these hormones on calcium and phosphate homeostasis.

Parathyroid Hormone

The most important endocrine regulator o calcium homeostasis is parathyroid hormone , an 84-amino acid peptide hormone secreted by the parathyroid glands. The secretion o PTH is f nely regulated in response to plasma calcium levels. Calcium-sensing receptors reside on the plasma membrane o chie cells in the parathyroid gland; when bound by extracellular calcium ions, these G protein-coupled receptors mediate increases in the level o intracellular ree calcium, which, in turn, decreases secretion o pre ormed PTH. By this mechanism, high plasma calcium levels suppress PTH secretion, while low plasma calcium levels stimulate PTH secretion. (Note: In many other secretory tissues, an increase in intracellular calcium enhances secretion. Thus, the parathyroid chie cell is unusual in its response to changes in intracellular calcium.) PTH acts on three organs to raise the plasma calcium concentration: it acts directly on kidney and bone and indirectly on the gastrointestinal (GI) tract (Fig. 32-4). The most rapid physiologic e ects o PTH are to increase reabsorption o calcium and decrease reabsorption o inorganic phosphate by the kidney tubules. These actions decrease renal clearance o calcium while increasing renal clearance o inorganic

584 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

TABLE 32-1 Summary of Endocrine Control of Calcium and Inorganic Phosphate Homeostasis HORMONE

TARGET ORGAN

MECHANISM

NET EFFECT

PTH

GI tract Kidney tubules Bone

↑ ↑ ↑ ↑

Vitamin D

GI tract Bone Parathyroid glands

↑ Ca 2 and ↑ Pi absorption ↑ Osteoclast number and activity ↓ PTH synthesis

↑ [Ca 2 ] ↑ [Pi] ↑ [Ca 2 ] ↑ [Pi]

FGF-23

Kidney tubule GI tract Bone

↓ Pi reabsorption; ↓ calcitriol secretion ↓ Ca 2 and ↓ Pi absorption via ↓ calcitriol secretion ↓ Mineralization o bone matrix

↓ [Pi] ↓ [Ca 2 ] ↓ [Pi]

Calcitonin

Bone Kidney tubule

↓ Osteoclast activity ↓ Ca 2 reabsorption (pharmacologic doses)

↓ [Ca 2 ] ↓ [Ca 2 ]

Glucocorticoids

GI tract Kidney tubule Bone

↓ ↓ ↑ ↑

Thyroid hormone

Bone

↑ Resorption

Gonadal steroids

Bone

↓ Osteoclast activity ↑ Osteoclast apoptosis ↓ Osteoblast apoptosis

Ca 2 and ↑ Pi absorption via vitamin D action Ca 2 reabsorption; ↓ Pi reabsorption Osteoclast activity — dominates i PTH 24 hr/day Osteoblast activity — dominates i PTH 3–5 hr/day

Ca 2 absorption Ca 2 and ↓ Pi reabsorption (pharmacologic doses) Osteoblast apoptosis, ↓ osteoblast activity Osteocyte apoptosis ↑ ormation

↑ ↑ ↑ ↓

↓ ↓ ↓ ↓

[Ca 2 [Ca 2 [Ca 2 [Ca 2

]↑ ]↓ ]↑ ]↓

[Pi] [Pi] [Pi] ↓ bone [Pi] ↑ bone

[Ca 2 ] ↓ [Pi] [Ca 2 ] ↓ [Pi] Bone Bone

↑ [Ca 2 ] ↓ bone ↓ [Ca 2 ] ↓ [Pi] ↓ Bone resorption

FGF-23, f broblast growth actor 23; GI, gastrointestinal; Pi, inorganic phosphate; PTH, parathyroid hormone.

phosphate. In this manner, PTH raises plasma calcium levels and decreases plasma inorganic phosphate concentrations. Another important, although slower, e ect o PTH results rom its direct actions on bone cells. Physiologic levels o PTH stimulate cell sur ace PTH receptors on osteoblasts, causing these cells to increase their expression o the osteoclast di erentiation actor RANKL (Fig. 32-3) and decrease their expression o its antagonist OPG. The resulting increase in osteoclastic activity increases bone resorption and thereby increases the release o calcium and inorganic phosphate into the circulation. PTH also induces bone marrow stromal cells to secrete cytokines such as IL-6, and these cytokines ultimately stimulate osteoclast proli eration and bone resorption. Finally, PTH raises plasma calcium levels by an indirect e ect on the intestine. PTH stimulates the kidney not only to increase calcium reabsorption and decrease phosphate reabsorption, as described above, but also to increase the enzymatic conversion o 25-hydroxy vitamin D to 1,25-dihydroxy vitamin D (calcitriol). This hydroxylation takes place in cells o the proximal renal tubules. Calcitriol, in turn, increases small intestinal absorption o calcium and (to a lesser extent) inorganic phosphate (discussed below). Although the release o skeletal calcium and inorganic phosphate could be considered catabolic, PTH simultaneously stimulates new bone ormation by promoting di erentiation o osteoblast precursors to mature osteoblasts and by enhancing osteoblast survival. Interaction o PTH with its receptor on mature osteoblasts stimulates G s, which increases adenylyl cyclase activity, which, in turn, increases intracellular cAMP. The PTH-induced increase in cAMP has an anti-apoptotic e ect on osteoblasts. In addition, the increase in cAMP promotes osteoblast release o IGF-1, which induces osteoblast precursor cells

in the bone marrow to di erentiate into mature osteoblasts (Fig. 32-3). The balance between PTH’s catabolic and anabolic e ects on bone depends on the length o time extracellular PTH remains in contact with PTH receptors on osteoblasts. Specif cally, intermittent, brie (1- to 3-hour) elevations in extracellular PTH increase bone ormation more than bone resorption and cause a net increase in bone mass. Consequently, intermittent PTH administration by once-daily injection or by some other drug delivery technology increases bone matrix production, bone mass, bone mineral density, and bone strength (see below). In contrast, continuous elevation o extracellular PTH increases bone resorption more than bone ormation and thereby causes net bone loss in patients with primary or secondary hyperparathyroidism.

Vitamin D

Despite its name, vitamin D3 is produced in the skin and is not required in the diet i sun exposure is generous. Because it is produced endogenously and travels in the blood to e ect responses in distant target tissues, vitamin D3 is more correctly considered a hormone. The term vitamin D applies to two related compounds, cholecalciferol and ergocalciferol. Cholecalci erol, or vitamin D3, is generated nonenzymatically in the skin when 7-dehydrocholesterol absorbs a photon o short ultraviolet light (UV-B; Fig. 32-5). Ergocalci erol, or vitamin D2, is produced when ergosterol in plants absorbs such a photon. Vitamins D2 and D3 are each added to dairy products and some other oods; each is available as a dietary supplement; and each is available (in much higher doses) as a prescription drug. Vitamins D2 and D3 have equal biological activities, and “vitamin D” in subsequent paragraphs re ers to both the D2 and D3 orms o the hormone.

C h a p t e r 3 2 Pharmacology of Bone Mineral Homeostasis

585

Thyroid gla nd

P a ra thyroid gla nds

H

H

HO

7-de hydrochole s te rol

P la s ma

P la s ma [Ca 2 +]

[Ca 2 +]

S kin

UV-B

P TH

H

Os te ocla s tic a ctivity libe ra te s P O 4 a nd Ca 2+ Bone HO

Kidne y

P O 4 / Ca 2+ re a bs orption Hydroxyla tion of 25(OH) vita min D to 1,25(OH)2 vita min D

Mucos a l Ca 2 + upta ke a nd tra ns port prote ins Ca 2 + a bs orption

Vita min D s tora ge

Inte s tine

FIGURE 32-4. Summary of the actions of PTH on bone, kidney, and intes2

tine. Decreased plasma [Ca ] is the primary stimulus for parathyroid hormone (PTH) secretion by the parathyroid glands. PTH raises plasma Ca 2 levels via its effects on bone, kidney, and intestine. In bone, PTH promotes increased differentiation of osteoclast precursors into mature osteoclasts. Osteoclasts resorb bone and thereby liberate inorganic phosphate (PO4) and Ca 2 into the plasma. In the kidney, PTH increases tubular reabsorption of Ca 2 and decreases proximal and distal tubular reabsorption of PO4. In addition, PTH stimulates proximal tubule cells to hydroxylate 25(OH) vitamin D, forming 1,25(OH)2 vitamin D. 1,25(OH)2 vitamin D then stimulates intestinal absorption of Ca 2 by increasing the expression of mucosal Ca 2 uptake and transport proteins. Note that the effect of PTH on the intestine is indirect, via increased renal synthesis of the active form of vitamin D. In a tightly controlled negative feedback loop, increased plasma [Ca 2 ] inhibits further PTH secretion by the parathyroid glands.

Vita min D3 (cholecalciferol) Circula tion Die ta ry Vita min D3 (a nima l s ource s ) Vita min D2 (pla nt s ource s )

S ide -cha in of Vita min D2 (ergocalciferol)

25-hydroxylase

Live r OH

H

HO

25-hydroxy vita min D (calcifediol)

1α-hydroxylase

P TH, hypophos pha te mia

Kidne y

Whether rom an endogenous (skin) or an exogenous (dietary) source, vitamin D travels to the liver, where it is either stored or converted to calci ediol [25-hydroxy vitamin D, or 25(OH)D] by the f rst o two enzymatic hydroxylation steps. The second enzymatic hydroxylation converts calci ediol to the f nal, active orm o vitamin D called calcitriol [1 ,25-dihydroxy vitamin D, or 1,25(OH)2D]. This second hydroxylation takes place in many tissues, particularly in the proximal tubule o the kidney (where it is PTH-dependent), but does not take place in the intestines because they lack the 1 -hydroxylase enzyme required or the second hydroxylation. Calcitriol’s primary effect on calcium balance is in the small intestine, where it increases the absorption of dietary calcium. Calcitriol enhances Ca2 absorption by acting on nuclear receptors in the enterocyte to up-regulate the expression o genes coding or multiple brush border proteins. Calcitriol also promotes the transcellular transport o Ca2 through the enterocyte by inducing the expression o (1) a calcium uptake pump on the luminal sur ace o the enterocyte; (2) calbindin, an intracellular Ca2 -binding

OH

H

HO

OH

1,25-dihydroxy vita min D (calcitriol)

FIGURE 32-5. Photobiosynthesis and activation of vitamin D. Both endogenous and exogenous vitamin D are converted to 25-hydroxy vitamin D in the liver and then to calcitriol in the kidney. Calcitriol is the active metabolite of vitamin D. Endogenous vitamin D3 is synthesized in the skin from 7-dehydrocholesterol in a reaction that is catalyzed by ultraviolet light (UV-B). Exogenous vitamin D can be provided as D3 (from animal sources) or as D2 (from plant sources); D3 and D2 have the same biological activity. Parathyroid hormone (PTH) increases the activity of 1 -hydroxylase in the kidney and thereby stimulates the conversion of 25-hydroxy vitamin D to calcitriol, as does hypophosphatemia.

586 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

protein; and (3) an ATP-dependent Ca2 pump that extrudes Ca2 rom the enterocyte into the surrounding capillaries. Because enterocytes do not express the 1 -hydroxylase enzyme needed to orm 1,25(OH)2D rom 25(OH)D, their absorption o calcium is regulated by blood levels o 1,25(OH)2D, which in turn depend on renal tubular unction and blood levels o PTH. Calcitriol has important e ects on other target organs, including the parathyroid glands, bone, kidneys, and immune system. Calcitriol binds to nuclear receptors in parathyroid cells and thereby inhibits PTH synthesis and release. In bone, calcitriol increases osteoclast number and activity, resulting in increased bone resorption. High blood levels o calcitriol, and lower levels o certain calcitriol analogues, increase bone ormation. In the distal tubule o the kidney, calcitriol increases the reabsorption o both calcium and phosphate. In the immune system, calcitriol production by macrophages may act as a local suppressant o adaptive immune cells; this observation has led to the use o calcitriol and calcitriol analogues in the treatment o psoriasis.

Fibroblast Growth Factor 23 and Phosphatonins

It has long been known that renal clearance o inorganic phosphate is increased by high-phosphate diets and decreased by low-phosphate diets in both normal and hypoparathyroid humans and animals. Recent research has begun to elucidate the mechanisms that regulate this homeostatic response. One responsible actor is f broblast growth actor 23 (FGF-23), a 251-amino acid protein. Injection o FGF-23 rapidly alters the activity o the renal tubular sodium-phosphate co-transporters NaPi-2a and NaPi-2c and thereby increases renal clearance of inorganic phosphate. FGF-23 also suppresses renal tubular 25-hydroxy vitamin D 1 -hydroxylase and induces renal tubular 25-hydroxy vitamin D 24-hydroxylase, thereby decreasing renal secretion o 1,25(OH)2D. The resulting decrease in blood 1,25(OH)2D reduces active transport o calcium and phosphate across the intestines and also increases parathyroid hormone secretion both directly and indirectly. Proteins with similar e ects on renal tubular clearance o phosphate and/or secretion o 1,25(OH)2D include secreted rizzled-related protein (sFRP-4), FGF-7, and matrix extracellular phosphoglycoprotein [MEPE, which does not suppress renal 1,25(OH)2D secretion]. These proteins, together with FGF-23, are collectively called phosphatonins . FGF-23 is expressed by many cells, including osteocytes. Animal models and humans with increased osteocyte expression o FGF-23 exhibit increased renal clearance o phosphate, hypophosphatemia, low or inappropriately normal blood levels o 1,25(OH)2D, and de ective mineralization o bone matrix. These f ndings suggest that osteocytes are a particularly important source o circulating FGF-23. Mutations that generate proteolysis-resistant FGF-23 produce a nearly identical syndrome in mice and humans (e.g., human autosomal dominant hypophosphatemic rickets, ADHR). A more common hereditary orm o FGF-23/phosphatonin excess in humans is X-linked hypophosphatemic rickets (XLH), caused by mutations in the endopeptidase PHEX. How PHEX mutations elevate blood FGF-23 and/or phosphatonin levels remains controversial. Nonhereditary causes o hypophosphatemia with increased renal clearance o phosphate, low or inappropriately normal blood levels o 1,25(OH)2D, and de ective mineralization o bone matrix include repeated intravenous

administration o saccharated iron (which elevates blood levels o FGF-23) and rare benign phosphatonin-secreting mesenchymal tumors that elevate blood levels o FGF-23 and/or other phosphatonins (oncogenic osteomalacia ). Cessation o saccharated iron therapy or ablation o the mesenchymal tumor rapidly reverses these two syndromes. Patients undergoing chronic dialysis or chronic renal insu f ciency have markedly elevated blood levels o FGF-23. The levels o FGF-23 are independently related to mortality, cardiac hypertrophy, and indices o endothelial dys unction, but it is unclear whether lowering or neutralizing blood FGF-23 in such patients prolongs their lives. Conditions associated with decreased levels o FGF-23 provide urther support or the importance o this hormone in calcium and phosphate homeostasis. Experimental deletion o the FGF-23 gene in mice causes hyperphosphatemia and high blood levels o 1,25(OH)2D, with resulting calcitriol intoxication (hypercalcemic renal ailure), extraskeletal calcium phosphate deposits (ectopic calcif cation, labeled tumoral calcinosis when severe), and de ective bone mineralization. Preventing calcitriol intoxication in these mice by dietary or genetic manipulations reveals that FGF-23 deletion impairs modulation o renal phosphate clearance in response to high-phosphate and low-phosphate diets. Other genetic manipulations, including disruption o FGF-23 glycosylation or inactivation o FGF-23 receptors, produce a similar e ect. When humans are ed high-phosphate or low-phosphate diets, their renal clearance o phosphate changes as described above. However, their blood levels o FGF-23 change less than expected, or sometimes not at all. Whether this discordance re ects the importance o other phosphatonins, or other variables, is not yet clear. It is also unclear whether serum inorganic phosphate regulates FGF-23 secretion and/or catabolism, because changes in serum inorganic phosphate are not consistently correlated with changes in serum FGF-23 levels. Although 1,25(OH)2D can increase FGF-23 secretion and blood levels, other mechanisms must be more important regulators o serum FGF-23, because serum FGF-23 is poorly correlated with serum 1,25(OH)2D.

Calcitonin, Glucocorticoids, Thyroid Hormone, and Gonadal Steroids PTH, vitamin D, and FGF-23 are the primary regulators o calcium and phosphate homeostasis, but several other endogenous hormones also have important e ects on bone mineral metabolism. These hormones include calcitonin, glucocorticoids, thyroid hormone, estrogens, and androgens. Calcitonin is important to calcium homeostasis in some animals but less important in humans. This hormone is a 32-amino acid peptide that is synthesized and released by para ollicular C cells o the thyroid gland in response to hypercalcemia. Calcitonin binds directly to receptors on osteoclasts; this binding inhibits the resorptive activity of the osteoclasts and thereby decreases bone resorption and plasma calcium levels. In adult humans, endogenous calcitonin has only weak e ects on plasma calcium levels, and the elimination o calcitonin secretion a ter thyroidectomy generally causes no signif cant changes in plasma calcium levels. Nevertheless, exogenous calcitonin is use ul in the emergency treatment o certain orms o hypercalcemia, as discussed below.

C h a p t e r 3 2 Pharmacology of Bone Mineral Homeostasis

Pharmacologic doses o glucocorticoids promote osteocyte and osteoblast apoptosis and inhibit osteoblast maturation and osteoblast activity, thereby decreasing bone ormation and, to a lesser extent, bone resorption. Chronic glucocorticoid use is a common cause o iatrogenic bone loss, osteoporosis, and ractures. When taking the history o a patient such as RS, it is important to determine whether she has ever taken glucocorticoids or months at a time, because this would be a signif cant risk actor or osteoporosis. Pharmacologic doses o glucocorticoids also decrease intestinal absorption o calcium and (at high doses) renal tubular reabsorption o calcium. The latter e ects would tend to lower plasma calcium levels; however, glucocorticoid use is not associated with hypocalcemia or changes in blood PTH, presumably because glucocorticoid-induced bone loss releases compensating amounts o skeletal calcium. Excess thyroid hormone also increases bone turnover. By stimulating bone resorption more than bone ormation, prolonged high levels o thyroid hormone can cause bone loss. In act, low bone mass is a common mani estation o hyperthyroidism. There ore, the evaluation o RS’s osteoporosis should include assessment o her thyroid status and measurement o her serum TSH level to rule out hyperthyroidism (see Chapter 28, Pharmacology o the Thyroid Gland). Estrogens and androgens inhibit osteoclastic activity and thereby slow the rate o bone turnover and bone loss. Among other e ects, these gonadal steroids inhibit the production o RANKL by immune cells and the production by osteoblasts o cytokines such as interleukin-6 that recruit and activate osteoclasts. Estrogen also has a pro-apoptotic e ect on osteoclasts and an anti-apoptotic e ect on osteoblasts and osteocytes. As described in more detail in Chapter 30, Pharmacology o Reproduction, estrogen exerts its actions principally by binding to the estrogen receptor (ER), which is a nuclear transcription actor. Binding o estrogen acilitates dimerization o the ER, allowing the estrogen–ER complex to recruit coactivator or corepressor molecules and bind to promoter regions o target genes. In this way, estrogen regulates the transcription o target genes encoding, or example, the cytokines that are important in bone turnover.

PATHOPHYSIOLOGY Bone turnover, including repeated cycles o bone resorption and bone ormation, is required to maintain the integrity o the skeleton. Osteoporosis and chronic kidney disease are two common disorders o bone mineral homeostasis. In osteoporosis, bone turnover is disrupted such that bone resorption exceeds bone ormation. In chronic kidney disease, the pathophysiology involves a complex interplay between decreased mineral absorption and secondary hyperparathyroidism. A summary o these and related diseases o bone mineral homeostasis—including their mechanisms, clinical eatures, and treatments—is provided in Table 32-2.

Osteoporosis Osteoporosis is a common condition in which bone mass is reduced and internal bone architecture is degraded throughout the skeleton due to decreased bone ormation, increased bone resorption, or both. The reduced bone mass and architectural deterioration make the bones ragile and predisposes them to ractures a ter minimal trauma. RS’s presentation is

587

typical, with minimal trauma and a sudden twisting motion leading to subsequent back pain rom a compression racture in a lumbar vertebra. Bone mineral density (BMD) measurements are the principal test used to determine racture risk. The lower the BMD, the higher the risk o racture. The mineral content o bones can be measured by their attenuation o x-rays and then adjusted or bone size by dividing the mineral content by the bone’s projected two-dimensional area on a simultaneous radiograph. The resulting ratio, termed areal bone mineral density (aBMD), di ers or di erent skeletal regions. To eliminate this variability, aBMD measurements are o ten expressed as standard deviations above or below the mean aBMD o that skeletal region in healthy young adults (T-score) or age-matched people (Z-score). Prospective observational studies have repeatedly shown that racture incidence in women age 55 or older approximately doubles or every 1.0 standard deviation decrease in T-score. Similarly, aBMD, T-score, and Z-score predict an excised bone’s resistance to destructive testing in vitro. Normal aBMD is def ned as the mean value measured in healthy young adults 1.0 standard deviation; osteopenia is def ned as aBMD values between 1.0 and 2.5 standard deviations below the mean or healthy young adults; and osteoporosis is def ned as aBMD values 2.5 standard deviations or more below the mean or healthy young adults. Peak bone mass is achieved in young adulthood and is determined by several actors, including dietary calcium, pubertal age, subsequent gonadal hormone status, physical activity, and the interplay o multiple genetic actors that are incompletely def ned. Once peak bone mass is attained, there is a very slow decline in bone mass during mid to late adult life. This decline probably results rom imper ections in the bone remodeling process: osteoblast-mediated bone ormation does not ully keep pace with osteoclast-mediated bone resorption. Moreover, with age, osteoblasts have a reduced capacity to proli erate, to synthesize organic bone matrix, and to respond to growth actors. As a result, there is an average loss o 0.7% o bone mass per year, and this rate is accelerated in the years around menopause (Fig. 32-6). Although the rate o bone remodeling increases in perimenopausal women, annual rates o bone loss do not change until such women are amenorrheic or intervals o 3 months or more (late perimenopause ). At that time, the lower estrogen levels lead to an increase in osteoclast activity and bone turnover rate, which causes an imbalance between bone ormation and bone resorption. The longer li espan (decreased apoptosis) o osteoclasts in the absence o estrogen allows these cells to excavate deeper cavities in trabecular bone, leading to bone remodeling characterized by widely spaced and thin trabeculae with ewer interconnections. These remodeled trabeculae are structurally weaker in weight-bearing regions than the well-connected, closely spaced, thick trabeculae characteristic o bone in premenopausal women. In cortical bone, deeper cavities coalesce to orm porous spaces. The lack o estrogen also leads to increased apoptosis o osteoblasts, rendering these cells unable to keep pace with the osteoclasts, and to increased apoptosis o osteocytes, impairing the mechanosensory network that detects microdamage and stimulates bone repair. Bone loss continues at the same rapid rate or several years a ter menses cease, a ter which the rate o annual bone loss decreases by about hal . By then, however, the increased

588 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

TABLE 32-2 Diseases of Bone Mineral Homeostasis: Mechanisms, Clinical Features, and Treatments DISEASE

MECHANISM

CLINICAL FEATURES

TREATMENT

Estrogen-def ciency bone loss

Bone resorption

ormation

Low bone mass and bone strength

Calcium, vitamin D; selective estrogen receptor modulator (SERM); estrogen; bisphosphonates; RANKL antagonists; calcitonin

Osteoporosis

Bone resorption

ormation

Low bone mass and bone strength, ragile bones

Calcium, vitamin D; SERM; estrogen; bisphosphonates; RANKL antagonists; daily subcutaneous parathyroid hormone (PTH); cathepsin K inhibitor (investigational); sclerostin antibodies (investigational)

Chronic kidney disease

↓ Excretion o phosphate ↓ Secretion o 1,25(OH)2D Secondary ↑ PTH

Ectopic calcif cation, hypocalcemia, osteomalacia, osteitis f brosa cystica

Phosphate restriction and binders; calcitriol or its analogues; calcimimetics

Hyperphosphatemiahyperostosis syndrome (HHS), tumoral calcinosis with hyperphosphatemia

Mutant FGF-23 or GALNT3 or Klotho gene; ↓ excretion o phosphate

Ectopic dermal and/or periarticular calcif cation; hyperostoses

Phosphate restriction and binders

Vitamin D def ciency

Inadequate sunlight or diet

Child: bone de ormity, pain, ragility Adult onset: bone pain, ragility

Calcium and vitamin D

D-dependent rickets, type I

Mutant 1-hydroxylase gene

Hypocalcemia, rickets

Calcitriol

D-dependent rickets, type II

Mutant calcitriol receptor

Hypocalcemia, rickets, alopecia

Calcitriol (megadose), intravenous calcium

Oncogenic osteomalacia

Hypersecretion o FGF-23

Elevated blood FGF-23, reduced or inappropriately normal 1,25(OH)2D

Ablate tumor secreting FGF-23

Hypophosphatemia induced by saccharated erric oxide

Iatrogenic hypersecretion o FGF-23

Elevated blood FGF-23, reduced or inappropriately normal 1,25(OH)2D

Withdraw saccharated erric oxide

X-linked hypophosphatemia (XLH)

Mutant PHEX protein

Rickets, hypophosphatemia, elevated blood FGF-23, reduced or inappropriately normal 1,25(OH)2D

Neutral potassium phosphate, calcitriol; calcimimetics (investigational)

Autosomal dominant hypophosphatemic rickets (ADHR)

Mutant FGF-23 (degradation-resistant)

Rickets, hypophosphatemia, elevated blood FGF-23, reduced or inappropriately normal 1,25(OH)2D

Neutral potassium phosphate, calcitriol; calcimimetics (investigational)

Autosomal recessive hypophosphatemia (ARHP)

Mutant DMP-1

Rickets, hypophosphatemia, elevated blood FGF-23, reduced or inappropriately normal 1,25(OH)2D, large pulp chambers in teeth

Neutral potassium phosphate, calcitriol; calcimimetics (investigational)

Hypophosphatemic rickets with hypercalciuria (HHRH)

Mutant NaPi2c phosphate transporter or DMP-1

Rickets, hypophosphatemia, hypercalciuria, elevated blood FGF-23 and 1,25(OH)2D, normal or low PTH

Neutral potassium phosphate

Primary hyperparathyroidism

Parathyroid tumor or parathyroid hyperplasia

Hypercalcemia, bone loss, bone pain, bone ragility, renal calculi

Bisphosphonate to stop bone loss; surgical removal; calcimimetics (investigational)

Familial hypocalciuric hypercalcemia (FHH)

Mutant (hypoactive) Ca 2 sensing receptor

Hypercalcemia, hypocalciuria, hypermagnesemia

Observation

Pseudohypoparathyroidism, type 1

Mutant G s ; impaired PTH action

Hypocalcemia, seizures, tetany, short metacarpals/tarsals, short height

Calcitriol or megadose vitamin D

Hypoparathyroidism due to CaSR

Mutant (hyperactive) Ca 2 sensing receptor

Low blood PTH, hypocalcemia, seizures, tetany

Calcitriol

Hypoparathyroidism

Parathyroid gland absence or decreased activity

Hypocalcemia, seizures, tetany

Calcitriol chlorthalidone; subcutaneous parathyroid hormone (PTH)

Paget’s disease

↑ Local bone turnover

Local bone pain and ragility, hearing loss, high-output heart ailure

Bisphosphonates; calcitonin (rarely)

chlorthalidone

C h a p t e r 3 2 Pharmacology o Bone Mineral Homeostasis Growth s purt

Increased production of cytokines Longer lifespan of osteoclasts ( apoptosis)

P e rime nopa us e in wome n

Shorter lifespan of osteoblasts ( apoptosis)

Activa tion of os te ocla s ts

Shorter lifespan of osteocytes ( apoptosis)

B

o

n

e

m

a

s

s

Me n

De e pe r, la rge r re s orption ca vitie s in bone

Wome n

More fra gile bone

0

25

50

75

589

Me cha nos e ns ing Microda ma ge in bone

100

Age (ye a rs )

Bone fra cture

FIGURE 32-7. Pathophysiologic basis o osteoporosis. Several interrelated FIGURE 32-6. Bone mass as a unction o age. In both men and women, bone mass increases with age until a peak is reached in young adulthood; the growth spurt begins earlier and peaks earlier in women compared to men (not shown). A ter the peak, bone mass gradually declines by approximately 0.7% per year. In women, the reduction in the requency o menses coincides with a sharp decline in bone mass, as the decrease in estrogen production leads to increased bone resorption. As bone mass decreases with age, the skeleton may become su f ciently ragile that minor trauma can cause ractures. The goal o antiresorptive agents is to arrest or slow the loss o bone. In contrast, bone anabolic agents can be used to reverse bone loss that has already occurred and restore bone mass and bone structure.

bone resorption and accumulation o microdamage have increased bone ragility. Patient RS, or example, was diagnosed with osteoporosis about 6 years a ter completing menopause. In summary, the bone of postmenopausal and late perimenopausal women is characterized by increased osteoclast activity and larger resorption cavities, increased but inadequate osteoblast activity, and impairment of the osteocyte mechanosensory network (Fig. 32-7). As discussed above, remodeling takes place to a greater degree in trabecular bone than in compact bone. Because appendicular bones contain trabecular bone only in their metaphyses while axial bones, such as the spine and pelvis, contain trabecular bone throughout, axial bones are more prone than appendicular bones to osteoporotic ractures. Within 25–35 years a ter menopause, women may lose as much as 35% o their cortical bone mass and as much as 50% o their trabecular bone mass. Therapeutic considerations regarding initiation o treatment or osteoporosis are discussed in Box 32-1. Certain systemic illnesses and medications induce secondary osteoporosis . Common predisposing causes include thyrotoxicosis, hyperparathyroidism, high doses o glucocorticoids, aromatase inhibitor therapy or breast cancer in women, androgen-deprivation therapy or prostate cancer in men, smoking, alcohol abuse, intestinal malabsorption and maldigestion syndromes, cirrhosis, and bone marrow abnormalities. Secondary osteoporosis is best treated by correcting the underlying cause.

Chronic Kidney Disease Chronic kidney disease causes secondary hyperparathyroidism (which enhances the resorption and ormation o bone),

actors contribute to the development o osteoporosis. Many o these actors are activated by the decline in estrogen levels in perimenopausal women. Disinhibited production o cytokines and other regulatory molecules leads to the activation o osteoclasts. Decreased estrogen allows these osteoclasts to have a longer unctional li espan; conversely, the lack o estrogen promotes apoptosis in osteoblasts and osteocytes. The resulting imbalance between osteoclast and osteoblast activity leads to the ormation o deep and large resorption cavities, which make the bone ragile and prone to racture. The relative paucity o osteocytes impairs the mechanosensory network on which repair o microdamage in bone depends. Increased microdamage also predisposes to bone ragility and eventual racture. Estrogen and raloxi ene reverse this pathophysiologic sequence o events by suppressing cytokine production, promoting osteoclast apoptosis, and inhibiting osteoblast and osteocyte apoptosis (not shown).

osteomalacia (an excess o unmineralized bone matrix), and osteitis f brosa cystica (increased osteoclastic resorption and

osteoblastic ormation o bone and replacement o hematopoietic cells by bone marrow stromal cells). Hyperparathyroidism in chronic kidney disease stems from the interplay of several factors, including hyperphosphatemia, increased blood FGF-23 levels, decreased production of 1,25(OH)2 vitamin D, and hypocalcemia (Fig. 32-8). Each o these actors originates as the result o a decrease in renal unction, mani ested as an impairment in both renal synthetic ability [important or, among other processes, the 1 -hydroxylation step in 1,25(OH)2 vitamin D synthesis] and renal tubular unction (important or phosphate excretion). Inadequate levels o 1,25(OH)2 vitamin D lead to inadequate intestinal absorption o calcium. The resulting hypocalcemia stimulates synthesis and secretion and suppresses degradation o PTH in parathyroid cells. The low levels o 1,25(OH)2D are also thought to cause a reduction in calcium receptor synthesis in the chie cells o the parathyroid gland. The decrease in calcium receptor number raises the set point or calcium regulation, so that a higher concentration o calcium is required to suppress PTH secretion. By this mechanism, hyperparathyroidism can persist even in the setting o hypercalcemia. In addition, evidence suggests that 1,25(OH)2D normally suppresses both growth o the parathyroid gland and transcription o the PTH gene. There ore, the def ciency o 1,25(OH)2D in chronic kidney disease causes secondary hyperparathyroidism by several di erent mechanisms. This understanding has led to the development o several treatments or the metabolic sequelae o chronic

590 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

B O X 3 2 - 1 Th e ra p e u tic Co n s id e ra tio n s fo r Tre a tm e n t o f Os te o p o ro s is The treatment o osteoporosis involves both nonpharmacologic (calcium, vitamin D, nutrition, exercise, and all prevention) and pharmacologic approaches. The decision to initiate pharmacologic therapy depends on bone mineral density (BMD) measurements, racture history, and racture risk. It also may depend on other actors, including concomitant medications that increase the risk o osteoporosis such as long-term glucocorticoid or aromatase-inhibitor therapy. Several years ago, the World Health Organization created a Fracture Risk Assessment tool (FRAX) that combines clinical risk actors with emoral neck BMD measurements. The FRAX tool estimates the 10-year probability or hip ractures and major osteoporotic ractures or an untreated person. The clinical risk actors include age, body mass index, history o previous ractures, parental hip ractures, glucocorticoid

kidney disease, including active vitamin D analogues— which bypass the requirement or 1 -hydroxylase activity in the kidney—and the calcimimetic cinacalcet—which adjusts the sensitivity o the calcium-sensing receptor on parathyroid chie cells (see below). Hyperphosphatemia, resulting rom decreased renal excretion o phosphate, urther exacerbates the hypocalcemia o chronic kidney disease. Hyperphosphatemia induces hypocalcemia by altering the equilibrium or hydroxyapatite ormation and dissolution, as described in Equation 32-1. Hyperphosphatemia also leads to the ormation o toxic calcium phosphate precipitates in extraskeletal tissues, as in tumoral calcinosis; in addition, in chronic kidney disease, hyperphosphatemia stimulates increased secretion o FGF-23. Elevated blood FGF-23 suppresses renal secretion o 1,25(OH)2D and has the toxic cardiovascular e ects described above. Paradoxically, osteomalacia may coexist with ectopic calcium phosphate deposits because bone matrix does not mineralize normally. Metabolic acidosis due to chronic kidney disease is one actor that inhibits bone mineralization, and other mineralization inhibitors are also involved but are not yet adequately def ned.

PHARMACOLOGIC CLASSES AND AGENTS Signif cant advances have occurred in recent years in the treatment o osteoporosis and chronic kidney disease. For osteoporosis, the relevant pharmacologic agents can be divided into two main categories: drugs that inhibit bone resorption (antiresorptive agents) and drugs that stimulate bone formation (anabolic agents). Antiresorptive agents consist o hormone replacement therapy (HRT), selective estrogen receptor modulators (SERMs), bisphosphonates, RANKL antagonists, calcitonin, and cathepsin K inhibitors (in development). Bone anabolic agents consist o uoride and parathyroid hormone. For chronic kidney disease, the relevant pharmacologic agents include drugs that lower plasma phosphate levels (oral phosphate binders) and drugs that decrease parathyroid hormone synthesis and secretion (vitamin D, vitamin D analogues, and calcimimetics).

therapy, current cigarette smoking, alcohol use, rheumatoid arthritis, and secondary osteoporosis. The National Osteoporosis Foundation has also published guidelines or treatment considerations in women and men age 50 and older. These include: 1. Hip or vertebral racture. 2. T-score 2.5 or below at the spine, emoral neck, or total hip. 3. T-score between 1.0 and 2.5 (low bone mass or osteopenia range) and high 10-year risk or ractures using the USadapted FRAX tool: treat i the 10-year risk is 3% or more or hip ractures or 20% or more or major osteoporotic ractures. 4. Patient pre erences may inf uence treatment or people with 10-year racture probabilities above or below these levels. ■

Oral calcium and vitamin D also have an important role in the prevention and treatment o osteoporosis, rickets, and hypoparathyroidism.

Antiresorptive Agents Antiresorptive agents prevent or arrest bone loss by suppressing osteoclastic bone resorption. However, because bone resorption and bone ormation are closely coupled processes, a decrease in one typically leads to a decrease in the other, via molecular mechanisms that remain to be elucidated. As a result, hormone replacement therapy (HRT), selective estrogen receptor modulators (SERMs), bisphosphonates, RANKL antagonists, and calcitonin induce little increase in bone tissue. The increase in bone mineral density seen during the f rst 12–18 months o therapy with these drugs represents f lling o resorption cavities produced during the previous period o excessive bone resorption, mineralization o this new bone, and completion o mineralization (secondary mineralization) in old bone ormed and partially mineralized during the 12–18 months preceding antiresorptive therapy. A ter the f rst 12–18 months o therapy with these agents, bone mineral density increases slowly, re ecting the slow ormation and mineralization o new bone when resorption is suppressed. Cathepsin K inhibitors are an unusual exception, because they appear to suppress osteoclastic bone resorption without suppressing bone ormation.

Hormone Replacement Therapy (HRT)

Estrogens reduce bone resorption by suppressing the transcription o genes coding or RANKL and cytokines such as IL-6 that induce osteoclast proli eration, di erentiation, and activation. Estrogen also promotes apoptosis o osteoclasts while inhibiting apoptosis o osteoblasts and osteocytes. Estrogen decreases bone ormation, as described above, but less so than more potent antiresorptive agents. Estrogen is usually administered with a progestational agent to reduce the risk o endometrial cancer in women with an intact uterus (see “Estrogens and Progestins” in Chapter 30). Estrogen also relieves postmenopausal hot ashes and vaginal dryness, and the primary indication or estrogen is the treatment o signif cant menopausal symptoms.

C h a p t e r 3 2 Pharmacology o Bone Mineral Homeostasis

591

Chronic kidne y dis e a s e Oral phosphate binders FGF-23

1,25(OH)2 D production Active vitamin D analogues

P hos pha te re te ntion

P la s ma phos pha te a nd binding to ca lcium

Active vitamin D analogues

GI Ca 2+ a bs orption

P la s ma [Ca 2+] S ynthe s is of P TH S e cre tion of P TH P a ra thyroid gla nd hype rpla s ia

Cinacalcet

S ynthe s is of P TH S e cre tion of P TH De gra da tion of P TH Ca 2+ re ce ptors on pa ra thyroid ce lls a nd S e t point for Ca 2+ re gula tion S ynthe s is of P TH P a ra thyroid gla nd hype rpla s ia Cinacalcet Hype rpa ra thyroidis m

Bone re s orption Os te oma la cia Os te itis fibros a cys tica

FIGURE 32-8. Pathophysiologic basis or osteomalacia and osteitis f brosa cystica in chronic kidney disease. In chronic kidney disease, compromised renal unction leads to decreased 1,25(OH)2 D synthesis and decreased phosphate excretion. The decrease in 1,25(OH)2 D causes decreased gastrointestinal (GI) absorption o Ca 2 , while the increased phosphate retention causes an increase in the levels o plasma phosphate, which complexes with Ca 2 . By these two mechanisms, chronic kidney disease leads to hypocalcemia. Hypocalcemia stimulates secretion o parathyroid hormone (PTH). Decreased levels o 1,25(OH)2 D stimulate PTH synthesis and parathyroid gland hyperplasia and lead to a decreased number o Ca 2 receptors on parathyroid gland chie cells and an elevated set point or Ca 2 regulation. Hyperphosphatemia may stimulate increased synthesis and secretion o PTH directly and also increase levels o FGF-23, leading to decreased levels o 1,25(OH)2 D. This combination o complex regulatory events leads to hyperparathyroidism, a syndrome characterized by increased bone resorption, increased amounts o unmineralized osteoid, and osteitis f brosa cystica. Oral phosphate binders lower plasma phosphate levels by preventing dietary phosphate absorption. Active vitamin D analogues bypass the de ect in renal 1 -hydroxylase activity that accompanies chronic kidney disease. Calcimimetics (cinacalcet) modulate the activity o the Ca 2 -sensing receptor on chie cells, such that the receptor is activated at lower plasma Ca 2 concentrations.

The adverse e ects o estrogen can cause patients to discontinue treatment; these include vaginal bleeding and breast tenderness. HRT also increases the risk o venous thromboembolism, in part because oral estrogen promotes the hepatic synthesis o clotting actors. For many women, the greatest concern regarding HRT is the increased longterm risk o breast cancer, which is statistically signif cant. HRT was once commonly prescribed or postmenopausal osteoporosis, but in 2002, a large US government-sponsored study concluded that the increased risks o breast cancer and stroke outweigh the potential benef ts o HRT on bone and other tissues. Because RS has two close relatives with breast cancer, she should strongly consider an alternative to HRT or treatment o her osteoporosis.

Selective Estrogen Receptor Modulators

Selective estrogen receptor modulators (SERMs) are a group o compounds that bind to the estrogen receptor (ER) and have tissue-selective e ects on the target organs o estrogen. Depending on the tissue, a SERM is capable o acting

as an estrogen agonist or an estrogen antagonist. These tissue-selective e ects occur because, in di erent tissues, SERM–ER complexes bind di erent tissue-specif c hormone response elements and/or di erent tissue-selective transcriptional corepressors and coactivators (see Chapter 30). The goal o SERM development is to retain the benef cial e ects o estrogen in one or more tissues while eliminating the undesirable e ects o estrogen in other tissues. Raloxi ene , or example, is an estrogen agonist in bone but an estrogen antagonist in the endometrium and breast (Fig. 32-9; see also Fig. 30-7). Raloxi ene is approved or prevention and treatment o osteoporosis because it increases vertebral and nonvertebral bone mineral density and decreases vertebral ractures. Raloxi ene is also approved or reduction in risk o invasive breast cancer in women with postmenopausal osteoporosis and in postmenopausal women at high risk or invasive breast cancer. Raloxi ene lowers low-density lipoprotein (LDL) cholesterol levels slightly but neither increases nor decreases the incidence o heart disease in postmenopausal women. Like estrogen, and to the same extent, raloxi ene

592 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

OH

-O HO

H

O

O

P

P

O

O-

-O

OH

HO

O

O

P

P

C R1

H

H

P yrophos pha te

OOH

R2

Bis phos phona te

HO Bisphosphonate

17β-e s tra diol

R1

R2

Etidrona te

OH

CH3

P a midrona te

OH

CH2

CH2

NH2

Ale ndrona te

OH

CH2

CH2

CH2

OH

S

HO

O O

N

Iba ndrona te

OH

CH2

CH2

Raloxifene

FIGURE 32-9. Structures of 17 -estradiol and raloxifene. Although raloxiene is not a steroid molecule, it is con ormationally similar to 17 -estradiol. Raloxi ene binds to the ligand binding domain o the estrogen receptor, allowing it to act as a partial estrogen agonist in some tissues (bone) and as an estrogen antagonist in other tissues (endometrium and breast). This selective action occurs because the raloxi ene-estrogen receptor complex can recruit transcriptional coactivator and/or corepressor actors in a tissue-specif c manner (see Chapter 30, Pharmacology o Reproduction, or urther details). The benzothiophene nucleus o raloxi ene is highlighted in a blue box.

N

NH2

CH3 (CH2 )4 CH3

N Ris e drona te

OH

CH2

Zole drona te

OH

CH2

Tiludrona te

H

S

N

N

Cl

FIGURE 32-10. Structures of pyrophosphate and various bisphosphonates.

increases the risk o venous thrombosis and pulmonary embolism. Raloxifene may be the preferred therapy for preventing osteoporosis in women with breast cancer or women with a family history of breast cancer. Because o her amily history o breast cancer, RS could potentially benef t rom raloxi ene. However, in adequately powered studies raloxi ene does not reduce nonvertebral ractures and may there ore not be su f ciently potent to protect RS, who has already had a vertebral racture and there ore has an increased risk o one or more nonvertebral ractures in the next 3–5 years, including an increased risk o hip racture.

Bisphosphonates

Bisphosphonates (BPs), currently the most widely used class o antiresorptive drugs, are analogues o pyrophosphate in which the readily hydrolyzable P-O-P bond is replaced by a nonhydrolyzable P-C-P bond. Five widely used bisphosphonates (the so-called amino-bisphosphonates)—alendronate , risedronate , ibandronate , pamidronate , and zoledronate — have a nitrogen-containing amino, pyridine, or imidazole moiety in the side chain, which greatly enhances their antiresorptive activity (Fig. 32-10). Because the oxygen atoms in the phosphonate groups coordinate with divalent cations such as calcium, BPs concentrate in mineralized tissues, where they are incorporated into the mineral and remain unmetabolized and biologically active. Once the bone is subsequently resorbed, the acids secreted by osteoclasts dissociate the bone mineral rom the BP, which is then excreted or deposited elsewhere in the skeleton or internalized by the osteoclasts. Within osteoclasts,

Note that the P-O-P structure o pyrophosphate is replaced with a P-C-P structure in bisphosphonate. This moti is conserved across the marketed bisphosphonates. The R1 and R2 side chains o bisphosphonates di er rom one drug to another: side chains containing a nitrogen atom have higher potency, and substitution o a hydroxyl group or a hydrogen atom at R1 enhances skeletal retention o the drug.

the amino-bisphosphonates block a step in the mevalonate pathway. Disruption o this process decreases prenylation, the covalent attachment o certain lipids ( arnesyl and geranylgeranyl moieties) to multiple proteins, including intracellular regulatory proteins such as GTPases. This, in turn, impairs several osteoclastic unctions (e.g., H -ATPase activity) and ultimately causes osteoclast apoptosis. BPs appear to inhibit the mevalonate pathway only in osteoclasts, in part because osteoclastic bone resorption greatly increases the concentration o BPs near osteoclasts, and perhaps because the acidic milieu beneath active osteoclasts protonates BPs and thereby acilitates their di usion across the osteoclast cell membrane. Intravenous pamidronate and zoledronate rapidly inhibit accelerated bone resorption caused by osteoclast hyperactivity, and these agents are approved or treatment o hypercalcemia associated with malignancy. This includes malignancies involving the bone marrow, malignancies metastatic to bone, and malignancies that secrete parathyroid hormone or parathyroid hormone-related peptide (PTHrP). PTHrP is a peptide structurally and unctionally similar to PTH, and it causes hypercalcemia by the same mechanisms as PTH. BPs are ine ective in treating hypercalcemia caused by intestinal hyperabsorption o calcium or

C h a p t e r 3 2 Pharmacology o Bone Mineral Homeostasis

hypercalcemia caused by impaired renal excretion of calcium (see Box 32-2). Some malignancies (e.g., certain lymphomas and some breast carcinomas) cause hypercalcemia by hypersecreting calcitriol. In such instances, intravenous bisphosphonates are less effective because the malignancyassociated hypercalcemia results from increased absorption of dietary calcium in addition to increased resorption of bone. Similarly, several randomized, double-blind, prospective clinical trials have shown that, although daily or weekly oral alendronate arrests bone loss and increases BMD in patients with mild primary hyperparathyroidism, it does not lower their plasma calcium levels. Neither oral nor intravenous bisphosphonates are approved for treatment of hypercalcemia due to nonmalignant conditions, but intravenous bisphosphonates are effective

593

therapy if hypercalcemia is caused by increased bone resorption (e.g., resorption associated with prolonged immobilization or paralysis, vitamin A intoxication, hyperthyroidism, or primary hyperparathyroidism with low-calcium diet). Although intravenous ibandronate is not approved for treatment of hypercalcemia, this agent has corrected hypercalcemia caused by increased bone resorption (with or without associated malignancy) in several randomized, double-blind, prospective clinical trials. Intravenous pamidronate and zoledronate also reduce skeletal complications (bone pain and fractures) in patients with osteolysis caused by bone metastases or multiple myeloma, and both agents are approved for this use. Intravenous bisphosphonates are not approved for this use in patients with other bone marrow malignancies (e.g., leukemia, lymphomas)

B O X 3 2 - 2 Tre a tm e n t o f Hyp e rca lce m ia a n d Hyp o ca lce m ia Hypercalcemia and Its Treatment Hypercalcemia is most commonly treated by one or more o three di erent approaches: decreasing intestinal calcium absorption, increasing renal calcium excretion, and inhibiting bone resorption. Emergency treatment o severe hypercalcemia starts with saline diuresis. In this treatment, intravenous saline is administered together with a loop diuretic that increases renal calcium excretion, such as urosemide. Calcium reabsorption in the kidney is, in part, passive, driven by the electrochemical gradient associated with sodium reabsorption. By inhibiting sodium reabsorption, loop diuretics decrease calcium reabsorption and, thus, increase renal calcium excretion. Saline diuresis is very e ective at rapidly reducing elevated plasma calcium levels. The saline in usion also rehydrates the patient and ensures adequate renal f ltration. Hypercalcemia can also be treated rapidly with calcitonin. As noted in the text, this agent decreases plasma calcium levels by inhibiting osteoclastic activity. The hypocalcemic e ects o calcitonin are rapid but limited in duration because tachyphylaxis develops within several days. The addition o a glucocorticoid can brie y delay tachyphylaxis. Granulomatous diseases, such as tuberculosis, sarcoidosis, and many others, can cause hypercalcemia because o excessive ectopic calcitriol production by activated mononuclear cells. The resulting increase in calcium absorption by the GI tract can be countered by eliminating dairy products and calcium- ortif ed orange juice and other calcium-rich oods rom the diet and blunted by the administration o oral phosphate, which orms insoluble complexes with dietary calcium and thereby decreases calcium absorption. Glucocorticoids (most commonly, prednisone) e ectively decrease the ectopic production o calcitriol and accelerate its catabolism. Some malignancies (especially certain lymphomas) cause hypercalcemia because o excessive ectopic calcitriol production, which is treated similarly. The hypercalcemia o most malignancies (e.g., cancer in the bone marrow or metastatic to bone) is treated by increasing renal calcium excretion and inhibiting bone resorption. Longterm management o such hypercalcemia can be achieved by

using intravenous bisphosphonates (BPs). These agents, like calcitonin, lower plasma calcium by inhibiting osteoclastic activity. Unlike calcitonin, BPs do not induce tachyphylaxis. Several BPs are e ective or this purpose, but pamidronate and zoledronate are most o ten used in the United States. Severe or symptomatic acute hypercalcemia (plasma calcium level 12 mg/dL) is generally treated with a combination o all three o the above approaches: saline diuresis with urosemide, calcitonin, and a bisphosphonate. The f rst two agents are typically e ective within the f rst 24 hours; bisphosphonates are typically e ective by the third day and may also have a prolonged e ect.

Hypocalcemia and Its Treatment Treatment o hypocalcemia depends on the etiology and the severity o the symptoms. Intravenous calcium gluconate should be used or (1) patients with severe symptoms (e.g., carpopedal spasm, tetany, seizures, and/or prolonged QT interval), (2) asymptomatic patients with an acute decrease in plasma calcium levels, and (3) patients with a corrected plasma calcium level o 7.5 mg/dLor lower. Patients with mild symptoms (e.g., tingling or circumoral paresthesias) may be able to be managed with oral calcium and vitamin D. Hypocalcemia can be caused by hypomagnesemia (which is typically due to severe GI or renal loss o magnesium) as a result o target-organ resistance to PTH and a decrease in PTH secretion. In the setting o hypomagnesemia, hypocalcemia is di f cult to correct until the plasma magnesium level is normalized. Patients with minimal to no symptoms may be treated with oral magnesium preparations (e.g., sustained-release magnesium chloride or oral magnesium oxide), although diarrhea may be a limiting actor. For patients with severe symptoms (e.g., tetany, arrhythmias, and/or seizures) or with very low plasma magnesium concentrations, intravenous magnesium sul ate may be necessary. Ultimately, as the plasma magnesium level increases, an increase in the plasma calcium level ollows. When the plasma magnesium concentration is normal (provided that the underlying etiology or hypomagnesemia is resolved), the treatment o hypocalcemia consists principally o oral calcium and vitamin D (either calcitriol or ergocalci erol), supplemented i necessary by chlorthalidone to lower renal clearance o calcium. ■

594 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

but are widely used or this purpose despite the lack o proven e f cacy in these other illnesses. Oral alendronate and risedronate and intravenous pamidronate and zoledronate decrease bone turnover and bone pain in patients with Paget’s disease and accelerate radiographic healing o the lytic bone lesions and cortical bone f ssures characteristic o Paget’s disease. All our bisphosphonates are approved or treatment o Paget’s disease that is symptomatic, associated with a high risk o complications ( racture, paralysis, and heart ailure), or associated with a serum alkaline phosphatase level at least double the upper limit o normal. Paget’s disease o bone that is widespread or associated with very severe osteolysis and/or very high serum alkaline phosphatase levels usually requires intravenous zoledronate, but milder Paget’s disease can usually be controlled by any o these our bisphosphonates. Randomized, double-blind, prospective clinical trials have also shown that BPs decrease bone resorption and prevent or arrest bone loss in patients with hyperthyroidism o all types and in skeletal regions immobilized by paraplegia, tetraplegia, hemiplegia, Guillain-Barré syndrome, etc., but no BP is approved or such uses. In well-controlled clinical trials, oral alendronate, risedronate, and ibandronate and intravenous ibandronate and zoledronate suppress bone resorption, arrest bone loss, and slightly increase spine and hip BMD in postmenopausal women, and all are approved or osteoporosis prevention and treatment in this population. In postmenopausal women, all our o these BPs decrease the risk o new vertebral ractures, and three o the our (all except or ibandronate in the marketed dose) reduce nonvertebral ractures and hip ractures. There ore, a BP would be a reasonable therapeutic option or RS, but ibandronate is not su f ciently potent because she is at signif cant risk o nonvertebral ractures. Generic alendronate is the least expensive choice. BPs also suppress bone resorption, arrest bone loss, and slightly increase spine and hip BMD in women with most other orms o hypogonadism (e.g., iatrogenic estrogen def ciency due to chemotherapy or aromatase inhibitors, or pituitary insu f ciency) but are not approved or such uses. Oral alendronate and risedronate and intravenous zoledronate also suppress bone resorption, arrest bone loss, and slightly increase spine and hip BMD in men with idiopathic or hypogonadal low BMD and are approved or such use. Ibandronate is not approved or prevention or arrest o bone loss in men. Oral alendronate and risedronate and intravenous zoledronate are approved or prevention or arrest o bone loss in patients o either sex taking chronic glucocorticoids in doses 7.5 mg prednisone-equivalent per day. Because o poor intestinal absorption, oral BPs must be swallowed in the morning a ter an overnight ast, and the patient must swallow nothing but water with the bisphosphonate and or the subsequent 30–60 minutes. Only therea ter can the patient swallow other medications, other liquids, or ood. Oral BPs can cause local esophagitis and esophageal erosion; or this reason, patients are advised to swallow bisphosphonates with at least 8 ounces (250 cc) o water and sit upright or stand or the next 30–60 minutes. Oral BPs are contraindicated in patients with delayed esophageal emptying. Intravenous BPs, especially pamidronate or zoledronate, can cause acute renal ailure or hepatitis, but this is rare. Renal impairment is less likely i the BP is in used slowly (e.g., zoledronate over 15 minutes or longer and pamidronate

over several hours). All bisphosphonates are excreted by glomerular f ltration. When renal unction is impaired, the dose o intravenous and oral BP must be reduced or withheld accordingly. No bisphosphonate is approved or use in patients with calculated glomerular f ltration rate (GFR) 30–35 mL/ min, and all BPs are contraindicated in patients with low plasma ionized calcium levels (measured or estimated by adjustment or the simultaneous plasma albumin level). Amino-bisphosphonates have been used to treat Paget’s disease o bone or more than 20 years and widely used to treat osteoporosis or more than 17 years. Serious adverse e ects are rare, but BPs are not metabolizable, and pharmacologically active BP accumulates progressively in the skeleton during chronic therapy. It is unclear whether this is therapeutically desirable (because it prevents bone resorption) or therapeutically undesirable. Prolonged inhibition o bone turnover can prevent repair o the microscopic cracks that normally develop in bone because o repetitive mechanical loads. The accumulation and coalescence o such cracks could, in theory, eventually reduce bone toughness and cause a late increase in ractures among patients treated or many years with potent bisphosphonates. Stopping BPs temporarily or permanently a ter 5–10 years may be appropriate or certain individuals. In one study in alendronatetreated women, alendronate was randomly withdrawn or continued a ter their f rst 5 years o alendronate use. During the next 5 years, symptomatic new spine ractures occurred more requently in the women randomly allocated to stop alendronate a ter 5 years. At the end o 10 years, however, there was no di erence in morphometric ractures between the two groups. Occasional patients treated chronically with bisphosphonates develop oversuppression o bone resorption (maniested as subnormal serum and/or urine indices o bone collagenolysis) or complete suppression o new bone ormation (as indicated by no new bone ormation in bone biopsies taken a ter in vivo tetracycline labeling). The latter f nding is typical o patients who develop a nonhealing stress racture or an atypical emoral sha t racture during chronic bisphosphonate therapy, but it is not yet clear whether such f ndings represent BP overdosage or impaired bone ormation intrinsic to these occasional patients. For example, many such patients are taking glucocorticoids chronically or other illnesses. It is now recognized that some patients taking potent bisphosphonates develop necrotizing osteomyelitis o the alveolar ridge (osteonecrosis o the jaw) a ter oral surgery. This occurs occasionally in patients taking intravenous bisphosphonates chronically to control hypercalcemia or other skeletal complications o malignancy but occurs rarely in patients taking intravenous or oral bisphosphonates chronically or osteoporosis. The BP doses used to treat patients with cancer are typically 9–10 times higher than those used to treat patients with osteoporosis. In addition, many patients with cancer have increased susceptibility to in ection because o chemotherapy, reduced ood intake, and other mani estations o widespread malignancy.

RANKL Antagonists

Denosumab is a synthetic, ully humanized monoclonal anti-

body directed against RANKL that reduces osteoclast numbers and bone resorption in humans and animal models o osteoporosis. Denosumab suppresses bone resorption, arrests

C h a p t e r 3 2 Pharmacology of Bone Mineral Homeostasis

bone loss, and slightly increases spine and hip BMD. These results have been observed in multiple patient populations, including women with postmenopausal osteoporosis, women with iatrogenic estrogen def ciency due to aromatase inhibitors, men with idiopathic or hypogonadal osteoporosis, and men with iatrogenic hypogonadism caused by prostate cancer therapy. Denosumab is approved in a dose o 60 mg subcutaneously every 6 months. With this dosing regimen, it is approved (1) or treatment o women with postmenopausal osteoporosis who are at high risk or racture, (2) to increase bone mass in men with osteoporosis, (3) to increase bone mass in men with non-metastatic prostate cancer receiving androgen deprivation therapy, and (4) in women receiving adjuvant aromatase inhibitor therapy or breast cancer who are at high risk or racture. Denosumab also reduces bone resorption and skeletal complications in patients with malignancies metastatic to bone or involving bone marrow. It is approved in a di erent dosing regimen, 120 mg every 4 weeks, or prevention o skeletal-related events in patients with bone metastases rom solid tumors. Denosumab also reduces bone resorption in patients with rheumatoid arthritis; it is not yet clear whether denosumab increases bone mass or reduces skeletal complications in this disease. The e ects o denosumab on hypercalcemia and Paget’s disease o bone are not yet clear. Recent studies have reported osteonecrosis o the jaw and atypical emur ractures with denosumab, although these adverse e ects are very rare.

Calcitonin

As discussed above, calcitonin binds to and activates a G protein-coupled receptor on osteoclasts, thereby decreasing the resorptive activity o these cells. Because o this action, exogenous calcitonin can be used to treat conditions characterized by high osteoclastic activity, such as certain orms o hypercalcemia, Paget’s disease o bone, and postmenopausal osteoporosis. The synthetic calcitonin marketed in the United States has an amino acid sequence native to salmon because that peptide has a higher a f nity or the human calcitonin receptor and a longer hal -li e than human calcitonin. Salmon calcitonin is a peptide and there ore is administered subcutaneously ( or Paget’s disease and hypercalcemia) or as a nasal spray ( or postmenopausal osteoporosis). Twice-daily subcutaneous injections are use ul in the rapid treatment o severe hypercalcemia (see Box 32-2). An important drawback to long-term calcitonin administration is the tachyphylaxis that can result rom desensitization o the receptor-signaling pathway. For the treatment o hypercalcemia, short-term administration o glucocorticoids can delay the onset o tachyphylaxis. Clinical trials in patients with Paget’s disease show that once-daily subcutaneous salmon calcitonin reduces bone turnover and bone pain and accelerates radiographic healing o osteolytic lesions. However, bisphosphonates have greater therapeutic e f cacy than calcitonin or treatment o Paget’s disease, particularly when the Paget’s disease is severe. Once-daily intranasal salmon calcitonin slows vertebral bone loss inconsistently in women who are less than 5 years postmenopausal (and there ore losing bone rapidly). In older women with postmenopausal osteoporosis, it reduces spine ractures inconsistently, ails to reduce nonspine ractures, and has inconsistent analgesic properties.

595

Because o its low e f cacy, intranasal salmon calcitonin is not a good therapeutic option or RS. It may be use ul in women at least 5 years postmenopausal who are unable or unwilling to take any o the more e ective alternatives (e.g., raloxi ene, estrogen, bisphosphonates, RANKL inhibitors, or teriparatide). However, there have been recent reports suggesting an increased incidence o cancer with calcitonin use.

Bone Anabolic Agents Antiresorptive agents slow the rate o bone loss but do not build new bone. For patients who have already lost a large amount o bone mass (BMD more than 3.0 standard deviations below normal) or who have experienced one or more osteoporotic ragility ractures, antiresorptive agents are not optimal therapies. This realization has led to the development o bone anabolic agents, which are drugs that actually increase bone mass and bone strength, not just prevent its loss.

Fluoride

The f rst bone anabolic agent was uoride, in doses substantially higher than those ingested in artif cially uoridated water. At such doses, uoride is a mitogen or osteoblasts and it increases trabecular bone mass while accelerating cortical bone loss. Use o uoride, however, leads to the conversion o hydroxyapatite to uoroapatite, which is denser and more brittle. It is unclear whether uoride prevents vertebral or nonvertebral ractures: studies to date have shown inconsistent results and uoride is not approved to treat osteoporosis.

Parathyroid Hormone

As noted above, a persistently elevated plasma concentration o PTH, such as occurs in hyperparathyroidism, leads to increased bone remodeling, with more bone resorbed than ormed. As a result, bone can become weak and susceptible to racture and osteitis f brosa cystica. In contrast, although intermittent exposure o bone cells to PTH also increases bone remodeling, more new bone is ormed than old bone resorbed. Thus, once-daily subcutaneous administration of PTH favors bone anabolism, while continuous exposure to PTH favors bone resorption. Native PTH is an 84-amino acid peptide, but N-terminal ragments containing the f rst 31–34 amino acids o PTH retain essentially all the important unctional properties o the native protein. The 1–34 ragment has been shown in clinical trials to act as a power ul anabolic agent that builds new bone. Because PTH(1–34) is a peptide, the bioavailability o this agent is close to zero when administered orally. The currently available ormulation is a subcutaneous injection that is designed to be sel -administered. Alternative dosage orms (e.g., transcutaneous) are in advanced stages o clinical development. Human PTH(1–34) is approved under the generic name teriparatide or the treatment o osteoporosis in postmenopausal women, idiopathic and hypogonadal osteoporosis in men, and glucocorticoid-induced osteoporosis in patients o either sex, and approved or the reduction o spine and nonspine ractures in postmenopausal osteoporotic women at high risk o racture. Full-length human PTH(1–84) is approved or such uses in some countries but not in the United States (because o hypercalcemia and other adverse e ects rom the marketed dose). Because prolonged treatment o rodents with either o these peptides causes dramatic bone overgrowth ollowed by osteosarcomas, teriparatide is used

596 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

only in patients at high risk o ractures. However, there is no evidence that any parathyroid hormone increases osteosarcomas in humans, and the drug has been approved and available or over 12 years. In humans, the anabolic skeletal e ects o teriparatide are attenuated by concomitant alendronate therapy. It is unclear whether concomitant therapy with other bisphosphonates, or prior therapy with any bisphosphonate, does the same.

Treatment of Secondary Hyperparathyroidism in Chronic Kidney Disease Three pharmacologic approaches are currently available to prevent and modi y the metabolic sequelae o chronic kidney disease—oral phosphate binders, calcitriol and its analogues, and calcimimetics.

Oral Phosphate Binders

In patients with chronic kidney disease, or chronic hyperphosphatemia o any cause, the increased plasma phosphate can complex with circulating calcium. The resulting decrease in plasma calcium concentration can lead to hyperparathyroidism, and the precipitation o calcium phosphate in extraskeletal tissues can impair their unction. Dietary phosphate restriction and oral phosphate binders can limit both processes. Aluminum hydroxide was one o the f rst agents used to treat hyperphosphatemia. Aluminum precipitates phosphate in the gastrointestinal tract, orming nonabsorbable complexes. Although e ective at lowering plasma phosphate levels, this approach has been abandoned (except in cases o re ractory hyperphosphatemia) because o aluminum toxicity: over the course o years, chronic use o aluminum-based phosphate binders can lead to chronic anemia, osteomalacia, and neurotoxicity. Oral preparations o calcium carbonate and calcium acetate can control plasma phosphate. These agents, when administered with meals, bind to dietary phosphate and thereby inhibit its absorption. At the doses required or phosphate binding, however, these agents can also cause iatrogenic hypercalcemia and may increase the risk o vascular calcif cation. Sevelamer is a nonabsorbable cationic ion-exchange resin that binds intestinal phosphate, thereby decreasing the absorption o dietary phosphate. Sevelamer also binds bile acids, leading to interruption o the enterohepatic circulation and to decreased cholesterol absorption. Its principal disadvantage is its expense. Sevelamer is used to treat hyperphosphatemia in patients with chronic kidney disease. Sevelamer is also used to correct hyperphosphatemia in patients with the hyperphosphatemia-hyperostosis syndrome (also known as tumoral calcinosis with hyperphosphatemia), who are def cient in FGF-23 secretion or action (Table 32-2).

Calcitriol and Its Analogues

Because impaired synthesis o 1 -vitamin D derivatives is one o the main homeostatic disturbances leading to secondary hyperparathyroidism in chronic kidney disease, vitamin D is a logical replacement therapy in this disease. Three active (i.e., 1 -hydroxylated) vitamin D congeners are approved or treatment o secondary hyperparathyroidism. All of these agents bypass the need for 1 -hydroxylation in the kidney and are therefore useful in the treatment of bone diseases that complicate renal failure. Active vitamin D increases dietary

absorption o calcium, and the resulting increase in plasma calcium level suppresses the secretion o PTH by chie cells o the parathyroid gland. In addition, these agents bind to and activate vitamin D receptors on the chie cells and thereby suppress PTH gene transcription and parathyroid hyperplasia. Care should be taken to avoid hypercalcemia when administering any o the active vitamin D congeners. Calcitriol [1,25(OH)2D3] is the dihydroxylated orm o vitamin D3. Calcitriol is available in oral and intravenous orms; some data suggest that the intravenous ormulation may be more e ective in patients on hemodialysis. Calcitriol should not be administered to patients with chronic kidney disease until hyperphosphatemia has been controlled with diet and/or drugs, because the addition o calcitriol can cause increased plasma levels o both calcium and phosphate. Paricalcitol [19-nor-1,25(OH)2D2] is a synthetic analogue o vitamin D. Doxercalciferol [1 -(OH)D2] is the 1 hydroxylated orm o vitamin D2; it is 25-hydroxylated to the ully active 1,25-dihydroxy orm in the liver. Both paricalcitol and doxercalci erol may lower plasma PTH levels without signif cantly raising plasma calcium levels.

Calcimimetics

Although vitamin D and its analogues can be e ective in the treatment o secondary hyperparathyroidism, these agents can also lead to unwanted hypercalcemia and hyperphosphatemia. The so-called calcimimetics—agents that modulate the activity o the calcium-sensing receptor on chie cells— are e ective treatments or hyperparathyroidism that do not cause these unwanted e ects. Cinacalcet, the f rst US Food and Drug Administration (FDA)-approved calcimimetic, binds to the transmembrane region o the calcium-sensing receptor and thereby modulates receptor activity by increasing its sensitivity to calcium. Because the cinacalcet-bound receptor is activated at lower calcium concentrations, PTH synthesis and secretion are also suppressed at lower calcium concentrations. As diagrammed in Figure 32-8, these e ects interrupt the pathophysiologic sequence o events leading rom chronic kidney disease to secondary hyperparathyroidism. Cinacalcet is approved or treatment o secondary hyperparathyroidism in patients with chronic kidney disease on dialysis and or treatment o hypercalcemia associated with parathyroid carcinoma. It is also approved or treatment o severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroid surgery. Unexpectedly, cinacalcet does not arrest or reverse bone loss in patients with either hyperparathyroidism or parathyroid carcinoma, or unclear reasons.

Calcium, Inorganic Phosphate, and Vitamin D Calcium

Oral calcium has both therapeutic and prophylactic utility. It is administered as a therapy or hypocalcemic states associated with disorders such as vitamin D-dependent rickets and hypoparathyroidism. In severe cases o hypocalcemia, calcium can be administered intravenously. Commonly used intravenous ormulations include calcium gluconate and calcium chloride . Calcium gluconate is pre erable because it produces less tissue irritation i extravasated. To prevent osteoporosis or treat mild hypocalcemia, calcium is typically administered orally as calcium citrate or calcium carbonate , although some other preparations are

C h a p t e r 3 2 Pharmacology of Bone Mineral Homeostasis

sometimes used. Calcium citrate is the more readily absorbed orm, but calcium carbonate is the more widely used because o its lower cost, higher ratio o calcium to total weight, wide availability (e.g., Tums®), and antacid properties. Calcium carbonate should be taken with meals. Since calcium carbonate requires gastric acid or absorption, while calcium citrate does not, calcium citrate should be used in patients receiving proton pump inhibitor therapy. Dietary calcium supplementation has been shown in clinical trials to reduce vertebral bone loss modestly in postmenopausal women, although its e ects on racture prevention are less clear. I RS had taken calcium regularly a ter menopause and during late perimenopause, her vertebral bone loss might have slowed, reducing her risk o a spine racture. She should now be counseled to take daily calcium (and vitamin D) supplementation as a component o her therapy or osteoporosis. In women over age 50, the total calcium intake ( ood supplement) should not exceed 1,100–1,200 mg calcium per day.

Inorganic Phosphate

Inorganic phosphate is administered as a therapy or hypophosphatemia caused by renal phosphate wasting, intestinal phosphate malabsorption, rapid bone remineralization, sepsis, or other disorders. Commonly used preparations are neutral potassium phosphate and neutral sodium phosphate. The potassium salt is usually pre erred because sodium can increase renal phosphate clearance by expanding extracellular uid volume. “Neutral” re ers to the pH o the salt when dissolved (acidic salts o inorganic phosphate complicate treatment undesirably). The ratio o phosphate to total weight varies by preparation, so inorganic phosphate should be prescribed by millimoles, not by weight. I hypophosphatemia is severe, potassium phosphate or sodium phosphate can be administered intravenously with care ul monitoring o plasma calcium levels. Oral phosphate overdosage causes diarrhea; intravenous phosphate overdosage causes hypocalcemia (see above).

Vitamin D

Vitamin D preparations include cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol [25(OH)D], and calcitriol [1,25(OH)2D3] (Fig. 32-5). Several synthetic vitamin D analogues are also available, as noted above. Vitamin D is used in the treatment o hypoparathyroidism, rickets, osteomalacia, osteoporosis, and chronic kidney disease. Calcitriol is pre erred because o its aster onset and o set (12 hours) and aster approach to steady state (72–96 hours). Because vitamin D increases both plasma calcium and plasma phosphate, the plasma levels o these minerals should be monitored care ully. In the case o hypoparathyroidism, calcitriol is used to increase intestinal absorption o calcium; concomitantly, a thiazide diuretic (pre erably chlorthalidone because o its prolonged action) is used to lower renal clearance o calcium. Once the plasma calcium level is normal, renal clearance o phosphate usually increases enough to lower plasma inorganic phosphate levels to normal. I not, oral phosphate binders should be added. Once plasma calcium and inorganic phosphate levels are near normal, urinary calcium excretion must be checked to guard against hypercalciuria. For type I vitamin D-dependent rickets (Table 32-2), calcitriol is used. Type II vitamin D-dependent rickets (Table 32-2) is re ractory to conventional doses o calcitriol,

597

but very high doses have been e ective or some patients with this disease. For nutritional rickets, vitamin D is used at low doses as a preventive measure and at higher doses as a treatment. In vitamin D-resistant rickets accompanied by hypophosphatemia, oral neutral potassium phosphate and calcitriol are administered. Vitamin D and dietary calcium supplements are used in combination to prevent as well as treat osteoporosis because many elderly individuals have poor calcium intake and are also vitamin D-def cient, especially i they lack sunlight exposure. Although di erences exist in the suggested amount o vitamin D supplements, the range o 800–1,000 international units daily is o ten accepted; some experts eel that higher doses are necessary. The combination o calcium and vitamin D prevents spine, nonspine, and hip ractures in some studies but not others; this inconsistency may re ect study-to-study di erences in the incidence o undiagnosed renal 25-OH vitamin D 1 -hydroxylase def ciency, which would require supplements o calcitriol rather than vitamin D. RS should be counseled to take a daily vitamin D supplement o at least 1,000 international units.

CONCLUSION AND FUTURE DIRECTIONS Bone is composed o organic and inorganic components. The organic component consists o cells (osteoblasts, osteoclasts, and osteocytes) and an organic matrix called osteoid (mainly type I collagen). The inorganic component consists primarily o the calcium phosphate salt hydroxyapatite. The dynamic structure o bone depends on the relative balance between anabolic and resorptive processes and on the physiologic regulators o calcium and phosphate homeostasis. The most important modulators o bone remodeling and bone mineral homeostasis are parathyroid hormone (PTH), calcitriol, and FGF-23. Through their actions on bone, kidney, and intestine, these hormones preserve bone mineral homeostasis, sometimes at the expense o bone integrity. Bone disorders can result rom abnormal levels o these hormones (e.g., high levels o PTH in hyperparathyroidism, low levels o vitamin D in nutritional rickets, and high levels o FGF-23 in hypophosphatemic rickets and oncogenic osteomalacia), increased rates o bone remodeling (e.g., unbalanced bone resorption in osteoporosis and increased ormation o disorganized bone in Paget’s disease), or ailure o organs that are important in maintaining mineral homeostasis (e.g., chronic kidney disease). Bone disorders usually lead to structurally weakened bone because o either (1) a reduction in bone mass because o increased bone resorption or decreased bone ormation or (2) the ormation o architecturally unsound bone because o excessively rapid bone ormation (woven bone) or def cient bone mineralization (rickets and osteomalacia). In turn, structural weakening o bone predisposes to bone racture or de ormity. Bone disorders can be treated by correcting the underlying hormonal or mineral imbalances (e.g., vitamin D and calcium) or by modulating bone remodeling (e.g., SERMs, bisphosphonates, and RANKL antagonists). Pharmacologic

598 PRINCIPLES OF ENDOCRINE PHARMACOLOGY

interventions directed at the physiology o bone remodeling can be divided into two main categories: antiresorptive agents and bone anabolic agents. The majority o drugs currently approved or the treatment o osteoporosis are antiresorptive agents. These drugs act by inhibiting osteoclastic bone resorption, thus slowing the loss o bone mass. However, these drugs do not stimulate new bone ormation and do not increase true bone mass (matrix plus mineral). Hence, antiresorptive agents do not represent optimal therapy or individuals who have already sustained severe loss o bone mass. The only FDA-approved bone anabolic agent is once-daily PTH, which acts by increasing bone ormation and is there ore the most benef cial agent or patients with very low bone mass. The structurally related natural protein, PTH-related protein, has similar e ects in animals, and a synthetic analogue o PTHrP increases bone mass in humans and is undergoing additional trials in humans. Most drugs that reduce bone resorption subsequently reduce bone ormation. Two important exceptions are currently undergoing clinical trials in humans: a ullyhumanized monoclonal antibody that neutralizes sclerostin (an osteocyte-derived glycoprotein that inhibits osteoblast activity) and an oral inhibitor o cathepsin K (a protease expressed in osteoclasts that helps to degrade the bone matrix). The ormer increases bone ormation without increasing bone resorption, and the latter decreases bone resorption without decreasing bone ormation. The action o these agents suggests that it may be possible to uncouple bone resorption rom bone ormation and thereby treat osteoporosis more e ectively.

Acknowledgment We thank Allen S. Liu and Ariel Weissmann or their valuable contributions to this chapter in the First and Second Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy, Robert M. Neer or his valuable contribution to the Third Edition, and Armen H. Tashjian, Jr. or his valuable contributions to the First, Second, and Third Editions.

Suggested Reading Andress DL. Vitamin D treatment in chronic kidney disease. Semin Dial 2005;18:315–321. (Reviews progression of chronic kidney disease and indications for vitamin D therapy.) Bergwitz C, Juppner H. Disorders o phosphate homeostasis and tissue mineralisation. Endocr Dev 2009;16:133–156. (Current understanding of the pathophysiology, diagnosis, and treatment of abnormal phosphate homeostasis and tissue mineralization.) Ebeling PR. Osteoporosis in men. N Engl J Med 2008;358:1474–1482. (Review of an underappreciated public health problem.) Lobo RA. Where are we 10 years a ter the Women’s Health Initiative? J Clin Endocrinol Metab 2013;98:1771–1780. (Discussion that updates many of the issues noted in the Women’s Health Initiative.) Maclean C, Newberry S, Maglione M, et al. Systematic review: comparative e ectiveness o treatments to prevent ractures in men and women with low bone density or osteoporosis. Ann Intern Med 2008;148:197–213, 423–425, 884–887. (Excellent overview of the comparative effectiveness of various agents for the treatment of osteoporosis.) National Osteoporosis Foundation. Clinician’s guide to prevention and treatment of osteoporosis. Washington, DC: National Osteoporosis Foundation; 2013. (An excellent review of evaluating, preventing, and managing osteoporosis.) Quer eld U. The therapeutic potential o novel phosphate binders. Pediatr Nephrol 2005;20:389–392. (Review of agents used to lower plasma phosphate levels.) Raisz LG. Pathogenesis o osteoporosis: concepts, con icts, and prospects. J Clin Invest 2005;115:3318–3325. (Current understanding of osteoporosis pathophysiology.) Rosen CJ. Postmenopausal osteoporosis. N Engl J Med 2005;353:595–603. (Succinct overview of the clinical management of osteoporosis.) Rosen CJ. Vitamin D insu f ciency. N Engl J Med 2011;364:248–254. (Discusses current understanding and uncertainties about vitamin D levels and supplementation.) Seeman E, Martin TJ. Co-administration o antiresorptive and anabolic agents: a missed opportunity. J Bone Miner Res 2015;30:753–764. (Discussion of potentially combining agents to stimulate bone formation and block bone resorption.) Steddon SJ, Cunningham J. Calcimimetics and calcilytics— ooling the calcium receptor. Lancet 2005;365:2237–2239. (New approaches to pharmacologic modulation of the calcium-sensing receptor.) Watts NB, Bilezikian JP, Camacho PM, et al. American Association o Clinical Endocrinologists medical guidelines or clinical practice or the diagnosis and treatment o postmenopausal osteoporosis. Endocr Pract 2010;16:1–37. (Detailed and extensive review with a large reference list.)

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V Principles of Chemotherapy

P e pt ptid idogg lyyca n ce ll wa llll Inhibi bito bi to rs o f c e ll wall s ynthe s is wa Fos fom omyc ycin Cyclos e rine V Va n co comy m ycin myci P e ni n cillins Ce pha los poo ri rins ns

Monoba ba ct ctaa ms m C a rba pe ne ms Etha mbb ut utol ol P yra zina ina mi m de Is onia zi zidd

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33

Principles of Antimicrobial and Antineoplastic Pharmacology THF

DHF

P rot rot ote te in in

In Inhibito rs o f tran tr ans c riptio n and transs lat latio ioo n

Ribb oss ome Ri o me m 50S

30S SDo n a ld

P urine s

P yrimi midi dine ne s

Inhi hibi bito to rs o f DNA A s ynthe s is a d inte g rity an

S ulfona m id idee s M . Co e n , Vid ya s a g a r Ko d u ri, a n d David E. Go la n Trim m e thoprim ho

Fida xo micin Fida i Chlora mphh e nicol i l Q uinolone s Rifa mpin Lincos a mide s Aminoglycos ide s S tre ptogra mi mins ns mRNA S pe ctinomycinn Oxa zolidinone s Ox Te tr Te traa cyc c ycc line s INTRODUCTION P le uromutilins & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 603–60 6044 M METHODS ME MET THO TH HOD OF OF TR T TRE TREATMENT REAT EATMEN ENT NT T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 6144 Glycylcyclinee s Comb mDNA inat MECH ME CHAN ANISMS OF SELECTIVE TARG RGET E ING. . . . . . . . . . . . . . . . . 604 DN Ation Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 Ma crolide s Prophylactic Chemooth ther erapeuti erap rapeu eeutics tics t cs . . . . . . . . . . . . . . . . . . . . . . . 6115 Uniq Un ique ue Drugg Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605 Ke tolide s Selective Inhibition off Si Simi mila larr Ta Targ rget etss . . . . . . . . . . . . . . . . . . . 605 05 INHIBITORS OF FO FOLATE ATE M METABOLISM: Common Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 6055 EXAMPLES OF O SELECTIVE TARGETING AND SYNE ERG RGIS ISTI TIC C DR DRUG INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . 615 PATHOGENS, CANCER CELL BIOLOGY, Folate Metab boli lism m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615 AND DRUG CLASSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Inhibitors of Folate Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . 61 6155 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Unique Drug Targets: Antimicrobial Fungi and Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Dihydropteroate Synthase Inhibitors . . . . . . . . . . . . . . . . 615 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608 Selective Inhibition of Similar Targets: Antimicrobial Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608 Dihydrofolate Reductase Inhibitors . . . . . . . . . . . . . . . . . 617 Carcinogenesis and Cell Proliferationn . . . . . . . . . . . . . . . . . 608 Common Targets: Antineoplastic Dihydrofolate Cytotoxic Chemotherapyy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610 Reductase Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617 Log Cell Kill Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611 Synergy of DHFR Inhibitors and Sulfonamides . . . . . . . . . . 618 MECHANISMS OF DRUG RESISTANCE . . . . . . . . . . . . . . . . . . . . 612 Genetic Causes of Drug Resistance . . . . . . . . . . . . . . . . . . . . . 612 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 618 Reduced Intracellular Drug Concentration . . . . . . . . . . . . . 613 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .619 Target-Based Mechanismss . . . . . . . . . . . . . . . . . . . . . . . . . . 613 Insensitivity to Apoptosiss . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 Practices That Promote Drug Resistance . . . . . . . . . . . . . . . . 614

INTRODUCTION While in ectious diseases and cancers have di erent underlying etiologies, rom a pharmacologic perspective, the broad principles o treatment are similar. One common thread in these pharmacologic strategies is the targeting of selective differences between the microbe or cancer cell and the normal host cell. A second common thread is that the goal of treatment is complete inhibition of growth of the microbe or cancer cell. Because both microbes and cancer cells can evolve resistance to drug therapies, the development o new treatments is also a continually evolving process. In ectious diseases and cancers are among the deadliest a f ictions plaguing human societies. The World Health Organization (WHO) has estimated that, in the year 2012, communicable disease accounted or 23% o 56 million deaths worldwide, and malignant neoplasms accounted or a urther 14.7%. Among the in ectious diseases, the most common causes o mortality worldwide included lower

respiratory in ections (5.5%), diarrheal diseases (2.7%), HIV/AIDS (2.8%), tuberculosis (1.7%), and malaria (1.2%). In the developed world, cancer (along with heart disease and stroke) is a more signi cant cause o death than in ectious disease. The deadliest cancers in the United States presently include lung cancer (159,260 estimated deaths in 2014), colon cancer (50,310), breast cancer (40,430), pancreatic cancer (39,590), and prostate cancer (29,480). Patterns o both in ectious and neoplastic diseases will likely change as increasingly e ective treatments and preventive measures are developed and distributed. This chapter ocuses on the principles o antimicrobial and antineoplastic pharmacology, but there are also many important and e ective nonpharmacologic strategies to combat microbes and cancer. These strategies include public health measures, vaccinations, and screening. Most public health and vaccination programs aim to prevent in ections rather than treat existing in ections. Smallpox, or example, was eradicated worldwide in 1977 through aggressive 603

604 PRINCIPLES OF CHEMOTHERAPY

In 19 3 5 , in Wu p p e rt a l, Ge rm a n y, 11-ye a r-o ld Hild e g a rd Do m a g k is d e s p e ra te ly ill w ith a s tre p to co cca l in e ctio n th a t s h e co n tra cte d a te r a n a ccid e n ta l p in p rick w ith a n e m b ro id e ry n e e d le . In d e s p e ra tio n , Hild e g a rd ’s a th e r, Dr. Ge rh a rd Do m a g k, in je cts h e r w ith p ro n to s il, a re d d ye w ith w h ich h e h a s b e e n e xp e rim e n tin g in h is la b o ra to ry. In w h a t s e e m s like a m ira cle , s h e m a ke s a co m p le te re co ve ry. Th is s to ry a ctu a lly b e g a n 3 ye a rs e a rlie r, w h e n Dr. Do m a g k o b s e rve d th a t p ro n to s il p ro te cte d m ice a n d ra b b its ro m le th a l d o s e s o s ta p hylo co cci a n d s tre p to co cci. He d is cove re d th is b y s cre e n in g th o u s a n d s o d ye s (w h ich a re , in a ct, ch e m ica ls th a t b in d to p ro te in s ) o r a n tib a cte ria l a ctivity. Wh e n h is d a u g h te r b e ca m e ill, h o w e ve r, Do m a g k wa s n o t s u re w h e th e r p ro n to s il’s a n tib a cte ria l e f ca cy in m ice

vaccination programs, although concerns have been raised about the potential use o this virus as a bioterror agent. A similar campaign to eradicate polio is ongoing. Reductions in smoking and other environmental carcinogens have had a major impact on cancer mortality. Cancer screening, through regular mammograms, colonoscopy, and other tests, is widely used to detect cancer in its early and more treatable stages. Early detection through the widespread use o Papanicolaou cytologic tests (Pap smears) has caused the mortality o cervical cancer to decrease by more than twothirds in the United States; cervical cancer has moved rom the primary to the 15th leading cause o cancer deaths in women. It is hoped that widespread vaccination against speci c types o human papilloma virus, the most common causative agent o cervical cancer, will urther reduce the mortality o this cancer. E ective strategies against disease, including drug therapy, also depend on socioeconomic actors. In a f uent countries, the widespread use o antimicrobial drugs and improvements in sanitation and nutrition have markedly reduced mortality rom in ectious diseases. This progress has only begun to reach the developing world, where otherwise treatable in ectious diseases remain major causes o mortality. Since 2012, a growing body o research has supported the use o combination therapy as preexposure prophylaxis (PrEP) in the prevention o HIV in ection. In May 2014, the Centers or Disease Control and Prevention (CDC) released new guidelines on the use o teno ovir-emtricitabine to prevent HIV in ection in populations at substantial risk,

w o u ld ca rry ove r to in e ctio n s in h u m a n s . He ke p t h is p e rs o n a l te s t o th e d ru g a s e cre t u n til d a ta ro m o th e r p hys icia n s in d ica te d th a t th e d ru g h a d b e e n s u cce s s u l in cu rin g o th e r p a tie n ts o th e ir in e ctio n s . In 1939, Ge rh a rd Do m a g k wa s a wa rd e d th e No b e l Prize in Phys io lo g y o r Me d icin e o r h is d is cove ry o th e th e ra p e u tic b e n e f t o p ro n to s il.

Questions 1 . What is the mechanism responsible for the antibacterial action of prontosil? 2 . Why does prontosil kill bacteria but not human cells? 3 . What has caused the utility of drugs such as prontosil to decline over the past 75 years? 4 . Why are drugs of the same class as prontosil now used in combination with other antibacterial agents?

including men who have sex with men, heterosexual men and women with in ected partners, and IV drug users. Despite the importance o public health measures, vaccinations, and screening procedures, drug therapy remains vital to the treatment o microbial disease and cancer. Understanding the general principles and mechanisms o antimicrobial and antineoplastic pharmacology is essential to the sa e and e ective prescribing o existing drugs and, especially in light o continually evolving mechanisms o resistance, to the discovery o new drugs.

MECHANISMS OF SELECTIVE TARGETING The goal o antimicrobial and antineoplastic drug therapy is selective toxicity: inhibiting pathways or targets that are critical or pathogen or cancer cell survival and replication at concentrations o drug lower than those required to a ect critical host pathways. Selectivity can be realized by attacking (1) targets unique to the pathogen or cancer cell that are not present in the host, (2) targets in the pathogen or cancer cell that are similar but not identical to those in the host, and (3) targets in the pathogen or cancer cell that are shared with the host—or are even host rather than pathogen gene products—but that vary in importance between pathogen and host and thus impart selectivity (Table 33-1). These selectively targeted di erences range rom structures that are unique to individual pathogens, such as the peptidoglycan

TABLE 33-1 Mechanisms of Selective Targeting by Chemotherapeutic Agents TYPE OF TARGETING

MECHANISM

EXAMPLE

Unique

Drug targets genetic or biochemical pathway that is unique to pathogen

Bacterial cell wall synthesis inhibitor

Selective

Drug targets protein isoform that is unique to pathogen or cancer cell

Dihydrofolate reductase (DHFR) inhibitor

Common

Drug targets a host protein or pathway that is more important to pathogen or cancer cell than to host cell

5-Fluorouracil

C h a p t e r 3 3 Principles of Antimicrobial and Antineoplastic Pharmacology 605

cell wall o bacteria, to di erences as slight as a single amino acid change in a signaling protein that is otherwise common to cancer cells and normal cells, such as the acquisition o activating mutations in the epidermal growth actor receptor (EGFR) in certain lung cancers. In principle, drugs exhibit the least toxicity to the host when they target unique di erences and the most toxicity when they target common pathways. For a given drug, the ratio o the toxic dose to the therapeutic dose is termed the therapeutic index (see Chapter 2, Pharmacodynamics) and is an indication o how selective the drug is in producing the desired e ects. A highly selective drug such as penicillin, which targets the peptidoglycan cell wall unique to bacteria, can be prescribed sa ely because o the large di erence between its therapeutic and toxic concentrations. Drugs such as erlotinib, which inhibits mutated EGFRs but cannot ully discriminate between wild-type and mutant receptors, have a much lower therapeutic index and a higher incidence o adverse e ects. As we learn more about the biology o pathogens and cancer cells, more selective drugs are being developed. For example, imatinib is a highly selective anticancer agent that targets the kinase domain o a usion protein that is produced by a novel gene rearrangement. This usion protein kinase is constitutively active and highly expressed in chronic myelogenous leukemia cells and is important or the growth and survival o these cells. In contrast, the drug has relatively little e ect on normal cells, in which the unrearranged protein kinase is less active, less highly expressed, and less critical or cell growth and survival (see Chapter 1, Drug–Receptor Interactions). That being said, it is important to recognize that many potential targets that seem attractive remain unexploited because o issues such as unexpected adverse e ects, un avorable pharmacokinetic properties, or prohibitive costs associated with experimental drugs that have been developed to date against these targets. In the section below, we discuss examples o targets o antimicrobial and antineoplastic drugs that illustrate the principles o selective toxicity.

Unique Drug Targets Unique drug targets include metabolic pathways, enzymes, and other gene products that are present in the pathogen or cancer cell but absent in the host. One attractive target or antibacterial drugs is the biochemical pathway that leads to synthesis o the bacterial peptidoglycan cell wall (see Chapter 35, Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis). This structure is both biochemically unique and essential or the survival o growing bacteria. Penicillin and other -lactam antibiotics inhibit the transpeptidase enzymes that catalyze the f nal cross-linking step in peptidoglycan synthesis. Without peptidoglycans, bacterial cell wall synthesis is atally compromised. Because o their unique specif city or bacterial transpeptidase proteins, the penicillins have minimal host toxicity—in act, allergic hypersensitivity is the major adverse reaction. Fungi also present a unique target that is exploited by the currently available anti ungal drugs. Like the bacterial cell wall, the ungal cell wall is biochemically unique and essential or survival. The echinocandins inhibit the synthesis o -(1,3)-D-glucan, an essential component o the ungal cell wall. Disruption o cell wall integrity can cause ungal cell lysis. The echinocandins are well tolerated compared to

other anti ungal drug classes, with adverse e ects unrelated to inhibition o their target, but are limited by their lack o oral bioavailability. Thus, drugs that act on unique targets are o ten highly selective, with high therapeutic indices.

Selective Inhibition of Similar Targets Many pathogenic organisms and cancer cells have metabolic pathways similar to those o normal human cells but because o evolutionary divergence or mutation possess enzyme or receptor iso orms that di er in sequence and structure rom their normal human counterparts. Drugs can take advantage o these di erences, even though they are sometimes subtle. However, the resulting therapeutic indices are usually smaller than with unique targets. Examples o this targeting strategy include an inhibitor o the B-Ra protein kinase and inhibitors o bacterial protein synthesis. B-Ra is part o a signaling pathway downstream o cell sur ace receptors. A mutant orm o B-Ra , with a single amino acid substitution, is requently expressed in the skin cancer melanoma. Despite this very subtle di erence in the protein kinase, the drug vemurafenib more potently inhibits the mutant B-Ra than it does the normal enzyme. Because the mutant kinase is required or the growth and survival o many melanomas, vemura enib treatment can cause dramatic responses in the disease with an acceptable toxicity prof le. In both humans and bacteria, protein synthesis is a multistep process that involves binding o tRNAs and mRNA to the ribosome, decoding o mRNA, synthesis o peptide bonds, translocation o the tRNAs relative to the mRNA, emergence o the polypeptide chain, and release o the polypeptide rom the ribosome. The bacterial protein synthesis machinery di ers rom its human counterpart in the use o a di erent set o ribosomal RNAs and proteins and, correspondingly, distinct ribosomes. Several drug classes, including the macrolides and aminoglycosides, exploit these di erences to selectively inhibit bacterial protein synthesis (see Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation). Macrolide antibiotics such as erythromycin bind to the 50S bacterial ribosomal subunit and block translation by preventing emergence o polypeptides rom the ribosome. Aminoglycoside antibiotics such as streptomycin and gentamicin bind to the 30S bacterial ribosomal subunit and disrupt the decoding o mRNA. More generally, the bacterial protein synthesis inhibitors include a wide variety o individual drugs with diverse mechanisms, and the selectivity and dose-limiting toxicities o these drugs are o ten class- and/or drug-specif c. For example, the macrolides rarely cause serious adverse e ects, whereas some o the aminoglycosides have dose-limiting ototoxicity and nephrotoxicity. Some adverse e ects appear to result rom drug binding to human mitochondrial ribosomes in addition to bacterial ribosomes. Thus, selective inhibition o similar targets, as exemplif ed by mutant B-Ra inhibitors and protein synthesis inhibitors, can result in e ects characterized by therapeutic indices that range rom low to high, depending on the individual drug or drug class under consideration.

Common Targets It is o ten the case that the host and pathogen or cancer share common targets. In these cases, selective toxicity can be achieved when the pathogen or cancer is more a ected than the host by inhibition o the target. One striking example is

606 PRINCIPLES OF CHEMOTHERAPY

the human chemokine receptor CCR5. This protein is essential or entry o certain strains o HIV into cells but is dispensable or human health as revealed by studies o individuals who, despite requent exposure to HIV, have remained uninected due to a deletion in the CCR5 gene. Accordingly, the antiviral drug maraviroc , which binds to CCR5 and prevents HIV entry into cells, can help suppress HIV replication with minimal toxicity to patients (see Chapter 38, Pharmacology o Viral In ections). The paradigm o common targets is most requently exemplif ed by antineoplastic drugs. Because tumor cells arise rom trans ormed normal cells, they share nearly all o the cellular machinery needed or growth and replication. However, tumor cells may be more dependent on certain o these pathways than normal cells and there ore can be more sensitive to their inhibition. Also, some o these pathways can be more sensitized in cancer cells than in normal cells to agents that stress the cells, or example, by damaging DNA. These di erences can be subtle, and anticancer drugs that attempt to exploit these di erences o ten have narrow therapeutic indices. There ore, selective inhibition o cancer cell growth remains a major challenge. Recent discoveries have identif ed a number o proteins that are mutant or overexpressed in cancer cells, and selective inhibitors o these proteins are entering clinical use with increasing requency (see Chapter 40, Pharmacology o Cancer: Signal Transduction, and discussion o vemura enib above). Nonetheless, it is still the case that the selectivity o many currently used antineoplastic drugs is based not on specif c mutant protein targets but rather on variations in cancer cell growth behavior and on the increased susceptibility o cancer cells to induction o apoptosis or senescence. Cancer, as a disease o persistent proli eration, requires continued cell division. There ore, cytotoxic drugs targeting processes involved in DNA synthesis, mitosis, and cell cycle progression may kill rapidly cycling cancer cells pre erentially over their normal relatives. (An important correlate to this statement is that many chemotherapeutic strategies are more success ul against rapidly growing than slowly growing cancers.) Antimetabolites such as 5-f uorouracil (5-FU) inhibit DNA synthesis in dividing cells (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance). 5-FU inhibits thymidylate synthase, the enzyme responsible or converting dUMP to dTMP, a pyrimidine building block o DNA. As a pyrimidine analogue, 5-FU is also incorporated into growing RNA and DNA strands, thereby interrupting the synthesis o these strands. By causing DNA damage, 5-FU induces the cell to activate its apoptotic pathway, resulting in programmed cell death. 5-FU is toxic to all human cells undergoing DNA synthesis and, thus, is particularly toxic both or rapidly cycling tumor cells (therapeutic e ect) and or high-turnover host tissues such as the bone marrow and gastrointestinal mucosa (adverse e ect). These examples illustrate the importance o studying the cell biology, molecular biology, and biochemistry o microbes and cancer cells to identi y specif c targets or selective inhibition. Clinically, an awareness o drug mechanisms and the basis o drug selectivity can help to explain the narrow or broad therapeutic indices that have an impact on drug dosing and treatment strategies. Understanding the selectivity o drugs or their targets is also important in combating drug resistance. Thus, the undamental pharmacologic

principles o drug–receptor interactions, therapeutic and adverse e ects, and drug resistance orm the basis or selective targeting in antimicrobial and antineoplastic drug therapy.

PATHOGENS, CANCER CELL BIOLOGY, AND DRUG CLASSES Pharmacologic interventions target specif c di erences between the host and the microbial pathogen or cancer cell. This section examines some o the distinctive characteristics that evolution has bestowed on organisms and the major drug classes that target these molecular di erences among host cells, pathogens, and cancer cells.

Bacteria Bacteria are organisms that o ten contain unique targets or pharmacologic intervention. Some o these drug targets have been discussed previously and are illustrated in Figure 33-1. Currently available drugs act to interrupt bacterial DNA replication and repair (this chapter and Chapter 34), transcription and translation (Chapter 34), and cell wall synthesis (Chapter 35). Depending on the role o the drug target in bacterial physiology, antibacterial drugs can produce bacteriostatic or bactericidal e ects. Drugs that inhibit the growth o the pathogen without causing cell death are called bacteriostatic . These drugs target metabolic pathways that are necessary or bacterial growth but not or bacterial survival. Most protein synthesis inhibitors have a bacteriostatic e ect (aminoglycosides are an important exception). The clinical e ectiveness o these drugs relies on an intact host immune system to clear the nongrowing (but viable) bacteria. In contrast, bactericidal drugs kill bacteria. For example, cell wall synthesis inhibitors such as penicillins and cephalosporins cause bacterial lysis when the bacteria grow in hypertonic or hypotonic environments. Bacterial in ections in immunocompetent hosts can o ten be treated with bacteriostatic drugs, whereas the treatment o bacterial in ections in immunocompromised hosts o ten requires bactericidal drugs. Bacteriostatic and bactericidal e ects must be considered when antibiotics are used in combination (see Chapter 41, Principles o Combination Chemotherapy). The combination of a bacteriostatic drug with a bactericidal drug can result in antagonistic effects. For example, the bacteriostatic drug tetracycline inhibits protein synthesis and thereby retards cell growth and division. The action o this drug antagonizes the e ects o a cell wall synthesis inhibitor, such as penicillin, which requires bacterial growth in order to be e ective. In contrast, the combination of two bactericidal drugs can be synergistic ; that is, the e ect o the combination is greater than the sum o the e ects o each drug alone (at the same doses o the two drugs). For example, a penicillin– aminoglycoside combination can have a synergistic e ect because inhibition o bacterial cell wall synthesis by the penicillin allows increased entry o the aminoglycoside. The combination o two bacteriostatic drugs can also be synergistic (see “Synergy o DHFR Inhibitors and Sul onamides” later in this chapter).

Fungi and Parasites Eukaryotes, which include pathogenic ungi (yeasts and molds) and parasites (protozoa and helminths) as well as all

C h a p t e r 3 3 Principles o Antimicrobial and Antineoplastic Pharmacology 607 P e ptidoglyca n ce ll wa ll Inhibitors of cell wall synthesis Fos fomycin Cyclos e rine Va ncomycin P e nicillins Ce pha los porins

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FIGURE 33-1. Sites o action o antibacterial drug classes. Antibacterial drug classes are o ten divided into three general groups. Drugs in one group inhibit specif c enzymes involved in DNA synthesis and integrity: sul onamides and trimethoprim inhibit the ormation or use o olate compounds that are necessary or nucleotide synthesis; quinolones inhibit bacterial type II topoisomerases. Drugs targeting transcription and translation inhibit bacterial processes that mediate RNA and protein synthesis: f daxomicin inhibits initiation o RNA synthesis by bacterial DNA-dependent RNA polymerase; ri ampin inhibits elongation o RNA chains by the same enzyme; aminoglycosides, spectinomycin, tetracyclines, and glycylcyclines inhibit the bacterial 30S ribosomal subunit; macrolides, ketolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins inhibit the bacterial 50S ribosomal subunit. A third group o drugs inhibits specif c steps in bacterial cell wall synthesis: os omycin and cycloserine inhibit early steps in peptidoglycan monomer synthesis; vancomycin binds to peptidoglycan intermediates, inhibiting their polymerization; penicillins, cephalosporins, monobactams, and carbapenems inhibit peptidoglycan cross-linking; and ethambutol, pyrazinamide, and isoniazid inhibit processes necessary or synthesis o the cell wall and outer membrane o Mycobacterium tuberculosis. Several clinically use ul antibacterial drugs do not f t into one o these three groups; one recent example is daptomycin. The development o resistance is a problem or all antibacterial agents. Many bacteria carry plasmids (small, circular segments o DNA) with genes that con er resistance to an antibacterial agent or class o agents. PABA, para-aminobenzoic acid; DHF, dihydro olate; THF, tetrahydro olate.

multicellular organisms, are more complex than bacteria. Cells in these organisms contain a nucleus and membranebound organelles, as well as a plasma membrane. Eukaryotic cells reproduce by mitotic division rather than binary f ssion. Because o the similarities among human, ungal, and parasitic cells, in ections caused by ungi and parasites can be more di f cult to target than bacterial in ections. However, the burden o disease rom these organisms is vast. Parasitic in ections caused by protozoa and helminths (worms) a ect some 3 billion people worldwide, especially in less developed countries where the consequences o in ection can be devastating. In both developed and less developed parts o the world, increasing numbers o patients are immunocompromised rom AIDS, cancer chemotherapy, organ transplants, and old age. Such patients are especially susceptible to ungal and parasitic in ections, which are becoming more prominent and will require greater attention in the uture. Currently available anti ungal drugs can be divided into our main classes. Polyenes (e.g., amphotericin, nystatin) and azoles (e.g., miconazole , f uconazole ) selectively target ergosterol in the ungal cell membrane, and echinocandins (e.g., caspo ungin, mica ungin) inhibit the synthesis o -(1,3)-D-glucans in the ungal cell wall. Pyrimidines such as 5-f uorocytosine inhibit DNA synthesis. Another class o miscellaneous anti ungals, mostly acids, is used only topically because o unacceptable systemic toxicity. As with antibacterials, anti ungals can be ungistatic or ungicidal; this

distinction is usually determined empirically. For example, the azoles inter ere with ungal cytochrome P450-mediated ergosterol metabolism. Many azoles (e.g., itraconazole and f uconazole ) are ungistatic. Newer azole agents (e.g., voriconazole and ravuconazole ) may have ungicidal activity against some ungal species. As compared to ungistatic drugs, ungicidal drugs are more e f cacious and aster acting and allow more avorable dosing regimens. Anti ungal drugs are discussed in urther detail in Chapter 36, Pharmacology o Fungal In ections. Parasites exhibit diverse and complex li e cycles and metabolic pathways, and the treatment o parasitic in ections utilizes a wide array o drugs (see Chapter 37, Pharmacology o Parasitic In ections). One important protozoal in ection is malaria, which is transmitted when the emale Anopheles mosquito deposits Plasmodia sporozoites in the human bloodstream. The parasites leave the circulation and develop into tissue schizonts in the liver. The tissue schizonts rupture, releasing merozoites that enter the circulation to in ect red blood cells (erythrocytes). The parasites then mature to trophozoites and, f nally, to mature schizonts. Crops o mature schizonts are released into the bloodstream when the erythrocytes rupture, causing the typical cyclic ever associated with malaria. Antimalarial drugs target di erent stages o the protozoal li e cycle. Aminoquinolines (such as the previous f rst-line drug, chloroquine ) inhibit the polymerization o heme within the erythrocyte; it is thought

608 PRINCIPLES OF CHEMOTHERAPY

that nonpolymerized heme is toxic to intraerythrocytic Plasmodia. Artemisinins are also thought to inter ere with heme metabolism in the parasite. Dihydro olate reductase inhibitors, protein synthesis inhibitors, and other classes o drugs are also used in malaria treatment. The choice o drugs o ten depends on the local pattern o resistance. As chloroquine resistance is now common and resistance to most antimalarial agents has increased, the WHO recommends against all single-agent treatment regimens in the rst line o therapy or malaria. Instead, combination therapies are now recommended as rst-line treatments. Resistance to artemisinin and its derivatives has been observed, and the WHO recommends artemisinin-based combination treatments both to increase the e cacy o therapy and to reduce the spread o drug-resistant malaria. Combinations o an artemisininbased drug with amodiaquine , mef oquine , or sul adoxinepyrimethamine are recommended. Malaria is an excellent example o a complex parasite that, while theoretically susceptible to numerous classes o drugs, is becoming resistant to many currently available therapies.

Viruses Viruses are noncellular organisms that typically consist o a nucleic acid core o RNA or DNA enclosed in a proteinaceous capsid. Some viruses also possess a host cell-derived lipid envelope containing viral proteins. Viruses lack the capability to synthesize proteins themselves, relying instead on the host cell machinery. Most viruses also encode distinct or even unique proteins not normally produced by human cells, however. Many o these proteins are involved in the viral li e cycle, mediating attachment and entry o the virus into the host cell, uncoating o the viral capsid, expression o viral genes, replication o the viral genome, assembly and maturation o the viral particle, and release o viral progeny rom the host cell. These virus-speci c processes are o ten targeted by antiviral drugs. A schematic diagram o the general viral li e cycle is presented in Figure 33-2 to illustrate the stages o viral replication that can be targeted by antiviral drugs. Because these targets are present only during active viral replication, viruses capable o latency have not been cured by any currently available antiviral drugs. The HIV protease is an excellent example o a viral protein that has served as a ruit ul target or drug development. This enzyme cleaves viral precursor proteins to generate the structural proteins and enzymes necessary or virus maturation. Without HIV protease, only immature and nonin ective virions (individual virus particles) are produced. HIV protease inhibitors structurally mimic natural substrates o the protease but contain a noncleavable bond. These drugs act as competitive inhibitors at the active site o the enzyme (see Chapter 38). In combination with other classes o antiHIV drugs, protease inhibitors helped to revolutionize the treatment o patients with HIV/AIDS, converting a nearly invariably atal disease to a chronic illness. Inf uenza viruses o er examples o other classes o proteins that have been success ully targeted. Zanamivir and oseltamivir target a viral neuraminidase that is vital or virion release rom host cells. Amantadine and rimantadine act on the inf uenza virus membrane protein M2 (a proton channel) to inhibit viral uncoating. These anti-inf uenza drugs are e ective inhibitors o their targets. However, at least partly because the immune system ordinarily clears inf uenza inections in less than a week, these agents have not had as

dramatic a clinical impact as anti-HIV drugs have had. This example illustrates the point that even selective inhibitors with high therapeutic indices do not necessarily become highly e ective drugs in the clinic. Currently, the most important class o antiviral drugs is the polymerase inhibitors . Most viruses use a viral polymerase, either an RNA or DNA polymerase, to replicate their genetic material. Polymerase inhibitors are especially e ective against human herpesviruses, HIV, hepatitis B virus, and hepatitis C virus. Two types o polymerase inhibitors are the nucleoside analogues and the nonnucleoside polymerase inhibitors. Nucleoside analogues (such as acyclovir, zidovudine , and so osbuvir) become phosphorylated and thereby activated by viral or cellular kinases (phosphorylating enzymes), at which point they competitively inhibit the viral polymerase and, in some cases, are incorporated into the growing DNA strand. Selectivity is dependent on the relative a nities o the nucleoside analogue or the viral and cellular kinases and polymerases and on the abilities o the enzymes to phosphorylate the drug or incorporate it into DNA, respectively. Nonnucleoside polymerase inhibitors (such as e avirenz) inhibit viral polymerase (in this case, HIV reverse transcriptase), preventing DNA replication, but bind to a di erent site than the nucleoside analogues do. Mutations in viral polymerase genes are a major mechanism o resistance to polymerase inhibitors. Whether these resistance mutations a ect the replicative capacity ( tness) o the virus can play a major role in determining the e ectiveness o the therapy. Chapter 38 provides a detailed discussion o the pharmacology o antiviral drugs.

Cancer Cells Cancer is a disease o cell proli eration in which normal cells are trans ormed into cells with dysregulated growth. Neoplastic cells compete with normal cells or energy and nutrition, resulting in deterioration o normal organ unction. Cancers also impinge on vital organs by mass e ects. Carcinogenesis, chemotherapy, and the log cell kill model o tumor regression are discussed below to provide an overview o cancer pharmacology. Chapters 39 and 40 should be read with these principles in mind, and Chapter 41 provides integrated examples o the clinical applications o combination antineoplastic chemotherapy.

Carcinogenesis and Cell Proliferation

Carcinogenesis occurs in three main steps—trans ormation, proli eration, and metastasis. Trans ormation denotes a change in phenotype rom a cell with normal growth controls to a cell with dysregulated growth. Nonlethal genetic changes (mutations) can be inherited in the germ line, can occur spontaneously, or can be caused by environmental agents such as chemicals, radiation, or viruses. I the change is not repaired, the mutated genes (e.g., genes involved in growth regulation and DNA repair) can express altered gene products that allow abnormal cell growth and proli eration. Among other e ects, mutations can activate growth-promoting genes, inactivate growth-inhibiting genes, alter apoptosis-regulating genes, con er immortalization, and inactivate DNA repair genes. Additionally, the expression o these genes can be altered by certain heritable changes that do not alter DNA sequence, such as changes in DNA methylation or modications to histones that package chromosomal DNA. Such epigenetic changes can also promote carcinogenesis.

C h a p t e r 3 3 Principles o Antimicrobial and Antineoplastic Pharmacology 609 Virus

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FIGURE 33-2. Stages of the viral life cycle targeted by antiviral drug classes. The viral li e cycle begins with attachment o the virus to a host cell receptor and entry o the virus into the cell. The virus then uncoats, sometimes in an endosomal compartment. The uncoated viral nucleic acid is either directly translated into proteins on host ribosomes or undergoes transcription (RNA synthesis) and the newly synthesized mRNAs are translated, the proteins are processed post-translationally, and the viral genome (DNA or RNA) is replicated. The replicated viral genome and viral proteins are assembled into a virion (viral particle), which is then released rom the host cell. The process o virion assembly and/or release is o ten accompanied by maturation o the virus into an in ective agent that is able to repeat this li e cycle with a new host cell. Depending on the particular virus, the various stages o the viral li e cycle may occur in a di erent order rom that shown in this general model. The anti-HIVdrugs maraviroc and en uvirtide (T-20) block the attachment and entry o HIVinto host cells. The ion channel blockers amantadine and rimantadine inhibit inf uenza virus uncoating. The anti-HCV NS3/4A protease inhibitors block viral gene expression by preventing post-translational processing o the viral polyprotein. Polymerase inhibitors are a large class o antiviral agents that include acyclovir, so osbuvir, and e avirenz; these drugs inhibit viral genome replication by inter ering with viral DNA polymerase (acyclovir), viral RNA polymerase (so osbuvir), and reverse transcriptase (e avirenz). The anti-hepatitis C virus (HCV) drug ledipasvir inhibits viral genome replication by inter ering with the viral NS5A protein. The anti-HIVdrugs raltegravir, elvitegravir, and dolutegravir inhibit viral genome replication by inter ering with the viral integrase. Anti-HIVprotease inhibitors, such as darunavir, atazanavir, and ritonavir, inhibit viral maturation. Neuraminidase inhibitors block the release o inf uenza virus particles rom the host cell.

Expression of altered gene products and/or loss of normal gene regulation can cause dysregulated growth. Most cancers are initially clonal (i.e., genetically identical to a single precursor cell) but evolve to heterogeneity as new mutations and epigenetic changes increase the variation among daughter cells. When progeny cells with higher survival capacity are selected, increased cell proliferation ensues, and the tumor progresses to greater and greater heterogeneity. Thus, carcinogenesis, the progression from a normal cell to a malignant tumor, is a multistep process that usually requires an accumulation of multiple alterations. As more is learned about the molecular basis for carcinogenesis, these differences can be targeted for selective drug therapy. The growth of transformed cells into a tumor requires proliferation, or an increase in the number of cells. Dividing human cells progress through a cell cycle (or mitotic cycle) consisting of distinct phases. The two key events in

the cell cycle are the synthesis of DNA during S phase and the division of the parent cell into two daughter cells during mitosis or M phase. The phase between cell division and DNA synthesis is called gap 1 (G1), and the phase between DNA synthesis and mitosis is called gap 2 (G2). Proteins called cyclins and cyclin-dependent kinases (CDKs) govern progression through the phases of the cell cycle. Mutations in cyclin and/or CDK genes can result in neoplastic transformation, and loss of normal cell cycle control can result in genetic instability, augmenting the transformed phenotype. After a cancer cell divides, a daughter cell has four potential fates: it can become quiescent by entering a resting phase called G0, enter the cell cycle and proliferate, enter a state of cell-cycle arrest called senescence, or die. The ratio of the number of cells that are proliferating to the total number of cells in the tumor is called the growth fraction. An average tumor growth fraction is about 20%, because only one

610 PRINCIPLES OF CHEMOTHERAPY

in f ve cells participates in the cell cycle at any given time. Many antineoplastic drugs are cytotoxic agents that target dividing cells. Hence, tumor cells in a quiescent (G0) state, such as nutrient-starved cells in the center o a large tumor, are not easily killed by cytotoxic chemotherapy. Small or rapidly growing cancers (i.e., cancers with high growth ractions, such as leukemias) o ten respond more avorably to cytotoxic chemotherapy than do large bulky tumors. Unortunately, cells in normal tissues characterized by high growth ractions, such as the bone marrow and gastrointestinal mucosa, are also killed by cytotoxic antineoplastic drugs, resulting in dose-limiting toxicities. While cell-autonomous processes (i.e., processes o the cancer cell itsel ) are well-understood drivers o the proli erative phenotype in a tumor cell, non-cell-autonomous processes also play vital roles in tumor maintenance and growth. Trans ormed cancer cells secrete and induce a variety o chemical mediators to create a specialized local environment. These chemical mediators include growth actors such as epidermal growth actor (EGF), and inhibitors o growth actor signaling have been developed or clinical use as targeted cancer chemotherapeutic agents. Some tumors create a protective f brous connective tissue stroma; or example, this property makes breast cancer nodules palpable. Most solid tumors also require the induction o blood vessel growth (angiogenesis) to deliver nutrients into the center o the tumor; or this reason, angiogenesis inhibitors represent a valuable class o antineoplastic drugs.

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Cancer cells may acquire the capability to invade tissues and metastasize throughout the body. In order to metastasize, tumor cells acquire mutations or epigenetic changes that allow invasion into tissues and vessels, seeding o cavities, spread through lymph or blood vessels, and growth in a new environment. Aggressive, rapidly growing primary tumors are generally more likely to metastasize than indolent, slowly growing tumors. In the process o metastasis, tumor cells can also evolve di erent receptor expression patterns and drug susceptibilities and can even “dedi erentiate” or change cell type (e.g., epidermal to mesenchymal transition). O ten, although the primary tumor may respond well to chemotherapy, the metastatic cells respond poorly. Thus, metastatic spread typically represents a poor prognostic sign.

Cytotoxic Chemotherapy

By the time a typical solid tumor is clinically evident, it contains at least 109 cells, has acquired signif cant genetic heterogeneity, and has developed a distinct surrounding stroma. The tumor may or may not have metastasized rom its site o origin (primary site) to one or more secondary sites. These actors can render the cancer di f cult to treat pharmacologically. Many cytotoxic (traditional) chemotherapeutic agents inter ere with cell proli eration and rely on rapid cell cycling and/or promotion o apoptosis or their relative selectivity against cancer cells (Fig. 33-3). This is because tumors are most sensitive to cytotoxic chemotherapy when they are growing rapidly and progressing through the cell cycle.

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Microtubule s mRNA

Inhibitors of microtubule function Vinca a lka loids Ta xa ne s

DNA DNA damaging agents Alkyla ting a ge nts Antitumor a ntibiotics P la tinum compounds Topois ome ra s e inhibitors

FIGURE 33-3. Cytotoxic antineoplastic drug classes. Many cancer cells divide more requently than normal cells, and cancer cells can o ten be killed pre erentially by targeting three critical processes in cell growth and division. DNA-damaging agents alter the structure o DNA and thereby promote apoptosis o the cell. These drugs include alkylating agents (which covalently couple alkyl groups to nucleophilic sites on DNA), antitumor antibiotics (which cause ree radical damage to DNA), platinum compounds (which cross-link DNA), and topoisomerase inhibitors (which induce damage to DNA by stabilizing topoisomerase-induced strand breaks). Inhibitors o DNA synthesis and integrity block intermediate steps in DNA synthesis; these agents include antimetabolites and olate pathway inhibitors (which inhibit purine and pyrimidine metabolism). Inhibitors o microtubule unction inter ere with the mitotic spindle that is required or cell division. This group o drugs includes vinca alkaloids, which inhibit microtubule polymerization, and taxanes, which stabilize polymerized microtubules. The classes o targeted antineoplastic agents—such as growth actor receptor and signal transduction antagonists, proteasome inhibitors, angiogenesis inhibitors, tumor-specif c monoclonal antibodies, and hormones—are not shown (see Chapter 40).

C h a p t e r 3 3 Principles o Antimicrobial and Antineoplastic Pharmacology 611

These metabolically active cells are susceptible to drugs that inter ere with cell growth and division (the mitotoxicity hypothesis ). Many cytotoxic antineoplastic drugs interere with the cell cycle at a particular phase; such drugs are called cell-cycle specif c . Other cytotoxic antineoplastic drugs act independently o the cell cycle and are called cellcycle nonspecif c (Fig. 33-4). Inhibitors o DNA synthesis, such as antimetabolites, are S-phase specif c. Microtubule poisons, such as taxanes and vinca alkaloids, inter ere with spindle ormation during M phase. Alkylating agents that damage DNA and other cellular macromolecules act during all phases o the cell cycle. These various classes o drugs can be administered in combination, using cell-cycle specif c drugs to target mitotically active cells and cell-cycle nonspecif c agents to kill both cycling and noncycling tumor cells (see Chapter 41). The mitotoxicity hypothesis o cytotoxic cancer therapy leaves some puzzles unresolved, however. Although cytotoxic chemotherapy is o ten toxic to the bone marrow, gastrointestinal mucosa, and hair ollicles, these tissues usually Inhibitors of microtubule function M

Antitumor antibiotics

Glucocorticoids G1

G2

S Topoisomerase inhibitors

Antimetabolites and folate pathway inhibitors

Alkylating agents Platinum compounds (ce ll-cycle nons pe cific)

FIGURE 33-4. Cell-cycle specif city o some cytotoxic antineoplastic drug classes. The cell cycle is divided into our phases. Cell division into two identical daughter cells occurs during mitosis (M phase). Cells then enter the gap 1 (G1) phase, which is characterized by active metabolism in the absence o DNA synthesis. Cells replicate their DNA during the synthesis (S) phase. A ter completion o S phase, the cell prepares or mitosis during the gap 2 (G2) phase. Some cytotoxic antineoplastic drugs exhibit specif city or di erent phases o the cell cycle, depending on their mechanism o action. Inhibitors o microtubule unction a ect cells in M phase; glucocorticoids a ect cells in G1; antimetabolites and olate pathway inhibitors a ect cells in S phase; antitumor antibiotics a ect cells in G2; topoisomerase inhibitors a ect cells in S phase and G2. Alkylating agents and platinum compounds a ect cell unction in all phases and are there ore cell-cycle nonspecif c. The di erential cell-cycle specif city o the various drug classes allows them to be used in combination to target di erent populations o cells. For example, cell-cycle specif c drugs can be administered to target actively replicating neoplastic cells, whereas cell-cycle nonspecif c agents can be used to target quiescent (nonreplicating) neoplastic cells. The classes o targeted antineoplastic agents—such as growth actor receptor and signal transduction antagonists, proteasome inhibitors, angiogenesis inhibitors, tumor-specif c monoclonal antibodies, and hormones—are not shown (see Chapter 40).

recover, while (in success ul treatment) cancers with similar growth kinetics are eradicated. It has now been established that almost all chemotherapeutic drugs also cause apoptosis o cancer cells. DNA damage is normally sensed by molecules, such as p53, that arrest the cell cycle in order to allow time or the damage to be repaired. I the damage is not repaired, a cascade o biochemical events is triggered, which can result in apoptosis (programmed cell death). There ore, a cancer cell that is de ective in its capability or DNA repair may undergo apoptosis, whereas a normal cell can repair its DNA and recover. Cancers that express wild-type p53, such as most leukemias, lymphomas, and testicular cancers, are o ten highly responsive to chemotherapy. In contrast, cancers that acquire a mutation in p53, including many pancreatic, lung, and colon cancers, are o ten minimally responsive or even resistant to DNA-damaging drugs, because DNA damage does not trigger apoptosis in these cells. Advances in cancer cell biology over the past several decades have led to the development o classes o therapeutic agents that more selectively target the molecular pathways responsible or the dysregulated growth o cancer cells. Because specif c cancers may become “addicted” to a particular growth actor or signal transduction pathway or their survival (independent o their proli eration rate), selective targeting o these pathways can provide a basis or selective killing o cancer cells. This concept and the many classes o targeted antineoplastic agents that have been developed— including growth actor receptor and signal transduction antagonists, proteasome inhibitors, angiogenesis inhibitors, and tumor-specif c monoclonal antibodies—are discussed in Chapter 40.

Log Cell Kill Model

The log cell kill model is based on experimentally observed rates o tumor growth and tumor regression in response to cytotoxic chemotherapy. Tumor growth is typically exponential, with a doubling time (i.e., time required or the total number o cancer cells to double) that depends on the type o cancer. For example, testicular cancer o ten has a doubling time o less than 1 month, whereas colon cancer tends to double every 3 months. In solid tumors, the cancer may grow exponentially until a clinically observable tumor size is achieved. The log cell kill model states that the cell destruction caused by cytotoxic cancer chemotherapy is f rst-order; that is, each dose o chemotherapy kills a constant raction o cells. I the tumor starts with 1012 cells and 99.99% are killed, then 108 malignant cells will remain. The next dose o chemotherapy will then kill 99.99% o the remaining cells, and so on. Unlike antibacterial drugs, which can o ten be used in a constant high dose until the bacteria are eradicated, most cytotoxic antineoplastic drugs must be used intermittently to reduce toxic side e ects. Intermittent dosing allows partial recovery o normal cells but also provides time or cancer cell regrowth and or evolution o drug resistance in the cancer cells. As shown in Figure 33-5, intermittent “cycles” o cytotoxic chemotherapy are administered until all the cancer cells are killed or the tumor develops resistance. Drug-resistant cells continue to grow exponentially despite treatment, eventually resulting in death o the host. Improvements in the rates o eradication o malignant cell populations by cytotoxic drugs are likely to require either higher doses o these agents (which are limited by toxicity) or initiation o therapy at a time when the tumor contains

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S urge ry Ra dia tion the ra py

Time

FIGURE 33-5. Log cell kill model o tumor growth and regression. The log cell kill model predicts that the e ects o cytotoxic antineoplastic chemotherapy can be modeled as a f rst-order process. That is, a given dose o drug kills a constant fraction o tumor cells, and the number o cells killed depends on the total number o cells remaining. The our curves (A–D) represent our possible outcomes o antineoplastic therapy. Curve A is the growth curve o untreated cancer. The cancer continues to grow over time, eventually resulting in the death o the patient. Curve B represents curative local treatment (surgery and/or radiation therapy) be ore metastatic spread o the malignancy. Curve C represents local treatment o the primary tumor, ollowed immediately by systemic cytotoxic chemotherapy administered in cycles (down arrows) to eradicate the remaining metastatic cancer cells. Note that each cycle o chemotherapy reduces the number o cancer cells by a constant raction (here, by about two “logs,” or about 99%) and that some cancer growth occurs as the normal tissues are given time to recover between cycles o cytotoxic chemotherapy. Curve D represents local treatment ollowed by systemic chemotherapy that ails when the tumor becomes resistant to the drugs or when toxic drug e ects occur that are intolerable to the patient. Note that 109 to 1010 cancer cells must typically be present or a tumor to be detectable; or this reason, multiple cycles o chemotherapy are required to eradicate the cancer, even when there is no detectable tumor remaining.

ewer cells (which o ten implies earlier detection). (In contrast, it seems that, or certain targeted antineoplastic agents, higher doses do not contribute urther to e f cacy once a su f ciently high dose is achieved to inhibit the target.) Adjuvant therapies, such as surgery and radiation, are other important modalities used to reduce the number o tumor cells be ore chemotherapy is initiated.

MECHANISMS OF DRUG RESISTANCE Drug resistance is a major problem in all o antimicrobial and antineoplastic pharmacology. Although resistance to current drug therapies is emerging relatively rapidly, the rate o introduction o new drugs (especially antimicrobial drugs) is relatively slow. Formerly curable acute diseases such as gonorrhea and typhoid ever are becoming more di f cult to treat due to drug resistance, and chronic or endemic in ections such as tuberculosis or malaria are growing increasingly resistant worldwide. In some parts o China, up to 99% o gonorrhea isolates are multidrug resistant. In the United States, 60% o hospital-acquired (nosocomial) inections due to Gram-positive bacteria are caused by drugresistant microbes. Tuberculosis, the ourth leading cause o in ectious disease deaths worldwide, currently has an estimated 5% overall multidrug resistance (MDR) rate, although the rate o MDR in new cases o tuberculosis is as

high as 20–30% in some Asian countries (e.g., Azerbaijan, Belarus, Estonia, Kazakhstan, Kyrgyzstan, Russian Federation, and Uzbekistan). The appearance o MDR tuberculosis in the United States is o special concern because o the airborne spread o this organism. Despite these ominous trends, only several new classes o antibiotics—exemplif ed by new transcription inhibitors (e.g., f daxomicin), glycylcyclines (tigecycline ), streptogramins (quinupristin/dal opristin), oxazolidinones (linezolid), pleuromutilins (retapamulin), and lipopeptides (daptomycin)—have entered clinical use in the past our decades, and some o these have only limited clinical utility. The numerous examples o rapidly emerging drug-resistant organisms suggest that this problem must be addressed promptly. Because pathogens and cancer cells are primed to evolve rapidly in response to adaptive pressure, resistance can eventually appear with the use o any antimicrobial or antineoplastic drug. In a population o microbes or trans ormed cells, cells that contain genetic changes promoting replication in the presence o the drug will survive. Thus, high cell number, rapid growth rate, and high mutation rate all promote the development o a heterogeneous population o cells that can acquire resistance through mutational escape. Additionally, the emergence o resistance is exacerbated when any o multiple mutations can con er resistance (target size) or when such mutations do not substantially reduce replicative capacity (f tness). Because the use o a drug inherently selects or pathogens or cancer cells that can survive in the presence o high concentrations o that drug, drug resistance is inevitable when a su f cient number o patients is treated and is common when the drug is continued or a su f ciently long period o time. In many cases, the emergence o drug resistance con ounds e ective treatment.

Genetic Causes o Drug Resistance Drug resistance, by def nition, implies a heritable change in an organism. The recent explosion in antimicrobial drug resistance has both genetic and nongenetic causes. Genetic mechanisms o resistance can arise rom chromosomal or extrachromosomal (episomal) changes and rom exchange o genetic material. Drug resistance should not be conused with the lack o susceptibility o an organism (e.g., Mycoplasma, which lack cell walls, are not susceptible to penicillins) or with other mechanisms by which organisms can become less susceptible to drugs (e.g., by adaptation in the absence o genetic change such as the ormation o biof lms). Table 33-2 lists the major mechanisms o drug resistance that can be caused by either chromosomal mutation or genetic exchange. Chromosomal mutations typically occur in genes that code or drug targets or in genes that code or drug transport or metabolism systems. These mutations can then be trans erred to daughter cells (vertical transmission) to create drug-resistant pathogens or cancer cells. Alternatively, bacteria can acquire resistance by gaining genetic material rom other bacteria (horizontal transmission). For example, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) are able to cause highly eared nosocomial in ections because these bacteria have acquired resistance genes. Bacteria acquire genetic material by three main mechanisms: conjugation, transduction, and trans ormation. In conjugation, chromosomal or plasmid DNA is trans erred directly between bacteria. DNA can

C h a p t e r 3 3 Principles o Antimicrobial and Antineoplastic Pharmacology 613

TABLE 33-2 Mechanisms of Genetic Drug Resistance MECHANISM

EXAMPLE: ANTIMICROBIAL

EXAMPLE: ANTINEOPLASTIC

Inactivate drug Prevent uptake o drug

Inactivation o -lactam antibiotics by -lactamase Prevention o aminoglycoside entry by altered porins

Promote e f ux o drug

E f ux o multiple drugs by multidrug resistance (MDR) membrane e f ux pump

Inactivation o antimetabolites by deaminase Decreased methotrexate entry by decreased expression o reduced olate carrier E f ux o multiple drugs by p170 membrane e f ux pump (MDR1)

Reduced Intracellular Drug Concentration

Target-Based Mechanisms Alter drug target Overexpress drug target Overproduce endogenous ligand or substrate Bypass metabolic requirement or target

Insensitivity to Apoptosis

Expression o altered peptidoglycan that no longer binds vancomycin Overexpression o dihydropteroate synthase or dihydro olate reductase (DHFR) Overproduction o substrate para-aminobenzoic acid (PABA) causes resistance to sul onamides Loss o inhibition o host kinase bypasses requirement or viral kinase, causing resistance to the investigational antiviral drug maribavir

Expression o mutant DHFR that no longer binds methotrexate Overexpression o DHFR, thymidylate synthase, or topoisomerase Overproduction o substrate asparagine and/or glutamine causes resistance to L-asparaginase (under investigation) Activation o alternative signaling pathway con ers vemura enib resistance in melanoma

Not applicable

p53 mutation or loss

also be trans erred rom one cell to another by a bacterial virus, or bacteriophage, in a process called transduction. In transformation, naked DNA in the environment is taken up by the bacteria. Drug resistance in bacteria is most o ten caused by the trans er o plasmids, which are extrachromosomal strands o DNA that contain drug resistance genes. Trans er o a DNA plasmid is especially important or drug resistance because this mechanism occurs at high rates both within and between bacterial species and because multiple drug resistance genes can be trans erred simultaneously.

Reduced Intracellular Drug Concentration

Drugs must reach their targets in order to be e ective. Both microbes and cancer cells have evolved mechanisms to reduce drug concentrations be ore the drugs reach their targets. One major mechanism entails the inactivation o drugs. Many bacteria acquire resistance to -lactam antibiotics, such as penicillins, cephalosporins, and carbapenems, through expression o a hydrolytic enzyme, -lactamase , which cleaves the -lactam ring and thereby inactivates the drug. A single -lactamase enzyme can hydrolyze 103 penicillin molecules per second, signi cantly reducing the intracellular concentration o active drug. As another example, cancer cells that overexpress a deaminase enzyme can rapidly inactivate purine or pyrimidine analogues such as 5-FU and make the drugs less e ective. Pathogens and cancer cells can also acquire mutations that prevent uptake o the drug into the cell or otherwise prevent access o the drug to the target molecule. For example, cancer cells with mutated olate-transport systems become resistant to olate analogues, such as methotrexate , that require active transport into cells in order to inhibit dihydro olate reductase (DHFR). Finally, both bacteria and cancer cells can acquire the ability to cause active drug e f ux rom the cell. Bacteria typically possess membrane pumps to transport lipophilic or

amphipathic molecules (such as antibiotics) in and out o the cells. Overproduction o these membrane proteins or their variants can mediate active pumping o an antibiotic out o the cell aster than the drug can enter the cell. Despite the achievement o therapeutic blood levels o the antibiotic, this active e f ux mechanism can cause intrabacterial drug concentrations to be ine ectively low. Similarly, the emergence o multidrug resistant cancers is o ten associated with cancer cell overexpression o membrane proteins, such as the P-glycoprotein (p170 or MDR1), which actively pump antineoplastic drugs out o the cell. These e f ux pumps are especially important because they are capable o pumping out more than one type o drug, thus allowing pathogens or cancer cells to become resistant to multiple drugs o di erent classes. It is worth noting that heritable changes are not the only class o mechanisms leading to reduced intracellular drug concentration. For example, the brain is protected by a blood– brain barrier that acts to exclude pathogens and toxins, but it also excludes many antibiotics and chemotherapeutic agents. This makes treatment o central nervous system in ections and brain cancers more di cult. Certain in ections are characterized by the ormation o abscesses, and the hard wall o the abscess can similarly act to exclude antibiotics.

Target-Based Mechanisms

Pathogens and cancer cells can also evolve to alter or overexpress drug targets or evolve other changes related to the drug target that result in drug resistance. One common mechanism or development o drug resistance is alteration o the target o a drug. In vancomycin-resistant enterococcus, the vanHAX genes encode a novel enzymatic pathway that alters sur ace peptidoglycan synthesis such that the sequence terminates in D-Ala-D-lactate instead o the normal D-Ala-D-Ala. This substitution does not a ect peptidoglycan cross-linking in the synthesis o the bacterial cell wall and thus does not alter the integrity o the cell wall, but it does lower by 1,000- old the binding a nity o vancomycin or the dipeptide.

614 PRINCIPLES OF CHEMOTHERAPY

Almost all examples o antiviral drug resistance are due to alteration o targets through mutation o the genes encoding the targets. For example, resistance to the anti-herpesvirus drug acyclovir occurs through mutations in the viral thymidine kinase gene, which encodes the enzyme that activates the drug, or the viral DNA polymerase gene, which encodes the enzyme inhibited by the triphosphate orm o the drug (see Fig. 38-7). In bacteria and cancer cells, both alteration and overexpression o the enzyme targets o cytotoxic antineoplastic drugs—such as DHFR, thymidylate synthase, and topoisomerase—can reduce the raction o the targets that bind drug, thus reducing potency and con erring drug resistance. Variations on this mechanism are to overproduce an endogenous ligand or substrate that competes with the drug or the drug target, as occurs with resistance o bacteria to sul onamides (see under “Inhibitors o Folate Metabolism”), or to alter the target, ligand, or substrate so that the target binds the ligand more tightly, processes the substrate more e ciently, or is less necessary or microbial or cancer cell growth and survival. For example, mutations that alter sites o cleavage by HIV protease so that they are processed more readily can contribute to resistance to protease inhibitors. Yet another target-based mechanism, which is becoming increasingly important in resistance to targeted anticancer drugs, is to bypass the metabolic requirement for the target. For example, as mentioned above, melanomas o ten depend on inappropriate signaling by a mutant B-Ra protein kinase or their growth and survival, and targeting o this mutant kinase by inhibitors such as vemura enib can result in dramatic regressions o the tumors. However, resistance arises requently. One resistance mechanism involves activation o an entirely di erent signaling pathway to drive proli eration o the melanoma cells.

Insensitivity to Apoptosis

Drug resistance in cancer cells can occur through chromosomal or extrachromosomal mutations or epigenetic changes that are then passed to daughter cells to create a resistant tumor. Although anticancer drugs act at a variety o molecular targets, most, i not all, ultimately cause cell death by inducing apoptosis. In general, drug-induced molecular lesions can lead to cell-cycle arrest, activation o repair processes, senescence, or apoptosis. Mutations in key proteins associated with the control o apoptosis, such as p53 and Bcl-2, can result in ailure to induce the apoptotic response to DNA damage and can thereby reduce the sensitivity o tumor cells to many anticancer drugs. As noted above, tumors with wildtype p53, such as many leukemias, lymphomas, and testicular cancers, are o ten highly responsive to chemotherapy. In contrast, many pancreatic, lung, and colon cancers have a high incidence o p53 mutations and are minimally responsive to chemotherapy. Thus, the causes o drug resistance include changes in chromosomal DNA or episomal DNA, epigenetic changes, and external acquisition o genetic material. Resistance can be caused by drug inactivation, decreased drug uptake, increased drug e f ux, alteration or overexpression o the target structure or pathway, bypass o the requirement or the target, repair o drug-induced lesions, and insensitivity to apoptosis. Resistance is probably the major limiting factor in the effective treatment of both infections and cancer. Drug therapy is a dynamic balance, an “evolutionary arms race,” between the design o new drugs and the evolution o changes leading to drug resistance.

Practices That Promote Drug Resistance One o the most important causes o drug resistance is the overprescription o antimicrobial drugs that are not indicated or the clinical situation. Overprescription is a problem not only in humans but also in the treatment and prophylaxis o animal in ections. Such widespread use promotes drug resistance, which can then be trans erred rom one microorganism to another by the mechanisms described above. Because low drug concentrations in vivo can permit multiple cycles o replication and selection or resistant variants, low patient adherence can also promote resistance, as can the erratic drug availability ound in parts o the developing world (and even in some communities in the developed world). International travel promotes a global disease community, ensuring that the multidrug-resistant tuberculosis ound in Russia or Peru will eventually emerge in hospitals in the United States. Finally, demographic shi ts and other trends have led to the ormation o large populations that are susceptible to in ections, such as immunocompromised cancer patients, AIDS patients, and the elderly population.

METHODS OF TREATMENT Combination Chemotherapy The development o drug resistance depends on such actors as the number o microorganisms or cancer cells in the pretreatment population, the rate o replication or “generation time” o the organism or cell, the intrinsic rate o mutation in the population, the target size or resistance mutations, and the replicative capacity ( tness) o the resistant organism or cell. Compared to treatment with a single agent, treatment with a combination o drugs can signi cantly decrease the probability that resistance will develop. Combination chemotherapy is the standard-o -care in tuberculosis and HIV therapy and most antineoplastic drug regimens. Despite potential or real drawbacks o increased toxicities and cost, there can be several major reasons to administer multiple drugs simultaneously in a combination chemotherapy regimen; the rationales are discussed in urther detail in Chapter 41. First, the use o multiple drugs with di erent mechanisms o action targets multiple steps in microbial or cancer cell growth, leading to the maximum achievable e ect. Second, provided that resistance to one drug does not also con er resistance to others in the combination, the use o combinations o drugs makes it more di cult or resistance to develop because, while the likelihood o development o a resistance mutation to one drug is relatively high, the concurrent emergence o separate mutations against several di erent drugs is less likely. The probability o resistance to multiple drugs is the product o the probabilities o resistance to the individual drugs in the combination; or example, i resistance to each drug has a probability o 10 3 (which would be high), then the probability o resistance to three drugs would be 10 9. Third, the use o lower doses o synergistically acting drugs in the combination can reduce drug-associated adverse e ects. This is especially important in antimicrobial chemotherapy, where synergistic activity o drug combinations has been clearly demonstrated. Fourth, because many cytotoxic antineoplastic drugs have distinct dose-limiting adverse e ects (toxicities), it is o ten possible to give each drug to its maximally tolerated dose while achieving increased overall cell killing.

C h a p t e r 3 3 Principles of Antimicrobial and Antineoplastic Pharmacology 615

Finally, the concept o combination chemotherapy is being redef ned as new treatments become available. In the uture, immunotherapies, hormone therapies, and biotherapies will become increasingly integrated into combination chemotherapy regimens (see Chapter 54, Protein Therapeutics).

Prophylactic Chemotherapeutics In most instances, antimicrobial and antineoplastic drugs are used to treat overt disease. These classes o drugs can also be used to prevent diseases rom occurring (chemoprophylaxis), both be ore a potential exposure and a ter a known exposure. The potential benef t o chemoprophylaxis must always be weighed against the risk o evolving drug-resistant pathogens or cancer cells and the potential or toxicity attributable to the chemoprophylactic agent. Antimicrobial chemoprophylaxis is requently used in high-risk patients to prevent in ection. Travelers to malaria-in ested areas, or example, o ten take prophylactic antimalarial drugs such as mef oquine (see Chapter 37). Chemoprophylaxis is also used in some types o surgery to prevent wound in ections. Antibiotics are commonly administered prophylactically during surgical procedures that could release bacteria into the wound site, such as colon resection. In certain situations, immunocompromised patients are given antibacterial, antiungal, antiviral, and/or antiparasitic drugs prophylactically to prevent opportunistic in ections. For example, acyclovir can protect previously in ected immunocompromised patients against disease caused by reactivation o latent herpes simplex virus. Chemoprophylaxis or preemptive therapy can also be used in healthy persons a ter exposures to certain pathogens. Prophylactic therapy a ter known or suspected exposure to gonorrhea, syphilis, bacterial meningitis, HIV, and other inections can o ten prevent disease. The risk o seroconversion a ter a single needle stick exposure to HIV-in ected blood is approximately 0.3% (95% conf dence interval [CI] 0.2–0.5%). Although limited data are available regarding the reduction o risk achievable with prophylaxis, the CDC currently recommends postexposure treatment with a three-drug antiretroviral therapy regimen (e.g., raltegravir, teno ovir [TDF], and emtricitabine [FTC]) or 4 weeks. Several antiretroviral drug combinations, such as zidovudine lamivudine and TDF FTC, have been shown to reduce maternal transmission o HIV, representing chemoprophylaxis or the etus (see Chapter 38).

INHIBITORS OF FOLATE METABOLISM: EXAMPLES OF SELECTIVE TARGETING AND SYNERGISTIC DRUG INTERACTIONS Folic acid is a vitamin that participates in multiple enzymatic

reactions involving the trans er o one-carbon units. These reactions are essential or the biosynthesis o DNA and RNA precursors; the amino acids glycine, methionine, and glutamic acid; the ormyl-methionine initiator tRNA; and other essential metabolites. Given the importance o olate metabolism in the biochemistry o the cell, it is not surprising that agents inhibiting olate biosynthesis and inter ering with the olate cycle have been used widely in the treatment o bacterial in ections, parasitic in ections, and cancer. These drug classes also provide excellent examples o the principles o chemotherapy.

Folate Metabolism The structure o olic acid contains three chemical moieties (Fig. 33-6A): a pteridine ring system, para-aminobenzoic acid (PABA), and the amino acid glutamate. (Because o its ability to absorb ultraviolet light, PABA is the active ingredient in many topical sunscreens.) For humans, olate is an essential vitamin that must be provided intact in the diet. In bacteria and certain protozoans, however, olate is synthesized rom precursors, as shown in Figure 33-7. Both dietary olate and olate synthesized rom precursors enter the olate cycle (Fig. 33-7). In this cycle, dihydro olate is reduced to tetrahydro olate by dihydro olate reductase (DHFR). Tetrahydro olate then participates in many metabolic interconversions that involve one-carbon trans ers. For example, tetrahydro olate is an essential donor o carbon atoms in the synthesis o inosine monophosphate (IMP) (leading to adenosine monophosphate [AMP] and guanosine monophosphate [GMP]) and in the conversion o deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) (see Fig. 39-2). In all o these reactions, tetrahydro olate donates a carbon atom and, in the process, is oxidized to dihydro olate. For urther rounds o one-carbon trans ers to occur, the dihydro olate must be reduced to tetrahydro olate by DHFR.

Inhibitors o Folate Metabolism Antimetabolites are agents that inhibit nucleotide and DNA

synthesis (see Chapter 39). This chapter uses one class o antimetabolites, the inhibitors o olate metabolism, to exempli y the basis or selective targeting o antimicrobial and antineoplastic drugs according to the distinctiveness o the drug target. As described above, selectivity can take the orm o (1) a protein or a biochemical pathway that is unique to the pathogen or cancer cell, (2) a structure (iso orm) o a protein that is specif c to the pathogen or cancer cell, or (3) a requirement or a host protein or pathway that is specif c to the pathogen or cancer cell. Where relevant, the ollowing discussion emphasizes the basis or selectivity o each therapeutic agent. Inhibitors o olate metabolism include inhibitors o dihydropteroate synthase and inhibitors o dihydro olate reductase. In each case, drugs that structurally resemble the physiologic substrate o the enzyme act as enzyme inhibitors.

Unique Drug Targets: Antimicrobial Dihydropteroate Synthase Inhibitors

Bacteria and certain protozoans are unable to take up olic acid rom the environment and there ore must synthesize the vitamin de novo rom PABA, pteridine, and glutamate using the enzyme dihydropteroate synthase (Fig. 33-7). Mammalian cells, in contrast, use olate receptors and olate carriers in the plasma membrane o the gastrointestinal lining to scavenge the intact vitamin. This undamental metabolic di erence between pathogen and host cells helps make dihydropteroate synthase an excellent target or antimicrobial therapy. The sul a drugs, such as sul amethoxazole and sul adiazine , are PABA analogues that competitively inhibit dihydropteroate synthase and thereby prevent the synthesis o olic acid in the pathogens. The lack o olic acid, in turn, prevents synthesis o purines, pyrimidines, and some amino acids and eventually results in cessation o pathogen growth. Sul a drugs are usually bacteriostatic (i.e., they usually prevent bacterial growth

616 PRINCIPLES OF CHEMOTHERAPY A Folic a cid

H2 N

N N

Ba cte ria

P te ridine + PABA Dihydropteroate synthase

N

Dihydropte roic a cid

H N

N

Gluta ma te

H N

OH P te ridine moie ty

PABA

COOH

O

Ba cte ria a nd huma ns

COOH

Dihydrofola te Dihydrofolate reductase (DHFR)

Gluta ma te

Te tra hydrofola te

B PABA a na logue s

NH2

O

O

S

N

O

S

O

O

NH

S

NH

O

N N

Sulfadiazine

Sulfamethoxazole

N N

N

P urine s

Me thionine Glycine fMe t-tRNA

Thymidine

DNA RNA

P rote ins

DNA

FIGURE 33-7. Folate synthesis and functions. Folate synthesis begins with

C Fola te a na logue s H2 N

5-Fluorouracil Flucytosine

N

N

NH2

Sulfanilamide

Trimethoprim Methotrexate Pyrimethamine

NH2

NH2

O

Sulfonamides

H N

NH2 O

COOH COOH

the ormation o dihydropteroic acid rom pteridine and para-aminobenzoic acid (PABA); this reaction is catalyzed by dihydropteroate synthase. Glutamate and dihydropteroic acid condense to orm dihydro olate (DHF). DHF is reduced to tetrahydro olate (THF) by dihydro olate reductase (DHFR). THF and its congeners (not shown) serve as one-carbon donors in numerous reactions necessary or the biosynthesis o DNA, RNA, and proteins. In each such reaction, the reduced olate (THF) becomes oxidized to DHF, and the THF must then be regenerated via reduction by DHFR. Inhibitors o olate metabolism target three steps in the olate pathway. Sul onamides inhibit dihydropteroate synthase; trimethoprim, methotrexate, and pyrimethamine inhibit DHFR; and 5-f uorouracil (5-FU) and f ucytosine inhibit thymidylate synthase (see Fig. 39-4). Note that bacteria and certain parasites synthesize olate de novo rom pteridine and PABA, whereas humans require dietary olate.

Methotrexate

but do not kill the bacteria). However, in some settings in which thymine is unavailable, such as in urine, they can be bactericidal (thymine-less death). There are two structural classes o sul a drugs: sul onamides and sul ones.

O H2 N

N

O

H2 N

N

Sulfonamides and Sulfones

N

N

O NH2

Trimethoprim

NH2

Cl

Pyrimethamine

FIGURE 33-6. Structures of folic acid, PABA analogues (sulfonamides), and folate analogues (dihydrofolate reductase inhibitors). A. Folic acid is ormed by the condensation o pteridine, para-aminobenzoic acid (PABA), and glutamate (see Fig. 33-7). Folate is the deprotonated orm o olic acid. B. PABA analogues (sul onamides) structurally resemble PABA. These drugs inhibit dihydropteroate synthase, the enzyme that catalyzes the ormation o dihydropteroic acid rom PABA and pteridine (see Fig. 33-7). C. Folate analogues (dihydro olate reductase inhibitors) structurally resemble olic acid. These drugs inhibit dihydro olate reductase, the enzyme that converts dihydro olate to tetrahydro olate.

As demonstrated in the case o Hildegard Domagk, sulfonamides were the f rst modern agents to be employed in the treatment o bacterial in ections (prontosil is a sul onamide precursor). Figure 33-6 shows the similarity in structure between PABA and the sul onamide analogues sulfanilamide , sulfadiazine , and sulfamethoxazole . Sul onamides are highly selective or dihydropteroate synthase. Because microbial growth requires activity o this enzyme, and the enzyme is not expressed in mammalian cells, this drug class has very ew adverse e ects (except in the special case o neonates, noted below). Despite the exquisite selectivity o the sul onamides, the development o resistance to these drugs has resulted in their diminished use. Resistance to sul onamides most requently develops because o (1) overproduction o the endogenous substrate, PABA, or (2) a mutation in the PABA binding site on dihydropteroate synthase, resulting in reduced a f nity o

C h a p t e r 3 3 Principles of Antimicrobial and Antineoplastic Pharmacology 617

the enzyme or sul onamides. Some resistant streptococci produce levels o PABA that are 70- old higher than the normal value. Because o the high incidence o sul onamide resistance in the bacterial and parasite populations, these drugs are rarely administered as single agents. Instead, they are commonly administered in combination with a synergistic drug such as trimethoprim or pyrimethamine , as discussed below. Sul onamides compete with bilirubin or binding sites on serum albumin and can cause kernicterus in newborns. Kernicterus , a condition characterized by markedly elevated concentrations o unconjugated ( ree) bilirubin in the blood o neonates, can lead to severe brain damage. For this reason, newborns should not be treated with sul onamides. Dapsone , a member o the sulfone class o dihydropteroate synthase inhibitors, is used in the treatment o leprosy and as a second-line agent in the prevention o Pneumocystis jiroveci pneumonia (PCP). The mechanism o action o dapsone is the same as that o the sul onamides, and dapsone and trimethoprim or pyrimethamine can also be used as a synergistic drug combination (see discussion below). Because dapsone is an oxidizing agent, approximately 5% o patients can develop methemoglobinemia a ter administration o the drug. Susceptible patients are typically def cient in the erythrocyte enzyme glucose-6-phosphate dehydrogenase, which is involved in the detoxif cation o endogenous and exogenous oxidizing agents.

Selective Inhibition of Similar Targets: Antimicrobial Dihydrofolate Reductase Inhibitors

Dihydro olate reductase (DHFR) is the enzyme that is used by all organisms to reduce dihydro olate (DHF) to tetrahydro olate (THF). Several drugs, including trimethoprim, pyrimethamine , and methotrexate , are olate analogues that competitively inhibit DHFR and prevent the regeneration o THF rom DHF (Figs. 33-6 and 33-7). By doing so, these drugs prevent the synthesis o purine nucleotides as well as the methylation o dUMP to dTMP (see above). Pharmacologic inhibition o DHFR is used both in the treatment o in ection and in cancer chemotherapy. Many inhibitors o DHFR have been developed. As shown in Table 33-3, methotrexate is a potent (sub-nanomolar) inhibitor o DHFR, although it exhibits little selectivity amongst the mammalian, bacterial, and protozoal iso orms o the enzyme. In contrast, inhibitors with structures that are more divergent rom that o olate, such as trimethoprim and pyrimethamine (see Fig. 33-6), show considerable selectivity o DHFR inhibition among the various iso orms o the enzyme. Trimethoprim is a potent and selective antibacterial agent; pyrimethamine is a potent and selective antiprotozoal drug; and methotrexate is an antineoplastic agent used to treat many di erent malignancies. Why are trimethoprim and pyrimethamine each selective or specif c iso orms o DHFR, while methotrexate is not? For example, a 50% reduction in the activity o bacterial DHFR can be achieved at a trimethoprim concentration o 0.007 M, while comparable inhibition o human DHFR requires a trimethoprim concentration o 350 M (Table 33-3). In part, this may be because methotrexate closely resembles dihydro olate, which is the normal substrate or DHFR. While the amino acid sequences o DHFRs amongst bacteria, protozoans, and humans vary substantially, these isoorms have been evolutionarily constrained to maintain their

TABLE 33-3 IC50 Values for Three Dihydrofolate Reductase Inhibitors DHFR Isoform DHFR Inhibitor

E. coli DHFR

Malarial DHFR

Mammalian DHFR

Trimethoprim

7

1,800

350,000

Pyrimethamine

2,500

0.5

1,800

Methotrexate

0.1

0.7

0.2

IC50 is the concentration of drug required for 50% enzyme inhibition. All values are reported in nM (10 9 M) units. Boldface indicates the DHFR isoform targeted for therapy. Trimethoprim and pyrimethamine are selective inhibitors of the E. coli and malarial isoforms of DHFR, respectively. In contrast, methotrexate is a nonselective inhibitor of all three DHFR isoforms. DHFR, dihydrofolate reductase.

enzymatic activity in the conversion o DHF to THF and there ore remain sensitive to drugs such as MTX that closely mimic the enzymatic substrate. In contrast, drugs such as trimethoprim and pyrimethamine, which are poorer mimics o the enzymatic substrate, are able to take advantage o these sequence di erences, which translate to structural di erences, and more selectively target individual iso orms. The basis or selectivity thus resides in di erences in enzyme structure that are largely irrelevant or binding o the natural substrate but that have an important role in analogue (drug) binding. Increased understanding o the structural basis or DHFR inhibition may lead to the development o still more selective agents. Trimethoprim Trimethoprim is a olate analogue that selectively inhibits bac-

terial DHFR (Fig. 33-6C; Table 33-3) and thereby prevents the conversion o DHF to THF. As with the sul onamides, trimethoprim is usually bacteriostatic, although it, too, can lead to thymine-less death in certain settings. Because trimethoprim is excreted unchanged in the urine, it can be used as a single agent to treat uncomplicated urinary tract in ections. For most in ections, however, trimethoprim is used in combination with sul amethoxazole. The rationale or this combination antibacterial chemotherapy is described below.

Pyrimethamine Pyrimethamine is a olate analogue that selectively inhibits

parasitic DHFR (Fig. 33-6C; Table 33-3). Pyrimethamine is currently the only e ective chemotherapeutic agent against toxoplasmosis; or this indication, it is typically administered in combination with sul adiazine. Pyrimethamine has also been used to treat malaria, although widespread resistance has limited its e ectiveness in recent years. Further discussion o the therapeutic applications o pyrimethamine and sul adiazine can be ound in Chapter 37.

Common Targets: Antineoplastic Dihydrofolate Reductase Inhibitors Methotrexate

As described above, methotrexate (MTX) is a olate analogue that reversibly inhibits DHFR. In mammalian cells, DHFR inhibition causes a critical shortage o intracellular supplies o tetrahydro olate, resulting in inhibition o de novo purine

618 PRINCIPLES OF CHEMOTHERAPY

and thymidylate synthesis and, there ore, cessation o DNA and RNA synthesis. Because the synthesis o DNA is halted, mammalian cells treated with methotrexate are arrested in the S phase o the cell cycle. The basis or the relative selectivity o methotrexate or cancer cells compared to normal cells is thought to be that rapidly growing cancer cells have an increased requirement or metabolites, such as purines and thymidylate, that are critical to DNA synthesis and that are generated by olatedependent enzymes. In addition, malignant cells may be more susceptible than normal cells to the apoptosis-inducing e ects o MTX (see discussion below). O note, the use ulness o high-dose MTX in cancer chemotherapy has been greatly improved by the application o folinic acid rescue . In this technique, olinic acid (N-5 ormyltetrahydro olate, also called leucovorin) is administered to the patient several hours a ter an otherwise lethal dose o methotrexate. The rationale or this technique is that the malignant cells are killed selectively, while the normal cells are “rescued” by the olinic acid. The molecular mechanism underlying olinic acid rescue is unclear. One hypothesis suggests that normal (nonmalignant) cells are able to concentrate the olinic acid (and, thus, to protect themselves rom the e ects o MTX), whereas malignant cells have a reduced rate o olinic acid transport (and, there ore, are pre erentially harmed by high doses o MTX). Another hypothesis suggests that high-dose MTX induces apoptosis in malignant cells but cell-cycle arrest in normal cells; the normal cells are then able to use the olinic acid to resume cell growth and division, while the malignant cells are already committed to programmed cell death. MTX is used to treat many tumor types, including carcinomas o the breast, lung, and head and neck, acute lymphoblastic leukemia, and choriocarcinoma. MTX is also used to treat psoriasis and certain autoimmune diseases such as rheumatoid arthritis. Methotrexate toxicity is mani ested primarily in rapidly dividing host cells, causing damage to the gastrointestinal mucosa and the bone marrow. These e ects are generally reversible a ter therapy is discontinued. MTX is extremely toxic to the etus because olic acid is essential or the proper di erentiation o etal cells and or closure o the neural tube. MTX has undergone clinical trials as an abortion-inducing agent, either alone or in combination with the prostaglandin analogue misoprostol, and is sometimes used o -label to terminate early-stage ectopic pregnancy.

Synergy of DHFR Inhibitors and Sulfonamides

Both trimethoprim and pyrimethamine can be used in combination with sul onamides to block sequential steps in the biosynthetic pathway leading to tetrahydro olate (Fig. 33-7). This type o combination chemotherapy, called sequential blockade , has been e ective in the treatment o certain parasitic in ections (pyrimethamine and sul adiazine) and bacterial in ections (trimethoprim and sul amethoxazole). One rationale or the use o a DHFR inhibitor and a sul a drug in combination is the marked synergistic interaction between these two classes o drugs (see Chapter 41). The sul onamide decreases the intracellular concentration o dihydro olate; this increases the e ectiveness o the DHFR inhibitor, which competes with dihydro olate or binding to the enzyme. The sul a/DHFR inhibitor combination can also be e ective in treating strains o bacteria and parasites that exhibit resistance to monotherapy with a DHFR inhibitor. Typically, this

drug resistance phenotype is caused by the expression o a structurally altered DHFR that has a lower a f nity or the inhibitor. The trade-o or the bacteria or parasite is o ten that the altered DHFR also has a lower a f nity or the natural ligand dihydro olate. In such strains, sul onamide treatment can decrease the intracellular concentration o dihydro olate to the point that the altered DHFR cannot meet the metabolic requirements o the cell. Another important rationale or the use o a combination such as trimethoprim/sul amethoxazole is that resistance to trimethoprim alone or sul amethoxazole alone develops rather quickly, whereas resistance to the drug combination develops much more slowly. As discussed earlier in the chapter, because the two drugs act on di erent enzymes, two di erent mutations would need to occur simultaneously or the bacteria to develop resistance to the drug combination, and the likelihood that two mutations will occur simultaneously is much lower than the likelihood that one mutation will occur (see Chapter 41).

CONCLUSION AND FUTURE DIRECTIONS Many o the principles underlying the pharmacologic treatment o microbial diseases and cancer are similar. Pharmacologic treatments o both in ection and cancer rely on selective inhibition o the pathogen or cancer cell to prevent its growth or survival, with a minimum o adverse e ects that could inter ere with host unction. Selective inhibition o a unique target, such as the bacterial cell wall, is, in principle, ideal. O ten, less selective therapies must be employed, targeting a molecule or pathway that is similar or even identical between the pathogen or cancer cell and the host. Even highly selective drugs aimed at an entirely unique target can be rendered ine ective i the microbe or cancer cell mutates to become resistant. Both microbes and cancer cells grow rapidly, with the potential or evolving or acquiring mutations that con er resistance. Physicians attempt to circumvent the development o resistance by initiating treatment early, using maximally tolerated doses o drugs, and administering multiple drugs in combination. Despite these strategies, however, resistance has become a major impediment to success ul therapy. As more is learned about the biology o microbes and cancer cells and more unique targets are discovered, it is hoped that treatments will become more selective, less toxic, and less prone to the development o drug resistance. Antimetabolite-based therapies, such as the inhibition o olate metabolism, have a long and storied history and have taught us a great deal about the principles o selective toxicity in the treatment o in ections and cancer. Despite the advent o novel anticancer therapies directed at newly identif ed driver mutations, transcription actors, or immune regulators, antimetabolite therapies remain both a cornerstone o current cancer treatment and the subject o active investigation. For example, a recent analysis o expression patterns o metabolic enzymes in matched tumor and normal tissue samples showed that tumors overexpress TSTA3, PYCR1, and MTHFD2. These enzymes play critical roles in ucosylation, proline synthesis, and mitochondrial one-carbon metabolism, respectively—metabolic processes that are currently less appreciated in cancer pathophysiology. Methylenetetrahydro olate dehydrogenase (NADP dependent) 2

C h a p t e r 3 3 Principles of Antimicrobial and Antineoplastic Pharmacology 619

(MTHFD2) is the mitochondrial paralog o an enzyme that plays a vital role in one-carbon trans ers. In the mitochondria, MTHFD2 acts to generate ormate (likely through glycine catabolism) that is exported to the cytosol, conjugated to THF by the cytosolic paralog MTHFD1, and then used or nucleotide synthesis. This f nding adds to our appreciation o the complexity o olate metabolism and, in particular, the role o mitochondria and mitochondrial paralogs o olate-dependent enzymes in nucleotide synthesis. Although much more work needs to be done, TSTA3, PYCR1, and MTHFD2 may together represent novel metabolic targets in the treatment o cancer and, in the case o MTHFD2, deepen our understanding o olate metabolism and open the door to the development o novel drugs. The work that Gerhard Domagk began 80 years ago continues to yield new knowledge and the promise o new therapies.

Acknowledgment We thank Heidi Harbison, Harris S. Rose, and Quentin J. Baca or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading American Cancer Society Statistics. http://www.cancer.org/docroot/STT /stt_0.asp. (Source of cancer statistics provided in this chapter.) Antimicrobial Resistance Prevention Initiative: proceedings o an expert panel on resistance. Am J Med 2006;119(6 Suppl 1):S1–S76. (Series of seven articles and discussion on mechanisms of antimicrobial drug resistance.) Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 8th ed. Philadelphia: Churchill Livingstone; 2014. (Authoritative textbook on clinical management of infectious diseases.)

Coen DM, Richman DD. Antiviral agents. In: Knipe DM, Howley PM, Cohen JI, et al., eds. Fields virology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2013:338–373. (Detailed review of the mechanisms and uses of antiviral drugs.) Fischbach MA, Walsh CT. Antibiotics or emerging pathogens. Science 2009;325:1089–1093. (Overview of the need for new antibiotics to treat infections with multidrug-resistant organisms and discussion of approaches to identifying novel classes of antibiotics.) Kuhar DT, Henderson DK, Struble KA, et al. Updated US Public Health Service guidelines or the management o occupational exposures to human immunodef ciency virus and recommendations or postexposure prophylaxis. Infect Control Hosp Epidemiol 2013;34:875–892. (Comprehensive guidelines for postexposure prophylaxis for the prevention of HIV infection, including an extensive reference list.) LaFemina R, ed. Antiviral research: strategies in antiviral drug discovery. Washington, DC: ASM Press; 2009. (Review of strategies used to discover antiviral drugs.) Moscow JA, Schneider E, Sikic BI, et al. Drug resistance and its clinical circumvention. In: Hong WK, Bast RC Jr, Hait W, et al., eds. HollandFrei cancer medicine. 8th ed. Hamilton, Ontario, Canada: BC Decker and American Association or Cancer Research; 2009:597–610. (Discusses mechanisms of resistance to antineoplastic agents.) Nilsson R, Jain M, Madhusudhan N, et al. Metabolic enzyme expression highlights a key role or MTHFD2 and the mitochondrial olate pathway in cancer. Nat Commun 2014;5:3128–3133. (Demonstrates role of MTHFD2 in cancer.) US Public Health Service. Preexposure prophylaxis or the prevention o HIV in ection in the United States – 2014: a clinical practice guideline. www.cdc.gov/hiv/pd /PrEPguidelines2014.pd . (Comprehensive guidelines for preexposure prophylaxis for the prevention of HIV infection, including an extensive reference list.) Vousden KH, Prives C. Blinded by the light: the growing complexity o p53. Cell 2009;137:413–431. (Review of p53 mechanisms, functions, and pharmacology.) Walsh CT. Antibiotics: actions, origins, resistance. Washington, DC: ASM Press; 2003. (Reviews structural and chemical basis of antibiotic action and resistance.) WHO Statistical In ormation System. http://www.who.int/whosis/. (Source of world health statistics provided in this chapter.)

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C h a p t e r 3 3 Principles o Antimicrobial and Antineoplastic Pharmacology 621

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INTRODUCTION The central dogma processes—DNA replication, transcription, and translation—are generally similar in bacteria and humans. DNA is replicated and transcribed into RNA, and messenger RNA is translated into protein. However, there are important di erences in the biochemistry o bacterial and human central dogma processes, and these di erences can be exploited or the development and clinical use o antibiotics. Three such di erences are targeted by the currently available antibacterial chemotherapeutic drugs: (1) topoisomerases, which regulate supercoiling o DNA and mediate segregation o replicated strands o DNA; (2) RNA polymerases, which transcribe DNA into RNA; and (3) ribosomes, which translate messenger RNA (mRNA) into protein. This chapter brief y reviews the biochemistry o central dogma processes in bacteria and discusses certain relevant di erences between these processes in bacteria and humans. With this background, the chapter discusses the mechanisms by which pharmacologic agents interrupt bacterial DNA replication, transcription, and translation.

BIOCHEMISTRY OF BACTERIAL DNA REPLICATION, TRANSCRIPTION, AND TRANSLATION The central dogma o molecular biology begins with the structure o DNA, which is the macromolecule that carries 622

genetic in ormation. To transmit all o the genetic in ormation in a cell to two progeny cells, the parental DNA must be copied in its entirety (replicated), and the two resulting copies must be segregated—one copy going to each progeny cell. In order to express the genes that are present in the DNA, these speci c portions o the DNA are copied (transcribed) into RNA. Some RNAs (mRNAs) are then read (translated) by the protein synthesis machinery in order to produce proteins. Certain other RNAs, such as trans er RNAs (tRNAs) and ribosomal RNAs (rRNAs), per orm complex unctions essential to protein synthesis. It is important to note that the ollowing discussion o these bacterial processes is vastly simpli ed in order to emphasize the steps that are inhibited by antibiotics.

DNA Structure DNA is composed o two strands o polymerized deoxyribonucleotides that wind around one another in a “double helix” con ormation. The 3 -hydroxyl group o each nucleotide’s deoxyribose ring is joined by a phosphate group to the 5 -hydroxyl group o the next nucleotide, thereby orming the phosphodiester backbone o each side o the double helical “ladder” (Figs. 34-1 and 34-2). The purines adenine (A) and guanine (G) and the pyrimidines thymine (T) and cytosine (C), which are covalently linked to the deoxyribose ring, associate with one another (A with T, G with C) via hydrogen bonds to orm the “rungs” o the ladder (Fig. 34-2). It is the

C h a p t e r 3 4 Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation

It is th e s u m m e r o 1976. Pa rticip a n ts re tu rn in g ro m a n Am e rica n Le g io n co n ve n tio n in Ph ila d e lp h ia a re a llin g s e ve re ly ill w ith a m ys te rio u s typ e o p n e u m o n ia . Th e o u tb re a k ce n te rs o n th e Be lle vu e S tra t o rd Ho te l, w h e re 150 h o te l o ccu p a n ts a n d 32 p a s s e rs b y co n tra ct “ Le g io n n a ire s ’ d is e a s e .” Tw e n ty-n in e victim s u ltim a te ly d ie . Co n ve n tio n a l s p u tu m s ta in s , cu ltu re s , a n d e ve n a u to p s y m a te ria l s h o w n o co n s is te n t p a th o g e n s . Th e te rro r o a n u n kn o w n e p id e m ic d is e a s e s p a rks ru m o rs a n d n e w s re p o rts o p o is o n g a s e s , ta in te d w a te r s u p p lie s , te rro ris ts , a n d d e a d ly viru s e s . Se ve ra l m o n th s la te r, la b o ra to ry a n d f e ld inve s tiga tio n te a m s ro m th e Ce n te rs o r Dis e a s e Co n tro l a n d Pre ve n tio n (CDC) id e n ti y th e ca u s a tive a e ro b ic Gra m -n e g a tive b a cte riu m a n d n a m e it Le g io n e lla p n e u m o p h ila . It is o b s e rve d th a t p a tie n ts tre a te d w ith e ryth ro m ycin , a m a cro lid e a n tib io tic, a n d

linear sequence of bases that encodes the genetic information of a cell. How the nucleotide precursors to these bases are synthesized is reviewed in Chapter 39, Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance. DNA structure is essentially the same between bacteria and

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FIGURE 34-1. Backbone structure of DNA. DNA is a polymer o nucleotides in which a phosphodiester bond connects the 2 -deoxyribose sugars o each neighboring nucleotide. The phosphodiester bond links the 3 -OH o one deoxyribose to the 5 -OH o the next deoxyribose, thus orming the backbone o the DNA strand.

623

te tra cyclin e h a ve b e tte r o u tco m e s th a n th o s e tre a te d w ith o th e r a g e n ts . To d a y, n e w e r m a cro lid e s , s u ch a s a zith ro m ycin , a n d u o ro q u in o lo n e s a re o te n u s e d o r tre a tin g Le g io n n a ire s ’ d is e a s e —a s w e ll a s m a ny ch la m yd ia l, s tre p to co cca l, a n d s ta p hylo co cca l in e ctio n s .

Questions 1 . Why are some antibiotics such as quinolones and aminoglycosides bactericidal, while other antibiotics such as tetracyclines and macrolides are bacteriostatic? 2 . Which step in translation is blocked by tetracyclines and which by macrolides? 3 . How do bacteria develop resistance to macrolide and f uoroquinolone antibiotics? Do they need to acquire exogenous DNA in order to become resistant to these antibiotics?

eukaryotes. However, bacterial chromosomes are usually circular DNAs, while eukaryotic chromosomes, including our own, are linear molecules.

DNA Replication, Segregation, and Topoisomerases The faithful replication and segregation of bacterial DNA to progeny cells involve numerous steps, many of which could make good targets for antibacterial drugs. To date, the enzymes in this process that have been most successfully targeted are topoisomerases . These enzymes perform several functions during DNA replication and segregation. During DNA replication in both bacterial and eukaryotic cells, complementary strands of DNA are synthesized bidirectionally, forming two so-called replication forks. To initiate this process, the two DNA strands that compose the double helix must unwind and separate. In so doing, the DNA strands form excess “supercoils” in which the helical polymer overtwists as it rotates in the same direction as the turn of the helix. Supercoils increase tension in DNA strands and thereby interfere with further unwinding. In the absence of a process to relieve the stress created by the supercoils, the entire chromosome would have to rotate; this process would be complex and energy-consuming and could entangle the entire molecule. Moreover, when DNA replication is completed, the two progeny DNA copies are wrapped around each other. In bacteria, because the chromosomes are circular, the intertwined progeny copies form interlocking rings (catenanes). These intertwined rings must be separated (resolved) before they can be segregated to the progeny cells. Topoisomerases perform both of these functions— removing excess DNA supercoils during DNA replication and separating intertwined progeny DNA. Topoisomerases catalyze these activities by breaking, rotating, and resealing DNA strands. There are two types of topoisomerases. Type I topoisomerases form and reseal single-stranded breaks in DNA to decrease positive supercoiling (Fig. 34-3). Type II topoisomerases form and reseal double-stranded

624 PRINCIPLES OF CHEMOTHERAPY A

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H

Gua nine

The mechanism o action o a type II topoisomerase proceeds in two steps. First, the enzyme binds a segment o DNA and orms covalent bonds with phosphates rom each strand, thereby nicking both strands. Second, the enzyme causes a second stretch o DNA rom the same molecule to pass through the break, relieving supercoiling (Fig. 34-4). This passage o double-stranded DNA through a doublestranded break permits separation o intertwined copies o DNA ollowing replication and, thereby, segregation o DNA into progeny cells. Two main type II topoisomerases are present in bacteria. The rst to be identi ed, DNA gyrase , is a type II topoisomerase that is unusual in that it can introduce negative supercoils be ore the DNA strands separate and thereby neutralize positive supercoils that orm during DNA unwinding. The second is topoisomerase IV. DNA gyrase is particularly crucial or segregation in some bacteria, while topoisomerase IV is the critical enzyme in other bacteria. Because supercoiling is important or transcription as well as segregation, topoisomerases inf uence this central dogma process as well. Given their multiple unctions, topoisomerases are usually engaged with DNA, and this is important or their roles as drug targets. These enzymes are important not only as antibacterial drug targets but also as targets or cancer chemotherapy (see Chapter 39).

Bacterial Transcription

C

bas e O

O H H O

H

O P

H

se

H

O

ba s e O

OH O

H

H

O

H

P

H

O

O-

FIGURE 34-2. Hydrogen bonding between DNA strands. A and B. The dashed lines indicate hydrogen bonds between complementary bases on opposite DNA strands. Adenine (A) and thymine (T) form two hydrogen bonds, while guanine (G) and cytosine (C) form three hydrogen bonds. C. These A-T and G-C base pairs form the “rungs” of the DNA double helical “ladder.” Note that the deoxyribose moieties and phosphodiester bonds are located on the outside of the DNA double helix, while the purine and pyrimidine bases stack in the center of the DNA molecule.

breaks (Fig. 34-4). Both types o topoisomerases can remove excess DNA supercoils during DNA replication. However, only type II topoisomerases can resolve intertwined copies o double-stranded DNA to permit segregation o the DNA to daughter cells. Type II enzymes are both more complex and more versatile than type I topoisomerases, and the type II enzyme is a more common molecular target or chemotherapeutic agents.

Gene expression begins with transcription, which involves the synthesis o single-stranded RNA transcripts rom a DNA template. Transcription is catalyzed by the enzyme RNA polymerase . In bacteria, ve subunits (2 , 1 , 1 , and 1 ) associate to orm the holoenzyme. As discussed below, the subunit is instrumental or initiating transcription, while the rest o the RNA polymerase enzyme—also known as the core enzyme—contains the catalytic machinery or RNA synthesis. The process of transcription occurs in three stages: initiation, elongation, and termination (Fig. 34-5). During initiation, the RNA polymerase holoenzyme binds to and then separates the strands o a short segment o doublehelical DNA a ter its subunit recognizes an upstream site. Once the double helix is unwound to orm a singlestranded template, RNA polymerase initiates RNA synthesis at a start site on the DNA. Initiation entails con ormational changes in the enzyme so that it opens and closes around DNA and it makes appropriate contacts with unwound DNA and the nascent RNA. During elongation, RNA polymerase synthesizes a complementary RNA strand by joining together ribonucleoside triphosphates via phosphodiester bonds. In the process, the subunit dissociates rom the holoenzyme. RNA synthesis proceeds in the 5 →3 direction, with the nascent RNA strand emerging rom an exit channel o the enzyme, until a termination sequence is reached. The RNA polymerase enzyme di ers between bacteria and humans and thus can serve as a selective target or antibacterial drug action. In bacteria, one RNA polymerase synthesizes all o the RNA in the cell (except or the short RNA primers needed or DNA replication, which are made by primase ). In contrast, eukaryotes express three di erent nuclear RNA polymerases, and each enzyme is considerably more complex in its subunit structure than the bacterial counterpart.

C h a p t e r 3 4 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation Strand-rotation mechanism

Strand-passage mechanism

Bind

Bind Me lt

Type I topoisomerase

Bre a k Rota te

Bre a k

Rota te J oin

Pass J oin

FIGURE 34-3. Regulation of DNA supercoiling by type I topoisomerases. Two mechanisms have been proposed for the action of type I topoisomerases. In the strand-rotation model, type I topoisomerase binds to opposite strands of the DNA double helix. The topoisomerase then nicks one strand and remains bound to one of the nicked ends (f lled green circle). The unbound end of the nicked strand is able to unwind by one or more turns and is then joined (religated) to its parent strand. In the strand-passage model, type I topoisomerase binding to the DNA double helix results in melting (separation) of the two DNA strands. The DNA-bound topoisomerase then introduces a nick into one strand, while remaining bound to each end of the broken DNA strand (f lled green circles). The broken strand is then passed through the helix and joined (religated), resulting in a net unwinding of the DNA. Camptothecins, which are used in cancer chemotherapy (see Chapter 39), inhibit the joining of the broken strand of DNA after strand passage.

Camptothecins

B

A

C

G-s e gme nt ATP a s e doma in

625

T-s e gme nt

B' doma in ATP

Type II topoisomerase

ATP

ATP

A' doma in ADP + P i F

E

ATP

ATP

D

ATP

ATP

ATP

ATP

Quinolones (inhibit ba cte ria l e nzyme ) Anthracyclines Epipodophyllotoxins (inhibit huma n e nzyme ) Amsacrine

FIGURE 34-4. Regulation of DNA supercoiling by type II topoisomerases. A. The type II topoisomerase enzymes contain A , B , and ATPase domains. The A and B domains engage a segment of the DNA double helix (G-segment). B. Interaction with the G-segment induces a conformational change in the type II isomerase, causing it to “lock” around the DNA G-segment. C. ATP binds to the ATPase domains of the topoisomerase, and a second segment of the DNA double helix (T-segment) enters and is “locked” into the B domains. D. Once the enzyme is engaged with both DNA segments, the topoisomerase cuts both strands of the G-segment DNA. E. This double-stranded break in the G-segment allows the T-segment to pass through the G-segment to the opposite side of the topoisomerase. F. The T-segment is released from the topoisomerase, and the G-segment break is resealed. ATP is hydrolyzed to ADP, ADP dissociates from the topoisomerase, and the cycle begins anew. The result of each cycle is to change the coiling of DNA or, when two separate circular DNA molecules are involved, to resolve catenanes. Quinolone antibiotics inhibit passage of the T-segment and resealing of the G-segment by bacterial type II topoisomerases. At therapeutic concentrations, quinolones also promote topoisomerase subunit dissociation, resulting in double-stranded breaks in the DNA and killing of the bacteria. Several classes of cancer chemotherapeutic agents, including the anthracyclines, epipodophyllotoxins, and amsacrine, inhibit passage of the T-segment and resealing of the G-segment by human type II topoisomerases, thereby causing double-stranded DNA breaks and inducing apoptosis of the cancer cells (see Chapter 39).

626 PRINCIPLES OF CHEMOTHERAPY RNA polyme ra s e (α2 ββ'σ holoe nzyme )

Fidaxomicin A Initia tion

S ta rt

α2 ββ' B Elonga tion

3'

Rifampin, rifabutin 5'

Elonga tion s ite Te mpla te s tra nd Na s ce nt RNA

σ

Move me nt of polyme ra s e C Te rmina tion

The ribosome o a representative bacterium, Escherichia coli, has a sedimentation coe f cient o 70S and is composed o a 30S subunit and a 50S subunit. The 30S subunit contains a single 16S rRNA molecule and 21 di erent proteins, while the 50S subunit contains two rRNA molecules—23S rRNA and 5S rRNA—and more than 30 di erent proteins. Importantly, it is the rRNAs rather than the protein components of the ribosome that are mainly responsible for the ribosome’s key activities, namely, decoding the mRNA, linking together amino acids, and translocating the translation machine. The 70S ribosome contains three sites that bind tRNAs during translation: the A or “aminoacyl” site (also known as the “acceptor” site ), which binds incoming tRNA molecules carrying the various amino acids; the P or “peptidyl” site , which contains the growing peptide chain; and the E or “exit” site, which binds the tRNAs that have been used during translation be ore they are ejected rom the ribosome (Fig. 34-6). Translation, like transcription, can be divided into three stages (Fig. 34-7). During initiation, the components o the translation system assemble together, abetted by proteins called initiation factors . First, the mRNA joins with the 30S subunit o the bacterial ribosome and with a specif c tRNA molecule linked to formylated methionine ( Met), the f rst amino acid encoded by every bacterial mRNA. The tRNA- ormylated methionine molecule ( Met-tRNA ) binds to its initiation codon (AUG) on the mRNA. Next, the 50S subunit joins with the 30S subunit to orm the complete 70S ribosome. The Met-tRNA now occupies the P site o the 70S ribosome. Elongation, abetted by elongation factors, involves the addition o amino acids to the carboxyl end o the growing polypeptide chain, as the ribosome moves rom the 5 -end to

RNA 70S ribos ome α2 ββ' 50 s ubunit 50S (23S rRNA, 5S rRNA, (2 more tha n 30 prote ins ) m E

FIGURE 34-5. Bacterial transcription. A. During initiation, the RNA polymerase holoenzyme ( 2 ) searches for and recognizes promoter sequences on DNA. The holoenzyme then separates the strands of the double helical DNA, exposing the start site for transcription, and begins synthesis of the new RNA strand. Fidaxomicin blocks this stage of transcription. B. During elongation, the core enzyme (without the subunit) extends the new RNA strand in the 5 → 3 direction, using the unwound DNA strand as a template. RNA polymerase separates the strands of the DNA double helix as it moves along the template strand, extruding the 5 end of the transcript behind it. Rifampin blocks elongation after the nascent transcript reaches a certain length. C. Upon reaching a termination sequence, the DNA, core enzyme, and newly synthesized RNA separate from one another.

Bacterial Protein Synthesis Once the mRNA transcripts are synthesized rom a DNA template, these transcripts are translated by the bacterial translational machinery. Although the overall process o translation is similar between bacteria and higher organisms, there are a number o pharmacologically exploitable di erences in the details o the mechanisms. In particular, the composition o the rRNA molecules di ers between bacterial and human ribosomes. Thus, bacterial ribosomes can also serve as selective targets or antibiotics.

P

A

Macrolides Ketolides Chloramphenicol Lincosamides Streptogramins Oxazolidinones Pleuromutilins 30 s ubunit 30S (16S rRNA, 21 prote ins ) (1

He a d

Aminoglycosides Spectinomycin Tetracyclines Glycylcyclines

FIGURE 34-6. The bacterial 70S ribosome. The bacterial 70S ribosome consists of a 30S subunit, which has a structural feature known as the head, and a 50S subunit. Each subunit is composed of ribosomal RNA (rRNA) and numerous proteins. The rRNAs are mainly responsible for most of the important activities of the ribosome and are the targets of antibiotic drugs that inhibit translation. Aminoglycosides, spectinomycin, tetracyclines, and glycylcyclines bind to 16S rRNA in the 30S subunit. Macrolides, ketolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins bind to 23S rRNA in the 50S subunit. A, aminoacyl site (site of binding of aminoacyl tRNA); P, peptidyl site (site of binding of tRNA that is covalently joined to the elongating peptide chain); E, exit site (site of binding of tRNA that has been ejected from the P site during translocation).

C h a p t e r 3 4 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation fMe t fMe t-tRNA mRNA

+

50S

30S

Initia tion comple x 70S ribos ome

E P A

Amino a cid Cha rge d tRNA

tRNA binding Tetracyclines (30S ) Glycylcyclines (30S )

tRNA E P A

Aminoglycosides (30S ) Chloramphenicol (50S , A s ite ) Lincosamides (50S , A a nd P s ite s ) Streptogramins (50S , A a nd P s ite s ) Oxazolidinones (50S , A s ite ) Pleuromutilins (50S , A a nd P s ite s )

De coding

P e ptide bond forma tion

E P A

Uncha rge d tRNA tRNA binding

Cha rge d tRNA

E P A

Tra ns loca tion a nd pe ptide move me nt

Spectinomycin (30S )

Macrolides (50S , polype ptide e xit tunne l) Ketolides (50S , polype ptide e xit tunne l) Streptogramins (50S , polype ptide e xit tunne l)

FIGURE 34-7. Bacterial translation. Bacterial translation begins with the assembly of an initiation complex containing a 30S ribosomal subunit, mRNA, formyl-methionine-linked tRNA, and a 50S ribosomal subunit. This assembly step is dependent on the binding of fMet-tRNAf to an initiator codon in the mRNA. The assembled 70S ribosome contains the aminoacyl (A), peptidyl (P), and exit (E) sites. The Asite contains a triplet codon of mRNAand allows the anticodon of an incoming amino acid-linked tRNA(i.e., charged tRNA) to bind to its corresponding codon. The decoding function of 16S rRNA helps ensure that binding of the mRNA codon to the correct tRNA, but not an incorrect tRNA, leads to conformational changes that position the amino acid on the charged Asite tRNAnear the amino acid on the charged P site tRNA within the peptidyl transfer center of the 23S rRNA in the 50S subunit. There, formation of a peptide bond occurs between the amino acid occupying the Asite and the carboxy terminus of the nascent peptide residing in the P site. Once the peptide bond has formed, the tRNA–mRNAcomplex translocates from the A site to the P site, the tRNAmolecule that had occupied the P site translocates to the E site, from which it dissociates, and the elongating polypeptide chain moves out through the exit tunnel. The Asite is now empty, and introduction of the next charged tRNAmolecule into the Asite completes the cycle. Translation continues until a stop codon is encountered in the mRNA, at which point the newly synthesized protein is released from the ribosome. Pharmacologic agents that inhibit translation interfere with the activities of the bacterial ribosome. Aminoglycosides bind to 16S rRNA in the 30S subunit and interfere with decoding, thus resulting in the synthesis of proteins containing incorrect amino acids; tetracyclines and glycylcyclines block aminoacyl tRNA binding to the A site; chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins inhibit the peptide bond formation activity of the 50S subunit. Spectinomycin inhibits translocation, and macrolides, ketolides, and streptogramins inhibit peptide movement through the exit tunnel of the ribosome.

627

the 3 -end o the mRNA that is being translated. tRNA molecules carrying specif c amino acids (aminoacyl tRNAs) enter the ribosomal A site, and their three base anticodons basepair to their complementary codons on the mRNA. Utilization o the correct tRNA requires not only anticodon–codon recognition between tRNA and mRNA, respectively, but also decoding unctions provided largely by the 16S rRNA in the decoding center o the 30S ribosomal subunit. Binding o the correct tRNA to its codon in the A site leads to several con ormational changes—in 16S rRNA within the decoding center, in a region o 23S rRNA o the 50S subunit that protrudes into the decoding center, in the A site tRNA, and in protein elongation actors. These movements culminate in the positioning o the tRNA-bound amino acid in the A site so that it is close to the Met (on the P site tRNA) in a region o the 50S subunit known as the peptidyl transferase center. This center, which is ormed largely by the 23S rRNA, catalyzes the ormation o a peptide bond between Met and the next amino acid. Once the peptide bond is ormed linking Met to the next amino acid, which, in turn, is linked to the tRNA in the A site, the tRNA in the A site is said to have “accepted” the Met. A ter the peptide bond has been ormed, the ribosome undergoes more con ormational changes, including rotation (“ratcheting”) o the 30S subunit relative to the 50S subunit and a swiveling motion o the “head” o the 30S subunit, so that both subunits advance three nucleotides toward the 3 -end o the mRNA. In this multistep process, the tRNA that was originally linked to the Met is ejected rom the P site and binds to the E site, the tRNA that is now linked to two amino acids shi ts rom the A site to the unoccupied P site, and the A site becomes available. This process is known as translocation. As these translocation events occur, the nascent polypeptide starts to move through the exit tunnel o the ribosome. In this manner, polypeptide chain elongation results rom multiple cycles o aminoacyl tRNA binding to the A site, peptide bond ormation, and translocation. During termination, proteins called release factors recognize the termination codon in the A site and activate discharge o the newly synthesized protein and dissociation o the ribosome–mRNA complex, with the 70S ribosome separating into its 50S and 30S subunits. Three general points are worth noting about bacterial translation. First, the two ribosomal subunits demonstrate segregated functions: the 30S subunit is largely responsible or aith ul decoding o the mRNA message, while the 50S subunit catalyzes peptide bond ormation. Translocation, however, involves both subunits. Second, these functions are performed mainly by the RNA components of the ribosome. Third, inhibitors of protein synthesis affect the process of translation at different steps and, indeed, have greatly helped dissect these steps.

PHARMACOLOGIC CLASSES AND AGENTS Three general categories o drugs target bacterial DNA replication, transcription, and translation: (1) drugs that target type II topoisomerases, (2) drugs that target RNA polymerase, and (3) drugs that target ribosomes. Quinolone antibiotics are broad-spectrum agents that not only inhibit certain topoisomerases but also convert these enzymes

628 PRINCIPLES OF CHEMOTHERAPY

into DNA-damaging agents. Fidaxomicin and ri amycin derivatives bind to and inhibit bacterial RNA polymerase. Fidaxomicin is used to treat colitis caused by Clostridium di f cile, and one ri amycin derivative, ri ampin, is a mainstay in the therapy o tuberculosis. Multiple classes o drugs bind bacterial ribosomes to inhibit protein synthesis. Speci cally, aminoglycosides, spectinomycin, tetracyclines, and glycylcyclines bind the 30S ribosomal subunit, while macrolides, ketolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins target the 50S ribosomal subunit. These inhibitors o protein synthesis generally act on both Gram-positive and Gram-negative organisms and are there ore in wide clinical use (see Chapter 35, Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis, or a discussion o Gram-positive and Gram-negative bacteria). Elucidation o the mechanisms o action o the agents described below has depended crucially on the eld o bacterial genetics. In particular, the molecular targets o antibiotics have been identi ed by (1) isolating bacteria that are resistant to the particular antibiotic (e.g., ri ampin), (2) showing that the target molecule (e.g., RNA polymerase) exhibits biochemical resistance to the antibiotic, and (3) demonstrating that the drug-resistance mutation lies within the gene encoding the target (e.g., the gene encoding the subunit o RNA polymerase). More recent work, using nuclear magnetic resonance spectroscopy and x-ray crystallography, has urther elucidated the structures o the targets as well as the molecular nature o the various drug–target interactions. Indeed, the 2009 Nobel Prize in Chemistry was awarded or crystallographic analyses o ribosomes and their binding to certain antibiotics.

Inhibitors o Topoisomerases: Quinolones Quinolones are a major class o bactericidal antibiotics that

act by inhibiting bacterial type II topoisomerases. One o the earliest quinolones to enter clinical use was nalidixic acid (Fig. 34-8), and the mechanism o action o the quinolones was elucidated largely by studying this drug. Fluoroquinolones (all ending in “-f oxacin”) have a f uorine atom at position 6 (Fig. 34-8), which increases the potency and spectrum o bacteria killed by these agents compared to nalidixic acid. The older f uoroquinolones, ciprof oxacin, norf oxacin, and of oxacin, are used to treat urinary tract in ections and gastrointestinal in ections caused by Gramnegative bacteria, including E. coli, Klebsiella pneumoniae, Campylobacter jejuni, and Enterobacter, Salmonella, and Shigella species. Newer f uoroquinolones, including gemif oxacin, moxif oxacin, and levof oxacin, retain activity against Gram-negative bacteria and also have activity against Streptococcus pneumoniae and bacteria that cause atypical pneumonia (Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila), so these drugs are commonly used to treat bacterial pneumonia. Levof oxacin and moxif oxacin have activity against Mycobacterium tuberculosis and are sometimes used in combination with other antimycobacterial drugs to treat tuberculosis. Fluoroquinolone resistance is common in staphylococci, so other antibiotics are usually used to treat in ections with these organisms. Bacteria typically evolve resistance to the quinolones through chromosomal mutations in the genes that encode type II topoisomerases or through alterations in

O COOH

N

N

Nalidixic acid O F

COOH

N

N

HN

Ciprofloxacin O F

COOH

N N

N O

Levofloxacin

FIGURE 34-8. Structures o antimicrobial drugs targeting bacterial topoisomerases. Nalidixic acid, ciprof oxacin, and levof oxacin are quinolone antibiotics that inhibit bacterial type II topoisomerases.

the expression o membrane porins and e f ux pumps that determine the concentration o drug inside the bacteria. Adverse e ects are in requent but can include nausea, vomiting, and diarrhea and, rarely, tendinitis, tendon rupture, and peripheral neuropathy. Quinolones act by inhibiting one or both o the two type II topoisomerases in sensitive bacteria: DNA gyrase (topoisomerase II) and topoisomerase IV. Selectivity o action results rom di erences in structure between the bacterial and eukaryotic orms o these enzymes. Quinolones primarily inhibit DNA gyrase in Gram-negative organisms, and they inhibit topoisomerase IV in Gram-positive organisms such as Streptococcus pneumoniae. The mechanism o action o the quinolones involves subverting the unction o bacterial type II topoisomerases. Ordinarily, type II topoisomerases bind to and break both strands o a DNA molecule, allowing another stretch o the same molecule to pass through the double-stranded DNA break (Fig. 34-4). Quinolones inhibit these enzymes be ore the second segment o DNA can pass through, thereby stabilizing the orm o the complex in which the DNA polymer is broken. At low concentrations, quinolones inhibit type II topoisomerases reversibly, and their action is bacteriostatic. At higher

C h a p t e r 3 4 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation

concentrations, however—which are readily achieved in patients—quinolones convert the topoisomerases into DNAdamaging agents by stimulating dissociation o the enzyme subunits rom the broken DNA. DNA with double-stranded breaks cannot be replicated (unless the breaks are repaired), and transcription cannot proceed through such breaks. The double-stranded breaks themselves and/or the bacterial response to the double-stranded breaks lead ultimately to cell death. Thus, at therapeutic doses, the quinolone antibiotics are bactericidal.

Inhibitors of Transcription: Fidaxomicin and Rifamycin Derivatives Fidaxomicin is a macrocyclic antibiotic with an 18-membered

macrolactone ring (Fig. 34-9). The use o daxomicin is limited to treatment o C. di f cile colitis. Fidaxomicin is as e ective as standard therapy with oral vancomycin or initial clinical cure o C. di f cile colitis, and patients treated with daxomicin are less likely to have recurrence o disease HO Cl

O

O

O

O

O

OH

O

HO

OH O

O

O O

HO

HO

Cl

O

OH

Fidaxomicin

CH3 COO CH3 O

OH

OH

OH

O

OH NH N N

O

O

O

N

OH

Rifampin

CH3 COO CH3 O

OH

OH

OH

O

O NH

O

NH

O O

N N

Rifabutin

FIGURE 34-9. Structures of antimicrobial drugs targeting bacterial RNA polymerases. Fidaxomicin inhibits bacterial DNA-dependent RNA polymerase at the initiation stage, while rifampin and rifabutin, which are derivatives of rifamycin B, inhibit at the elongation stage. The rifamycin B backbone of rifampin and rifabutin is shown in blue.

629

within 4 weeks o initial cure. The selectivity o daxomicin stems rom its high potency and bactericidal action against C. di f cile; it is much less potent against the Gram-negative bacteria that compose the desirable intestinal f ora. The mechanism by which daxomicin inhibits transcription is not well understood, but it di ers rom the mechanism by which ri amycin derivatives act (see below). Fidaxomicin acts at the initiation stage o RNA synthesis, a ter binding o RNA polymerase to DNA and be ore separation o the strands o the DNA double helix (Fig. 34-5). Fidaxomicin resistance mutations encode amino acid substitutions in a region o the enzyme that is involved in the con ormational changes that occur during initiation; this region also makes contact with unwound DNA and nascent RNA. Fidaxomicin-resistant bacteria remain susceptible to ri ampin, which is consistent with the conclusion that the two drugs act through di erent mechanisms. Because very little daxomicin is absorbed rom the gastrointestinal tract, it can be administered orally to achieve concentrations in the colon that greatly exceed those needed to kill C. di f cile. Fidaxomicin is generally well tolerated, but adverse e ects can include nausea, vomiting, abdominal pain, gastrointestinal hemorrhage, neutropenia, and anemia. Acute hypersensitivity reactions to daxomicin have occasionally been reported. Rifampin (also known as rifampicin) and its structural relative, rifabutin, are two semisynthetic derivatives o the naturally occurring antibiotic ri amycin B (Fig. 34-9). Although ri ampin can be used or prophylaxis o meningococcal disease and or treatment o some other bacterial in ections, its major use is in the treatment o tuberculosis and other mycobacterial in ections. Ri ampin is particularly e ective against phagosome-dwelling mycobacteria because it is bactericidal or intracellular as well as extracellular bacteria. Furthermore, ri ampin increases the in vitro activity o isoniazid, another rst-line drug used in the combination therapy o tuberculosis (see Chapter 35 and Chapter 41, Principles o Combination Chemotherapy). Ri amycin derivatives exert their bactericidal activity against mycobacteria by orming a highly stable complex with the DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis. The drugs target the subunit o bacterial RNA polymerase. Ri ampin permits the initiation o transcription but then blocks elongation once the length o the nascent RNA reaches two to three nucleotides. Exactly how this occurs or all ri amycin derivatives and all bacterial RNA polymerases has not been completely resolved; however, or one bacterial RNA polymerase, there is crystallographic evidence that ri ampin occludes the exit channel by which the nascent RNA emerges rom the enzyme. Ri ampin displays high selectivity or bacteria, as mammalian polymerases (even those o mitochondria, which are considered bacteria-like) are inhibited by ri ampin only at ar higher concentrations. Hence, ri ampin is generally well tolerated, and the incidence o adverse e ects (typically, rash, ever, nausea, vomiting, and jaundice) is low. Because the rapid emergence o resistance makes single-drug therapy o tuberculosis not only ine ective but also counterproductive, ri ampin is administered in combination with other antituberculosis drugs. In vitro experiments show that 1 out o every 106 to 108 tubercle bacilli can develop resistance to ri ampin via a one-step mutational process that

630 PRINCIPLES OF CHEMOTHERAPY

appears to a ect the binding site o the drug on the polymerase. However, as a component o a multidrug therapeutic regimen, ri ampin can markedly reduce the li etime rate o reactivation o latent tuberculosis (see Chapter 41).

Inhibitors of Translation Three general considerations apply to inhibitors o bacterial translation. First, translation inhibitors target either the 30S or 50S subunit o the bacterial ribosome. The ollowing discussion o translation inhibitors is presented in terms o 30S versus 50S inhibition (Table 34-1). The second consideration concerns selectivity. In addition to their inhibitory e ects on bacterial ribosomes, protein synthesis inhibitors can a ect mammalian mitochondrial TABLE 34-1 Sites and Mechanisms of Action of Antibacterial Translation Inhibitors DRUG OR DRUG CLASS

SITE OF ACTION

MECHANISM OF ACTION

Drugs targeting the 30S ribosomal subunit Aminoglycosides

16S rRNA

Induce misreading; halt protein synthesis at higher concentrations

Spectinomycin

16S rRNA

Inhibits translocation by blocking swiveling of the head of the 30S subunit

Tetracyclines and glycylcyclines

16S rRNA

Block aminoacyl tRNA binding to A site

Drugs targeting the 50S ribosomal subunit Macrolides and ketolides

23S rRNA

Inhibit translocation by blocking the exit tunnel from which the growing polypeptide chain emerges

Chloramphenicol

23S rRNA

Inhibits peptide bond formation by interfering with positioning of the aminoacyl moiety in the A site of the peptidyl transferase center

Lincosamides

23S rRNA

Inhibit peptide bond formation by binding to the A site and, possibly, the P site in the peptidyl transferase center

Streptogramins

23S rRNA

Inhibit peptide bond formation by binding to the A site and P site in the peptidyl transferase center and block the polypeptide exit tunnel

Oxazolidinones

23S rRNA

Inhibit peptide bond formation by blocking productive binding of the aminoacyl moiety in the A site of the peptidyl transferase center

Pleuromutilins

23S rRNA

Inhibit peptide bond formation by binding to the A site and P site in the peptidyl transferase center

ribosomes, cytosolic ribosomes, or both. Inhibition o host ribosomes is one common mechanism by which these drugs cause adverse e ects. For some antibiotics, such as chloramphenicol, inhibition o mammalian ribosomes represents a major drawback and can lead to serious, even lethal, adverse e ects. Tetracyclines can also inhibit mammalian ribosomes in vitro; ortunately, however, this class o drugs is concentrated selectively in bacterial cells. Certain other translation inhibitors exhibit little or no inhibition o mammalian ribosomes at clinically relevant concentrations; or these agents, the dose-limiting toxicities appear to be attributable to other mechanisms. As with most orally available, broad-spectrum antibiotics, gastrointestinal adverse events appear to be due to elimination o normal gut f ora. An interesting twist on the issue o selectivity emerged in the 1990s. It was discovered that certain aminoglycoside, macrolide, and lincosamide antibiotics demonstrate some e cacy against eukaryotic microorganisms (e.g., protozoan parasites) that cause opportunistic in ections in patients with AIDS and in other immunocompromised individuals. In these microorganisms, it appears that the activity o the antibiotics can be attributed to their inhibition o organellar protein synthesis in the microorganism (see Chapter 37, Pharmacology o Parasitic In ections). The third consideration is that complete inhibition o protein synthesis is not su f cient to kill a bacterium. Bacteria can generate several responses to various growth-stif ing treatments that allow them to remain dormant until the treatment is removed. One o these responses permits the bacteria to survive complete inhibition o protein synthesis. As a result, most inhibitors o protein synthesis are bacteriostatic. Aminoglycosides are the major exception to this rule.

Antimicrobial Drugs Targeting the 30S Ribosomal Subunit Aminoglycosides Aminoglycosides are used mainly to treat in ections caused

by Gram-negative bacteria. Aminoglycosides are also used in synergistic combinations to treat some serious in ections with Gram-positive bacteria. These antibiotics are not active against obligate anaerobic bacteria. Aminoglycosides are charged molecules that are not orally bioavailable, so they must be administered parenterally. The aminoglycosides include streptomycin (the rst aminoglycoside, discovered in 1944), neomycin, kanamycin, tobramycin, paromomycin, gentamicin, netilmicin, and amikacin (streptomycin and gentamycin are shown in Fig. 34-10). O these, gentamicin, tobramycin, and amikacin are the most widely used agents because o their lower toxicity and broader coverage o Gram-negative organisms. Aminoglycosides bind at low concentrations to the 16S rRNA o the 30S subunit. At these concentrations, aminoglycosides induce ribosomes to misread mRNA during elongation, leading to synthesis o proteins containing incorrect amino acids. It is logical to in er rom this e ect that aminoglycosides inter ere with the mRNA-decoding unction o the 30S subunit. Crystal structures o 30S-aminoglycoside complexes have greatly aided our understanding o the decoding process. How aminoglycosides a ect decoding has been studied structurally mainly or paromomycin. There are two current models or the induction o misreading by paromomycin binding to the 30S subunit. In one model, paromomycin binding causes con ormational changes in

C h a p t e r 3 4 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation

631

NH HO NH OH

HO O

O CHO HO HO HO

HN

OH O

HO HO

H2 N

NH H2 N

O

O

NH2

O HO

H2 N NH2 O

NHCH3

O

HO

OH

HO

NHCH3

Streptomycin

H N

HO

H

O

H

Gentamicin OH H

OH

OH N H

O

OH OH O

OH OH O

O

O

Tetracycline

N OH NH2

OH

OH NH2

Spectinomycin

H

N

O

O H HO O NH

HO

H

O

Doxycycline

H N

H

H

N

O N H

OH NH2

OH

O

OH OH O

O

Tigecycline

FIGURE 34-10. Structures of antimicrobial drugs targeting the 30S ribosomal subunit. Streptomycin and gentamicin are aminoglycosides. Spectinomycin is a structural relative of the aminoglycosides. Tetracycline and doxycycline are tetracyclines. Tigecycline is a glycylcycline.

particular nucleotides o the 16S rRNA that monitor codon– anticodon interactions, thereby mimicking changes caused by binding o the correct (cognate) tRNA anticodon to an mRNA codon in the A site. It is thought that these con ormational changes induce the subsequent con ormational changes that result in peptide bond ormation, even when the incorrect tRNA is present in the A site. (Consistent with this model, streptomycin also induces misreading, and it binds at a di erent, but nearby, site to stabilize the 30S subunit in a state similar to that induced by binding the correct tRNA.) A second model stems rom the observation that crystal structures o 70S ribosomes containing either incorrect or correct tRNAs exhibit the same con ormational changes thought to lead to peptide bond ormation. When added to these ribosomes, paromomycin induces a con ormational change in a 16S nucleotide that promotes movement o a region o 23S rRNA that protrudes into the decoding center. This model posits that the con ormational change in 23S rRNA allows retention o the incorrect tRNA in the A site, so that peptide bond ormation and thus misreading can occur. At higher concentrations, aminoglycosides completely inhibit protein synthesis. Exactly how this occurs is not understood, although in vitro, there is evidence that at least some aminoglycosides inhibit translocation and, in act, stimulate tRNA movement in the opposite direction (reverse translocation). Certain aminoglycosides can bind at high concentrations to the 23S rRNA o the 50S subunit and prevent the dissociation o 70S ribosomes into individual subunits ollowing termination o translation. In treated bacteria, not only does misreading occur, but ribosomes also become

trapped at the AUG initiation codons o mRNA. Eventually, translation halts, despite the presence o ribosomes that are not bound to drug. In contrast to other protein synthesis inhibitors, aminoglycosides are bactericidal. This is an important eature in the treatment o serious in ections. Although the precise mechanism or bactericidal activity is not known, one appealing model, developed by the late Bernard Davis, has gained some acceptance (Fig. 34-11). The Davis model rames bacterial cell death in terms o the concentration-dependent e ects o aminoglycosides. When drug rst enters the cell, it is poorly transported across bacterial membranes. At these initial low concentrations, misreading occurs, leading to synthesis o mis olded proteins. Some o these proteins insert into membranes and cause the ormation o membrane pores, which allow aminoglycosides to f ood the cell and halt protein synthesis completely. As a result, the damage to the membrane cannot be repaired, and leakage o ions and, later, larger molecules leads to cell death. An alternative model also invokes misreading and protein mis olding as key steps in bactericidal action but posits that the mis olded proteins activate a stress-response sensor that contributes to membrane damage and DNA damage, and thus to cell death. Another important aspect o aminoglycoside activity is that these drugs act synergistically with agents that inhibit peptidoglycan synthesis, such as -lactams and glycopeptide antibiotics. There ore, aminoglycosides and -lactams are commonly used in combination (see Chapter 41). The explanation most commonly suggested or this synergy is that inhibition o cell wall synthesis increases the entry o

632 PRINCIPLES OF CHEMOTHERAPY Ce ll wa ll A

Inne r me mbra ne

Oute r me mbra ne

Incorre ct a mino a cid incorpora te d

P olys ome

Aminoglycos ide

C

B

Me mbra ne pore (a bnorma l prote in)

D Aminoglycos ide "monos ome " (nonfunctiona l)

FIGURE 34-11. Davis model for the bactericidal activity of aminoglycosides. The Davis model o aminoglycoside action proposes that low concentrations o aminoglycosides induce protein misreading and that the misread (abnormal) proteins allow higher concentrations o aminoglycosides to enter the cell and halt protein synthesis. A. Initially, aminoglycosides are present at low concentrations inside the bacterial cell, despite therapeutic (high) extracellular concentrations o drug, because the drug molecules are taken up poorly by bacteria. B. Low intracellular concentrations o aminoglycoside bind to bacterial ribosomes and cause incorporation o incorrect amino acids (misreading) into nascent polypeptides. C. The abnormal, mis olded proteins insert into the bacterial membranes, orming pores and causing membrane damage. D. The damaged membranes allow additional aminoglycoside molecules to f ood into the cell, causing complete inhibition o ribosome activity. The e ect is irreversible, perhaps because o trapping o drug inside the cell (“caging”). The membrane damage cannot be repaired because new proteins cannot be synthesized, and cell death ensues.

aminoglycosides into the bacteria. The synergy between lactams and the aminoglycosides contrasts sharply with the antagonism between -lactams and the bacteriostatic inhibitors o protein synthesis discussed below. Three general mechanisms have been established or resistance to aminoglycosides. First, and clinically most common, is the plasmid-encoded production o a trans erase enzyme or enzymes that inactivate aminoglycosides by adenylation, acetylation, or phosphorylation. Second, drug entry into the cell can be impaired, perhaps by alteration or elimination o porins or other proteins involved in drug transport. Third, the drug target on the 30S ribosomal subunit can become resistant to drug binding by virtue o mutation or the activity o a plasmid-encoded enzyme. In addition to several general types o toxicity, such as hypersensitivity reactions and drug-induced ever, aminoglycosides can cause three specif c adverse e ects: ototoxicity, nephrotoxicity, and neuromuscular blockade. O these, ototoxicity (mani esting as either auditory or vestibular damage that is usually irreversible) is the most important actor restricting aminoglycoside use. The aminoglycosides accumulate and persist in the perilymph and endolymph o the inner ear and, at high concentrations, they damage hair cells. There is excellent evidence rom human genetic studies that ototoxicity is caused, at least in part, by aminoglycoside inhibition o host mitochondrial ribosomes. An alternative view is based on the f nding that aminoglycosides bind to phospholipids in the cell membrane and to iron. Drug binding to iron can generate ree radicals, which may be agents o damage in the inner ear. Aminoglycosides also cause acute renal failure that is usually reversible. Aminoglycoside concentrations can reach

high levels in renal proximal tubular cells due to receptormediated endocytosis via a specif c receptor called megalin in clathrin-coated pits. The biochemical mechanism responsible or the subsequent toxicity is poorly understood, although both mitochondrial poisoning and perturbation o the plasma membrane are suspected. At very high concentrations, aminoglycosides can produce reversible nondepolarizing neuromuscular blockade , potentially causing respiratory paralysis. This e ect is thought to result rom drug competition with calcium at presynaptic sites, leading to reduction in acetylcholine release, ailure o the postsynaptic end-plate to depolarize, and muscle paralysis. Spectinomycin Spectinomycin (Fig. 34-10) also binds to the 16S rRNA o

the 30S ribosomal subunit but at a location di erent rom those bound by aminoglycosides. Spectinomycin permits ormation o the 70S ribosome but inhibits translocation. This appears to be due to inhibition o swiveling o the head o the 30S subunit during translocation. Unlike the aminoglycosides, spectinomycin does not induce codon misreading and is not bactericidal. Spectinomycin is administered parenterally and is used clinically only as an alternative therapy or gonorrheal in ections.

Tetracyclines and Glycylcyclines

Tetracyclines have been used clinically or many years. Four tetracyclines are available or use in humans in the United States: tetracycline , demeclocycline , doxycycline , and minocycline . All are close structural relatives and can be considered as a group (tetracycline and doxycycline are shown

C h a p t e r 3 4 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation

in Fig. 34-10). Although di erences in clinical e cacy are minor, doxycycline and minocycline are most o ten used because they can be administered at less requent dosing intervals than tetracycline and because, unlike tetracycline, their absorption is not signi cantly reduced when taken orally with ood. Tetracyclines are bacteriostatic broad-spectrum antibiotics that are used widely. Tetracyclines bind reversibly to the 16S rRNA o the 30S subunit and inhibit protein synthesis by blocking the binding o aminoacyl tRNA to the A site on the mRNA–ribosome complex. This action prevents the addition o urther amino acids to the nascent peptide. However, inhibition o protein synthesis does not account entirely or the high bacterial selectivity o tetracyclines, because these drugs can also halt eukaryotic protein synthesis in vitro at concentrations not much higher than those required to inhibit bacterial protein synthesis. Rather, the high selectivity of tetracyclines derives from the active accumulation of these drugs in bacteria but not in mammalian cells. Tetracyclines enter Gram-negative bacteria by passive di usion through porin proteins in the outer membrane, ollowed by active (energy-dependent) transport across the inner cytoplasmic membrane. Uptake into Gram-positive bacteria occurs similarly via an energydependent transport system. In contrast, mammalian cells lack the active transport system ound in susceptible bacteria. Since the bacterial selectivity o tetracyclines results rom drug-concentrating mechanisms, it ollows that resistance can occur through increased drug e f ux or decreased drug inf ux. In act, plasmid-encoded e f ux pumps represent the most widespread mechanism employed by tetracycline-resistant microbes. A second orm o resistance arises through the production o proteins that bind the ribosome and inter ere with the binding o tetracyclines. A third mechanism o resistance involves the enzymatic inactivation o tetracyclines. An important pharmacokinetic eature o the tetracyclines is the interaction o these drugs with oods and oral drugs containing divalent and trivalent cations. Absorption o oral tetracycline is inhibited by about hal i taken with ood (particularly calcium-rich dairy products), so tetracycline should be taken when the stomach is empty. In contrast, doxycycline and minocycline may be taken with ood. Intestinal absorption o all tetracyclines is inhibited by medicines containing divalent and trivalent cations, such as antacids. The same interaction with cations—speci cally calcium—causes sequestration o the drug in developing bones and teeth, potentially leading to reduced skeletal growth and permanent brown discoloration o the teeth in children. Gastrointestinal distress and renal toxicity are the two most problematic adverse e ects o the tetracyclines. Nausea, vomiting, and diarrhea occur with all tetracyclines but are more common with tetracycline (which is taken on an empty stomach) than with doxycycline and minocycline. All tetracyclines are excreted in both urine and bile. Urine is the primary route o excretion or tetracycline, and this drug is associated with renal toxicity in people with preexisting renal ailure. Lower ractions o doxycycline and minocycline are eliminated via the kidney, making these drugs sa er or use in patients with kidney disease. Finally, a red rash occurs on sun-exposed skin (photosensitivity) in some patients taking tetracyclines. Tigecycline is the rst member o a newer class o antibiotics—glycylcyclines . The our-ring structure o tigecycline resembles that o the tetracyclines (Fig. 34-10), as

633

does its mechanism o action. Tigecycline has a broader spectrum o activity than tetracyclines and has been approved or intravenous administration in the treatment o serious skin and abdominal in ections and o community-acquired pneumonia caused by susceptible organisms. The US Food and Drug Administration (FDA) warns that tigecycline should be reserved or use only when alternative treatments are not suitable, because o a higher rate o mortality in patients treated with tigecycline than with other antibiotics.

Antimicrobial Drugs Targeting the 50S Ribosomal Subunit The clinically available antibiotics that target the 50S subunit —macrolides, ketolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins—bind to a small region o 23S rRNA near the peptidyl trans erase active center. Small di erences in their binding sites and di erences in their chemical structures are responsible or di erences in their detailed mechanisms o action. Macrolides and Ketolides

Macrolides are named or their large lactone rings. Attached to these rings are one or more deoxy sugars. Erythromycin (Fig. 34-12) is the best-known member o this group. Two semisynthetic derivatives o erythromycin, azithromycin and clarithromycin, are broader in spectrum and better tolerated than erythromycin and are there ore growing in use. As illustrated in the introductory case, macrolides have proven to be especially important in the treatment o pulmonary in ections, including Legionnaires’ disease. These agents display excellent lung tissue penetration, and just as important, they have intracellular activity against Legionella. Macrolides are bacteriostatic antibiotics that bind to a speci c segment o 23S rRNA. They act during the elongation stage o protein synthesis by blocking the exit tunnel rom which nascent peptides emerge, as clearly illustrated by a crystal structure o erythromycin bound to the 50S subunit (Fig. 34-13A). As a result, short nascent peptides are synthesized (akin to the short nascent transcripts synthesized by bacterial RNA polymerase in the presence o ri amycin derivatives), and then translation stops. Interestingly, however, certain proteins can be synthesized even in the presence o macrolides. Evidently, the drugs do not completely block the exit tunnel, and some polypeptides can “slither” past the block. Macrolide use is complicated by the problem o resistance, which is usually plasmid encoded. One mechanism employed by some resistant Enterobacteriaceae is the production o esterases or phosphotrans erases that modi y macrolides. Modi cation o the ribosomal binding site by chromosomal mutation represents a second mechanism o resistance. Some bacteria reduce the permeability o their membrane to macrolides or (more commonly) increase active drug e f ux. Production o a methylase that modies the 23S rRNA target site o the macrolides, leading to decreased drug binding, accounts or the vast majority o resistance to macrolides in Gram-positive organisms. Constitutive production o methylase also con ers resistance to the structurally unrelated compounds clindamycin and streptogramin B, which bind 23S rRNA close to the macrolide target site (see discussion below). Adverse reactions to erythromycin typically involve the gastrointestinal tract or the liver. Gastrointestinal intolerance represents the most requent reason or discontinuing erythromycin, as the drug can directly stimulate gut motility and

634 PRINCIPLES OF CHEMOTHERAPY O

HO

OH OH

OH

N

O OH

O

CHCl2

HN O2N

O

HO

O

O

O OH

O

O

Chloramphenicol

Erythromycin O

CH3 N

H C

CH3 CH2 CH2

O

H

H NH HO H

C

Cl

N

N

O

CH3

N

HN

O

CH O H OH H H

O

O

H

O

S CH3

HO

N

O H N

S

N O O

NH N

OH

Clindamycin

Quinupristin O OH

N O

O O

N

N

O

O

F

N

N

H N O

Linezolid

N O

S O

O

O

O

Dalfopristin

OH N

O H

S O

O

Retapamulin

FIGURE 34-12. Structures of antimicrobial drugs targeting the 50S ribosomal subunit. Chloramphenicol, erythromycin (a macrolide), clindamycin (a lincosamide), quinupristin (a streptogramin), dalfopristin (a streptogramin), linezolid (an oxazolidinone), and retapamulin (a pleuromutilin) each inhibit bacterial translation by targeting the 50S ribosomal subunit.

cause nausea, vomiting, diarrhea, and sometimes anorexia. Erythromycin can also produce acute cholestatic hepatitis (with ever, jaundice, and impaired liver unction), probably as a hypersensitivity reaction. Metabolites o erythromycin can inhibit certain cytochrome P450 isozymes in the liver and thus increase the plasma concentration o numerous drugs that are also metabolized by these liver enzymes. Azithromycin and clarithromycin are generally well tolerated, although these drugs can also cause liver impairment. Telithromycin, a third semisynthetic derivative o erythromycin, was approved by the FDA in 2004. Formally known

as a ketolide rather than a macrolide, telithromycin has a mechanism o action similar to that o the macrolides, but with a higher a f nity or the 50S ribosomal subunit due to its ability to bind an additional site on 23S rRNA. Its higher a f nity or the 50S subunit and its lack o induction o expression o some genes mediating resistance allows telithromycin to be used in treating in ections due to some Gram-positive cocci that are resistant to macrolides. Telithromycin is also bactericidal against certain Gram-positive bacteria, but it is not clear why. Telithromycin can be involved in numerous drug–drug interactions and in rare cases o ulminant hepatic

C h a p t e r 3 4 Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation A

B

Ma crolide s Clinda mycin

Chlora mphe nicol CC-puromycin CHCl2 P -s ite tRNA

A-s ite tRNA

FIGURE 34-13. Mechanism of action of erythromycin, clindamycin, and chloramphenicol revealed by crystallographic analysis of drug binding to the 50S ribosomal subunit. A. Erythromycin (red) binds to a specif c segment o 23S rRNA and blocks the exit tunnel rom which nascent peptides emerge. B. Clindamycin and macrolides have partially overlapping binding sites on the 50S ribosomal subunit, as do clindamycin and chloramphenicol. The positions o the A-site tRNA and P-site tRNA are also shown. The exact binding locations and con ormations o the drugs vary in crystal structures o drug-bound ribosomes rom di erent species.

necrosis. Telithromycin is contraindicated in patients with myasthenia gravis since it exacerbates weakness and can lead to respiratory compromise in patients with this disease. Chloramphenicol Chloramphenicol (Fig. 34-12) is a bacteriostatic broad-

spectrum antibiotic that is active against both aerobic and anaerobic Gram-positive and Gram-negative organisms. The most highly susceptible organisms include Haemophilus in uenzae, Neisseria meningitidis, and some strains o Bacteroides. However, the potential or serious toxicity has limited the systemic use o chloramphenicol. The drug is still used occasionally in the treatment o typhoid ever, bacterial meningitis, and rickettsial diseases but only when sa er alternatives cannot be used, as in the case o resistance or serious drug allergy.

635

Chloramphenicol binds to 23S rRNA and inhibits peptide bond ormation, apparently by occupying a site that inter eres with proper positioning o the aminoacyl moiety o tRNA in the A site in the peptidyl trans erase center (Fig. 34-13B). Microbes have developed resistance to chloramphenicol by two major mechanisms. Low-level resistance has emerged in large chloramphenicol-susceptible populations by the selection o mutants with decreased permeability to the drug. The more clinically signi cant type o chloramphenicol resistance has arisen rom the spread o speci c plasmid-encoded acetyltransferases (at least three types o which have been characterized) that inactivate the drug. The undamental mechanism underlying the toxicity o chloramphenicol appears to involve inhibition o mitochondrial protein synthesis. One mani estation o this toxicity is the gray baby syndrome , which can occur when chloramphenicol is administered at high doses to newborn in ants. Because newborns lack an e ective glucuronic acid conjugation mechanism or the degradation and detoxi cation o chloramphenicol, the drug can accumulate to toxic levels and cause vomiting, f accidity, hypothermia, gray color, respiratory distress, and metabolic acidosis. More requently, chloramphenicol causes dose-related, reversible depression o erythropoiesis and gastrointestinal distress (nausea, vomiting, and diarrhea). Aplastic anemia , a rare but potentially atal toxicity, occurs via an idiopathic mechanism that is unrelated to dose. O special interest are the adverse e ects that chloramphenicol can cause in tandem with other drugs. Like the macrolides, chloramphenicol increases the hal -li e o certain drugs, such as phenytoin and war arin, by inhibiting the cytochrome P450 enzymes that metabolize these drugs. Chloramphenicol also antagonizes the bactericidal e ects o penicillins and aminoglycosides, as do other bacteriostatic inhibitors o microbial protein synthesis. Lincosamides The major lincosamide in clinical use is clindamycin (Fig. 34-12).

Clindamycin blocks peptide bond ormation, apparently through interactions with the A site (like chloramphenicol) (Fig. 34-13B), and, possibly, also through interactions with the P site. The most important indication or clindamycin is the treatment o serious intra-abdominal or gynecological in ections that are likely to include penicillin-resistant Bacteroides ragilis and other intestinal anaerobes. Clindamycin is a cause o pseudomembranous colitis caused by Clostridium di f cile overgrowth. An in requent member o the normal ecal f ora, C. di f cile is selected or during the administration o clindamycin or other broad-spectrum oral antibiotics to which it is not susceptible. C. di f cile elaborates a cytotoxin that can cause colitis characterized by mucosal ulcerations, severe diarrhea, and ever. This serious adverse e ect is a major concern with the use o oral clindamycin.

Streptogramins

In 1999, the FDA approved the rst drug in the streptogramin class o protein synthesis inhibitors. The drug is a mixture o two distinct chemicals: dalfopristin, a group A streptogramin, and quinupristin, a group B streptogramin (Fig. 34-12). Dal opristin/quinupristin was approved or the treatment o serious or li e-threatening in ections caused by vancomycinresistant Enterococcus aecium or Streptococcus pyogenes. Common adverse e ects o dal opristin/quinupristin include elevated bilirubin, pain with administration, and joint and muscle pain.

636 PRINCIPLES OF CHEMOTHERAPY

Streptogramins inhibit protein synthesis by binding to the peptidyl trans erase center o bacterial 23S rRNA. Mutations and modif cations a ecting this region can con er resistance. The A component binds to a location overlapping both the A site and the P site in the peptidyl trans erase center, and it can inhibit peptidyl trans erase in vitro. The binding site or the B component overlaps with that o the macrolides, and it is thought that, like the macrolides, quinupristin blocks the emergence o nascent peptides rom the ribosome. When dal opristin binds the ribosome, the ribosome changes conormation such that it binds quinupristin with higher a f nity, leading to synergy between the two streptogramin components. Streptogramins are unusual among the 50S antibiotics in that they are bactericidal against many, but not all, susceptible bacterial species. A clear explanation or this phenomenon remains elusive; the current hypothesis is that, unlike the other 50S antibiotics, the streptogramins induce a conormational change in the ribosome that is reversible only a ter subunit dissociation. Oxazolidinones

In 2000, the FDA approved linezolid (Fig. 34-12), the f rst drug in the oxazolidinone class o antibacterial agents. In 2014, tedizolid phosphate , a prodrug o tedizolid, became the second oxazolidinone to be approved. Both drugs are orally available and demonstrate excellent activity against drug-resistant Gram-positive bacteria, including methicillinresistant S. aureus (MRSA), penicillin-resistant streptococcus, and vancomycin-resistant enterococcus (VRE), but have very little activity against Gram-negative bacteria. Although there was initially controversy regarding the precise mechanism o action o oxazolidinones, crystallographic analyses have located the binding site o linezolid in a pocket o the A site where the amino acid moiety in aminoacyl tRNA normally binds. Moreover, mutations in 23S rRNA can con er drug resistance. These results and those o biochemical studies suggest that oxazolidinones block productive interactions o aminoacyl tRNAs with the A site in the peptidyl trans erase center. Linezolid has occasionally been associated with serious adverse e ects, including myelosuppression and neuropathy, but tedizolid appears to be associated with lower rates o these adverse e ects. Pleuromutilins

In 2007, the FDA approved retapamulin (Fig. 34-12), the f rst drug in the pleuromutilin class o antibiotics. This drug is used as a topical treatment or minor bacterial skin in ections (impetigo), and its mechanism o action is relatively well understood. Like linezolid, pleuromutilins bind to a pocket in the A site o the peptidyl trans erase center where aminoacyl tRNA normally binds. Distinct rom linezolid, pleuromutilin binding also extends into the P site. Thus, the binding site o pleuromutilins is similar to that o group A streptogramins. The locations o mutations con erring

resistance to pleuromutilins are consistent with this binding site. These compounds inhibit peptide bond ormation, but once elongation is underway and the A and P sites are occupied, pleuromutilins are no longer active. Adverse e ects o retapamulin are minor and largely limited to local irritation and pruritus at the site o use. The act that three o the most recently developed antibiotic classes inhibit the ribosome emphasizes the continuing value o this complex structure as a target or new drug development. There is much continuing e ort to discover new protein synthesis inhibitors, and this work is aided by the availability o structures o ribosomes bound to drugs.

CONCLUSION AND FUTURE DIRECTIONS Multiple classes o antibiotics target the bacterial machinery responsible or the central dogma processes, disrupting these processes at multiple steps. Most o these drugs demonstrate selective binding to bacterial enzymes or RNAs and have relatively ew adverse e ects. All are associated with some degree o toxicity, however, and some (e.g., chloramphenicol) have limited clinical use because o their potential to cause li e-threatening adverse e ects. Several o these antibiotic classes—the quinolones, ri amycin derivatives, f daxomicin, and a ew o the protein synthesis inhibitors— can be bactericidal, but most protein synthesis inhibitors are bacteriostatic. Drug resistance is a persistent and serious problem or all o these agents. Although the emergence o resistance is an expected consequence o antibiotic use, judicious drug administration, multidrug therapies, and the continued development o new antibacterial agents can combat the emergence o resistance. The development o f daxomicin and the newer classes o bacterial ribosome inhibitors represents an important advance in the search or drugs that are e ective against resistant bacteria. Further elucidation o the mechanism o action o these drugs will both in orm basic biology and def ne new biochemical targets or pharmacologic intervention.

Suggested Reading Kannan K, Vazquez-Laslop N, Mankin AS. Selective protein synthesis by ribosomes with a drug-obstructed exit tunnel. Cell 2012;151:508–520. (This report changed thinking about the mechanisms o macrolide and ketolide antibiotics by showing that they permit the synthesis o subsets o bacterial proteins despite blocking the polypeptide exit tunnel.) Louie TJ, Miller MA, Mullane KM, et al. Fidaxomicin versus vancomycin or Clostridium di f cile in ection. New Engl J Med 2011;364:422–431. (Clinical trial evaluating use o f daxomicin or C. di f cile in ection.) Walsh CT. Antibiotics: actions, origins, resistance. Washington, DC: ASM Press; 2003. (Reviews antibiotic synthesis, action, and mechanisms o resistance.) Wilson DN. Ribosome-targeting antibiotics and mechanisms o bacterial resistance. Nat Rev Microbiol 2014;12:35–48. (Reviews molecular mechanisms o action o and resistance to these antibiotics.)

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640 PRINCIPLES OF CHEMOTHERAPY

HO HO HO

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NH

O

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P EP A ur M

NA ADP H ur M

B

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L-Ala , D-Glu, L-Ly Lyss , ATP ATP ATP

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35

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Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

Pa rk k nucle otidd e

B C om omp p le tion of mure in monome r s ynthe s is is , a nd mure in mon onom omee r exp p or o t, p o lyme riza tion, a nd c ros s -linking

NAG NAM L-Ala -γ-D-Glu-L-L γ ys -D-Ala -D-Ala y

β-lac tams : Pe nic illins C phalo s po rins Ce M noo ba Mo bacc tam tams Carb rbap ape ne ms

(L-Gly)5

NAG

O

NH D-Ala

LL (L-Gly) ly)5 L-Lys γ γ-D-Gl u L-Ala

Vanc ycm in m o n d David W. Ku b ia k, Ra m yD-Ala A. Arn a o u t , a nD-Ala d S a raV h P.o m Ha

NAG

D-Al -Alaa D-Ala (L-Gly)5 L-Lys γ γ-D-Gl u

NAM L-Ala -γ-D-Glu-L-L γ yys -D-Ala -D-Ala NAG

(L-Gly)5 NH2

L Ala L-Ala

NH

HO HO HO P 2 O 7 -3

O AcHN N

Pe rip ipla la s m

D a D-Al Lys (L-Gly)5 L-L γ γ-D-Gl u L-Ala L Ala

D-Ala (L-Gly)5 L-Lys

D- a D-Al (L-Gly)5 L-Lys

γ γ-D-Gl u

γ γ-D-Gl u

L Al L-Ala

L-Ala

INTRODUCTION TR & CASE . .NH . . . . . . . . . . . . . . . . . . . . TP . .P. . . . . 641–642 64 t . . . . . . . . .P. GT . . . . . . . . . . . 64 6499 Polymerization NH O O Vanc a nco ncom oom myc ycin in, Tela Tee la lavvanc va n in, ODal Dalbavancin, and Oritavancin n . 649 BIOCHEMISTRY OC C H OF BACTERIAL TER R IA CELL WALL SYNTHESIS . . . . . 641 O HO HO O O HOof Po HO HO r Cr O Structure HO Inhibit InOhibi Inhi bito tors Polymer Cross-Linking nking . O. . . . . . . . . . . . . . . . .HO .O. . 665 6500O O O HO O H HO Cell Wall c tu u OaandOOFunction Functio O Ors of O . . . . . . . . . . . . . . . . . . . . . . . . 641 O ucture O HO O HO HO N -Lactam AcHN AcHN A HN lycan HOtics: am Antib Antibi bioti ics: ic cAcHN Gen Gener eneral ral al C Consi onsiderations . . . 650 6AcH AcHN O s . . . . . .HO O AcHN Pept Pe ptidogl Biosynthesis B Bi isAcHN . . O. . . .O. . . . . . . . . . . . . . . . . . . 643 AcHN A A AcHN 3 O O P O P 2O7 O O P O -Lactam Antibiotics: Specif c Agentss -. . . .- . . . . . . . . . . . . 65 6522 Synthesis o Murein MonomerssO -. . .O.-. . . . . . . . . . . . . . . . . . 643 O O Inhibitors In nhhiiibbitto orrss of of Cell Cell Membrane Ce Me mb mbrraannee Stability Staabbility St y . . . . . . . . . . . . . . . . . . . . 65 6654 544 Glycan G lyycccaan lyc ly an P Polymerization Po oly olym ol lym yme ri rizzzaaati riza attio tioon n. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 6645 4455 Anti cte te ria ial al A Agents ge nnts ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 6654 54 54 Cytopla Cyt Cyto Cy yyt yto toopppl pla la s m mic ic A m etimbra me m bcobacterial rabne ra n ect Polymer P ollyym ym meer er C Cross-Linking Cr Cro rro ros oos oss sss ssss--Liinnkkiing inng g . . . . . . . . . . . . . . . . . . . . . . . . . .C Ethambutol, Ethambutol E Et tth hhaaam mb m buto uutto tol ol P Pyrazinamide Pyrazinamide, Pyr Py yyrrraaazzzin inam in a mi mid m iid ddee and a nndd IIs an Isoniazid s on so onnia niia iaz a zziiid d . . . . . . . . . . . . . . 65 6654 54 54 Mycobacterial M yco yc c oob bbaaacccttteeerrria ia l C Cell Ce eell llll W Wall Wa aalllll S Synthesis Sy yynnt nth thhees e ssiis . . . . . . . . . . . . . . . . . . . . . . 64 6646 4466 Autolysins and Cell Wall Degradation . . . . . . . . . . . . . . . . . . . 648 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 655 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 648 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .655 Inhibitors of Murein Monomer Synthesis . . . . . . . . . . . . . . . . 648 Fos omycinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648 Cycloserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649 Bacitracinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649

INTRODUCTION In 1928, Alexander Fleming made a chance discovery that would revolutionize the treatment o bacterial in ections. He observed that certain molds produce a compound that inhibits the growth o bacteria. The compound he isolated was penicillin, the f rst in a long line o antibiotics that act by inhibiting the biosynthesis o peptidoglycan, the major component o the bacterial cell wall. The unique chemical and structural properties o peptidoglycan make it an attractive and prominent target or antibacterial chemotherapy. The emergence and spread o antibiotic resistance increasingly complicate the clinical use o cell wall synthesis inhibitors, however. This chapter reviews the biochemistry o peptidoglycan synthesis and describes the mechanisms o action, uses, and limitations o the antibiotics that interere with this pathway. These limitations include resistance, toxicity, and drug–drug interactions. Antibiotics that target other essential components o the bacterial cell wall are also discussed.

rNHof Murein Inhibitors O

D-Ala

BIOCHEMISTRY OF BACTERIAL CELL WALL SYNTHESIS Cell Wall Structure and Function Peptidoglycan, named or its peptide and sugar composition, is a three-dimensional meshwork o peptide-cross-linked sugar polymers that surrounds the bacterial cell just outside its cytoplasmic membrane (Fig. 35-1). Peptidoglycan is also known as murein, a ter the Latin murus (wall). Nearly all clinically important bacteria produce peptidoglycan. The major exceptions are Mycoplasma pneumoniae, which can cause atypical pneumonia, and the intracellular orm (or “reticulate body”) o Chlamydia trachomatis, which can cause a sexually transmitted in ection. Peptidoglycan is critically important or the survival o bacteria, which experience large uctuations in osmotic pressure depending on their environment. The peptidoglycan meshwork wrapped around the cell provides the tensile strength required to withstand high turgor pressures that would otherwise cause the plasma

641

NH O O HO O

O AcHN O

O

O

642 PRINCIPLES OF CHEMOTHERAPY

S a m a n th a T is a 50-ye a r-o ld w o m a n w h o p re s e n ts to a n u rg e n t ca re ce n te r w ith a s o re a re a o n h e r le t m e d ia l th ig h . S h e re p o rts th a t s h e s cra tch e d h e r th ig h o n s o m e e xe rcis e e q u ip m e n t a t h e r lo ca l g ym , w h e re s h e w o rks o u t 6 d a ys a w e e k. Th e a re a s lo w ly b e ca m e re d , wa rm , a n d te n d e r ove r th e n e xt 4 d a ys . Ove r th e p a s t 24 h o u rs , s h e h a s e lt th a t th e s o re s p o t is “ re a d y to b u rs t.” On e xa m in a tio n , Ms . T h a s a 2- 2-cm , ro u n d , f u ctu a n t a re a o n th e le t th ig h , w ith a s u rro u n d in g p a tch o e ryth e m a th a t cove rs a 67-cm a re a o th e th ig h . S h e u n d e rg o e s in cis io n a n d d ra in a g e o th e a b s ce s s in th e u rg e n t ca re o ce a n d th e p u ru le n t m a te ria l is s e n t o r cu ltu re , w h ich la te r g ro w s m e th icillin -s e n s itive S ta p hylo co ccu s a u re u s (MS SA). S h e is tre a te d w ith d icloxa cillin . Ms . T re tu rn s to th e u rg e n t ca re clin ic 10 d a ys la te r a n d re p o rts th a t th e le t th ig h w o u n d is n e a rly co m p le te ly h e a le d b u t th a t s h e n o w h a s p ro u s e d ia rrh e a a n d e ve r. S h e e e ls lig h t-h e a d e d w h e n s ta n d in g . S h e re p o rts th a t s h e h a s b e e n livin g w ith

membrane to rupture. Since peptidoglycan is essential or bacterial survival, its biosynthesis is a major target or antibiotics. The largest and most widely used class o bacterial cell wall synthesis inhibitors, the beta-lactam ( -lactam) antibiotics, inhibit the transpeptidase enzymes that mediate peptide cross-linking o the sugar polymers. Bacteria are conventionally divided into two groups, Gram-positive and Gram-negative , based on the relative ability o the bacteria to retain the purple color o the gentian violet component o the Gram stain a ter being washed with an organic solvent such as acetone. Gram-positive bacteria

Gram-positive bacteria

h e r e ld e rly a th e r, w h o h a s s e ve ra l ch ro n ic m e d ica l a ilm e n ts a n d wa s re ce n tly tre a te d o r Clo s trid iu m d if cile in e ctio n . Ms . T is re e rre d to h e r lo ca l e m e rg e n cy ro o m o r tre a tm e n t o d e hyd ra tio n , a n d s h e is tre a te d w ith in tra ve n o u s f u id s . A s to o l s a m p le te s ts p o s itive o r Clo s trid iu m d if cile toxin . Ms . T is tre a te d w ith o ra l va n co m ycin a n d h e r d ia rrh e a re s o lve s ove r th e co u rs e o th e n e xt w e e k.

Questions 1 . Which test can help determine the cause o Ms. T’s skin and so t tissue in ection be ore the culture result is available? 2 . What type o antibiotic is dicloxacillin and what is its mechanism o action? 3 . Is dicloxacillin an appropriate drug or treatment o an MSSA skin in ection? 4 . What is the mechanism o action o vancomycin? 5 . Why is vancomycin administered orally instead o intravenously to treat C. di f cile in ection?

retain the stain and appear purple, whereas Gram-negative bacteria lose the stain and appear pink rom the subsequently applied sa ranin counterstain. Gram staining is requently used to help identi y the bacteria present in a specimen o body f uid such as urine, sputum, or pus. The Gram stain is one test that the urgent care clinician used to determine what type o organism was causing Ms. T’s skin abscess and cellulitis in the introductory case. The ability to retain Gram stain results rom two distinguishing characteristics o cell wall architecture (Fig. 35-1). First, Gram-negative bacteria possess an outer membrane, an asymmetric bilayer in

Gram-negative bacteria

Mycobacteria

Lipopolys a ccha ride Mure in

P ore

P ore Oute r me mbra ne Lipoprotein P e ripla s m

Extra cta ble phos pholipids Mycolic a cids Ara binoga la cta n Mure in

Mure in Cytopla s mic me mbra ne

Cytopla s mic me mbra ne

Cytopla s mic me mbra ne

FIGURE 35-1. Bacterial cell wall architecture. In Gram-positive bacteria (left), the cell wall is composed o a thick layer o murein, through which nutrients, waste products, and antibiotics can di use. Lipoteichoic acids in the outer leaf et o the cytoplasmic membrane intercalate through the cell wall to the outer sur ace o Gram-positive bacteria (not shown); the hydrophilic side chains o these molecules are involved in bacterial adherence, eeding, and evasion o the host immune system. In Gram-negative bacteria (center), the murein layer is thinner and is surrounded by a second, outer lipid bilayer membrane. Hydrophilic molecules cross this outer membrane through channels, which are ormed by a cylindrical arrangement o pore proteins (porins). Gram-negative bacteria have lipopolysaccharide (LPS) in the outer membrane; LPS is a major antigen or the immune response to Gram-negative organisms. The cell wall o mycobacteria (right), which include the causative agents o tuberculosis (M. tuberculosis) and leprosy (M. leprae), is analogous to that o Gram-negative bacteria. The main di erence between the sur ace architecture o mycobacteria and that o Gram-negative bacteria is in the lipid structures outside the murein layer. In mycobacteria, the outer membrane contains arabinogalactan-linked mycolic acids, extractable phospholipids, and other lipid components. One simpli ed model or the organization o the mycobacterial outer membrane is shown.

C h a p t e r 3 5 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

which the outer lea et is composed o lipopolysaccharide. This structurally unusual membrane orms a permeability barrier that excludes a wide variety o molecules and limits the penetration o Gram stain into the periplasm, the space between the inner and outer membranes where the peptidoglycan layer is located. Second, Gram-positive bacteria have a very thick murein layer, whereas Gram-negative bacteria have only a thin layer. Since Gram stain binds to peptidoglycan, and the binding capacity and accessibility o the thick murein layer are much greater in Gram-positive than Gramnegative organisms, the Gram-positive bacteria stain purple. The outer membrane o Gram-negative bacteria not only limits the penetration o Gram stain into the periplasm, but it also prevents the penetration o many other molecules, including antibiotics that target peptidoglycan synthesis—such as vancomycin and bacitracin. Hence, although Gram-negative organisms express the molecular targets or these antibiotics, they are not susceptible. To enable uptake o hydrophilic nutrients and excretion o hydrophilic waste products, Gramnegative bacteria have outer membrane porins —beta-barrel proteins that traverse the outer membrane and allow certain molecules to pass in and out (see Fig. 35-1). Porins are important pharmacologically because it is through these pores that most hydrophilic antibiotics with activity against Gramnegative organisms gain access to the murein layer and to the structures beneath this layer. Also important pharmacologically are the lipopolysaccharides (LPS) that compose the outer lea et o the outer membrane o Gram-negative bacteria. Lipopolysaccharides are amphipathic molecules that protect bacteria rom toxic hydrophilic host molecules such as bile salts. Lipopolysaccharides are also important or bacterial adherence to host cells and or evasion o the host immune response. Polymyxin is a topically used antibiotic that acilitates its own entry into the periplasm by binding to LPS and disrupting the integrity o the outer membrane. Once in the periplasm, polymyxin permeabilizes the inner membrane, discharging the membrane potential so that bacterial cells no longer generate the energy required or survival. Although polymyxin is too toxic or systemic use in people, its mechanism o action suggests that it may be possible to develop less toxic molecules that breach the outer membrane and allow the passage o antibiotics to their molecular targets in Gram-negative bacteria. Gram-positive bacteria do not have an outer membrane; the extracellular enzymes involved in cell wall synthesis are there ore accessible to a wider range o antibiotics than those that can penetrate Gram-negative organisms. However, the cell wall o Gram-positive organisms is not composed simply o peptidoglycan; there is also a set o other cell wall polymers that play important roles in adherence to host tissue and other aspects o pathogenicity. These include lipoteichoic acids and wall teichoic acids , anionic polymers that are typically composed o acyclic sugar-phosphate repeats unctionalized with D-alanine and cyclic sugars such as glucose. Lipoteichoic acids are anchored in the bacterial membrane and extend into the peptidoglycan layers. Wall teichoic acids are covalently attached to peptidoglycan and extend through and beyond its outermost layer. These polymers are important or bacterial in ection o the host, and the pathways o teichoic acid biosynthesis are possible targets or antibiotics. In some Gram-positive organisms, including Staphylococcus aureus, the peptidoglycan layers are also unctionalized with proteins that are required or pathogenesis. These proteins

643

are covalently attached to uncross-linked peptides in peptidoglycan by enzymes called sortases . Sortases have also been suggested as possible targets or antibiotics. These important structural di erences between the cell envelopes o Gram-negative and Gram-positive bacteria lead to di erential access o antibiotics to cellular targets and also present di erent opportunities or the development o new antibiotics. Despite this, peptidoglycan biosynthesis, which is conserved among Gram-negative and Grampositive organisms, remains the most important antibacterial cell envelope target. In act, the peptidoglycan biosynthetic pathway is one o a very small number o broad-spectrum antibacterial targets that exist in bacterial pathogens. The other broad-spectrum targets include DNA synthesis, RNA synthesis, and protein synthesis (see Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation). O these processes, only peptidoglycan biosynthesis is unique to bacteria.

Peptidoglycan Biosynthesis Peptidoglycan biosynthesis occurs in three major stages. The f rst stage is intracellular and involves the synthesis o murein monomers rom amino-acid and sugar building blocks; the second and third stages involve the export o these murein monomers to the sur ace o the inner membrane, ollowed by their polymerization into linear peptidoglycan polymers and their cross-linking into two-dimensional lattices and three-dimensional mats (Fig. 35-2). Because the details o bacterial cell wall synthesis can be daunting, it is help ul to keep in mind the three major stages—monomer synthesis, glycan polymerization, and polymer cross-linking—in the discussion that ollows. In principle, any o the biochemical steps in peptidoglycan biosynthesis could be a target or antibiotics; in practice, clinically used antibiotics target only a ew o the steps in these stages. A vast number o secondary metabolites produced by soil and marine microorganisms also block peptidoglycan biosynthesis, providing a reservoir o structurally and unctionally novel compounds or possible clinical development as our existing antibiotics ail due to the spread o resistance.

Synthesis of Murein Monomers

The murein monomer is a disaccharide comprising N-acetylglucosamine connected via a beta linkage to the C4 hydroxyl o N-acetyl muramic acid, which is unctionalized on the C3 lactate moiety with a peptide (Fig. 35-2). The f rst phase o peptidoglycan synthesis takes place in the cytoplasm and involves the conversion o UDP-N-acetylglucosamine (UDP-NAG), a nucleotide-sugar used as a building block in many cell wall polymers, to UDP-N-acetyl muramic acid pentapeptide (UDP-NAM-peptide; also known as the Park nucleotide). The f rst two enzymes in this process, MurA and MurB, convert the C3 hydroxyl o NAG to lactate. MurA, also known as enolpyruvate transferase, trans ers enolpyruvate rom phosphoenolpyruvate (PEP) to UDP-NAG to orm UDP-NAG pyruvate enol ether (Box 35-1). The avoenzyme MurB (also known as UDP-NAG-enolpyruvate reductase) then reduces the double bond to produce UDP-NAM, which has a ree carboxylate to serve as the handle or the peptide chain. UDP-NAM is a sugar unique to bacteria, and its biosynthesis thus provides opportunities or selective antibiotics. One clinically used antibiotic that blocks the biosynthesis o UDP-NAM is fosfomycin, a PEP analogue that inhibits MurA.

644 PRINCIPLES OF CHEMOTHERAPY A Mure in monome r s ynthe s is (cytopla s mic pha s e )

HO HO HO

O O

NH

O O AcHN O P O P O O- O-

O

N

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OH OH

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NADP H B ur M

HO

O

L-Ala , D-Glu, L-Lys , ATP ATP ATP

O O

NH

O O AcHN O P O P O O- O-

O

N

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HN

OH OH

UDP -NAG

HO HO O

UDP -NAM

HO

O

O O

NH

O O AcHN O P O P O O- O-

O

N

O

Cycloserine H N

Fosfomycin O

HO

MurF, ATP

D-Ala -D-Ala

HO

DdlB, ATP D-Ala + D-Ala

NH2

O

O

NH

O O AcHN O P O P O O- O-

O

N

O

OH OH NH

O

Alanine racemase L-Ala + L-Ala

O

O

HN

O

OH OH NH

O

HO HO O

H N O

NH2 O H N

HN

O OH

O

P a rk nucle otide

B Comple tion of mure in monome r s ynthe s is , a nd mure in monome r e xport, polyme riza tion, a nd cros s -linking β-lactams: Penicillins Cephalosporins Monobactams Carbapenems

NAG NAM L-Ala -γ-D-Glu-L-Lys -D-Ala -D-Ala (L-Gly)5

NAG

O

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D-Ala

(L-Gly)5 L-Lys L-Ala O O

O HO O

AcHN

O AcHN

HO O HO

P 2 O 7 -3

O

O HO O

AcHN

HO HO HO

O O O AcHN O P O P O O- O-

O

L-Ala

PGT

NH

O

O

γ-D-Glu

L-Ala

NH

TP

(L-Gly)5 L-Lys

γ-D-Glu

L-Ala

NH

D-Ala

(L-Gly)5 L-Lys

γ-D-Glu

NH2

L-Ala

NH HO HO HO

NAG

γ-D-Glu

D-Ala

D-Ala

(L-Gly)5 L-Lys

(L-Gly)5

Glycopeptides

D-Ala

D-Ala

NAM L-Ala -γ-D-Glu-L-Lys -D-Ala -D-Ala

(L-Gly)5 L-Lys

γ-D-Glu

D-Ala

NAG

D-Ala

P e ripla s m

NH

O O HO O

AcHN P 2 O 7 -3

HO O HO

O AcHN

O O HO O

O AcHN

O O O AcHN O P O P O O- O-

HO HO HO

O HO O

O AcHN

O O O AcHN O P O P O O- O-

Cytopla s mic me mbra ne P 2 O 7 -3

HO P O 4 -2 Ba ctopre nol HO O phos pha te

Dephosphorylase

HN L-Ala

O

O O O O AcHN O P O P O O- O-

NH O

N

OH OH

γ-D-Glu

Bacitracin

L-Lys

HO HO O

O

MraY

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O O AcHN O P O P O O- O-

MurG

O AcHN

HO O O

HN

L-Ala γ-D-Glu

P a rk nucle otide

O

HO HO HO

O

O

HO HO HO

O O O AcHN O P O P O O- O-

Staph FemABX

L-Ala

UDP -NAG

UDP

L-Lys

D-Ala

D-Ala

D-Ala

D-Ala

D-Ala

D-Ala

AcHN

HO O O

HN

O

O O O AcHN O P O P O O- O-

L-Ala

Gly-tRNA tRNA

γ-D-Glu

L-Lys

O

γ-D-Glu L-Lys

Lipid II

D-Ala

(L-Gly)5

Mure in monome r

D-Ala

Cytopla s m

FIGURE 35-2. Bacterial cell wall biosynthesis and its inhibition by pharmacologic agents. Bacterial cell wall biosynthesis can be divided into three major stages. A. In the cytoplasmic phase o murein monomer synthesis, glucose is amidated and phosphorylated to glucosamine-1-phosphate (not shown), which is acetylated and conjugated to a uridine diphosphate (UDP) nucleotide by the enzyme GlmU (not shown) to orm UDP-N-acetylglucosamine (UDP-NAG). Addition o phosphoenolpyruvate (PEP) by enolpyruvate trans erase (MurA) and reduction o the resulting product by MurB orm UDP-N-acetyl muramic acid (UDP-NAM). NAG and NAM are the two sugar building blocks or subsequent cell wall synthesis. MurC, MurD, and MurE sequentially add the amino acids L-alanine, D-glutamate, and L-lysine to UDP-NAM. In some bacteria, diaminopimelic acid (DAP) is added instead o L-lysine. Alanine racemase converts L-alanine to D-alanine, and D-Ala-D-Ala ligase B (DdlB) orms the dipeptide D-Ala-D-Ala. This dipeptide is then added to the L-Ala-D-Glu-L-Lys (or L-AlaD-Glu-DAP) tripeptide by MurF, resulting in a UDP-NAM molecule linked to f ve amino acids (Park nucleotide). Fos omycin is a selective inhibitor o MurA. Cycloserine inhibits both alanine racemase and D-Ala-D-Ala ligase B, thereby preventing the addition o alanine residues to the growing peptide chain. B. The NAM–pentapeptide complex is trans erred rom UDP to the lipid carrier bactoprenol by the enzyme MraY, and NAG is added rom UDP-NAG by MurG. In some bacteria, one to f ve amino acids can then be added to L-lysine or DAP to orm a branched peptidoglycan; the amino acids are added rom amino acyl tRNA. (Here, as an example, f ve glycine residues are added rom glycyl-tRNA.) This completes the synthesis o the murein monomer. In the murein monomer export and polymerization stage, the bactoprenol–peptidoglycan complex is transported rom the bacterial inner membrane to the periplasmic space, where peptidoglycan glycosyltrans erases (PGTs) join the murein monomer to the growing peptidoglycan chain. Simultaneously, the bactoprenol is liberated to acilitate another round o murein monomer translocation. Bactoprenol diphosphate is dephosphorylated to bactoprenol phosphate by dephosphorylase, regenerating the orm o the lipid carrier that can react with the Park nucleotide. In the f nal stage o cell wall biosynthesis, adjacent glycopeptide polymers are cross-linked in a reaction catalyzed by bacterial transpeptidases (TPs). In the example shown, a transpeptidase cross-links a glycine pentapeptide on one peptidoglycan chain to a D-Ala residue on an adjacent peptidoglycan chain; as shown in detail in Figure 35-3, the terminal D-Ala residue is displaced in this reaction. Bacitracin inhibits bactoprenol dephosphorylation and thereby interrupts murein monomer synthesis and export. The glycopeptides vancomycin, telavancin, dalbavancin, and oritavancin bind the D-Ala-D-Ala terminus o the bactoprenol-conjugated murein monomer unit and thereby prevent the PGT-mediated addition o murein monomer to the growing peptidoglycan chain. The -lactam antibiotics (penicillins, cephalosporins, monobactams, and carbapenems) inhibit the transpeptidase enzymes that cross-link adjacent peptidoglycan polymers.

C h a p t e r 3 5 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

645

B O X 3 5 - 1 En zym e s o f Ce ll Wa ll Bio s yn th e s is Like most enzymes, the enzymes of cell wall biosynthesis are known by multiple names. The Mur naming convention used here is the emerging standard, but the enzymes are also still known by the following descriptive names (among others): GlmU MurA MurB MurC MurD

Diamine N-acetyltransferase Enolpyruvate transferase UDP–NAG-enolpyruvate reductase UDP–NAM-L-Ala synthetase UDP–NAM-L-Ala-D-Glu synthetase

The peptide component o UDP-NAM-peptide is assembled on the C3 lactate rom amino acids and dipeptides by a series o ATP-dependent ligases. MurC, MurD, and MurE sequentially add the amino acids L-alanine, D-glutamate, and a diamino acid—either L-lysine or diaminopimelic acid (DAP)—to UDP-NAM. DAP di ers rom lysine in having a carboxyl group as well as an amine on the side chain. Most Gram-positive bacteria use L-lysine, whereas a minority o Gram-positive and all known Gram-negative bacteria use DAP. This is noteworthy because m-DAP is not ound in humans, and there ore, it o ers a unique target or uture drug development. Peptide ormation continues with the addition o a D-alanyl-D-alanine dipeptide (D-Ala-D-Ala) to the growing chain. The dipeptide is synthesized rom two molecules o L-alanine in two reactions. Because amino acids are usually available in the environment in the L-con ormation—which is the con ormation ound in most mammalian proteins—the f rst reaction requires the trans ormation o two molecules o L-alanine into D-alanine. This reaction is catalyzed by the enzyme alanine racemase . (Similarly, a glutamate racemase converts L-glutamate to D-glutamate to provide the building block or the second amino acid in the peptide chain.) In the second reaction, an ATP-dependent enzyme called D-Ala-D-Ala synthetase (or D-Ala-D-Ala ligase B [DdlB]) joins the two D-alanines together a ter f rst activating one as the AMP-ester. The resulting D-Ala-D-Ala dipeptide is added to the UDP-NAM-tripeptide by MurF to orm UDP-NAML-Ala-D-Glu-L-Lys (or m-DAP-)-D-Ala-D-Ala, a molecule re erred to as the Park nucleotide (Fig. 35-2A). The second phase o peptidoglycan synthesis takes place on the inner sur ace o the cytoplasmic membrane and begins with the trans er o UDP-NAM-peptide to a phospholipid carrier embedded in the membrane (Fig. 35-2B). This carrier is called bactoprenol phosphate or, alternatively, undecaprenol phosphate in recognition o the act that it is assembled rom 11 f ve-carbon isoprene units. Bactoprenol phosphate (BP) is called a carrier because murein monomers as well as many other cell wall precursors are assembled on it, delivered by it to the sur ace o the plasma membrane, and then released in a process that regenerates the carrier or urther cycles o reaction and precursor transport. The reaction by which UDP-NAM-peptide is anchored to the carrier lipid is mediated by an integral membrane protein called MraY. This enzyme catalyzes a diphosphate exchange reaction, so called

MurE

UDP–NAM-L-Ala -D-Glu-2,6-dia minopime la te synthetase MurF UDP–NAM-tripeptide-D-Ala-D-Ala synthetase MraY UDP–NAM-pentapeptide:undecaprenol-phosphate transferase MurG Unde c a pre noldiphos pho-NAM-pe nta pe ptide : NAG transferase Note: Undecaprenol is another name for bactoprenol. ■

because the uridine diphosphate linkage to the NAM-peptide is replaced with an undecaprenol diphosphate linkage, in a chemical exchange reaction illustrated in Figure 35-2B. This reaction is thermodynamically neutral since the products contain the same types o bonds as the starting materials, and indeed, MraY is a readily reversible enzyme. Once the NAM-peptide is anchored to the carrier lipid on the cytoplasmic sur ace o the membrane, a membrane-associated enzyme called MurG catalyzes the trans er o N-acetyl glucosamine to the C4 hydroxyl o the NAM sugar to produce a lipid-anchored NAM-NAG disaccharide commonly known as Lipid II. Finally, in some Gram-positive bacteria, including S. aureus, a linker peptide, typically composed o f ve glycine residues, is usually added to the lysine (or DAP) side chain amine. The additional amino acids in the branching peptide are not added in the same ashion as those in the main peptide chain. Rather than being activated as AMPesters or attack by nucleophilic amines, these amino acids are activated by ester bonds to tRNA molecules. In S. aureus, three di erent enzymes (FemA, FemB, and FemX) assemble the glycine pentapeptide branch rom the appropriately charged tRNAs. FemX, which attaches the f rst glycine, is essential or survival. FemA and FemB (which add the next our glycines) are not essential or survival, but their deletion compromises the viability o the organism by a ecting cross-linking and integrity o the cell wall. Thus, these enzymes are potential targets or antibiotic development. In Gram-negative bacteria, the murein monomers are usually cross-linked to one another directly, without the use o a branching peptide. These steps complete the synthesis o a murein monomer. Be ore the f nal stages o cell wall synthesis can take place, the murine monomer must be trans erred rom the inner surace o the cytoplasmic membrane to the outer sur ace. How this is accomplished is an active area o investigation and may be a potential target or new antimicrobial agents.

Glycan Polymerization

Murein monomers on the external sur ace o the cytoplasmic membrane undergo polymerization to make long glycan chains through several rounds o glycosylation. Polymerization is catalyzed by enzymes called peptidoglycan glycosyltransferases (PGTs, or ormerly, transglycosylases). These enzymes catalyze several rounds o elongation by addition o disaccharide subunits to the reducing end o

646 PRINCIPLES OF CHEMOTHERAPY

the growing polymer without releasing it. With each glycosylation reaction, bactoprenol diphosphate is released and returns to the inner sur ace o the cytoplasmic membrane, where it loses a phosphate group to orm bactoprenol phosphate; this step is catalyzed by a dephosphorylase . Bactoprenol phosphate is now ready to accept another Park nucleotide (Fig. 35-2B). The PGTs are o ten ound as N-terminal catalytic domains in bi unctional proteins that also have a C-terminal transpeptidation domain; however, they can also be ound as mono unctional PGTs (known as MGTs). Most bacteria have a number o structurally related PGTs, some bi unctional and some mono unctional. Their enzymatic activities are similar in vitro, but they are presumed to play di erent roles in cells. For example, in rod-shaped organisms, some PGTs are dedicated to the synthesis o side wall peptidoglycan, whereas others are dedicated to the synthesis o septal peptidoglycan. Nevertheless, these enzymes can partially substitute or one another, complicating a detailed understanding o their specif c roles. One possible way o understanding this biological complexity is that bacteria have evolved to have multiple overlapping systems to ensure survival should specif c problems arise in an individual machine. This partial redundancy can be a disadvantage rom the standpoint o antibiotic treatment.

Polymer Cross-Linking

In the f nal stage o cell wall synthesis, murein chains are cross-linked to one another by enzymes called transpeptidases (TPs). Because transpeptidases were f rst identif ed as the molecular targets o penicillin, they are also called penicillin-binding proteins (PBPs). The PGT domain couples murein monomers to produce glycan strands. These oligosaccharide chains must then be cross-linked through their stem peptides to produce the murein ound in bacterial cell walls. The transpeptidation reaction takes place in two steps: activation and coupling. In the activation step, a serine hydroxyl in the active site o a TP enzyme attacks the D-AlaD-Ala amide bond o one o the stem peptides on the glycan polymer, orming a covalent enzyme–peptidoglycan intermediate and releasing alanine. In the coupling step, a ree amino group on the terminal amino acid o the interbridge peptide (glycine or many Gram-positive bacteria) or on DAP (Gram-negative bacteria) then attacks this intermediate, producing a new amide bond cross-link between the two stem peptides and regenerating the active enzyme (Figs. 35-2B and 35-3). Penicillin, a -lactam, apparently mimics the terminal D-Ala-D-Ala substrate: it binds in the TP active site, where it then reacts with the serine nucleophile to orm a covalent enzyme–penicillin complex (Fig. 35-3). This modif cation inactivates the enzyme, thereby resulting in lower degrees o cell wall cross-linking; in turn, this compromises the integrity o the cell wall and eventually causes cell lysis (see discussion below). Bacteria typically contain several TPs with di erent but overlapping specif cities. As described above or PGTs, these di erent enzyme iso orms are used to build di erent parts o the wall. Escherichia coli, or example, has six transpeptidases, some o which build the cylindrical middle o this rod-shaped bacteria, and others o which build its hemispherical ends. It is believed that di erences in the number and type o cross-links and glycan chain length give each bacterial species its characteristic shape and size and the cell

wall o each species its characteristic thickness. Consistent with this hypothesis, it has been ound that the complement o transpeptidases di ers rom species to species and especially between rods such as E. coli and C. perfringens and spherical cocci such as streptococci and staphylococci. It is thought that, in some cases, bacteria exploit the presence o multiple TPs to develop antibiotic resistance in the clinic. A major orm o resistance develops in S. aureus when strains acquire a resistant TP that is capable o crosslinking peptidoglycan even when exposed to the -lactam methicillin, which typically inactivates TPs in a manner similar to penicillin (see discussion below). The cell wall produced by methicillin-resistant S. aureus (MRSA) in the presence o drug has lower levels o cross-linking than in the absence o drug, which is presumed to be due to the ine f ciency o the resistant TP. One possible strategy or overcoming MRSA is to urther weaken the cross-linking ability o this resistant TP.

Mycobacterial Cell Wall Synthesis The cell wall structures described above are ound in the vast majority o clinically relevant bacteria, including Grampositive cocci such as streptococci and staphylococci, Gramnegative rods such as E. coli and Pseudomonas aeruginosa, and Gram-positive rods such as C. perfringens. However, a discussion o cell wall structure would not be complete without mentioning the unusual cell envelopes o the Corynebacteriae, a group o bacteria that includes the important pathogens Mycobacterium tuberculosis and Mycobacterium leprae. These bacteria are classif ed as high G C (i.e., a high percentage o guanine and cytosine in their DNA) Gram-positives, but their cell envelopes have characteristics o both Gram-positive and Gram-negative bacteria. Unlike other Gram-positives, the Corynebacteriae have an outer membrane. The NAM sugars in the peptidoglycan layer that surrounds the cytoplasmic (inner) membrane have covalently attached NAG-arabinogalactan polymers to which are attached mycolic acids. The mycolic acids have long alkyl chains containing as many as 90 carbons, and these alkyl chains orm a waxy layer that make the bacteria resistant to acid decolorization (acid- ast). The mycolic acids are essential or the assembly o the outer membrane, but the organizational details are unclear. In addition to mycolic acids, the outer membrane o mycobacteria contains secreted phospholipids called extractable lipids (see Fig. 35-1). Mycobacteria have outer membrane porins, but their structures are di erent rom the structures o the porins ound in Gram-negative bacteria. The synthesis o NAG-arabinogalactan begins with the trans er o a molecule o NAG phosphate rom UDP-NAG to mycobacterial bactoprenol phosphate. Next, a molecule o the sugar rhamnose is added, ollowed by the addition o the several galactose and arabinose units that make up arabinogalactan. Arabinosyl transferase catalyzes the addition o the arabinose units. Mycolic acid is a long, complex, branched atty acid. The starting materials or its synthesis include long, saturated hydrocarbon chains that are synthesized rom two-carbon units carried by acetyl CoA. The enzyme fatty acid synthetase 1 (FAS1) catalyzes the ormation o these saturated hydrocarbon chains, and the enzyme fatty acid synthetase 2 (FAS2) catalyzes the linkage o these chains. The linked product then undergoes several enzymatic trans ormations

C h a p t e r 3 5 Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis

Normal transpeptidation

Two pe ptidoglyca n cha ins

HO O HO

HO O O

O AcHN

O

Penicillin action HO O HO

O

AcHN

AcHN

HO

γ-D-Glu

O

H N

N H

O AcHN

L-Ala

Ser Enzyme

OH

D-Ala -D-Ala

S N

O

COOH

HO Ser

Enzyme

(L-Gly)4 L-Lys

N H

O

O

γ-D-Glu

O H2 N

O

H N

R

O

HN

L-Ala (L-Gly)5 L-Lys

HO O O

O

O

HN

647

D-Ala D-Ala

Gly

Activation step HO O HO

HO O O

O AcHN

Enzyme pe ptidoglyca n inte rme dia te

O

HO O HO

O

AcHN

AcHN

O

HN

HN

L-Ala

γ-D-Glu

O

(L-Gly)5 L-Lys

N H

O

H2 N

Ser

N H

Enzyme

O

H2 N

(L-Gly)4 L-Lys

O

R

AcHN

O O Ser

NH S O HN COOH

Enzyme

D-Ala D-Ala

Gly

O

O

O

L-Ala

γ-D-Glu

+

HO O O

O

“De a d-e nd” e nzyme pe nicillin comple x

OH

Dis pla ce d D-Ala

Coupling step HO O HO

Cros s -linke d pe ptidoglyca n cha ins

O AcHN

HO O O

O

O

AcHN

O

HN

HO O HO

AcHN

HO O O

HN

L-Ala

N H

H N O

γ-D-Glu

O N H

D-Ala -L-Gly cros s -link

O

O

AcHN

O

L-Ala

γ-D-Glu (L-Gly)5 L-Lys

O

(L-Gly)4 L-Lys

+ HO

Ser Enzyme

D-Ala D-Ala

FIGURE 35-3. Transpeptidase action and its inhibition by penicillin. The le t side o the f gure shows the mechanism by which transpeptidases catalyze transpeptidation, a reaction that occurs in bacteria but not in mammalian cells. Top panel: A nucleophilic hydroxyl group in the active site o the transpeptidase (Enzyme) attacks the peptide bond between the two D-Ala residues at the terminus o a pentapeptide moiety on one peptidoglycan chain. Middle panel: The terminal D-alanine residue is displaced rom the peptidoglycan chain, and an enzyme-D-alanine-peptidoglycan intermediate is ormed. This intermediate is then attacked by the amino terminus o a polyglycine pentapeptide linked at its carboxy terminus to L-lysine or diaminopimelic acid on an adjacent peptidoglycan chain (see Fig. 35-2). Bottom panel: As the enzyme is liberated rom the intermediate, a new peptide bond (cross-link) is ormed between the terminal glycine residue on one peptidoglycan chain and the enzyme-activated D-alanine residue on the adjacent peptidoglycan chain. The ree enzyme can then catalyze another transpeptidation reaction. The right side o the f gure shows the mechanism by which penicillin inter eres with transpeptidation, leading to the ormation o a penicilloyl-enzyme “dead-end” complex. In this orm, the enzyme is incapable o catalyzing urther transpeptidation (cross-linking) reactions.

to become mycolic acid. Mycolic acid is eventually added to NAG-arabinogalactan, which, in turn, is attached to NAM to organize and form a major component of the mycobacterial outer membrane (Figs. 35-1 and 35-4). In principle, any step in this process is susceptible to pharmacologic intervention. As discussed below, standard antimycobacterial treatment regimens include antibiotics that target both the synthesis of NAG-arabinogalactan and the early reactions of mycolic acid synthesis.

The mycobacterial cell envelope is thick, asymmetric, and highly impermeable to both hydrophilic and hydrophobic substances. M. tuberculosis is among the most challenging pathogens to eradicate because (1) its cell envelope resists entry of many antibiotics and (2) the organism grows very slowly; note that cellwall-active antibiotics are typically most effective against bacteria that are actively growing and making new cell wall rapidly. Special treatment regimens, involving long-term therapy with combinations of antibiotics, are required to cure tuberculosis.

648 PRINCIPLES OF CHEMOTHERAPY

O

O N

S CoA

NH 2

Ace tyl CoA

N Pyrazinamide

FAS1

O

O N H

NH2

R

N

OH

Fa tty a cids Isoniazid

FAS2

Mycolic a cids

P hos pholipids

stem peptide rom murein during cell division to promote daughter cell separation. Loss o these three amidases causes a noticeable de ect in cell division, while loss o one usually has little or no e ect. New murein synthesis and autolysin-mediated murein destruction must be care ully balanced or bacteria to survive. Indeed, studies have shown that unilaterally blocking murein synthesis (e.g., by drugs like penicillin) results in autolysin-mediated autolysis and cell death. The molecular events that initiate autolysis are poorly understood. The current belie is that specif c proteins recruit the degradative machinery only a ter the cell has assembled the machinery responsible or cell wall synthesis. This ordered recruitment ensures that degradation does not occur unless new cell wall is being made. The bactericidal effect o cephalexin, a f rstgeneration cephalosporin, has been shown to involve targeting o the synthetic machinery by specif cally inhibiting the transpeptidation step o cell wall synthesis and subverting this regulatory mechanism (see discussion below). Cephalexin does not perturb the normal assembly o the synthetic machinery but instead simply inactivates this complex by inhibiting transpeptidase enzymes. Apparently, the regulatory mechanisms o the cell can determine only that the machinery or new cell wall synthesis is present and not whether it is unctional. As a result, the cell recruits the degradative machinery without ongoing synthesis, and lysis ensues. Many o the -lactams discussed in this chapter inter ere with the balance between cell wall synthesis and degradation.

PHARMACOLOGIC CLASSES AND AGENTS FIGURE 35-4. Mycolic acid biosynthesis and antimycobacterial drug action. Mycolic acids are produced by the cross-linking o atty acid chains derived rom acetyl coenzyme A (Acetyl CoA). Each o the arrows in this simplif ed representation denotes multiple synthetic steps; the ocus is on the atty acid synthetases (FAS1 and FAS2) because o their importance as drug targets. Specif cally, FAS1 is inhibited by pyrazinamide, and FAS2 is inhibited by isoniazid.

Autolysins and Cell Wall Degradation Although the cell wall provides stability, it is a dynamic structure; it is continuously modif ed by synthetic and degradative enzymes that are f nely tuned to allow the sacculus to grow and divide without lysing. For bacteria to grow, bacterial cell walls must expand; or expansion to occur, new murein units must be incorporated into the existing cell wall. This is di f cult to accomplish in a “f nished” cell wall, which is composed o specif c lengths o glycan polymers with particular degrees o cross-linked stem peptides. In addition, or a bacterium to divide, its cell wall must at some point be broken to allow two daughter cells to separate. Bacteria address these issues by using highly regulated autolysins . These enzymes punch small holes in the cell wall that allow or remodeling and expansion. Di erent autolysins exhibit pre erences or di erent bonds in murein. Similar to the synthetic enzymes, many are unctionally redundant but play necessary roles in the cell. For example, in E. coli, three autolysins called NAM-L-alanine amidases cleave the

The pharmacology o the drug classes that inhibit bacterial cell wall synthesis is discussed in the same order as the biochemistry o cell wall synthesis (Fig. 35-2). Although drugs have been identif ed that inhibit a number o steps in the biochemistry o cell wall synthesis, the polymer crosslinking (transpeptidation) step is, by ar, the most clinically important biochemical target. For this reason, most o the discussion ocuses on the panoply o agents that inhibit the cross-linking o peptidoglycan polymers.

Inhibitors of Murein Monomer Synthesis Fosfomycin

Fosfomycin (also written phosphomycin) is a phosphoenol-

pyruvate (PEP) analogue that inhibits bacterial enolpyruvate trans erase (also known as MurA) by covalent modif cation o the enzyme’s active site. Given that PEP is a key intermediate in (mammalian) glycolysis, it may come as a surprise that this agent does not inter ere with carbohydrate metabolism in human cells; this selectivity o antibacterial action likely results rom structural di erences between the mammalian and bacterial enzymes that act on PEP. Thus, os omycin has no appreciable e ect on human enolase, pyruvate kinase, or carboxykinase, and the drug is relatively nontoxic. Fos omycin enters the cell via transporters or glycerophosphate or glucose-6-phosphate that are normally used by bacteria to take up these nutrients rom the environment. Fos omycin is especially e ective against Gram-negative bacteria that in ect the urinary tract, including E. coli, because it is excreted unchanged in the urine. A single 3-g dose

C h a p t e r 3 5 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

o oral os omycin tromethamine has been shown to be as e ective as multiple doses o other agents in the treatment o uncomplicated urinary tract in ections. As a rule, os omycin is less e ective against Gram-positive bacteria because these bacteria generally lack selective glycerophosphate and glucose-6-phosphate transporters, although it is o ten active against Enterococcus aecalis. Resistance to os omycin is typically caused by mutations in these transporters; in addition, a temperature-sensitive E. coli strain has been ound in which a mutation in enolpyruvate trans erase results in reduced a f nity o the enzyme or both PEP and os omycin. Adverse e ects o os omycin are uncommon; between 1% and 10% o patients develop headache, diarrhea, or nausea. Signif cant drug interactions are also rare. The absorption o oral os omycin can be decreased by co-administration with promotility agents such as metoclopramide, and probenecid can reduce renal clearance o the drug. Intravenous disodium os omycin (which has not been approved by the US Food and Drug Administration [FDA] or use in the United States) has been shown to have antibacterial synergy in vitro with -lactams, aminoglycosides, and uoroquinolones.

Cycloserine

Cycloserine , a structural analogue o D-Ala, is a second-

line agent used to treat multidrug-resistant M. tuberculosis in ection (Fig. 35-5). Cycloserine inhibits both the alanine racemase that converts L-Ala to D-Ala and the D-Ala-D-Ala ligase that joins together two D-Ala molecules (Fig. 35-2A). Cycloserine is an irreversible inhibitor o these enzymes and, in act, binds these enzymes more tightly than does their natural substrate, D-Ala. Resistance to cycloserine occurs by multiple mechanisms, some o which are still unknown; known mechanisms include overexpression o alanine racemase and mutations in the alanine uptake system. As with many small molecules, including os omycin, cycloserine is excreted in the urine. Adverse e ects include seizures, psychosis, and neurological syndromes such as peripheral neuropathy. Patients with neuropsychiatric disease, alcoholism, and chronic kidney disease should avoid the drug. Alcohol, isoniazid, and ethionamide potentiate its toxicity; pyridoxine may mitigate cycloserine-induced peripheral neuropathy. Cycloserine inhibits the hepatic metabolism o phenytoin.

Bacitracin

So named because it was f rst identif ed in a species o Bacillus, bacitracin is a peptide antibiotic that inter eres with the dephosphorylation o bactoprenol diphosphate, rendering the bactoprenol lipid carrier useless or urther rounds o murein monomer synthesis and export (Fig. 35-2B). Bacitracin is

OH

OH

H2 N N

H2 N

O

O D-Cycloserine

D-Ala nine

FIGURE 35-5. Structure of cycloserine. Cycloserine is a structural analogue of D-alanine that inhibits the racemic interconversion of L-alanine to D-alanine by alanine racemase. Cycloserine also inhibits the activity of D-Ala-D-Ala ligase B, the enzyme that catalyzes the formation of the D-Ala-D-Ala dipeptide that is subsequently utilized in the synthesis of murein monomers (see Fig. 35-2A).

649

there ore notable among the cell wall synthesis inhibitors or having a lipid, rather than a protein or peptide, as its target. Bacitracin inhibits dephosphorylation by orming a complex with bactoprenol diphosphate that involves the drug’s imidazole and thiazoline rings. This interaction requires a divalent metal ion, usually Zn2 or Mg2 ; hence, drugs that act as metal chelators could inter ere with the activity o bacitracin. Due to its signif cant kidney, neurological, and bone marrow toxicity, bacitracin is not used systemically. It is most commonly used topically or superf cial dermal or ophthalmologic in ections. Because bacitracin is not absorbed when administered orally, the antibiotic remains within the gut lumen and is thus used occasionally or gut decontamination prior to colorectal surgery.

Inhibitors of Murein Polymerization Vancomycin, Telavancin, Dalbavancin, and Oritavancin Vancomycin, the agent used to treat C. di f cile in ection in

the introductory case, is a glycopeptide with bactericidal activity against Gram-positive rods (such as Clostridia) and Gram-positive cocci. Telavancin, dalbavancin, and oritavancin are related lipoglycopeptides with a similar spectrum o action. Gram-negative rods are resistant to the action o these drugs. These agents interrupt cell wall synthesis by binding tightly to the D-Ala-D-Ala terminus o the murein monomer unit, inhibiting peptidoglycan polymerization and thereby blocking the addition o murein units to the growing polymer chain. Telavancin and oritavancin have, in addition to the D-Ala-D-Ala-binding moiety, a lipid side chain that interacts with the bacterial cell membrane; this lipid anchor both enhances drug binding to the D-Ala-D-Ala terminus and e ects depolarization o the bacterial membrane, resulting in higher antibacterial potency than vancomycin. Dalbavancin is synthesized rom a natural glycopeptide. The amidation o the carboxyl group o the peptide provides enhanced activity against staphylococci, including coagulase-negative staphylococci (CoNS). In addition, the lack o an acetylglucosamine group in dalbavancin allows or increased activity against resistant enterococci. Oritavancin is also derived rom a naturally occurring glycopeptide. The addition o an N-alkyl-p-chlorophenylbenzyl substituent on the disaccharide sugar results in increased activity against enterococci, including vancomycin-resistant strains. Dalbavancin and oritavancin have prolonged elimination hal lives, allowing or once-weekly dosing. Intravenous vancomycin is most commonly used to treat serious in ections such as pneumonia, bacteremia, and endocarditis caused by methicillin-resistant Staphylococcus aureus (MRSA) (see discussion below). Intravenous telavancin is used to treat serious skin in ections and pneumonias caused by susceptible strains o staphylococci (including MRSA) and streptococci. Dalbavancin and oritavancin are used to treat serious skin and so t tissue in ections caused by susceptible stains o staphylococci (including MRSA) and streptococci; both drugs also have utility in treating enterococcal in ections. Oral vancomycin is commonly used to treat gastrointestinal in ections caused by C. di f cile; when administered orally, it is poorly absorbed and there ore remains within the gastrointestinal tract. The adverse e ects o vancomycin include skin ushing or rash—the so-called red man syndrome —which is due to release o histamine and can be avoided by decreasing the rate o intravenous in usion or preadministering antihistamines.

650 PRINCIPLES OF CHEMOTHERAPY

Vancomycin has also been associated with nephrotoxicity and rarely ototoxicity, particularly when other nephrotoxic or ototoxic medications such as gentamicin are co-administered. Patients with underlying renal dys unction need adjusted dosing and measurement o drug levels in order to mitigate nephrotoxicity. Drug ever, hypersensitivity rash, and drug-induced neutropenia can also occur. Telavancin appears to have a toxicity prof le similar to that o vancomycin, with a slightly lower risk o in usion-related reactions but a higher incidence o nephrotoxicity. Dalbavancin does not appear to cause inusion-related reactions or nephrotoxicity; the most common adverse event in clinical trials was mild gastrointestinal upset. Oritavancin has similar toxicities to vancomycin except or a much lower risk o renal toxicity. Telavancin and oritavancin can bind to artif cial phospholipid sur aces in some commercially available anticoagulation assays, resulting in alsely prolonged prothrombin, activated partial thromboplastin, and activated clotting times (PT, aPTT, and ACT, respectively). This also results in a alsely elevated international normalized ratio (INR). In order to minimize this inter erence, blood drawn or these assays should be taken near the time o the trough plasma concentration o telavancin or oritavancin. Resistance to vancomycin, telavancin, dalbavancin, and oritavancin most commonly arises through the acquisition o DNA encoding enzymes that catalyze the ormation o D-Ala-D-lactate instead o D-Ala-D-Ala. As with D-AlaD-Ala, D-Ala-D-lactate is incorporated into the murein monomer unit and participates readily in the transpeptidase reaction, but the D-Ala-D-lactate dipeptide is not bound by vancomycin and related glycopeptides. Two enzymes mediate the synthesis o D-Ala-D-lactate: VanH, a dehydrogenase that generates D-lactate rom pyruvate, and VanA, a ligase that links D-Ala to D-lactate. VanH and VanA are encoded on a transposable element that can be ound on either the bacterial chromosome or an extrachromosomal plasmid. This element also encodes enzymes that degrade D-Ala-DAla, thereby removing any residual targets o vancomycin. In clinical practice, bacteria resistant to vancomycin (such as vancomycin-resistant enterococci [VRE]) are o ten resistant to most other antibacterials; plasmid-mediated spread o vancomycin resistance is there ore a serious medical problem. A ew cases o vancomycin-resistant S. aureus (VRSA) due to acquisition o enterococcal resistance genes have been reported. Vancomycin-intermediate S. aureus (VISA) has also been described; these organisms have a thicker murein layer in which increased amounts o ree D-Ala-D-Ala act as a decoy target or vancomycin. Dalbavancin and oritavancin are more stable than vancomycin and telavancin against the development o resistance to Staphylococcus and Streptococcus species. Oritavancin may be active against Enterococcus strains that exhibit resistance to the other glycopeptides.

Inhibitors of Polymer Cross-Linking -Lactam Antibiotics: General Considerations

With more than 30 di erent agents currently in use, including the original penicillin and the dicloxacillin used in the introductory case, the -lactams are the largest and most widely prescribed class o antibiotics that inhibit bacterial cell wall synthesis. The various agents in this class di er in chemical structure (Fig. 35-6) and consequently in spectrum o action, but all -lactams share the same antibiotic mechanism o action: inhibition of murein polymer cross-linking.

A

B H N

R O

S N

O

H N

R O

S N

O

COOH

COOH

Penicillins

H N

R1 O

β-la cta ma s e s cle a ve this bond

S N

O

R2

N

O

COOH

COOH

Clavulanic acid

Cephalosporins

O

H N

R O

OH

O

S N

O

S O3H

N O

COOH

Monobactams

Sulbactam

OH

O

O

O

SR

N

COOH Carbapenems

H2 N

N N O

OS O 3 H Avibactam

FIGURE 35-6. Structural features of -lactam antibiotics and -lactamase inhibitors. A. The -lactam family members (penicillins, cephalosporins, monobactams, and carbapenems) differ from one another in their backbone structures; individual drugs within these subclasses also differ in their R groups. Note the four-membered -lactam ring that is common to all four families (blue boxes); it is this ring that gives the agents their ability to block the transpeptidation reaction (and also their name). B. Bacteria expressing -lactamases are able to cleave the -lactam bond (blue line) that is required for antibiotic action. The -lactamase inhibitors clavulanic acid, sulbactam, and avibactam act as decoys by binding to (and thereby inhibiting) -lactamase enzymes. Note the structural similarity between the -lactamase inhibitors and the -lactam antibiotics.

Chemically, the key to this mechanism o action is the presence o a our-membered -lactam ring (Fig. 35-6). This ring makes every -lactam a structural analogue o the terminal D-Ala-D-Ala dipeptide o the Park nucleotide and hence a substrate or one or more bacterial transpeptidases. As with the Park nucleotide, the -lactam reacts covalently with the active-site serine in the transpeptidase, thereby orming an acyl enzyme intermediate. Unlike the Park nucleotide in the normal substrate reaction, however, the -lactam ring renders the carboxy terminal end o the -lactam unable to be cleaved rom the rest o the molecule. As a result, the incoming amino terminal end o the adjacent peptide cannot attack the acyl enzyme intermediate, and the transpeptidase reaches a “dead-end” complex (Fig. 35-3). (This mode o irreversible enzyme inhibition is sometimes called suicide substrate inhibition.) Provided that the cells are growing, transpeptidase inhibition results in autolysin-mediated autolysis and cell death. Hence, as a rule, -lactams are bactericidal or actively dividing bacteria.

C h a p t e r 3 5 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

The various subclasses o -lactam agents all into our amilies—the penicillins , the cephalosporins (which are urther subdivided into f ve “generations”), the monobactams , and the carbapenems . Each o these subclasses di ers structurally in the chemical substituents that are attached to the -lactam ring (see Fig. 35-6). In general, the development o these amilies resulted rom pharmacologists’ e orts in the laboratory to improve on penicillin’s antibiotic spectrum of action and to stay ahead o the spread o antibiotic resistance . Recall that spectrum o action re ers to the number and variety o bacterial species against which an antibiotic shows bactericidal or bacteriostatic activity. Hence, broad-spectrum -lactams are typically active against Gram-negative as well as Gram-positive bacteria, whereas narrow-spectrum lactams are typically e ective only against Gram-positive organisms. Bacterial transpeptidases are located in the periplasmic space between the cytoplasmic membrane and the cell wall. Hence, to exert their e ects, -lactams must traverse the cell wall and, in the case o Gram-negative bacteria, the outer membrane as well. A -lactam’s spectrum o action is determined by two actors: its ability to penetrate the outer membrane and cell wall and, once in the periplasmic space, its ability to inhibit specif c transpeptidases. Both hydrophilic and (to a lesser extent) hydrophobic agents di use through the thick murein layer o Gram-positive bacteria, but hydrophilic agents pass through the outer membrane pores o Gram-negative bacteria much more readily than do hydrophobic agents. As a result, hydrophilic agents such as ampicillin, amoxicillin, and, especially, piperacillin and ticarcillin tend to have broad spectra o action, whereas hydrophobic agents such as oxacillin, cloxacillin, dicloxacillin, nafcillin, methicillin, and penicillin G tend to have narrow spectra o action (see discussion below or details). This means that some Gram-negative bacteria are inherently resistant to narrow-spectrum -lactams simply by virtue o the permeability barrier presented by their outer membrane. (Similarly, intracellular bacteria , that is, bacteria that live within human cells, such as Chlamydia, are, in general, also inherently resistant to -lactams, both because mammalian cells tend to lack -lactam uptake mechanisms and because these bacteria tend either to have unique cell wall architectures or to lack cell walls altogether.) The second actor that determines a -lactam’s spectrum o action is the extent to which the drug, a ter accessing the periplasmic space, inhibits a particular transpeptidase. In large part, this is determined by the -lactam’s a f nity or the transpeptidase. As noted above, bacteria typically have several transpeptidase enzymes that di er subtly in their substrate specif city and cross-linking activity; these di erences are especially prominent between rods and cocci. Most lactams have selectivity or several di erent transpeptidases; others, such as the penicillin analogue methicillin that was previously used against S. aureus, are specif c or just one. Antibiotic resistance can be encoded by either chromosomal (intrinsic ) or acquired (extrinsic ) genes. For -lactams, chromosomal resistance in Gram-positive bacteria is most commonly con erred by a chromosomally encoded mutation in a transpeptidase-encoding gene that abolishes the transpeptidase’s ability to bind a particular -lactam or by acquisition o a gene encoding a transpeptidase with low a f nity or the -lactam. This mechanism is the cause o resistance to methicillin in S. aureus, as mentioned above, and

651

the mechanism by which pneumococci acquire resistance to penicillin. However, resistance to -lactams by genetically altered transpeptidases is the exception, not the rule, because most -lactams are active against multiple transpeptidases that would all need to be mutated in order to abolish the drugs’ e ectiveness. Most resistance to -lactams is con erred by proteins called -lactamases that are encoded on the chromosome or on extrachromosomal DNA plasmids . As their name implies, -lactamases are enzymes that inactivate -lactams via (hydrolytic) cleavage o the -lactam ring. More than 100 di erent -lactamases have been identif ed, each with activity against a particular -lactam or set o -lactams. -Lactamases are secreted in Gram-positive bacteria; in Gram-negative bacteria, these enzymes are retained in the periplasmic space between the cell wall and outer membrane. Gram-negative bacteria produce much less -lactamase than Gram-positive bacteria do, but because the Gram-negatives concentrate the -lactamase where it is needed in the periplasmic space, the -lactamase is more e ective at con erring resistance. This concentration e ect, coupled with the strong permeability barrier to penicillins a orded by the bacterial outer membrane, makes Gram-negative bacteria largely re ractory to penicillin therapy. That many -lactamases are encoded on plasmids is o special clinical importance. Because plasmids are easily trans erred by conjugation rom one bacterium to another, the resistance con erred by the plasmid can sweep rapidly through a bacterial population. Moreover, plasmids can “jump strains,” spreading resistance rom one strain to another. Organisms such as Klebsiella pneumoniae and E. coli may also produce extended-spectrum -lactamases (ESBLs) and carbapenemases that render them resistant to most -lactam antibiotics, including penicillins, cephalosporins, the monobactam aztreonam, and the carbapenems. Other bacteria, such as Enterobacter species, may overexpress a chromosomally encoded -lactamase that produces similarly broad resistance to -lactams. Pharmacologists have responded to the challenge o -lactamases in two ways. First, as noted above, new amilies o -lactams have been developed with structures that make them less susceptible to cleavage by existing -lactamases. Second, -lactamase inhibitors have been developed that can be co-administered with -lactam antibiotics. -Lactamase inhibitors are -lactam-like molecules that bind to the active site o -lactamases and thereby prevent the -lactamases rom destroying the -lactam antibiotics with which the lactamase inhibitors are co-administered. Four examples o -lactamase inhibitors are clavulanic acid (clavulanate ), sulbactam, tazobactam, and avibactam (Fig. 35-6). Avibactam is a novel -lactamase inhibitor recently developed or use in combination with ce tazidime to provide enhanced activity against resistant Gram-negative bacteria. -Lactams act synergistically with aminoglycosides , the bactericidal inhibitors o protein synthesis discussed in Chapter 34. (For more on synergy, see Chapter 41, Principles o Combination Chemotherapy.) Aminoglycosides inhibit protein synthesis by binding to the 30S ribosomal subunit in the cytoplasm o the cell. To access the cytoplasm, aminoglycosides must di use passively across the cell wall be ore being transported actively across the cytoplasmic membrane. It is thought that the cell walls o some bacteria, such as enterococci, are poorly permeable to aminoglycosides when these drugs are

652 PRINCIPLES OF CHEMOTHERAPY

Antibody N β-la cta m a ntibiotic NH2

Modifie d huma n prote in (a ntige nic)

Huma n prote in (non-a ntige nic)

FIGURE 35-7.

-Lactam toxicity. In the absence o modif cation, human proteins are generally nonantigenic. -Lactams can modi y amino groups on human proteins, creating an immunogenic -lactam hapten. This new antigenic determinant can be recognized as “nonsel ” by antibodies o the host immune system.

administered as single agents. Because -lactams increase cell wall permeability, co-administration o a -lactam acilitates the uptake o an aminoglycoside and thus enhances its e ect. Hypersensitivity reactions are the most common adverse e ects o -lactams. As small molecules, -lactams would not be expected to stimulate immune responses by themselves, and indeed they do not. However, -lactam rings can react with amino groups on human proteins to create a hapten– carrier complex (Fig. 35-7). The -lactam–protein conjugate can then provoke a hypersensitivity response. The most serious o these reactions is anaphylaxis , which typically occurs within an hour o drug administration and leads to bronchospasm, angioedema, and/or cardiovascular collapse. Urticaria, morbilli orm drug rash, serum sickness, and drug ever may also occur. Proteins on the sur ace o red blood cells can also be modif ed by penicillin, leading to drug-induced autoimmune hemolytic anemia. Rarely, -lactam antibiotics cause drug-induced lupus. For most individuals, this process is strongly dose dependent: the likelihood o a hypersensitivity reaction increases with each administration o a -lactam. -Lactams o a given class o ten cross-react with each other, but -lactams o one class are less o ten cross-reactive with -lactams o another class. Patients with a penicillin allergy should not receive ampicillin or other penicillins due to the high risk o cross-reactivity. Patients with a penicillin allergy other than serum sickness or anaphylaxis generally may receive a cephalosporin or a carbapenem. Aztreonam (a monobactam) is unique in that it has no cross-reactivity with either penicillins or carbapenems; however, cross-reactivity between aztreonam and ce tazidime (a cephalosporin), presumably due to a shared side chain, has been reported. Although allergic reactions to carbapenems can occur in patients with penicillin allergy, they are rare.

-Lactam Antibiotics: Specif c Agents Penicillins

As noted above, there are our structurally distinct subclasses o -lactam antibiotics (see Fig. 35-6A). The f rst o these subclasses, the penicillins, can be urther divided into f ve groups according to their spectra o action.

The f rst group o penicillins includes penicillin G, which is intravenously administered, and penicillin V, its gastric acid-stable oral counterpart. Penicillin V is used to treat dental in ections and to prevent recurrent rheumatic ever in patients with a prior episode and recurrent streptococcal cellulitis in patients with lymphedema. Penicillin G is used to treat serious in ections with Gram-positive bacteria such as pneumococcus and S. pyogenes (some strains o each), Gram-negative diplococci such as Neisseria species (except penicillinase-producing N. gonorrhoeae), Gram-positive rods o the genera Clostridium and Actinomyces, and spirochetes such as syphilis and Leptospira. High-dose penicillin G may cause seizures, in addition to the already mentioned hypersensitivity reactions and rash. All penicillins can cause acute interstitial nephritis. Drug–drug interactions are rare, but the anticoagulant e ects o war arin may be potentiated by concomitant penicillin administration. The second group consists o the antistaphylococcal penicillins , including oxacillin, cloxacillin, dicloxacillin, nafcillin, and methicillin. These drugs are structurally resistant to staphylococcal -lactamase, which is encoded by plasmid genes in most clinical isolates. Because o their relative hydrophobicity, however, antistaphylococcal penicillins lack activity against Gram-negative bacteria. (Recall also that methicillin binds to only a single transpeptidase.) Thus, oral antistaphylococcal penicillins are used mostly or skin and so t tissue in ections. Intravenous antistaphylococcal penicillins are predominantly used or more serious methicillin-sensitive S. aureus skin and so t tissue in ections or other serious in ections, such as bacteremia, endocarditis, or osteomyelitis. Use o the oral antistaphylococcal penicillins (cloxacillin and dicloxacillin) is somewhat limited by their gastrointestinal adverse e ects (nausea and antibioticassociated diarrhea) and occasionally by secondary development o C. di f cile in ection. Adverse e ects o intravenous na cillin include phlebitis at the injection site; agranulocytosis and acute interstitial nephritis occur at a higher rate than with the other penicillins. Na cillin is an inducer o hepatic isoenzyme CYP3A4 and can decrease the plasma levels o drugs that are substrates or CYP3A4. Oxacillin can cause hepatotoxicity, which is reversible with discontinuation o the drug. The utility o antistaphylococcal penicillins in treating S. aureus has been compromised by the emergence o MRSA strains. Patients with MRSA in ection are typically treated with vancomycin or with non-cell wall-active agents such as trimethoprim-sul amethoxazole, doxycycline, linezolid, or tedizolid. Ampicillin and amoxicillin are members o the third group o penicillins, the amino penicillins , which have a positively charged amino group on the R side chain (see Fig. 35-6A). This positive charge enhances di usion through porin channels but does not con er resistance to -lactamases. These agents are e ective against a variety o Gram-positive cocci, Gram-negative cocci such as Neisseria gonorrhoeae and N. meningitidis, and Gram-negative rods such as E. coli and Haemophilus in uenzae, but their spectrum is limited by sensitivity to most -lactamases. Intravenous ampicillin is used most commonly to treat invasive enterococcal in ections and Listeria meningitis; oral amoxicillin is used to treat uncomplicated ear, nose, and throat in ections, to prevent endocarditis in high-risk patients undergoing dental work, and as a component o combination therapy or Helicobacter pylori in ection. Rash is the most common adverse e ect.

C h a p t e r 3 5 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

The spectrum o both agents is broadened when they are co-administered with -lactamase inhibitors such as clavulanic acid (with amoxicillin) or sulbactam (with ampicillin) to treat -lactamase-producing organisms such as S. aureus, H. inf uenzae, E. coli, Klebsiella, and anaerobes. Sulbactam itsel has activity against Acinetobacter. Agents in the ourth group o penicillins, the carboxy penicillins , are also broad in spectrum. The carboxyl group on the R side chain provides a negative charge that con ers resistance to some -lactamases but is less e ective than a positively charged amino group in acilitating di usion through porin channels. To overcome this limitation in di usion, high doses are used. Resistance to the chromosomally encoded -lactamases o Enterobacter and Pseudomonas adds these organisms to the spectrum o the carboxy penicillins. This group includes ticarcillin. A f th group, the ureido penicillins , is represented by piperacillin. This drug has both positive and negative charges on the R side chain and is generally more potent than the carboxy penicillins. Its spectrum o action is similar to that o the carboxy penicillins; in addition, ureido penicillins have activity against Klebsiella and enterococci. Piperacillin is currently available only in combination with the -lactamase inhibitor tazobactam. Cephalosporins

Cephalosporins di er structurally rom penicillins by having a six-membered rather than a f ve-membered accessory ring attached to the -lactam ring (Fig. 35-6A). First-generation cephalosporins (cefazolin, cephalexin, and cefadroxil) are active against Gram-positive species as well as the Gram-negative rods Proteus mirabilis and E. coli, both o which cause urinary tract in ections, and Klebsiella pneumoniae, which causes pneumonia in addition to urinary tract in ections. These agents are sensitive to many -lactamases but are not degraded by the chromosomally encoded -lactamase o K. pneumoniae and the common staphylococcal -lactamase. Cephalexin and ce adroxil are both administered orally and are used to treat skin and so t tissue in ections and streptococcal pharyngitis. The intravenous agent ce azolin is also used to treat serious skin and so t tissue in ections and is used or surgical prophylaxis and treatment o serious in ections due to MSSA in individuals unable to take an antistaphylococcal penicillin. Second-generation cephalosporins can be divided into two groups. Cefuroxime , which represents the f rst group, has increased activity against H. inf uenzae compared to the f rst-generation cephalosporins; cefotetan and cefoxitin, which represent the second group, demonstrate increased activity against Bacteroides. In general, second-generation cephalosporins are resistant to more -lactamases than are f rst-generation cephalosporins. Thus, ce uroxime can be used to treat community-acquired pneumonia, and ce otetan is used to treat intra-abdominal and pelvic in ections, including pelvic in ammatory disease. Adverse e ects o these agents include diarrhea, mild liver enzyme elevation, and hypersensitivity reactions; rarely, agranulocytosis or interstitial nephritis can occur. Third-generation cephalosporins (ceftriaxone , cefotaxime , and cefpodoxime ) are resistant to many -lactamases and are thus highly active against Enterobacteriaceae (E. coli, indole-positive Proteus, Klebsiella, Enterobacter, Serratia, and Citrobacter) as well as Neisseria and H. inf uenzae.

653

The third-generation cephalosporins are less active against Gram-positive organisms than are the f rst-generation drugs; despite that, they have good activity against penicillin-intermediate S. pneumoniae (although cephalosporin resistance can occur). Common uses include treatment o pneumonia, community-acquired meningitis due to S. pneumoniae, uncomplicated gonococcal in ection, culture-negative endocarditis, and Lyme disease involving the central nervous system or joints. In addition to the adverse e ects already mentioned, ce triaxone can cause cholestatic hepatitis, albeit uncommonly. Ceftazidime is another commonly used third-generation cephalosporin; its spectrum di ers rom the other agents in that it has signif cant activity against Pseudomonas aeruginosa and minimal activity against Gram-positive organisms. It is used predominantly to treat hospital-acquired Gram-negative bacterial in ections and documented in ections with P. aeruginosa and as empiric therapy or neutropenic patients with ever. Gram-negative bacteria that have acquired extended-spectrum -lactamase activity are resistant to third-generation cephalosporins. Cefepime is the only currently available ourth-generation cephalosporin. Like ce triaxone, it is highly active against Enterobacteriaceae, Neisseria, H. inf uenzae, and Grampositive organisms; additionally, it is as active as ce tazidime against P. aeruginosa. Ce epime is also more resistant to the chromosomally encoded -lactamases o Enterobacter than are third-generation cephalosporins. Unlike ce tazidime, however, ce epime is not approved or treatment o meningitis. An uncommon adverse e ect is the development o autoantibodies against red blood cell antigens, typically without signif cant hemolysis. In addition, ce epime can in requently cause neurotoxicity including myoclonus and encephalopathy, particularly in the elderly and in patients with impaired renal unction; or the latter patients, the antibiotic requires dose adjustment. Ceftaroline is a f th-generation cephalosporin. This drug is distinct in having antimicrobial activity against multidrug-resistant S. aureus—including methicillin-resistant, vancomycinintermediate S. aureus and vancomycin-resistant strains—as well as S. pneumoniae and respiratory Gram-negative pathogens such as Moraxella catarrhalis and H. inf uenzae, including -lactamase-expressing strains. Ce taroline is only available intravenously and is approved or treatment o community-acquired pneumonia and skin in ections. Ceftolozane is a novel intravenous cephalosporin currently available in combination with the -lactamase inhibitor tazobactam. It is FDA approved or the treatment o complicated urinary tract in ections and complicated intraabdominal in ections. Ce tolozane is structurally similar to ce tazidime but has a more heavily substituted pyrazole at the 3-position side chain that con ers greater stability against -lactamase-producing Pseudomonas aeruginosa compared to ce tazidime. It has antimicrobial activity against many Gram-negative aerobes, -lactamase-producing Enterobacteriaceae, and Pseudomonas aeruginosa. It lacks activity against carbapenemase-producing Klebsiella pneumoniae. While it is active against most streptococci, it has limited activity against staphylococci. The addition o tazobactam broadens ce tolozane’s activity against various anaerobes, such as Bacteroides species and Prevotella species, but not Clostridium species. As noted above, cephalosporins can generally be used in patients with non-li e-threatening allergic reactions to

654 PRINCIPLES OF CHEMOTHERAPY

penicillins. Nevertheless, cephalosporins can cause hypersensitivity reactions themselves and should be used with caution in patients with known cephalosporin hypersensitivity. Interestingly, ce otetan and ce operazone (another thirdgeneration cephalosporin) contain an N-methylthiotetrazole (NMTT) side chain that causes two unique adverse e ects. The f rst is an alcohol intolerance syndrome known as the disulf ram-like reaction (disulf ram is a drug that inhibits alcohol metabolism; see Chapter 19, Pharmacology o Drugs o Abuse). The second involves an e ect on vitamin K metabolism that results in decreased synthesis o vitamin K-dependent coagulation actors; thus, ce otetan and ce operazone should be used with caution in patients taking war arin and in patients with underlying coagulation abnormalities (see Chapter 23, Pharmacology o Hemostasis and Thrombosis). Ce otetan, like most o the cephalosporins, can also cause antibody-mediated hemolysis. Monobactams and Carbapenems

The only available monobactam, aztreonam, is active against most Gram-negative bacteria, including P. aeruginosa, but it has no activity against Gram-positive organisms. Aztreonam is particularly use ul in patients with serious penicillin allergy who have in ections due to resistant Gram-negative organisms because o its lack o cross-allergenicity with penicillins; however, Gram-negative bacteria with extendedspectrum -lactamases are resistant to the drug. It is available intravenously or treatment o systemic in ections and in an inhaled orm or prevention o pulmonary exacerbations in patients with cystic f brosis, who are commonly colonized with P. aeruginosa. Four carbapenems are currently used in clinical practice: imipenem, meropenem, doripenem, and ertapenem. All our are broadly active against most Gram-positive, Gramnegative, and anaerobic organisms. None is active against MRSA, VRE, or Legionella; and Gram-negative bacteria with carbapenemases (especially K. pneumoniae) exhibit resistance to these drugs. Importantly, ertapenem is much less active against P. aeruginosa and Acinetobacter than the other three agents; the benef t o ertapenem is its once-daily dosing. Because imipenem is inactivated by the human renal enzyme dehydropeptidase I, this drug is co-administered with the dehydropeptidase inhibitor cilastatin. Neither meropenem, doripenem, nor ertapenem is inactivated by the renal enzyme. All our agents are typically administered intravenously, but ertapenem and imipenem can also be administered intramuscularly. No oral carbapenems are currently available. All carbapenems can cause hypersensitivity reactions and IV site phlebitis; at high plasma drug levels, imipenem and meropenem can cause seizures. Probenecid can increase meropenem levels, and all carbapenems can decrease valproate levels.

Inhibitors o Cell Membrane Stability Daptomycin is a cyclic lipopeptide antibiotic. Its exact mech-

anism o action is unclear, but the drug appears to integrate into the membranes o Gram-positive bacteria. Oligomerization o daptomycin may then result in the ormation o pores, leading to potassium e ux, membrane depolarization, and cell death. Daptomycin is administered intravenously and is approved or the treatment o complicated skin in ections and bacteremia (including bacteremia rom right-sided endocarditis) caused by Staphylococcus aureus.

Daptomycin also has therapeutic e f cacy in the treatment o Gram-positive bacterial in ections and specif cally may be active in the treatment o methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcal in ections. Adverse e ects include myopathy and eosinophilic pneumonia. Because o the association with myopathy, caution should be exercised when co-administering statins with daptomycin. Daptomycin can bind to artif cial phospholipid sur aces in some commercially available anticoagulation assays, resulting in alsely prolonged PT and aPTT. In order to minimize this inter erence, blood drawn or these assays should be taken near the time o the trough plasma concentration o daptomycin.

Antimycobacterial Agents Ethambutol, Pyrazinamide, and Isoniazid

Ethambutol, pyrazinamide , and isoniazid (INH) are three o

the f ve f rst-line agents used to treat tuberculosis (ri ampin and streptomycin, discussed in Chapter 34, are the other two). Patients with active tuberculosis and without a history o prior therapy are started on a our-drug regimen i the local prevalence o isoniazid resistance is higher than 4%. I isoniazid resistance is rare, a three-drug regimen without ethambutol can be used (see Chapter 41). Ethambutol, a bacteriostatic agent, decreases arabinogalactan synthesis by inhibiting the arabinosyl trans erase that adds arabinose units to the growing arabinogalactan chain. Pyrazinamide and INH inhibit mycolic acid synthesis. Pyrazinamide is a prodrug; it must be converted to its active orm, pyrazinoic acid, by the enzyme pyrazinamidase. Pyrazinoic acid inhibits FAS1, the enzyme that synthesizes the atty acid precursors o mycolic acid. Isoniazid and the related second-line agent ethionamide target the FAS2 complex and are bactericidal, although the exact mechanism o bacterial killing is unknown. The targets o pyrazinamide and isoniazid are summarized in Figure 35-4. Treatment o active tuberculosis requires multidrug therapy. Since resistance to antimycobacterial agents usually occurs by mutation, a power ul argument in avor o this strategy is based on the requency o resistance mutations and the number o bacteria present in a clinical in ection. Each tuberculous lesion in an in ected lung can contain 108 bacteria. The requency o mutants resistant to any single antimycobacterial drug is about 1 in 106 bacteria. This requency means that, in each tuberculous lesion, an average o about 100 bacteria will already be resistant to an antimycobacterial drug, even be ore that drug is administered. Combination therapy with just two drugs reduces the likelihood o encountering preexisting resistance to just 1 bacterium in 1012; treatment with our drugs lowers this probability to 1 in 1024 (see Chapter 41). Antimycobacterial agents can cause serious adverse e ects. Ethambutol is associated with optic neuritis; patients report impaired visual acuity, loss o color discrimination, constricted visual f elds, and/or central and peripheral scotomata. Symptoms usually occur a ter more than a month o therapy and are reversible; however, sudden-onset irreversible blindness has been reported. There ore, patients taking ethambutol must be seen regularly or eye examination by an ophthalmologist to assess both visual acuity and color discrimination. Pyrazinamide is associated with arthralgias and (usually asymptomatic) hyperuricemia; more importantly, it commonly causes hepatotoxicity that can be severe and

C h a p t e r 3 5 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis

irreversible. Whereas patients who experience mild hepatotoxicity due to INH may be rechallenged with the drug, patients who experience pyrazinamide-induced hepatotoxicity should not be rechallenged. Isoniazid is associated with hepatitis as well as peripheral neuropathy. INH-induced hepatotoxicity can be mild, mani esting only as minor liver enzyme elevation not requiring cessation o the drug (occurs in 10–20% o patients), or it can be severe, leading to symptomatic hepatitis (occurs in 0.1% o patients overall, with increased risk in older patients with underlying liver disease who are also taking ri ampin). Neurological maniestations o INH toxicity include paresthesias, peripheral neuropathy, and ataxia; this toxicity is due to INH’s competitive inhibition o pyridoxine in neurotransmitter synthesis and can be prevented by pyridoxine supplementation. Isoniazid can also inhibit or induce cytochrome P450 enzymes and thereby interact with multiple other drugs, including ri ampin, the antiseizure medications carbamazepine and phenytoin, azole-type anti ungals, and alcohol. Isoniazid is a weak monoamine oxidase inhibitor; administered with serotonergic agents such as meperidine or uoxetine, it may cause serotonin syndrome. Resistance to these drugs, and to antimycobacterial agents in general, results rom chromosomal mutations. Ethambutol resistance most o ten results rom mutations in the arabinosyl trans erase gene, some o which cause overexpression o the target enzyme. Resistance to isoniazid usually results rom an inactivating mutation in the mycobacterial enzyme catalase-peroxidase , which converts isoniazid into its antimycobacterial orm. Mutations in the inhA gene, which is required or mycolic acid synthesis, also con er resistance to INH. Resistance to pyrazinamide is generally due to mutations in the pyrazinamidase gene, which result in the inability to convert the prodrug into its active orm.

CONCLUSION AND FUTURE DIRECTIONS The bacterial cell wall presents unique antibacterial targets. This structure consists o a three-dimensional mat o cross-linked peptide-sugar polymers called murein and is synthesized in three stages: (1) synthesis o murein monomers, (2) polymerization o monomers into murein polymers, and (3) cross-linking o polymers to complete the wall. Antibacterial agents act in all three stages o cell wall synthesis: os omycin and cycloserine act in the f rst stage; vancomycin, telavancin, dalbavancin, oritavancin, and bacitracin act in the second stage; and the -lactams, the largest and most important group, act in the third stage. -Lactams—which include the penicillins, cephalosporins, monobactams, and carbapenems—are bactericidal; autolytic cell death most likely results rom the unopposed action o wall remodeling proteins called autolysins. Structural and chemical di erences among the -lactams determine their spectra o activity against bacteria with di erent cell wall architectures. Resistance to -lactam antibiotics is generally con erred by plasmid-encoded -lactamases. Pharmacologists have addressed this mechanism o resistance by (1) developing new -lactam agents, or example, the second- and thirdgeneration cephalosporins that are resistant to degradation by many -lactamases, and (2) co-administering -lactam

655

“decoys,” such as clavulanic acid and sulbactam, that serve as -lactamase inhibitors. Because -lactamases can be encoded on plasmids, they can spread through bacterial (and human) populations with great speed, making antibiotic development an ongoing “arms race.” Antimycobacterial agents act by blocking various steps in the synthesis o molecules, such as mycolic acid and arabinogalactan, that are unique to the mycobacterial cell wall. Resistance to these agents is typically due to chromosomal mutation, but combination therapy is critically important to avoid the development o mutational resistance. Future innovations will likely include the development o new agents directed against the additional unique molecular targets that are presented by the biochemistry o the bacterial cell wall.

Acknowledgment We thank Robert R. Rando, Anne G. Kasmar, Tania Lupoli, David C. Hooper, Daniel Kahne, and Suzanne Walker or their valuable contributions to this chapter in the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Bush K. Alarming -lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr Opin Microbiol 2010;13:558–564. (Reviews -lactam resistance in Gram-negative bacteria, ocusing on recent reports o ESBL- and carbapenemase-mediated resistance.) Drawz SM, Papp-Wallace KM, Bonomo RA. New -lactamase inhibitors: a therapeutic renaissance in an MDR world. Antimicrob Agents Chemother 2014;58:1835–1846. (Reviews approved and investigational drugs or the treatment o extended-spectrum -lactamase-producing organisms.) El Zoeiby A, Sanschagrin F, Levesque RC. Structure and unction o the Mur enzymes: development o novel inhibitors. Mol Microbiol 2003;47:1–12. (Reviews the structure, catalytic action, and inhibition o MurA–MurF.) Favrot L, Ronning DR. Targeting the mycobacterial envelope or tuberculosis drug development. Expert Rev Anti In ect Ther 2012;10:1023–1036. (Reviews the structure o the mycobacterial cell wall and its potential targets or drug development.) Gale EF, Cundli e E, Reynolds PE, Richmond MH, Waring MJ. The molecular basis o antibiotic action. 2nd ed. London: John Wiley; 1981. (Classic treatise on antibiotics that describes the experiments leading to the determination o many o the mechanisms o action discussed in this chapter.) Guskey MT, Tsuji BT. A comparative review o the lipoglycopeptides: oritavancin, dalbavancin, and telavancin. Pharmacotherapy 2010;30:80–94. (Discusses glycopeptide agents recently approved or use in the United States) Howden BP, Davies JK, Johnson PD, Stinear TP, Grayson ML. Reduced vancomycin susceptibility in Staphylococcus aureus: resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev 2010;23: 99–139. (Reviews VISA and VRSA, including def nitions, risk actors, and mechanisms o resistance.) Jacoby GA, Munoz-Price LS. The new beta-lactamases. N Engl J Med 2005; 352:380–391. (Reviews the pharmacology o -lactamases.) Mdluli K, Kaneko T, Upton A. The tuberculosis drug discovery and development pipeline and emerging drug targets. Cold Spring Harb Perspect Med 2015;5:a021154. (Reviews approved and investigational drugs or the treatment o tuberculosis.) Rattan A, Kalia A, Ahmad N. Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives. Emerg In ect Dis 1998;4:195–209. (Discusses the problem o resistance in tuberculosis.) Shahid M, Sobia F, Singh A, et al. Beta-lactams and beta-lactamase-inhibitors in current- or potential-clinical practice: a comprehensive update. Crit Rev Microbiol 2009;35:81–108. (Discusses novel beta-lactamase inhibitors and their combinations with beta-lactams.) Terico AT, Gallagher JC. Beta-lactam hypersensitivity and cross-reactivity. J Pharm Pract 2014;27:530–544. (Reviews hypersensitivity and crossreactivity o -lactams.)

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660 PRINCIPLES OF CHEMOTHERAPY

36 Pharmacology of Fungal Infections Ch e ls e a M a a n d Ap ril W. Arm s t ro n g

Miito totic s pindle Gris e o fulvin

Ce ll wa lll Ec hi h noo c and a ndin ins

DNA s ynthe y s is Fluc yto s ine mbra INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . .P. la . . s. .ma . . . me . 66 661–662 2 ne Inhibi bito tors rs off Squa uallene le ne Epo p xidase: Allylamines o te te ric ric inand B B)) Benzylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 BIOCHEMISTRY OF TH THE FUNGAL MEMBRANEPo lye ne s (ampho Inhibitors of 144 -Sterol Demethylase: Imida midazoles AND CELL WALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661 OH H a d Triazoless . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 an 65 PATHOPHYSIOLOGY OF FUN UNGA UN GAL IN GAL INFECTIONS N . . . . . . . . . . . . . . 662 OH O Inhibitors Fung ngal Membrane Stability: Polyenes . . . . . . . . 667 OH off Fu O PHAR PH ARMA ARM AR MACOLOGIC ACOLOGIC CLASSES AND D AG AGENTS A ENTS . . . . . . . . . . . . . . . . 663 Inhibitors of Fungal Wall Inhib ll Synt Synthe h sis: Echinocandins . . . . . . . 668 Inhi In hibi b tor of Fungal u Nucleic u Acid c Syn ynthesis: Chelator h of Polyvalent Cations: Ciclopir irox ox . . . . . . . . . . . . . . . . 66 668 Flu Flucytosin ucyt O . . . . . .OH . . . . . . .O . .H. . . . . . . . . . . . .OH . . . . . . .OH . . . . . 663 HO OH O OH CONCLUSION O CLU US USIO AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 6688 IInhibitor n r oof Fungal Mitosis: Griseofulvin . . . . . . . . . . . . . . . . . . 66 6655 C H st Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .669 Suggested Inhibitors of the Ergosterol Synthesis Pathway Pathw . . . . . . . . . . . 66 6655 S O

INTRODUCTION

O NH2 OH H

O

Fungi are ree-living microorganisms that exist ist aas yyeasts east aasts tts ((single-cell, siingle l cell ll roundd ungi) ungi), i) mold molds lds ((multicellular multi lticell llular l f llamentous amenttous ungi), or a combination o the two (so-called dimorphic ungi). All ungi are eukaryotic organisms. Because o their phylogenetic similarity, ungi and humans have homologous metabolic pathways or energy production, protein synthesis, and cell division. Consequently, there is greater di f culty in developing selective anti ungal agents than in developing selective antibacterial agents. The success o many antibacterial agents has resulted rom the identif cation o unique molecular targets in bacteria, emphasizing the necessity or identi ying unique ungal targets that can be exploited. Certain patient populations are particularly susceptible to ungal in ections (mycoses). These populations include surgical and intensive care unit (ICU) patients, patients with prostheses, and patients with compromised immune deenses. In the past three to our decades, the extensive use o broad-spectrum antibiotics, the wider use o long-term intravenous catheters, and in ection with human immunodef ciency virus (HIV) have correlated with an increasing incidence o opportunistic and systemic mycoses. Additionally, the successes o organ transplantation, immunosuppressive therapy, and cancer chemotherapy have contributed to an increasing number o chronically immunosuppressed patients, who are particularly susceptible to ungal in ections. Traditionally, the diagnosis o ungal in ections has relied on culture-based methods and direct examination o specimens under light microscopy. However, the indolent growth o ungi makes culturing ine f cient, while direct microscopic examination may not be reliable or provide def nitive speciation. These disadvantages have important clinical implications because prognosis o ten correlates inversely

with the duration o time rom clinical presentation to accurate cura cu rate te ddiagnosis. iagn ia gnos osis is. Co Consequently, Cons nseq eque uent ntly ly, on onee m major ajjor ocus o m modern odern d mycology is the development o rapid, nonculture-based methods h d o early l ddiagnosis. i i N New di diagnostic i techniques h i rely l on the polymerase chain reaction (PCR), western blot, antigen detection, and identif cation o ungal metabolites. Because many o these techniques are still investigational, they must be per ormed in parallel with traditional culture-based methods. The treatment options or opportunistic and systemic ungal in ections were once thought to be limited. These options are now expanding, however. Fungal processes that have been exploited in the development o anti ungal agents include nucleic acid synthesis, mitosis, and membrane synthesis and stability. Traditional anti ungal agents, such as azoles and polyenes, are directed against molecular targets involved in the synthesis and stability o the ungal membrane. The echinocandins, a relatively newer class o anti ungal agents, target an enzyme complex involved in the synthesis o the ungal cell wall. As the emergence o resistant ungi increases, it will become increasingly important to identi y and exploit new molecular targets or anti ungal therapy.

BIOCHEMISTRY OF THE FUNGAL MEMBRANE AND CELL WALL Although ungi have a cellular ultrastructure similar to that o animal cells, there are a number o unique biochemical di erences that have been exploited in the development o anti ungal drugs. To date, the most important biochemical di erence lies in the principal sterol used to maintain plasma membrane structure and unction. Mammalian cells use cholesterol or this purpose, whereas ungal cells use 661

662 PRINCIPLES OF CHEMOTHERAPY

J a m e s F, a 31-ye a r-o ld HIV-p o s itive m a n , p re s e n ts to h is p h ys icia n w ith a 3-w e e k h is to ry o e ve r, co u g h , a n d ch e s t p a in a te r to u rin g So u th e rn Ca lio rn ia . His h is to ry is n o ta b le o r p a s t in tra ve n o u s d ru g u s e . Clin ica l e va lu a tio n a n d ch e s t x-ra y re ve a l a le t lo w e r lo b e in f ltra te a n d le t p a ra tra ch e a l a d e n o p a thy. S p u tu m cu ltu re s a re p o s itive o r Coccidioides immitis, a n d b lo o d te s ts a re n o ta b le o r a n e le va te d tite r o a n tib o d ie s d ire cte d a ga in s t th is u n ga l p a th o g e n . Th e p hys icia n m a ke s a p re lim in a ry d ia g n o s is o p u lm o n a ry co ccid io id o m yco s is a n d p re s crib e s a co u rs e o a m p h o te ricin B. Ove r th e n e xt s e ve ra l d a ys , h o w e ve r, Mr. F d o e s not im prove. He goes to the em ergency departm ent w ith e ve r, ch ills , s w e a ts , co u g h , a tig u e , a n d h e a d ach es. His tem peratu re is 100°F, b ut he sho ws no eviden ce o m e nin gitis o r pe riphe ra l ad eno pathy. Lung e xa m in a tio n re ve a ls d i u s e w h e e zin g ove r th e le t lung f elds, noted o n b oth insp iration and expiration.

the structurally distinct sterol ergosterol. The biosynthesis o ergosterol involves a series o steps, two o which are targeted by currently available anti ungal drugs (Fig. 36-1). The enzymes that catalyze ergosterol synthesis are localized in ungal microsomes, which contain an electron transport system nearly identical to that ound in mammalian liver microsomes. The f rst targeted step, the conversion o squalene to lanosterol, is catalyzed by the enzyme squalene epoxidase . This enzyme is the molecular target o the allylamine and benzylamine anti ungal agents. The ungus-specif c cytochrome P450 enzyme 14 -sterol demethylase mediates the key reaction in the second targeted step, the conversion o lanosterol to ergosterol. Imidazole and triazole anti ungal agents inhibit 14 -sterol demethylase. There ore, allylamine, benzylamine, imidazole, and triazole anti ungal agents all inhibit the biosynthesis o ergosterol. Because ergosterol is necessary or the maintenance o plasma membrane structure and unction, these agents compromise ungal membrane integrity. Ergosterol synthesis inhibitors suppress ungal cell growth under most circumstances (fungistatic e ect), although they can sometimes cause ungal cell death (fungicidal e ect). Fungal cells are surrounded by a cell wall, a rigid structure that has been studied intensively as a new and important target or anti ungal therapy. The major components o the ungal cell wall are chitin, -(1,3)-D-glucan, -(1,6)-D-glucan, and cell wall glycoproteins (especially proteins containing complex mannose chains, or mannoproteins ). Chitin is a linear polysaccharide consisting o more than 2,000 N-acetylglucosamine units joined by -(1,4) linkages; these chains are bundled into microf brils that orm the undamental sca old o the cell wall. -(1,3)-D-glucan and -(1,6)-D-glucan, which are polymers o glucose units joined by -(1,3) and -(1,6) glycosidic linkages, respectively, are the most abundant components o the cell wall. These glucan polymers are covalently linked to the chitin sca old. The cell wall glycoproteins comprise a diverse group o proteins that are noncovalently associated with other

Bronchoscopy shows narrowing o the tracheal lum en by num erous m ucosal granulom as rom the le t m ainstem bronchus to the level o the m idtrachea. Fu ngal cu ltu re grows Coccidioides immitis, a d ef nitive diag no sis o chronic pulm onary coccidioidom ycosis is m ade, the granulom as are bronchos copically rem oved, and a m p h o te ricin B is co n tin u e d . A w e e k la te r, Mr. F’s s ym p tom s be gin to s u b s id e , a m p h o te ricin B is d is con tinued, and a course o uconazole is initiated.

Questions 1 . What actors predisposed Mr. F to ungal in ection? 2 . What are the mechanisms o action o amphotericin B and f uconazole? 3 . What adverse e ects could Mr. F experience as a consequence o treatment with amphotericin B and f uconazole?

cell wall components or are covalently linked to chitin, glucan, or other cell wall proteins. Because mammalian cells do not have cell walls, drugs directed against the ungal cell wall would be expected to have a high therapeutic index. Echinocandin anti ungal agents target -(1,3)-D-glucan synthase , the enzyme that adds glucose residues rom the donor molecule UDP-glucose to the growing polysaccharide chain. By inhibiting cell wall biosynthesis, echinocandins disrupt ungal cell wall integrity. Echinocandins o ten have ungicidal activity, although these agents are ungistatic under some circumstances (see “Suggested Reading”). Fungal adhesion represents a third potential target or antiungal drugs. Adhesion to host cells is mediated by the binding o ungal adhesins to host cell receptors. In yeasts, or example, aspartyl proteases and phospholipases mediate adhesion. Compounds that block adhesive interactions between ungal cells and mammalian cells are currently under development.

PATHOPHYSIOLOGY OF FUNGAL INFECTIONS Mycoses ( ungal in ections) can be divided into superf cial, cutaneous, subcutaneous, systemic or primary, and opportunistic in ections. Few ungi possess su f cient virulence to be considered primary pathogens capable o initiating serious in ections in immunocompetent hosts. However, immunocompromised hosts can develop serious systemic in ections with ungi that are not pathogenic in normal individuals. In the introductory case, Mr. F’s HIV in ection likely increased his risk o in ection with Coccidioides immitis. Thus, the pathogenesis o ungal in ections is based on the interplay between a host’s immune system and the pathogenicity o the particular ungal organism. Polymorphonuclear leukocytes, cell-mediated immunity, and humoral immunity are all important components o the host immune de ense against ungal pathogens.

C h a p t e r 3 6 Pharmacology o Fungal In ections Ace tyl CoA HMG CoA

Me va lona te Allylamines Benzylamines

S qua le ne Squalene epoxidase

Two re pre s e nta tive tria zole s : OH N

HO

N

N

N

N

F

N

H

La nos te rol F

Imidazoles Triazoles

14α-sterol demethylase

Fluconazole F

OH N N

N N

F

N

F

Voriconazole H

H

HO

Ergos te rol

Me mbra ne s ynthe s is

FIGURE 36-1. Ergosterol synthesis pathway. Ergosterol is synthesized in ungal cells rom acetyl CoA building blocks. One o the intermediates, squalene, is converted to lanosterol by the action o squalene epoxidase. Allylamines and benzylamines inhibit squalene epoxidase. 14 -Sterol demethylase, a cytochrome P450 enzyme not expressed in mammalian cells, catalyzes the f rst step in the conversion o lanosterol to the unique ungal sterol ergosterol. Imidazoles and triazoles inhibit 14 -sterol demethylase and thereby prevent the synthesis o ergosterol, which is the principal sterol in ungal membranes. Fluconazole and voriconazole are two representative triazoles.

The pathogenesis o ungal in ections is only partly understood, and di erent ungi possess distinct virulence actors that are unique to the pathogen. Adhesion is an initial step in the early stages o in ection. Adhesion and localization can occur on skin, mucosal, and prosthetic device sur aces. For example, Candida species adhere to a variety o sur aces via a combination o specif c ligand–receptor interactions as well as nonspecif c orces such as van der Waals and electrostatic interactions. Virulent pathogens are subsequently able to invade the colonized sur ace and proli erate in deep tissue, sometimes reaching the systemic circulation. Systemic dissemination can be accelerated by local tissue injury, such as that caused by cancer chemotherapy, ischemia, or the presence o a prosthetic device. In addition, some pathogens secrete lytic enzymes to enable invasive growth and systemic dissemination. C. immitis breaches the respiratory mucosa by producing an alkaline proteinase capable o digesting structural proteins in lung tissue. C. immitis also produces a 36-kDa extracellular proteinase capable o degrading human elastin, collagen, immunoglobulins, and hemoglobin.

663

Fungal cell wall composition plays an important role in the pathogenesis o ungal in ections. Pathogens such as Blastomyces dermatitidis, Histoplasma capsulatum, and Paracoccidioides brasiliensis modulate the complement o glycoproteins in their cell walls in response to host immune system interactions. For example, the cell wall o B. dermatitidis contains a 120-kDa glycoprotein, BAD-1 ( ormerly WI-1), which elicits a potent humoral and cellular immune response. Avirulent strains o B. dermatitidis have increased expression o BAD-1, which is recognized by the host immune system and leads to elimination o the pathogen through phagocytosis. In contrast, the cell wall o virulent strains o B. dermatitidis contains high levels o -(1,3)-glucan, which is inversely correlated with the amount o BAD-1 detectable on the cell sur ace. It is speculated that the increased amount o -(1,3)-glucan in the cell wall e ectively masks the BAD-1 sur ace glycoprotein, thereby allowing the virulent strains to evade host immune detection and destruction. The ability o a ungal pathogen to change rom one morphotype to another is termed phenotype switching. By responding to changes in the microenvironment, Candida species are capable o undergoing yeast-to-hyphae transormation. The hyphal orms o Candida species possess a “sense o touch” that allows them to grow in crevices and pores, thereby increasing their inf ltrative potential. Similarly, B. dermatitidis undergoes trans ormation rom conidia (small, asexual reproductive structures) to the larger yeast orms. The larger orms o er an important survival advantage because they are capable o resisting the phagocytic action o neutrophils and macrophages.

PHARMACOLOGIC CLASSES AND AGENTS The ideal anti ungal agent would possess our characteristics: broad spectrum o action against a variety o ungal pathogens, low drug toxicity, multiple routes o administration, and excellent penetration into the cerebrospinal uid (CSF), urine, and bone. With the recent expansion in identiying novel targets o anti ungal therapy, treatment options or superf cial and deep ungal in ections are improving. Some anti ungal agents can be used to treat both superf cial and deep mycoses (in some cases, using di erent ormulations), while others are restricted to narrower indications. In this section, the currently available anti ungal drugs are categorized according to their molecular targets and mechanisms o action. The primary molecular targets or anti ungal therapy are enzymes and other molecules involved in ungal DNA synthesis, mitosis, plasma membrane synthesis, and cell wall synthesis (Fig. 36-2). Because the clinical trials used to support regulatory approval o new drugs o ten exclude children and women o childbearing potential (see Chapter 52, Clinical Drug Evaluation and Regulatory Approval), the sa ety o some o the newer anti ungal agents is not precisely determined in these patient populations. The treating physician must there ore weigh the risks o treatment against the expected benef ts.

Inhibitor of Fungal Nucleic Acid Synthesis: Flucytosine Flucytosine is the name o

the uorinated pyrimidine 5- uorocytosine. Flucytosine is selectively taken up by ungal

664 PRINCIPLES OF CHEMOTHERAPY

as a single agent, resistance emerges rapidly due to mutations in ungal cytosine permease or cytosine deaminase. Although ucytosine has no intrinsic activity against Aspergillus, synergistic killing o Aspergillus by the combination o ucytosine and amphotericin B can be demonstrated experimentally. The mechanism o this synergistic interaction appears to involve enhancement o ucytosine uptake by ungal cells due to amphotericin-induced damage to the ungal plasma membrane. The spectrum o activity o ucytosine as a single agent is limited to candidiasis, cryptococcosis, and chromomycosis. Combination treatment with amphotericin B is recommended in acute cryptococcal meningitis in HIV-in ected adults. A pharmacokinetic advantage o ucytosine is its large volume o distribution, with excellent penetration into the central nervous system (CNS), eyes, and

Endopla s mic re ticulum (inhibit e rgos te rol s ynthe s is ) Allylamines Benzylamines Imidazoles Triazoles

Nucle us

NH2 Mitotic s pindle Griseofulvin DNA s ynthe s is Flucytosine

OH

O O

OH

OH

OH

OH

O

Flucytosine Cytos ine pe rme a s e

OH

O

N N H

P la s ma me mbra ne Polyenes (amphotericin B) OH

HO

F

Ce ll wa ll Echinocandins

Ce ll me mbra ne

OH H

O

Cytosine deaminase O NH2 OH

O

O

OH

F

Amphotericin B

NH

FIGURE 36-2. Cellular targets o anti ungal drugs. The currently available

N H

anti ungal agents act on distinct molecular targets. Flucytosine inhibits ungal DNA synthesis. Griseo ulvin inhibits ungal mitosis by disrupting mitotic spindles. Allylamines, benzylamines, imidazoles, and triazoles inhibit the ergosterol synthesis pathway in the endoplasmic reticulum. Polyenes bind to ergosterol in the ungal membrane and thereby disrupt plasma membrane integrity. Amphotericin B is a representative polyene. Echinocandins inhibit ungal cell wall synthesis.

cells via cytosine-specif c permeases that are expressed only in ungal membranes. Lacking these transporters, mammalian cells are protected. Inside the ungal cell, the enzyme cytosine deaminase converts ucytosine to 5- uorouracil (5-FU). (5-FU is itsel an antimetabolite that is used in cancer chemotherapy; see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance.) Subsequent reactions convert 5-FU to 5- uorodeoxyuridylic acid monophosphate (5-FdUMP), which is a potent inhibitor o thymidylate synthase . Inhibition o thymidylate synthase results in inhibition o DNA synthesis and cell division (Fig. 36-3). Flucytosine appears to be ungistatic under most circumstances. Although mammalian cells lack cytosine-specif c permeases and cytosine deaminase, ungi and bacteria in the intestine can convert ucytosine to 5- uorouracil, which can cause adverse e ects in host cells. Flucytosine is typically used in combination with amphotericin B to treat systemic mycoses; when the drug is used

O

5-Fluoroura cil (5-FU)

O F

NH N

O -

O

P O

O

O

O

-

H

H

H

OH

H

H

5-Fluorode oxyuridylic a cid monophos pha te (5-FdUMP )

dUMP

Thymidylate synthase

dTMP

FIGURE 36-3. Mechanism o action o f ucytosine. Flucytosine enters the ungal cell via a transmembrane cytosine permease. Inside the cell, cytosine deaminase converts f ucytosine to 5-f uorouracil (5-FU), which is subsequently converted to 5-f uorodeoxyuridylic acid monophosphate (5-FdUMP). 5-FdUMP inhibits thymidylate synthase and thereby blocks the conversion o deoxyuridylate (dUMP) to deoxythymidylate (dTMP). In the absence o dTMP, DNA synthesis is inhibited.

C h a p t e r 3 6 Pharmacology of Fungal Infections

urinary tract. Dose-dependent adverse e ects include bone marrow suppression leading to leukopenia and thrombocytopenia, nausea, vomiting, diarrhea, and hepatic dys unction. Flucytosine is contraindicated during pregnancy.

Inhibitor o Fungal Mitosis: Griseo ulvin Derived rom Penicillium griseofulvum in the 1950s, griseo ulvin inhibits ungal mitosis by binding to tubulin and a microtubule-associated protein and thereby disrupting assembly o the mitotic spindle. The drug is also reported to inhibit ungal RNA and DNA synthesis. Griseo ulvin accumulates in keratin precursor cells and binds tightly to keratin in di erentiated cells. The prolonged and tight association o griseo ulvin with keratin allows new growth o skin, hair, or nail to be ree o dermatophyte in ection. Griseo ulvin appears to be ungistatic under most circumstances. The therapeutic use o oral griseo ulvin is currently limited, due to the availability o topical anti ungal medications as well as other oral anti ungal agents with ewer adverse e ects. Griseo ulvin can be used to treat ungal in ection o the skin, hair, and nail due to Trichophyton, Microsporum, and Epidermophyton. The drug is not e ective against yeast (such as Pityrosporum) and dimorphic ungi. Doses should be taken at 6-hour intervals because blood levels o griseoulvin can be variable; absorption is enhanced i the drug is taken with a atty meal. It is important to continue treatment until the in ected skin, hair, or nail is completely replaced by normal tissue. Griseo ulvin use is not associated with a high incidence o serious adverse e ects. A relatively common (up to 15%) adverse e ect is headache, which tends to disappear as therapy continues. Other nervous system e ects include lethargy, vertigo, and blurred vision; these adverse e ects can be exacerbated by the consumption o alcohol. Occasionally, hepatotoxicity or albuminuria without renal insu f ciency can be observed. Hematologic adverse e ects—including leukopenia, neutropenia, and monocytosis—can occur during the f rst month o therapy. Serum sickness, angioedema, ex oliative dermatitis, and toxic epidermal necrolysis are extremely rare but potentially li e-threatening adverse e ects. Chronic use can sometimes result in increased ecal protoporphyrin levels. Concurrent administration with barbiturates decreases the gastrointestinal absorption o griseo ulvin. Because griseo ulvin induces hepatic cytochrome P450 enzymes, it can increase the metabolism o war arin and potentially reduce the e f cacy o low-estrogen oral contraceptive medications. Griseo ulvin should be avoided during pregnancy, since etal abnormalities have been reported.

Inhibitors o the Ergosterol Synthesis Pathway Inhibitors of Squalene Epoxidase: Allylamines and Benzylamines

In the ergosterol synthesis pathway (Fig. 36-1), squalene is converted to lanosterol by the action o squalene epoxidase . Inhibitors o squalene epoxidase prevent the ormation o lanosterol, which is a precursor or ergosterol. These drugs also promote accumulation o the toxic metabolite squalene in the ungal cell, making them ungicidal under most circumstances. The anti ungal agents that inhibit squalene epoxidase can be divided into allylamines and benzylamines based on their chemical structures: terbinaf ne and na tif ne are allylamines, whereas butenaf ne is a benzylamine.

665

Terbinaf ne is available in both oral and topical ormula-

tions. When taken orally, the drug is 99% protein-bound in the plasma, and it undergoes f rst-pass metabolism in the liver. Because o this f rst-pass metabolism, the oral bioavailability o terbinaf ne is 40%. The drug’s elimination hal -li e is extremely long, approximately 300 hours, because terbinaf ne accumulates extensively in the skin, nails, and at. The oral orm o terbinaf ne is used in the treatment o onychomycosis, tinea corporis, tinea cruris, tinea pedis, and tinea capitis. Terbinaf ne is not recommended in patients with renal or hepatic ailure or in pregnant women. Very rarely, the oral orm o terbinaf ne can lead to hepatotoxicity, Stevens-Johnson syndrome, neutropenia, and exacerbation o psoriasis or subacute cutaneous lupus erythematosus. Liver unction enzymes should be monitored during the treatment course. Plasma levels o terbinaf ne are increased by co-administration with cimetidine (a cytochrome P450 inhibitor) and decreased by co-administration with ri ampin (a cytochrome P450 inducer). Topical terbinaf ne is available in cream or spray orm and is indicated or tinea pedis, tinea cruris, and tinea corporis. Similar to terbinaf ne, na tif ne is a squalene epoxidase inhibitor that has broad-spectrum anti ungal activity. Na tif ne is only available topically as a cream or gel; it is e ective in tinea corporis, tinea cruris, and tinea pedis. Butenaf ne , a benzylamine, is a topical anti ungal agent with a mechanism o action and spectrum o anti ungal activity similar to that o the allylamines. Topical allylamines and benzylamines are more e ective than topical azole agents against common dermatophytes, especially those causing tinea pedis. However, topical terbinaf ne and butenaf ne are less e ective than topical azoles against Candida skin in ections (see below).

Inhibitors of 14 -Sterol Demethylase: Imidazoles and Triazoles

Another important molecular target in the ergosterol synthesis pathway is 14 -sterol demethylase , a microsomal cytochrome P450 enzyme that catalyzes the f rst step in the conversion o lanosterol to ergosterol. The azoles are anti ungal agents that inhibit ungal 14 -sterol demethylase. The resulting decrease in ergosterol synthesis and accumulation o 14 -methyl sterols disrupt the tightly packed acyl chains o the phospholipids in ungal membranes. Destabilization o the ungal membrane leads to dys unction o membrane-associated enzymes, including those in the electron transport chain, and may ultimately lead to cell death. Azoles are not completely selective or the ungal P450 enzyme, however, and they can also inhibit hepatic P450 enzymes. While the extent o hepatic P450 enzyme inhibition varies among the azoles, drug– drug interactions are an important consideration whenever an azole antifungal agent is prescribed. For example, cyclosporine is an immunosuppressive drug used to prevent gra t rejection in recipients o allogeneic kidney, liver, and heart transplants. It is metabolized by hepatic P450 enzymes and excreted in the bile. To minimize the risk o cyclosporineassociated nephrotoxicity and hepatotoxicity, patients concomitantly receiving an azole anti ungal agent should be treated with lower doses o cyclosporine. As a group, the azoles have a wide range o anti ungal activity and are clinically use ul against B. dermatitidis, Cryptococcus neoformans, H. capsulatum, Coccidioides species, P. brasiliensis, dermatophytes, and most Candida species. Azoles

666 PRINCIPLES OF CHEMOTHERAPY

have intermediate clinical activity against Fusarium, Sporothrix schenckii, Scedosporium apiospermum, and Aspergillus species. Pathogens mediating zygomycosis (invasive ungal in ections caused by Zygomycetes species) and Candida krusei are resistant to azoles. The azoles are generally ungistatic rather than ungicidal against susceptible organisms. The azole anti ungal agents can be categorized into two broad classes, imidazoles and triazoles , which share the same mechanism o action and similar anti ungal spectrum. Because systemically administered triazoles tend to have less e ect than systemically administered imidazoles on human sterol synthesis, recent drug development has ocused primarily on triazoles. The imidazole anti ungal class includes ketoconazole , clotrimazole , luliconazole , miconazole , econazole , butoconazole , oxiconazole , sertaconazole , sulconazole, and tioconazole . Ketoconazole was introduced in 1977 as the prototypic drug in this class. Ketoconazole is available in both oral and topical ormulations. Its broad spectrum o action includes C. immitis, C. neoformans, Candida species, H. capsulatum, B. dermatitidis, and a variety o dermatophytes. The pharmacokinetic and adverse e ect prof les o ketoconazole limit its clinical utility. (In act, oral ketoconazole has been replaced by itraconazole or the treatment o many mycoses; see discussion below.) Gastrointestinal absorption o oral ketoconazole depends on conversion o the drug to a salt in the acidic environment o the stomach. Thus, ketoconazole cannot be used i the patient has achlorhydria or is receiving bicarbonate, antacids, H2-blockers, or proton pump inhibitors. Ketoconazole has little penetration into the CSF and urine, which limits its e f cacy in CNS and urinary tract in ections. In approximately 20% o patients, the drug causes nausea, vomiting, or anorexia; hepatic dys unction occurs in 1–2% o patients. Ketoconazole potently inhibits hepatic P450 enzymes and there ore a ects the metabolism o many other drugs. At therapeutic doses, it also inhibits the P450 enzymes 17, 20-lyase and side-chain cleavage enzyme in the adrenal gland and gonads, thereby decreasing steroid hormone synthesis. Persistent adrenal insu f ciency has been reported in association with ketoconazole therapy; at high doses o the drug, signif cant inhibition o androgen synthesis can result in gynecomastia and impotence. This dose-dependent adverse e ect has been exploited therapeutically by some clinicians, who prescribe ketoconazole to inhibit androgen production in patients with advanced prostate cancer and to inhibit corticosteroid synthesis in patients with advanced adrenal cancer. Topical ketoconazole is widely used to treat common dermatophyte in ections and seborrheic dermatitis. Topical ketoconazole has been shown to have anti-in ammatory activity comparable to that o hydrocortisone. The cream ormulation contains sulf tes and there ore should be avoided in patients with sulf te hypersensitivity. Clotrimazole , luliconazole , miconazole , econazole , butoconazole , oxiconazole , sertaconazole , sulconazole , and tioconazole are topical imidazole anti ungal agents used to treat superf cial ungal in ections o the stratum corneum, squamous mucosa, and cornea. All o these agents are comparable to one another in e f cacy. In addition to inhibiting 14 -sterol demethylase, miconazole a ects atty acid synthesis and inhibits ungal oxidative and peroxidase enzymes. The currently available topical azoles are generally not e ective against hair or nail ungal in ections, and topical azoles should not be used to treat subcutaneous or systemic

mycoses. Topical azole agents are available or cutaneous and vaginal application, and selection o a particular agent should be based on cost and availability. Rare adverse e ects o these agents include itching, burning, and sensitization. The triazole class o anti ungal agents includes itraconazole , f uconazole , voriconazole , terconazole , posaconazole , and isavuconazole ; one additional member o this class, ravuconazole , is currently in clinical trials. Itraconazole is available in both oral and intravenous ormulations. Given its broad spectrum o activity, itraconazole has largely replaced oral ketoconazole or the treatment o many mycoses. The absorption o oral itraconazole is maximized in an acidic gastric environment. However, because the oral bioavailability o itraconazole is unpredictable, intravenous administration is sometimes pre erred. Itraconazole is oxidized in the liver to the active metabolite hydroxyitraconazole, which is more than 90% bound to plasma protein. Hydroxyitraconazole inhibits ungal 14 -sterol demethylase. Compared to ketoconazole and uconazole, itraconazole shows increased activity in aspergillosis, blastomycosis, and histoplasmosis. Itraconazole is not e f ciently transported into the CSF, urine, or saliva; however, itraconazole can be used in certain meningeal ungal in ections due to the high drug levels achieved in the meninges. Hepatotoxicity is the major adverse e ect associated with itraconazole therapy. Other adverse e ects include nausea, vomiting, abdominal pain, diarrhea, hypokalemia, pedal edema, and hair loss. Posaconazole is an oral triazole developed rom itraconazole. Posaconazole is ungistatic against most species o Candida, Cryptococcus, Trichosporon, and some species o Fusarium. It is also active against multidrug-resistant Candida, Aspergillus, and Zygomycetes isolates. Posaconazole is used primarily in the prophylaxis and treatment o invasive ungal in ections. The most common adverse e ects are nausea, vomiting, diarrhea, rash, hypokalemia, thrombocytopenia, and abnormal liver unction tests. Interactions may occur with co-administration o cimetidine, ri abutin, and phenytoin, and these drugs should be avoided in patients on posaconazole. Moreover, cyclosporine, tacrolimus, and midazolam dosages should be reduced in patients taking posaconazole. Fluconazole is currently the most widely used anti ungal drug. Fluconazole is a hydrophilic triazole that is available in both oral and intravenous ormulations. The bioavailability o oral uconazole is nearly 100%, and, unlike ketoconazole and itraconazole, its absorption is not in uenced by gastric pH. Once absorbed, uconazole di uses reely into CSF, sputum, urine, and saliva. Fluconazole is excreted primarily by the kidneys. Its relatively low adverse e ect prof le (see below) and excellent CSF penetration make uconazole the drug o choice or systemic candidiasis and cryptococcal meningitis. Due to the morbidity associated with intrathecal amphotericin B administration, uconazole is also the drug o choice or coccidioidal meningitis. While uconazole is active against blastomycosis, histoplasmosis, and sporotrichosis, it is less e ective than itraconazole against these in ections. Fluconazole is not e ective against aspergillosis. Fungal resistance to uconazole develops readily, and Candida species are the most notable pathogens to develop resistance (e.g., C. glabrata). Mechanisms o drug resistance include mutation o ungal P450 enzymes and overexpression o multidrug e ux transporter proteins. Numerous drug interactions have been noted with uconazole. As examples, uconazole can increase the levels o

C h a p t e r 3 6 Pharmacology of Fungal Infections

amitriptyline, cyclosporine, phenytoin, and war arin, and the levels and e ects o uconazole can be decreased by carbamazepine, isoniazid, and phenobarbital. Adverse e ects o uconazole include nausea, vomiting, abdominal pain, and diarrhea in about 10% o patients, as well as reversible alopecia with prolonged oral therapy. Rare cases o StevensJohnson syndrome and hepatic ailure have been reported. Ravuconazole , a uconazole derivative that is currently in clinical trials, demonstrates an expanded spectrum o anti ungal activity in vitro against multiple ungal species, including Aspergillus and the relatively resistant Candida species C. krusei and C. glabrata. Voriconazole is a triazole anti ungal agent that is available in both oral and parenteral orms. It is the drug o choice in the treatment o invasive aspergillosis and other molds such as Fusarium and Scedosporium. Voriconazole is ungicidal against essentially all species o Aspergillus, and its spectrum o activity also includes Candida species (including C. krusei and C. glabrata) and a number o newly emerging ungi. It is ine ective in the treatment o zygomycosis. Compared to amphotericin, voriconazole is associated with signif cantly better outcomes, particularly in di f cult-to-treat cases such as allogeneic bone marrow transplant recipients, patients with CNS in ections, and patients with disseminated in ections. Voriconazole inhibits hepatic P450 enzymes to a signif cant extent, and lower doses o cyclosporine or tacrolimus are used when these drugs are combined with voriconazole. Due to accelerated voriconazole metabolism, co-administration with ritonavir, ri ampin, and ri abutin is contraindicated. The intravenous ormulation o voriconazole should not be used in patients with renal ailure because the cyclodextrin excipient accumulates and can cause CNS toxicity. Hepatic toxicity is common but can usually be managed by decreasing the dose. Unusual visual symptoms (photophobia and colored lights) can occur at peak plasma concentrations o voriconazole; typically, these symptoms last or 30–60 minutes. Isavuconazole is a triazole anti ungal agent approved by the US Food and Drug Administration (FDA) in March 2015 or the treatment o invasive aspergillosis and invasive mucormycosis. It is available in oral and parenteral ormulations with equivalent bioavailability. Terconazole is a topical triazole used to treat vaginal candidiasis. Its mechanism o action and spectrum o anti ungal activity are similar to those o the other topical azoles. Terconazole is available as a vaginal suppository that is inserted at bedtime.

Inhibitors of Fungal Membrane Stability: Polyenes Amphotericin B, nystatin, and natamycin are polyene macro-

lide anti ungal agents. These drugs act by binding to ergosterol and disrupting ungal membrane stability. The three agents are natural products derived rom Streptomyces species. For decades, amphotericin B provided the only e ective treatment or systemic mycoses, including candidiasis, cryptococcal meningitis, invasive aspergillosis, zygomycosis, coccidioidomycosis, blastomycosis, and histoplasmosis. Both its therapeutic e ect and its toxicity are related to its a f nity or plasma membrane sterols. Fortunately, the a f nity o amphotericin B or ergosterol is 500 times greater than its a f nity or cholesterol. The binding o amphotericin B to ergosterol produces channels or pores that alter ungal

667

membrane permeability and allow or leakage o essential cellular contents, leading ultimately to cell death. The concentration o membrane-associated ergosterol in a given ungal species determines whether amphotericin B is ungicidal or ungistatic or that species. Resistance to amphotericin B, although less requent than with other anti ungal agents, is attributable to a decrease in the ergosterol content o the ungal membrane. In addition to its pore- orming activity, amphotericin B appears to destabilize ungal membranes by generating toxic ree radicals upon oxidation o the drug. Because amphotericin B is highly insoluble, it is supplied as a bu ered deoxycholate colloidal suspension. This suspension is poorly absorbed rom the gastrointestinal tract and must be administered intravenously. Once in the bloodstream, more than 90% o the drug binds rapidly to tissue sites, while the remainder binds to plasma proteins. Penetration o amphotericin B into the CSF is extremely low (2–4%). Hence, intrathecal therapy may be necessary or treatment o serious meningeal disease. The drug also di uses poorly into vitreous humor and amniotic uid. The toxicity o amphotericin B limits its clinical use. Adverse e ects are divided into three groups: immediate systemic reactions, renal e ects, and hematologic e ects. Systemic reactions can include cytokine storm, in which amphotericin B elicits release o tumor necrosis actor-alpha (TNF- ) and interleukin-1 (IL-1) rom cells o the host immune system. In turn, TNF- and IL-1 cause ever, chills, rigors, and hypotension within the f rst several hours a ter drug administration. These responses can usually be minimized by decreasing the rate o drug administration or by pretreatment with antipyretic agents (e.g., acetaminophen, nonsteroidal anti-in ammatory drugs [NSAIDs], or hydrocortisone). Renal toxicity o amphotericin B is a serious adverse e ect. The mechanism o renal toxicity is unknown but may be related to amphotericin-mediated vasoconstriction o a erent arterioles, leading to renal ischemia. Renal toxicity is o ten the limiting actor in determining the extent o the therapeutic response to amphotericin B. It may be necessary to discontinue therapy temporarily i the blood urea nitrogen (BUN) exceeds 50 mg/dL or the serum creatinine exceeds 3 mg/dL. (BUN and creatinine are surrogate measures o renal unction.) Renal tubular acidosis, cylindruria (the presence o renal cell casts in the urine), and hypokalemia can occur to such a degree that electrolyte replacement is required. In the introductory case, treatment with amphotericin B was discontinued as soon as Mr. F’s acute symptoms resolved in order to prevent renal toxicity. Hematologic toxicity o amphotericin B is also common; anemia is probably secondary to decreased production o erythropoietin. The renal and hematologic toxicities o amphotericin B are cumulative and dose-related. Therapeutic measures that can minimize these toxicities include avoidance o other nephrotoxic drugs, such as aminoglycosides and cyclosporine, and maintenance o euvolemia to provide adequate renal per usion. Attempts to reduce nephrotoxicity have also led to the development o lipid ormulations o amphotericin B. The strategy is to package amphotericin B in liposomes or other lipid carriers, with the goal o preventing high drug exposure to the proximal tubule o the nephron. Amphotec ®, Abelcet®, and AmBisome ® are all FDA-approved lipid-containing preparations o amphotericin B. They are equal in e f cacy to each other and to native amphotericin deoxycholate.

668 PRINCIPLES OF CHEMOTHERAPY

These ormulations are less toxic than the native compound but more expensive. Nystatin, a structural relative o amphotericin B, is a polyene anti ungal agent that also acts by binding ergosterol and causing pore ormation in ungal cell membranes. The drug is used topically to treat candidiasis involving the skin, vaginal mucosa, and oral mucosa. Nystatin is not absorbed systemically rom the skin, vagina, or gastrointestinal tract. Natamycin, another polyene anti ungal agent that binds ergosterol in the ungal cell membrane, is primarily used to treat Aspergillus or Fusarium corneal in ections. It is also indicated in the treatment o blepharitis and conjunctivitis. Natamycin accumulates in the corneal stroma but not in intraocular uid and is e ective at low concentrations.

Inhibitors of Fungal Wall Synthesis: Echinocandins The key components o the ungal cell wall are chitin, -(1,3)D-glucan, -(1,6)-D-glucan, and cell wall glycoproteins. Because human cells do not have a cell wall, ungal cell wall components represent unique targets or anti ungal therapy, and anti ungal agents directed at these targets are likely to be relatively nontoxic. Echinocandins are a class o anti ungal agents that target ungal cell wall synthesis by noncompetitively inhibiting the synthesis o -(1,3)-D-glucans. Disruption o cell wall integrity results in osmotic stress, lysis o the ungal cell, and ultimately ungal cell death. The three anti ungal agents in the echinocandin class are caspofungin, micafungin, and anidulafungin; all are semisynthetic lipopeptides derived rom natural products. The echinocandins have in vitro and in vivo anti ungal activity against Candida and Aspergillus species. All three echinocandins are ungicidal against Candida species, including C. krusei and C. glabrata, and ungistatic against Aspergillus species. They have poor activity against zygomycetes. All three agents are available only in parenteral orm because they are insu f ciently bioavailable or oral use. Caspofungin was the f rst echinocandin to be approved. The drug is used as primary therapy or esophageal candidiasis and candidemia, as salvage therapy or Aspergillus in ections, and as empiric therapy or ebrile neutropenia. Like the other echinocandins, caspo ungin is highly protein-bound (97%) in the plasma; it is metabolized in the liver via peptide bond hydrolysis and N-acetylation; and it penetrates poorly into the CSF (although animal data indicate that the echinocandins do have some activity in the CNS). Caspo ungin does not require dose adjustment or renal insu f ciency, but dose adjustment is required or patients with moderate hepatic dys unction. Because coadministration with cyclosporine signif cantly increases plasma concentrations o caspo ungin and elevates liver unction enzymes, this drug combination is generally not recommended unless the expected benef ts outweigh the risks. Similarly, co-administration with tacrolimus signif cantly increases plasma concentrations o tacrolimus. To achieve therapeutic plasma concentrations, caspo ungin dosing may need to be increased in patients receiving nelf navir, e avirenz, phenytoin, ri ampin, carbamazepine, or dexamethasone. Micafungin is approved or the treatment o esophageal candidiasis and as anti ungal prophylaxis or recipients o hematopoietic stem cell transplants. It is also e ective against

candidemia and pulmonary aspergillosis. Anidulafungin is approved or the treatment o esophageal candidiasis and candidemia. Several small case series have reported the use o echinocandins in combination with amphotericin B, ucytosine, itraconazole, or voriconazole in patients with re ractory ungal in ections. Aminocandin is an investigational echinocandin with a spectrum o activity similar to that o the other echinocandins. It has a hal -li e three old to our old greater than that o the other echinocandins, thus permitting less requent administration. Echinocandins are generally well tolerated; their adversee ect prof le is comparable to that o uconazole. Because echinocandins contain a peptide backbone, symptoms related to histamine release can be observed (see “Suggested Reading”). Other adverse e ects include headache, ever (more common with caspo ungin), rash, abnormal liver unction tests, and, rarely, hemolysis.

Chelator of Polyvalent Cations: Ciclopirox Ciclopirox is a synthetic hydroxypyridone anti ungal agent

whose mechanism o action is poorly understood. In experimental studies, the drug chelates the polyvalent cations Fe 3 and Al 3. Chelation o these ions inhibits numerous metal-dependent enzymes responsible or electron transport, DNA and RNA synthesis, energy production, catalase activity, and peroxide degradation within ungal cells. Ciclopirox also demonstrates mild anti-in ammatory properties that may be due to inhibition o 5-lipoxygenase and cyclooxygenase. Ciclopirox is approved or the treatment o seborrheic dermatitis, tinea versicolor, tinea corporis, tinea pedis, cutaneous candidiasis, and onychomycosis. It is administered as a topical cream, gel, lotion, shampoo, or lacquer. No serious adverse e ects have been associated with topical use o ciclopirox. Common adverse e ects include a burning sensation with application, pruritus, and contact dermatitis.

CONCLUSION AND FUTURE DIRECTIONS The development o anti ungal agents has progressed signif cantly since the introduction o amphotericin B. As the population o immunocompromised patients increases, opportunistic ungal in ections that are resistant to conventional anti ungal therapy pose new challenges to researchers and clinicians. For example, new anti ungal therapy is greatly needed in the treatment o zygomycosis. E ective topical anti ungal agents are eagerly sought or the treatment o nail and hair dermatophytosis, because oral therapies or these superf cial ungal in ections carry risks such as hepatotoxicity. The development o protease inhibitors and phospholipase inhibitors represent new rontiers in the treatment o Candida and Cryptococcal species, respectively. As novel and unique molecular targets are identif ed in ungal pathogens, newer anti ungal agents will be developed with the goal o minimizing mechanism-based (“on-target”) toxicity while expanding anti ungal spectrum o action.

Acknowledgment We thank Lorne W. Murray, Robert H. Rubin, Charles R. Taylor, and Ali Alikhan or their valuable contributions

C h a p t e r 3 6 Pharmacology of Fungal Infections

to this chapter in the First, Second, and Third Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Gauwerky K, Borelli C, Korting HC. Targeting virulence: a new paradigm for antifungals. Drug Discov Today 2009;14:214–222. (Discusses virulence factors of fungi and their inhibitors, with an emphasis on new options for antifungal development, including inhibitors of the secreted aspartyl protease of C. albicans.) Miceli MH, Kauffman CA. Isavuconazole: a new broad-spectrum triazole antifungal agent. Clin Infect Dis 2015;61:1558–1565. (Discusses the use of this recently approved agent.) Naeger-Murphy N, Pile JC. Clinical indications for newer antifungal agents. J Hosp Med 2008;4:102–111. (Discusses the use of echinocandins and triazoles in several common and/or important clinical situations.)

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Ostrosky-Zeichner L, Casadevall A, Galgiani JN, Odds FC, Rex JH. An insight into the antifungal pipeline: selected new molecules and beyond. Nat Rev Drug Discov 2010;9:719–727. (Discusses development of polyenes, azoles, echinocandins, and investigational antifungal drugs, including vaccines and antibody-based immunotherapy.) Patterson TF. Advances and challenges in management of invasive mycosis. Lancet 2005;366:1013–1025. (Focused discussion of fungal pathogens that occur in immunocompromised hosts and management strategies for these opportunistic pathogens.) Ruiz-Herrera J, Elorza MV, Valentin E, Sentandreu R. Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity. FEMS Yeast Res 2006;6:14–29. (Comprehensive review of the fungal cell wall.) Scher RK, Nakamura N, Tavakkol A. Luliconazole: a review of a new antifungal agent for the topical treatment of onychomycosis. Mycoses 2014;57:389–393. (Discusses the development and therapeutic potential of luliconazole, the latest FDA-approved antifungal agent.)

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C h a p t e r 3 6 Pharmacology o Fungal In ections 673

P la s modium m foo oodd va cuo c uolle

37

ADP AD P

Hee m oglob b in

Pharmacology of Parasitic Infections ATP

Prot P roteo eolytic enzymes Plasmepsins F Falcipain F Falcilysin

P rotonn ATP Pa s e

H+

Chllo ro quine Ch

Lo u is e C. Ive rs a n d Ed w a rd T. Rya n

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+ Fe rriprotoporphyr y in IX X (hee m (h mee )

Proo to to nate n d ro armacol quine i INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674 c hlooPro logyy of An Antiprotozoal Agents . . . . . . . . . . . . . . . . . . 683

MALARIAL PLASMOD DIA & CASES 1–22 . . . . . . . . . . . . . . . . . 674–67 74 75 Physiology gy off Ma Mala la rial i Plasmodia . . . . . . . . . . . . . . . . . . . . . . . 674 Life Li fe Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66744 Heme Metabolism m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 676 Electron Transport Chainn . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676 76 Ph harma maco colo logy gy of An Anti timalarial Agents . . . . . . . . . . . . . . . . . . . 6777 Inhi hibbitors of He H me Metab etabol olis ism. . . . . . . . . . . . . . . . . . . . . . . 677 Inhibitors of Electron Traa ns nspo port rt. . . . . . . . . . . . . . . . . . . . . . . 6799 Inhibitors of Translationn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680 Inhibitors of Fo Fola late te Met etab abol olis ism. . . . . . . . . . . . . . . . . . . . . . . 681 Antimalarial Drug Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . 681 OTHER PROTOZOA & CASE 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682 Physiology of Luminal Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . 682 Life C Lif Cycle l off E Entamoeba b hi histolytica l i . . . . . . . . . . . . . . . . . . . 682 Fermentation Pathwayss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682

INTRODUCTION More than one billion people worldwide are in ected with parasites. Parasites o medical importance include protozoa (such as the organisms that cause malaria, toxoplasmosis, giardiasis, amebiasis, leishmaniasis, and trypanosomiasis) and helminths (“worms”). Worms that in ect humans include cestodes (“f atworms” or “tapeworms,” such as the worm that causes taeniasis), nematodes (“roundworms,” which cause lariasis, strongyloidiasis, and ascariasis), and trematodes (“f ukes,” such as the worms that cause schistosomiasis). Ideally, antiparasitic drugs should be targeted to structures or biochemical pathways present or accessible only in parasites. Many antiparasitic drugs act by unknown or poorly de ned mechanisms o action, however. This chapter ocuses on a number o the better de ned agents, including those active against Plasmodia species (which cause malaria), Entamoeba histolytica (which causes amebiasis), and Onchocerca volvulus (which causes onchocerciasis, a larial in ection re erred to as river blindness). In each o these cases, antiparasitic agents inter ere with metabolic requirements o the parasite: the dependence o malarial plasmodia on heme metabolism, the dependence o luminal parasites on speci c ermentation pathways, and the dependence o helminths on neuromuscular activity. These three examples 674

Metroniddaz azole o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683 Tini Ti nida dazo da zole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684 Nitazoxanidee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684 Other Antiprotozoal ro Agentss . . . . . . . . . . . . . . . . . . . . . . . . . . 684 He mozoin moz mozoo4 HELMINTHS & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 (polyme (pol (po ymee rri rize i e d hhee me ) Physiology of Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 Life Cyc y le of On Onch choc ch ocer erca ca volvu llvulu ulus . . . . . . . . . . . . . . . . . . . . 686 Neu Ne N uromuscular Activityy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686 Ph harmacology arma maco colo logy gy of of Antihelmin Anti An tihe h lm lminthic m Agents . . . . . . . . . . . . . . . . . 686 Agentss That Th t Interru Interrupt Int Neuromuscular Activityy . . . . . . . . . 68 6866 Other Antihelminthic Agentss . . . . . . . . . . . . . . . . . . . . . . . . . 687 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 687 Sugg Su gges este tedd Re Read adin ingg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68 6888

are not all-inclusive but rather emphasize opportunities to use or design pharmacologic agents to interrupt metabolic requirements speci c to parasites.

MALARIAL PLASMODIA Each year, approximately 200 million individuals in more than 90 countries develop malaria, and over a hal million individuals die o malaria. Malaria is the most important parasitic disease o humans and one o the most important in ections o humans. Human malaria is caused by one o ve species o plasmodial parasites: Plasmodium falciparum, P. vivax, P. ovale , P. malariae , and P. knowlesi. The most serious type o malaria is that caused by P. falciparum.

Physiology of Malarial Plasmodia Life Cycle

The li e cycle o malaria involves a parasite, a mosquito vector, and a human host (Fig. 37-1). An Anopheles spp. mosquito can ingest sexual orms o malarial parasites (gametocytes) when taking a blood meal rom an in ected human. A ter usion o the male and emale gametocytes and maturation o the zygote within the mosquito, sporozoites are released rom an oocyst. The sporozoites, which migrate to the mosquito’s salivary glands, can be inoculated into the blood o another human host

C h a p t e r 3 7 Pharmacology o Parasitic In ections

675

Bin a ta , a 3-ye a r-o ld g irl livin g in a re m o te a re a o a ce n tra l A rica n co u n try, is in g o o d h e a lth w h e n , o n e d a y, s h e b e g in s to e e l h o t, h a s s w e a ts a n d s h a kin g ch ills , s to p s e a tin g , a n d b e co m e s in te rm itte n tly lis tle s s a n d le th a rg ic. S e ve ra l d a ys la te r, th e s e s ym p to m s clim a x in a s e izu re a n d co m a , p ro m p tin g Bin a ta ’s p a re n ts to ru s h h e r to th e lo ca l h e a lth ca re clin ic. In th e clin ic, th e u n co n s cio u s ch ild ’s n e ck is s u p p le , b u t s h e is e b rile to 103°F. He r lu n g s a re cle a r to a u s cu lta tio n , a n d th e re is n o ra s h . A s m e a r o Bin a ta ’s p e rip h e ra l b lo o d d is clo s e s P. fa lcip a ru m rin g tro p h o zo ite s in

a p p ro xim a te ly 10% o h e r e ryth ro cyte s . Bin a ta is g ive n th e o n ly a n tim a la ria l m e d icin e s a va ila b le a t th e clin ic, ch lo ro q u in e a n d p yrim e th a m in e /s u l a d o xin e ; h o w e ve r, th e ch ild d o e s n o t im p ro ve , a n d s h e d ie s w ith in 24 h o u rs .

Mr. G is a 36-ye a r-o ld m a rrie d s o twa re e n g in e e r w h o wa s b o rn a n d ra is e d in In d ia . He co m e s to th e Un ite d S ta te s a n d is co m p le te ly w e ll o r 6 m o n th s . He th e n b e g in s to e xp e rie n ce e p is o d e s o e ve rr, h e a d a ch e , a n d b o d y a ch e s . On e w e e k la te r, h e g o e s to h is p hys icia n , w h o e xa m in e s a s m e a r o Mr. G’s b lo o d , d ia g n o s e s m a la ria , a n d p re s crib e s ch lo ro q u in e o r tre a tm e n t. Th e ra p y w ith ch lo ro q u in e re s o lve s h is s ym p to m s co m p le te ly. Ho w e ve r, Mr. G

n o te s re cu rre n ce o e ve rs a n d th e o th e r s ym p to m s 3 m o n th s la te r a n d re tu rn s to h is d o cto r ’s o f ce .

during a subsequent blood meal. In the human, sporozoites leave the blood and multiply in the liver, orming tissue schizonts . This exoerythrocytic hepatic stage is asymptomatic. In a typical P. falciparum in ection, 1–12 weeks a ter the in ective bite, the liver cells release parasites into the bloodstream as merozoites . A single sporozoite can produce more than 30,000 merozoites. Merozoites invade erythrocytes, multiply asexually, and orm blood schizonts . This is the erythrocytic stage. In ected erythrocytes eventually rupture, releasing another generation o merozoites that continue the erythrocytic cycle. Rare merozoites also mature into gametocytes. Ingestion o these circulating gametocytes by an appropriate mosquito completes the li e cycle. The clinical symptoms o malaria, most distinctively ever, are caused by the intravascular lysis o erythrocytes and subsequent release o merozoites into the blood. The evers that Binata and Mr. G experienced were associated with these hemolytic episodes. Binata, un ortunately, developed cerebral malaria due to P. falciparum. P. falciparum-in ected erythrocytes express “knobs” on their sur ace that are composed o both host and parasite proteins. Parasite proteins include P EMP-1, a protein amily composed o approximately 100–150 gene products that mediate attachment o in ected erythrocytes to cellular receptors—including CD36, ICAM-1, ELAM-1, and chondroitin sul ate—on endothelial sur aces in the human host. This intravascular binding during a malarial episode occurs only during P. falciparum in ection and contributes to intravascular “sludging” o erythrocytes. Endothelial attachment lessens the amount o time during which in ected erythrocytes circulate systemically, thereby decreasing the likelihood that in ected erythrocytes will be cleared via splenic

Questions 1 . Why did Binata die? 2 . Why did Binata not improve a ter receiving antimalarial drugs? 3 . How o ten does a child die o malaria?

Questions 4 . What is a likely explanation or the return o Mr. G’s ever? 5 . How can Mr. G’s treatment be modif ed so that his illness will not return?

sequestration. Sludging also accounts, in large part, or the pathophysiology o malaria caused by P. falciparum. Sludging can a ect any organ, including the brain, lungs, and kidneys; damage to these organs leads to tissue hypoxia, ocal necrosis, and hemorrhage. In Binata’s case, the brain was involved (so-called cerebral malaria). Untreated, cerebral malaria is almost uni ormly atal, and, even with optimal treatment, cerebral malaria has a case atality rate exceeding 20%. Binata was treated with two drugs that have historically been quite important in treating individuals with malaria but which, un ortunately, are now ine ective in most places in the world because o widespread drug-resistant P. falciparum. Largely because o their low cost and availability, these drugs (chloroquine and a f xed combination o pyrimethamine and sul adoxine) were widely used in many developing areas o the world to treat older children and adults with partial immunity to malaria, but these agents have little clinical use in treating nonimmune individuals such as Binata. Due to the increasing ine ectiveness o these older agents, it is now recommended that individuals in sub-Saharan A rica with malaria be treated with an artemisinin derivative in combination with a second agent (see below). Un ortunately, Binata’s story has been all too common. On average, worldwide, a child dies o malaria every 75 seconds; o these deaths, more than 90% occur in sub-Saharan A rica, more than 90% occur in children under 5 years o age, and more than 95% are caused by P. falciparum in ection. No pharmacologic agent has yet been developed that inter eres with the recently elucidated role o P EMP-1 in the endothelial attachment o malarially in ected erythrocytes.

676 PRINCIPLES OF CHEMOTHERAPY

polymerize, it causes lysosomal membrane damage and toxicity to the malarial parasite. Quinoline antimalarials (see below) are believed to act by inhibiting heme polymerization, thereby creating an environment that is toxic to intraerythrocytic plasmodia.

Infe ction S porozoite s

Electron Transport Chain Live r

Circula tion

Me rozoite s

Tra ns mis s ion to mos quito

Malarial plasmodia also possess mitochondria with a tiny genome (approximately 6 kb) that encodes only three cytochromes (large protein complexes involved in electron transport and oxidative phosphorylation). These cytochromes, together with a number o mitochondrial-targeted proteins

As e xua l cycle

P la s modium food va cuole

Ga me tocyte s

FIGURE 37-1. Life cycle of malaria. Malarial plasmodia have a complex life cycle that relies on both humans and Anopheles spp. mosquitoes. Gametocytes from an infected human are transferred to a mosquito during a blood meal. In the mosquito stomach, a zygote forms and matures to become an oocyst on the outside wall of the stomach (not shown). Sporozoites released from the oocyst migrate to the salivary glands. During its next blood meal, the mosquito transfers Plasmodium spp. sporozoites from its saliva to another human. Sporozoites enter the host’s bloodstream and travel to the liver. Sporozoites replicate in the liver and then lyse infected hepatocytes, releasing merozoites into the circulation. Merozoites infect erythrocytes, undergoing asexual cycles of erythrocytic infection and lysis. Some merozoites differentiate into gametocytes, which can be ingested by another mosquito and thereby continue the cycle of infection. P. vivax and P. ovale can also form dormant hypnozoites, which can remain in infected hepatocytes for months to years before release into the circulation (not shown).

In Mr. G’s case, a peripheral blood smear showed P. vivax parasites inside his erythrocytes. Because P. falciparum and P. malariae in ections involve only one cycle o hepatic cell invasion, drugs that eliminate these species rom erythrocytes are usually su f cient to clear the in ection. Un ortunately, P. vivax and P. ovale also have dormant hepatic orms (hypnozoites ) that release merozoites over months to 1–2 years. There ore, individuals in ected with P. vivax or P. ovale should be treated with agents that are e ective against not only blood-stage plasmodia but also liver-stage parasites (see below). Because chloroquine does not eliminate hepatic orms o P. vivax and P. ovale, Mr. G’s P. vivax in ection recurred.

Heme Metabolism

Plasmodia have a limited capacity or de novo amino acid synthesis; instead, they rely on amino acids released rom ingested host hemoglobin molecules. Within red blood cells, plasmodia degrade hemoglobin in a digestive vacuole, which is an elaborate lysosome with an acidic pH (Fig. 37-2). Hemoglobin is sequentially degraded to its constituent amino acids by plasmodial aspartic proteases (plasmepsins), a cysteine protease ( alcipain), and a metalloprotease ( alcilysin). Degradation o hemoglobin releases protonated basic amino acids and a toxic heme metabolite, erriprotoporphyrin IX. Ferriprotoporphyrin IX is detoxif ed by polymerization to crystalline hemozoin. I erriprotoporphyrin IX does not

ADP

He moglobin ATP

Proteolytic enzymes Plasmepsins Falcipain Falcilysin

P roton ATP a s e

H+ Chloroquine

Amino a cids

P fCRT

+ Fe rriprotoporphyrin IX (he me ) Protonated chloroquine

He mozoin

(polyme rize d he me )

FIGURE 37-2. Proposed mechanisms of heme metabolism in the plasmodial food vacuole. Malarial plasmodia possess a specialized food vacuole that maintains an acidic intravacuolar environment by the action of a proton ATPase in the vacuolar membrane. Within the vacuole, human hemoglobin is used as a food source. Hemoglobin is proteolyzed to amino acids by several plasmodial-derived proteolytic enzymes, including plasmepsins, falcipain, and falcilysin. Protonated amino acids are then removed from the food vacuole through the PfCRT transporter. Degradation of hemoglobin also releases heme (ferriprotoporphyrin IX). Free ferriprotoporphyrin IX can react with oxygen to produce superoxide (O2 ); oxidant defense enzymes, which may include plasmodial-derived superoxide dismutase and catalase, convert the potentially cytotoxic superoxide to H2O (not shown). Plasmodia polymerize ferriprotoporphyrin IX into the nontoxic derivative hemozoin; evidence suggests that polymerization requires the activity of positively charged histidine-rich proteins (not shown). The iron moiety in ferriprotoporphyrin IX can also be oxidized from the ferrous (Fe 2 ) to the ferric (Fe 3 ) state, with concomitant production of hydrogen peroxide (H2O2). Many antimalarial agents are thought to disrupt the process of malarial heme metabolism; proposed mechanisms of drug action include inhibition of heme polymerization, enhancement of oxidant production, and reaction with heme to form cytotoxic metabolites. The inhibition of ferriprotoporphyrin IX polymerization by protonated chloroquine is shown.

C h a p t e r 3 7 Pharmacology o Parasitic In ections

Pharmacology o Antimalarial Agents

Dihydroorota te Orota te

DHOD (oxidized)

DHOD (reduced) H+

H+

e-

Outs ide Mitochondria l me mbra ne

677

Q

e-

e-

Cyt c

e-

Cyt bc1

Cyt c oxida s e

Ins ide

H+ Atovaquone

2e -+

2H+

+ 1/2O 2

H+

H2 O

FIGURE 37-3. The mitochondrial electron transport chain in plasmodia. The electron transport chain consists o a series o oxidation/reduction steps that culminate in the donation o electrons to oxygen, orming water. In plasmodia, the electron transport chain acts as an electron acceptor or reduced dihydroorotate dehydrogenase (DHOD), an enzyme that is essential or plasmodial pyrimidine synthesis. In this cascade, reduced ubiquinone (Q) trans ers electrons to the cytochrome bc 1 complex (Cyt bc 1), which then passes electrons to cytochrome c (Cyt c) and, f nally, to cytochrome c oxidase (Cyt c oxidase). In a 4-electron reduction o molecular oxygen (shown here as the half-reaction), cytochrome c oxidase donates electrons to oxygen to orm water. This chain o electron trans ers also involves the pumping o protons across the mitochondrial membrane by Cyt bc 1 and Cyt c oxidase; the resulting electrochemical gradient o protons is used to generate ATP (not shown). Atovaquone antagonizes the interaction between ubiquinone and the plasmodial cytochrome bc 1 complex, thereby disrupting pyrimidine synthesis by preventing the regeneration o oxidized DHOD.

derived rom the plasmodial nuclear genome, make up a rudimentary electron transport chain similar in organization to that ound in mammals (Fig. 37-3). In this electron transport chain, integral proteins o the mitochondrial inner membrane are reduced and then oxidized as they transport electrons rom one intermediate protein to another. The energy liberated by electron transport is used to drive proton pumping across the mitochondrial membrane, and the energy stored in the proton gradient drives ATP synthesis. In this electron transport chain, oxygen is the f nal electron acceptor, resulting in the reduction o oxygen to water. Plasmodia derive most o their ATP directly rom glycolysis and probably do not use mitochondrial electron transport as a signif cant source o energy. However, plasmodia do rely on electron transport or the oxidation o key enzymes involved in nucleotide synthesis. For example, dihydroorotate dehydrogenase (DHOD), the enzyme that mediates an early step in pyrimidine synthesis (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance), catalyzes the oxidation o dihydroorotate to orotate. As part o this reaction, DHOD is reduced, and the enzyme must be reoxidized be ore it can continue with another cycle o catalysis. Ubiquinone , an integral membrane protein located near the beginning o the electron transport chain, accepts electrons rom reduced DHOD, thus regenerating the oxidized orm o DHOD necessary or pyrimidine synthesis. Because plasmodia depend on de novo pyrimidine synthesis or DNA replication, interrupting the ability o ubiquinone to oxidize DHOD can disrupt plasmodial DNA replication (see below).

The currently available antimalarial agents target our physiologic pathways in plasmodia: heme metabolism (chloroquine , quinine , mef oquine , and artemisinin), electron transport (primaquine and atovaquone ), protein translation (doxycycline , tetracycline , and clindamycin), and olate metabolism (sul adoxine-pyrimethamine and proguanil). The ollowing section discusses the pharmacologic agents that target these pathways. Clinically, antimalarials can be classif ed into agents used or prophylaxis (to prevent malaria in individuals residing in or traveling through a malaria zone), agents used or treating individuals with acute blood-stage malaria, and agents used to eliminate hypnozoite liver-stage malarial in ections. Generally, agents used or prophylaxis must be well tolerated and easy to administer.

Inhibitors of Heme Metabolism

For many centuries, agents that disrupt intraerythrocytic malarial parasites have been the oundation o antimalarial treatment regimens. Most o these compounds are congeners o quinoline and, as a result, are all believed to possess similar mechanisms o action. Artemisinin, discussed at the end o this section, is also thought to act by inhibiting heme metabolism, although its structure is di erent rom that o the quinolines. Chloroquine

For the past 2,000 years, humans have used the roots o Dichroa ebri uga or the leaves o hydrangea in the treatment o individuals with malaria. More recently, the bark o the cinchona tree was ound to be a more e ective remedy. In all these plants, a quinoline compound is the pharmacologically active antiplasmodial agent. Chloroquine , a 4-aminoquinoline, was introduced in 1935 or use in the treatment o malaria. Chloroquine is a weak base that, in its neutral orm, reely di uses across the membrane o the parasite’s ood vacuole. Once inside the acidic environment o the vacuole, chloroquine is rapidly protonated, making it unable to di use out o the vacuole. As a result, protonated chloroquine accumulates to high concentrations inside the parasite’s ood vacuole, where it binds to erriprotoporphyrin IX and inhibits the polymerization o this heme metabolite. Accumulation o unpolymerized erriprotoporphyrin IX leads to oxidative membrane damage and is toxic to the parasite. Chloroquine thus poisons the parasite by preventing the detoxif cation o a toxic product o hemoglobin catabolism (Fig. 37-2). Chloroquine is concentrated by as much as 100- old in parasitized erythrocytes compared to unin ected erythrocytes. In addition, the concentration o chloroquine required to alkalinize lysosomes o mammalian cells is much higher than that needed to raise the pH in malarial ood vacuoles. There ore, chloroquine is relatively nontoxic to humans, although the drug commonly causes pruritus in darkly pigmented individuals, and it can exacerbate psoriasis and porphyria. Taken in supratherapeutic doses, however, chloroquine can cause vomiting, retinopathy, hypotension, conusion, and death. In act, chloroquine is used globally in suicides each year (largely because it is inexpensive, available, and toxic at high doses), and accidental ingestion by children can be atal. When initially introduced, chloroquine was a f rst-line drug used against all types o malaria; however, it is now

678 PRINCIPLES OF CHEMOTHERAPY

ine ective against most strains o P. falciparum in A rica, Asia, and South America (Fig. 37-4). Hypotheses regarding the mechanisms responsible or chloroquine resistance are based on the nding that chloroquine-resistant plasmodia accumulate less chloroquine inside ood vacuoles than chloroquine-sensitive plasmodia do. In the ood vacuole, protonated amino acids are generated by the parasite as it degrades hemoglobin. These protonated amino acids exit the lysosome by means o a transmembrane protein called PfCRT, encoded by pfcrt on P. falciparum chromosome 7. A number o mutations in P CRT have been associated with chloroquine resistance; or example, a substitution o threonine or lysine at position 76 (K76T) is highly correlated with chloroquine resistance. This mutated P CRT probably pumps protonated chloroquine out o the ood vacuole. This altered pump action could also be detrimental to the parasite, perhaps because o altered amino acid export and/or changes in vacuole pH. Many P. falciparum strains with mutations in pfcrt carry a second mutation in the gene pfmdr1 encoding Pgh1, a ood vacuole membrane protein involved in pH regulation. It is speculated that this second mutation provides a “corrective” action that allows chloroquine-resistant P. falciparum to continue growth in the presence o a pfcrt mutation. Strains o P. vivax with decreased susceptibility to chloroquine are now reported with increasing requency in areas o Papua New Guinea, Indonesia, and other ocal areas o Oceania and Latin America, although the exact mechanism o decreased susceptibility to chloroquine in these strains has not been ully established. Despite concerns regarding increasing resistance, chloroquine remains a drug o choice or treating most individuals with malaria caused by P. vivax, P. ovale, P. malariae, and P. knowlesi and by chloroquinesensitive strains o P. falciparum. It can also be used prophylactically to prevent malaria caused by sensitive strains o plasmodia.

Re s is ta nce to sulfadoxine— pyrimethamine

Re s is ta nce to chloroquine

Quinine and Quinidine Quinine is an alkaloid that consists o a quinoline ring linked

by a secondary carbinol to a quinuclidine ring. Its optical isomer, quinidine , has identical pharmacologic actions. Because o quinine’s structural similarity to other antimalarial quinolines, quinine is thought to attack plasmodia by the mechanism described above. Quinine has also been shown to intercalate into DNA through hydrogen bonding, thus inhibiting DNA strand separation, transcription, and translation. The overall e ect is a decrease in the growth and replication o the erythrocytic orm o plasmodia. Quinine and quinidine have been used to treat individuals with acute blood-stage malaria but are not used prophylactically. Use o quinine can cause cinchonism, a syndrome that includes tinnitus, dea ness, headaches, nausea, vomiting, and visual disturbances. Quinine and quinidine can also prolong the cardiac QT interval (see Chapter 24, Pharmacology o Cardiac Rhythm).

Mef oquine Mef oquine is a quinoline compound that is structurally re-

lated to other antimalarial agents. Unlike quinine, mef oquine does not bind to DNA. Its exact mechanism o action is unknown, although mef oquine appears to disrupt polymerization o heme in intraerythrocytic malarial parasites. Mef oquine has a number o adverse e ects, including nausea, cardiac conduction abnormalities (including bradycardia, prolongation o the QT interval, and arrhythmia), and neuropsychiatric e ects, including vivid dreams/nightmares, insomnia, anxiety, depression, hallucinations, seizures, and, rarely, psychosis. The mechanism(s) responsible or these adverse e ects is unknown. In 2013, the US Food and Drug Administration (FDA) issued a black box warning related to these neurologic and psychiatric adverse e ects. Mef oquine can be used both therapeutically and prophylactically. Strains o P. falciparum resistant to both chloroquine and mef oquine have been reported in areas o Southeast Asia.

Re s is ta nce to sulfadoxine— pyrimethamine

Re s is ta nce to sulfadoxine— pyrimethamine

Re s is ta nce to sulfadoxine— pyrimethamine, mefloquine, and halofantrine; incre a s ing re s is ta nce to artemisinin

FIGURE 37-4. Geographic distribution o drug-resistant Plasmodium falciparum. Historically, chloroquine was the drug o choice or prophylaxis and treatment o individuals with P. falciparum malaria. Un ortunately, P. falciparum is now resistant to chloroquine in most areas o the world (blue shading). In many areas, P. falciparum is also resistant to other antimalarial agents, including sul adoxine–pyrimethamine, mef oquine, and halo antrine. (Halo antrine is associated with potentially lethal cardiac toxicity and is there ore seldom used.)

C h a p t e r 3 7 Pharmacology of Parasitic Infections

679

and a second agent with a longer hal -li e (re erred to as artemisinin combination therapy [ACT]). Combinations include artemether–lume antrine, artesunate–mef oquine, artesunate– amodiaquine, and dihydroartemisinin–piperazine. Oral, parenteral, and rectal suppository ormulations are available. The WHO now recommends that ACT should be used as the rst-line treatment or chloroquine-resistant P. falciparum malaria. In comparison to quinine, artesunate is superior and is associated with a decreased risk o death, more rapid parasite clearance, and lower incidence o adverse events. In vitro resistance to artemisinin has been associated with mutations in the parasite calcium pump P ATP6 (see above). Un ortunately, increasing resistance to artemisinin has now been reported in a number o countries in Southeast Asia, although clinical response to ACT remains acceptable. Overall, artemisinin and its derivatives are better tolerated than most other antimalarial agents. In laboratory animals, intramuscular injection o oil-based ormulations o artemisinin has been shown to cause brainstem neuropathy; this potentially lethal e ect has not been observed in humans, but some studies have ound evidence suggesting that artemisinins may be associated with auditory impairment and other neurotoxic e ects in humans. Hypoglycemia occurs less o ten than with quinine-based therapy. Sa ety data in pregnancy are lacking.

Artemisinin Artemisinin, rom the wormwood plant Artemisia annua, has

been used in China (where it is known as qinghao) or centuries in the treatment o individuals with ever. Artemisinin derivatives have now become the rst-line drug or treating individuals with alciparum malaria. The compound is both a sesquiterpene lactone and a cyclic endoperoxide. When activated by ree or heme-bound iron, it orms a carbon-centered ree-radical compound (Fig. 37-5). This ree radical has the ability to alkylate many proteins as well as heme. The mechanism o speci city o the drug or plasmodia-in ected erythrocytes is unknown—potential sources o speci city include artemisinin’s requirement or heme or ree radical ormation and artemisinin’s pre erential accumulation in plasmodia. Drug action may relate to ree radical production in the ood vacuole o the parasite and subsequent inhibition o P ATP6, the parasite Ca2 ATPase that is the ortholog o the mammalian SERCA calcium pump (see Chapter 25, Pharmacology o Cardiac Contractility). Administration o artemisinin and its derivatives (artesunate , artemether, dihydroartemisinin) is associated with a rapid decrease in the level o malaria parasites in the blood o an in ected individual and rapid resolution o symptoms in patients with blood-stage malaria. Unlike many o the other antimalarials, artemisinins a ect blood-stage gametocytes and thus can decrease transmission o malaria rom an in ected human. Artemisinin is not e ective as a prophylactic agent against malaria. Due to the short hal -li e o artemisinins and the subsequent risk o recrudescence o malaria, and to decrease the likelihood o drug resistance, the World Health Organization (WHO) strongly recommends against use o artemisinins as monotherapy. Artemisinins should be used as xed combinations, usually including a rapidly acting artemisinin

Inhibitors of Electron Transport

Although the electron transport chain is a ubiquitous eature o eukaryotic cells, two agents have been developed that appear to selectively interrupt the plasmodial electron transport chain. This selectivity is due to di erent molecular structures o the same biochemical target rather than the presence o a unique enzymatic pathway in plasmodia Drug-heme adduct

Fe

Fe

He me

Activa tion

Artemisinin

Alkyla tion

Fe (fre e or he me -bound)

Arte mis inin (fre e ra dica l or e le ctrophillic inte rme dia te )

H O O

Drug-protein adduct

O H

H

O

P rote in O

FIGURE 37-5. Proposed mechanism of action of artemisinin. Artemisinin is a cyclic endoperoxide that forms a free radical after activation by iron (Fe). The mechanism of action of artemisinin is not known with certainty but may involve alkylation of macromolecules such as heme and proteins, resulting in the formation of artemisinin–heme adducts and artemisinin–protein adducts that are toxic to plasmodia. One such adduct may involve PfATP6, a parasite Ca 2 ATPase (not shown).

680 PRINCIPLES OF CHEMOTHERAPY

(see Chapter 33, Principles o Antimicrobial and Antineoplastic Pharmacology). Primaquine Primaquine was approved in 1952 or the treatment o ma-

laria. Because primaquine attacks the hepatic stage o malaria caused by P. vivax and P. ovale, it is used to prevent recrudescence o these in ections and is the only standard drug currently available or this use. In Mr. G’s case, primaquine can be prescribed to clear the hepatic stage o his malaria and prevent recurrence o his symptoms. Primaquine severely disrupts the metabolic processes o plasmodial mitochondria. The antimalarial activity is probably attributable to quinone , a primaquine metabolite that inter eres with the unction o ubiquinone as an electron carrier in the respiratory chain. Another potential mechanism o action involves the ability o certain primaquine metabolites to cause nonspecif c oxidative damage to plasmodial mitochondria. Primaquine is predominantly used to clear hepatic hypnozoites rom individuals with malaria caused by P. vivax or P. ovale. Strains o P. vivax have intrinsic variability in their susceptibility to primaquine. For example, the Chesson strain, f rst isolated rom an American soldier in Papua New Guinea in the 1940s, is less susceptible than other strains to primaquine. Because o this variability, an increased dose o primaquine (compared to the most common dose administered historically) is now recommended as standard treatment. Primaquine may also be used as a prophylactic agent. Individuals with glucose-6-phosphate dehydrogenase (G6PD) def ciency have a limited ability to protect their erythrocytes against oxidative damage. G6PD is needed to reduce NADP to NADPH, which converts oxidized glutathione to reduced glutathione. Reduced glutathione protects erythrocytes by catalyzing the breakdown o toxic oxidant compounds. The administration o primaquine causes signif cant oxidative stress because o the ormation o numerous oxidized compounds. As a result, primaquine can induce massive and potentially atal hemolysis in individuals with G6PD def ciency. There ore, primaquine should never be administered to an individual without f rst conf rming adequate G6PD activity in that individual’s erythrocytes. Primaquine should never be administered to pregnant women, because primaquine crosses the placenta and can induce atal hemolysis in etal erythrocytes independent o maternal G6PD status. Primaquine can also cause gastrointestinal disturbances, methemoglobinemia, and, very rarely, neutropenia, hypertension, arrhythmias, and neurological symptoms.

Atovaquone Atovaquone is a structural analogue o ubiquinone, the shut-

tling protein in the electron transport chain. Under physiologic conditions, the trans er o two electrons rom reduced ubiquinone to the cytochrome bc1 complex oxidizes the ubiquinone (Fig. 37-3). Atovaquone inhibits the interaction between reduced ubiquinone and the cytochrome bc1 complex and thereby disrupts electron transport. Because plasmodia depend on the electron transport chain to regenerate oxidized dihydroorotate reductase, treatment with atovaquone disrupts pyrimidine synthesis and thereby prevents plasmodia rom replicating their DNA. It is likely that inhibition o the electron transport chain also disrupts other steps in the intermediary metabolism o the parasite that depend on oxidation/reduction cycling o proteins.

The cytochrome bc1 complex is a ubiquitous eature o eukaryotic organisms. The selectivity o atovaquone or plasmodia likely relies on di erences in the sequences o amino acids between human and plasmodial ubiquinone–cytochrome bc1 binding regions. Atovaquone inhibits the activity o plasmodial cytochrome bc1 with approximately 100- old selectivity compared to the human orm o the protein. However, this selectivity is easily disrupted; a single point mutation in the cytochrome bc1 complex can render plasmodia resistant to atovaquone. For this reason, atovaquone is not used as a single agent. Atovaquone can be co-administered with doxycycline , a protein synthesis inhibitor, or as a f xed combination with proguanil, a dihydro olate reductase inhibitor (see discussion below). Proguanil and atovaquone are synergistic in their antimalarial activity. Interestingly, this synergy may not be related to proguanil’s action as an anti olate, because other inhibitors o dihydro olate reductase do not have synergistic e ects with atovaquone. Instead, when administered with atovaquone, proguanil may act as an uncoupling agent in mitochondrial membranes, thereby enhancing atovaquonemediated mitochondrial depolarization. Atovaquone is generally well tolerated; its use is associated with a low incidence o adverse gastrointestinal e ects and an occasional rash. In combination with a second antimalarial drug, atovaquone can be used both therapeutically and prophylactically.

Inhibitors of Translation Doxycycline, Tetracycline, and Clindamycin

Agents that disrupt parasite protein synthesis include doxycycline , tetracycline , and clindamycin. Doxycycline is a structural isomer o tetracycline and is produced semisynthetically rom oxytetracycline or methacycline. Doxycycline inhibits parasite protein synthesis by binding to the 30S ribosomal subunit, thereby blocking the binding o aminoacyl tRNA to mRNA (see Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation). By virtue o its high lipophilicity, doxycycline penetrates well into body tissues, has a large volume o distribution, and is reabsorbed in renal tubules and the gastrointestinal tract, resulting in a long hal -li e. Doxycycline’s oral bioavailability and long hal -li e make it a use ul drug (in combination with artesunate or quinine) or the treatment o chloroquine-resistant P. alciparum. Doxycycline should not be used as a single antimalarial agent. Adverse e ects include cutaneous photosensitivity, tooth discoloration in children, and vaginal candidiasis; gastrointestinal e ects (including nausea, diarrhea, and dyspepsia) are typically mild, although esophageal ulceration can occur rarely. Tetracycline and doxycycline have similar pharmacologic prof les, but tetracycline must be taken our times a day. Tetracycline may be used in combination with quinine or the treatment o chloroquine-resistant malaria; however, it is not recommended as a malaria chemoprophylactic. Clindamycin inhibits protein synthesis by binding to the 50S ribosomal subunit (see Chapter 34). Clindamycin may be used in combination with artesunate or quinine or the treatment o malaria when tetracycline or doxycycline is contraindicated ( or example, in pregnant women or children less than 8 years o age). Clindamycin is usually well tolerated, especially in children; its major adverse e ect is an increased risk o antibiotic-associated diarrhea caused by Clostridium di f cile. Clindamycin is not used as a malaria chemoprophylactic.

C h a p t e r 3 7 Pharmacology of Parasitic Infections

Inhibitors of Folate Metabolism

Folic acid is a vitamin involved in the trans er o one-carbon units in a variety o biosynthetic pathways, including those o DNA and RNA precursors and certain amino acids (see Chapter 33). In humans, olate is an essential vitamin and must be ingested in the diet. In parasites and bacteria, olate is synthesized de novo, providing a use ul target or selective drug action. Inhibition o olate metabolism can result in success ul treatment o parasitic in ections. In the context o malaria, anti olate drugs act against parasite-speci c iso orms o dihydropteroate synthetase and dihydro olate reductase. Combination therapies that include a sul onamide and pyrimethamine were historically used. Two antimalarial ormulations are available, sulfadoxine–pyrimethamine and the less requently used sulfalene–pyrimethamine . Sulfadoxine–Pyrimethamine Sulfadoxine is a para-aminobenzoic acid (PABA) analogue

that competitively inhibits parasite dihydropteroate synthetase, an essential enzyme in the olic acid synthesis pathway. Pyrimethamine is a olate analogue that competitively inhibits parasite dihydro olate reductase, the enzyme that converts dihydro olate to tetrahydro olate (see Figs. 33-6 and 33-7). In combination, sul adoxine and pyrimethamine act synergistically to inhibit growth o the malarial parasite. Sul onamide–pyrimethamine combinations were initially highly e ective against blood schizont stages o P. falciparum malaria, but not against gametocytes, and were less e ective against other species o malaria. Both drugs are highly protein-bound, resulting in long elimination hal lives. The long hal -li e o the combination provided selective pressure or the development o drug resistance in areas with high-level malaria transmission, and increasing resistance to this combination made it ine ective or treatment and prophylaxis in many parts o the world (Fig. 37-4). Individuals in ected with sensitive strains o malaria may be treated with sul adoxine–pyrimethamine as a convenient single dose. The most serious drug reactions involve hypersensitivity to the sul onamide component o the combination. Severe skin reactions such as Stevens-Johnson syndrome or erythema multi orme have been reported, but the incidence o these adverse e ects is rare a ter single-dose therapy or malaria. Adverse hematologic e ects include megaloblastic anemia, leukopenia, and thrombocytopenia. Sul onamide–pyrimethamine is not used as a chemoprophylactic agent against malaria.

Proguanil Proguanil is a derivative o pyrimidine and, like pyrimethamine,

is an inhibitor o dihydro olate reductase. Proguanil acts against the hepatic, pre-erythrocytic orms o P. falciparum and P. vivax. Proguanil has been used or prophylaxis in combination with chloroquine in areas o the world where chloroquine resistance is not widespread. However, other prophylactic agents are signi cantly more e ective, and this combination is not recommended. Proguanil may be used in a synergistic combination with atovaquone or both treatment and prevention o malaria (discussed above). Proguanil is usually well tolerated, but it has been associated with oral ulcerations, pancytopenia, thrombocytopenia, and granulocytopenia.

Antimalarial Drug Resistance Antimalarial drug resistance is a major public health problem and a signi cant barrier to the e ective treatment o

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individuals with malaria. In association with the collapse o e ective prevention e orts, lack o political will, and socioeconomic actors, the waning e cacy o antimalarial drugs contributed signi cantly to the increased burden o malaria morbidity and mortality observed worldwide rom the 1980s to the early 2000s. Chloroquine was the standard therapy or treating individuals with malaria or many years a ter its introduction in 1946. Resistance was rst reported in the 1950s and steadily increased since then; at present, resistance has been reported everywhere in the world except on the island o Hispaniola and in ocal parts o Central America, South America, and Asia. The chloroquine-resistance P. falciparum haplotype has recently been detected in Haiti, but clinical resistance has not yet been reported. Childhood mortality doubled in eastern and southern A rica in the 1980s and 1990s as chloroquine and sul adoxine–pyrimethamine resistance increased; chloroquine resistance was associated with an overall doubling o childhood mortality rom malaria during this period, with increases as high as 11- old in certain areas. Similarly, P. vivax resistance to chloroquine was unknown until 1989 but is now endemic in Indonesia and Papua New Guinea. Reports o chloroquine-resistant P. vivax have also emerged in South America, Brazil, Myanmar, and India. Resistance to sul adoxine–pyrimethamine was reported a ter the combination was introduced in 1971 as a second-line therapy or treating individuals with chloroquine-resistant P. falciparum. Resistance to sul adoxine– pyrimethamine was initially reported in Southeast Asia but is now relatively widespread in South America and prevalent in A rica as well. Strains o P. falciparum resistant to mef oquine were noted in Southeast Asia ollowing the widespread introduction o this agent in the 1980s. Mef oquine resistance has not spread more widely as yet, in large measure due to the act that the drug is not now routinely used to treat individuals with malaria. Strains o P. falciparum with increasing resistance to artemisinin were rst reported in Cambodia and have also been reported in Vietnam, Myanmar, and Thailand. Many actors contribute to the development o drug resistance by malaria parasites, including inappropriate and/or unsupervised drug use, inconsistent drug availability, poor adherence to treatment regimens due to adverse e ects and other actors, inconsistent quality o drug manu acturing, presence o counter eit drugs, and prohibitive drug costs. Combining therapies to reduce the development o resistance is a strategy that has long been employed in the treatment o individuals with tuberculosis, leprosy, and HIV in ection, and this approach is strongly recommended in the treatment o individuals with malaria. The WHO has demanded cessation o production o all stand-alone artemisinin products and has requested that only two-drug, xed-combination, artemisinin-containing products be manu actured. Although rapidly acting artemisinins reduce parasite burden by a actor o 104 with every treatment cycle, resulting in rapid clearance o parasites rom the bloodstream, the short hal li e o artemisinins avors the possibility o recrudescence o in ection and the risk o selective pressure or drug resistance. To counter these risks, the WHO recommends combining an artemisinin with a slowly eliminated blood schizonticidal agent.

682 PRINCIPLES OF CHEMOTHERAPY

Mr. S , a 29-ye a r-o ld Am e rica n jo u rn a lis t, re tu rn s ro m a trip to S o u th e a s t As ia . He e e ls f n e o r 5 w e e ks b u t th e n b e g in s to e xp e rie n ce m ild d ia rrh e a , a b d o m in a l p a in , a n d m a la is e . He d o e s n o t a ttrib u te h is s ym p to m s to th e trip , b e ca u s e th e y d e ve lo p e d w e ll a te r h e re tu rn e d h o m e . Fu rth e rm o re , Mr. S ’s w i e s h a re d th e s a m e o o d a n d wa te r d u rin g th e trip , a n d s h e re m a in s w e ll. As a re s u lt, Mr. S ig n o re s th e s ym p to m s o r a w e e k, b u t h e e ve n tu a lly g o e s to h is p hys icia n w h e n th e s ym p to m s d o n o t a b a te s p o n ta n e o u s ly. Phys ica l e xa m in a tio n re ve a ls te n d e rn e s s in th e rig h t u p p e r q u a d ra n t o th e a b d o m e n . Blo o d te s ts a re n o ta b le o r e le va te d live r

OTHER PROTOZOA In addition to plasmodium, other medically important protozoa include Entamoeba histolytica, the organism that causes amebiasis; Giardia lamblia, the organism that causes giardiasis; Cryptosporidium hominis/parvum, the organism that causes cryptosporidiosis; Trypanosoma brucei rhodesiense and T. b. gambiense, the causative agents o A rican sleeping sickness; Trypanosoma cruzi, the causative agent o Chagas’ disease; and Leishmania spp., the causative agents o leishmaniasis. Because more is known about E. histolytica, the ollowing physiology section ocuses on this parasite; however, the pharmacology section includes not only agents e ective against amebiasis but also agents e ective against A rican sleeping sickness, Chagas’ disease, and leishmaniasis.

Physiology of Luminal Protozoa The enteric protozoa Entamoeba dispar and E. histolytica are morphologically indistinguishable, although these two species can be di erentiated using specif c monoclonal antibodies. E. dispar does not cause invasive disease (i.e., it does not compromise the gut epithelium), but E. histolytica can cause an asymptomatic carrier state, invasive colitis, or so-called metastatic in ections (usually hepatic abscesses). Five percent to 10% o individuals who live in poverty in the developing world have serologic evidence o previous E. histolytica in ection. It is estimated that 50 million cases o dysentery are caused by E. histolytica each year, resulting in tens o thousands o deaths. Because Mr. S’s wi e shared the same ood and water with her husband, she was also likely in ected with E. histolytica. For unclear reasons, she excreted E. histolytica asymptomatically, while her husband developed invasive disease.

Life Cycle of Entamoeba histolytica

Colonic in ection with E. histolytica occurs as a result o ingestion o cysts through the ecal–oral route, or example, drinking contaminated water. Whether intestinal invasion occurs may be a unction o the number o cysts ingested, the strain o the parasite, the motility o the host gastrointestinal tract, and the presence o appropriate enteric bacteria to

e n zym e s , a n d a co m p u te d to m o g ra p h y (CT) s ca n re ve a ls a live r a b s ce s s . S to o l e xa m in a tio n is p o s itive o r h e m e a n d o r E. h is to lytica cys ts . He is p re s crib e d m e tro n id a zo le o r 10 d a ys , a te r w h ich h e ta ke s p a ro m o m ycin o r a n a d d itio n a l w e e k. Fo llo w-u p im a g in g co n f rm s re g re s s io n o Mr. S ’s live r a b s ce s s .

Questions 6 . Why is Mr. S’s wife asymptomatic? 7 . Wha t a re the potentia l a dve rs e effe cts of metronidazole? 8 . Why was Mr. S prescribed paromomycin after a course of metronidazole?

serve as nourishment or the ameba. Disease results when active trophozoites invade the intestinal epithelium, and secondary spread to the liver can occur via the portal circulation (Fig. 37-6). As its name implies, E. histolytica lyses and destroys human tissue. Trophozoites typically multiply superf cial to the muscularis mucosae o the intestines and spread laterally. They may also penetrate more deeply, occasionally per orating the intestinal wall and spreading locally. Seeding o the liver is also common. In Mr. S’s case, a CT scan revealed involvement o the liver with ormation o an abscess. E. histolytica exists in two orms: the inactive but in ective cyst and the active trophozoite . Cysts are ingested in contaminated ood or water. Excystation occurs in the small intestine, where the trophozoites mature. The trophozoite orm is capable o invading host tissue. Inside the human body, the trophozoites move using pseudopods and ingest bacteria, other protozoa, and host red blood cells. A trophozoite can convert to a binucleated cyst orm, which matures into a tetranucleated cyst that travels through the colon but is not capable o mucosal invasion (Fig. 37-6). Symptoms due to amebiasis vary rom diarrhea and abdominal cramps to ulminant dysentery and hepatic abscess ormation. Fewer than 40% o individuals with amebic dysentery develop ever, and microscopic evaluation o the stool typically discloses ew neutrophils. The onset o symptoms can range rom a ew days to a year a ter exposure, or symptoms may never occur. Mr. S’s symptoms did not develop until at least a month a ter exposure, giving him reason not to attribute his symptoms to his travels.

Fermentation Pathways

E. histolytica and other luminal parasites are a diverse group o eukaryotes with novel adaptations to their anaerobic niche. For example, E. histolytica lacks fermentation enzymes (lactate dehydrogenase and pyruvate decarboxylase) that are present in yeast and other eukaryotes. Ameba also lack enzymes o oxidative phosphorylation, the Krebs cycle, and pyruvate dehydrogenase. Instead, ameba (and many anaerobic organisms) utilize novel enzymes to provide a source or the electron trans ers that drive metabolism. Ameba are obligate fermenters of glucose to ethanol (Fig. 37-7). Many o these ermentation enzymes, which are missing in humans, yeast, and most eubacteria, contain a set o

C h a p t e r 3 7 Pharmacology of Parasitic Infections Nucle us

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is extremely rare between bacteria and higher eukaryotes, which (except or parasites such as E. histolytica that share environmental niches with bacteria) maintain their gametes in a sterile environment.

Ka ryos ome

Pharmacology of Antiprotozoal Agents Excre tion in fe ce s

Cys t

Inge s tion by huma n of conta mina te d food or wa te r

P s e udopodium Encys ta tion in colon

Excys ta tion in s ma ll inte s tine

Nucle us

Va cuole

Trophozoite

As ymptoma tic coloniza tion

Inte s tina l a me bia s is

Inte s tina l pe rfora tion a nd/or live r a bs ce s s

FIGURE 37-6. Manifestations of amebiasis. Ingestion of Entamoeba histolytica cysts can result in several different clinical outcomes, ranging from asymptomatic excretion of the cysts to invasive disease. Asymptomatic infection occurs when the ingested cysts excyst (mature) in the small intestine but do not invade the intestinal mucosa. These trophozoites then encyst in the colon, and excretion occurs in the feces. Invasive disease results when active trophozoites invade the intestinal epithelium. This invasion can result in asymptomatic colonization, intestinal amebiasis (amebic dysentery)— which is characterized by diarrhea and abdominal cramps—or intestinal perforation. Spread of infection via the portal vein can cause liver abscesses.

iron–sul ur centers called ferredoxins that trans er electrons under strongly reducing (anaerobic) conditions. This activity is in contrast to that o heme and cytochromes, which use iron centers to trans er electrons under oxidizing (aerobic) conditions. Pyruvate-ferredoxin oxidoreductase (PFOR), which contains a single erredoxin domain, catalyzes the decarboxylation o pyruvate to acetyl CoA, with the production o CO2. PFOR activity also produces reduced erredoxin, which can reduce protons to orm hydrogen gas or reduce NADP to NADPH. Acetyl CoA is reduced to ethanol via alcohol dehydrogenase E (ADHE), with the recovery o two NAD co actors. Anaerobic bacteria (e.g., Helicobacter spp. and Clostridia spp.) express PFORs, erredoxins, and ADHEs similar to those o luminal protozoa. Indeed, phylogenetic analyses suggest that most o the genes encoding parasite ermentation enzymes, and many o the genes encoding parasite enzymes involved in core energy metabolism, have been laterally trans erred rom anaerobic bacteria. Although lateral gene trans er is extraordinarily requent between bacteria, it

Metronidazole

Metronidazole is inactive until it is reduced within host or

microbial cells that possess a large negative redox potential; such redox potentials are present in many anaerobic or microaerophilic luminal parasites. Activation can occur by interaction with reduced erredoxin or with speci c nitroreductases (Fig. 37-7). Activated metronidazole orms reduced cytotoxic compounds that bind to proteins, membranes, and DNA in target cells, causing severe damage. Metronidazole sensitivity is directly related to the presence of PFOR activity. Most eukaryotes and eubacteria lack PFOR and there ore ail to activate metronidazole. However, in poorly oxygenated tissues such as abscesses, metronidazole can be activated. Because PFOR is expressed in protozoa but has no counterpart in mammalian systems, the drug is selectively toxic or ameba and anaerobic organisms. The widespread use o metronidazole has led to drug resistance in Helicobacter pylori, a common bacterial cause o gastritis and peptic ulcers (see Chapter 47, Integrative Inf ammation Pharmacology: Peptic Ulcer Disease). This resistance is due to a null mutation in the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase. Low-level resistance to metronidazole has also been observed in a number o anaerobic protozoa, including trichomonads (caused by decreased expression o erredoxin), Giardia (caused by decreased PFOR activity and decreased drug permeability), and ameba (caused by increased expression o superoxide dismutase ). Metronidazole resistance among luminal parasites has not yet become clinically important, however. There are three explanations or the slow development o resistance to metronidazole among luminal parasites. First, luminal parasites are generally diploid, so a single mutation will not typically con er resistance. This contrasts with the case o haploid bacteria and certain haploid stages o P. falciparum, in which resistance develops more quickly. Second, luminal parasites have ew metabolic alternatives to PFOR activity. Third, metronidazole is hydrophilic, so overexpression or modi cation o P-glycoprotein, which con ers resistance to hydrophobic drugs, does not increase metronidazole e f ux. Adverse e ects o metronidazole include gastrointestinal discom ort, headaches, occasional neuropathy, a metallic taste, and nausea. Metronidazole also causes nausea and f ushing when taken concomitantly with alcohol (a socalled disul ram-like e ect, caused by inhibition o ethanol metabolism). Metronidazole is active against E. histolytica trophozoites in tissues, but it has much less activity against intraluminal ameba (probably, in large part, because o the drug’s extensive absorption in the upper gastrointestinal tract, leading to its low drug concentration in the lumen o the colon, where the ameba live). There ore, individuals with invasive amebiasis are typically treated rst with metronidazole (to eradicate trophozoites that are actively invading human tissue) and then with a second agent that has more intraluminal activity, such as iodoquinol or paromomycin. The latter two agents kill ameba by unknown mechanisms but are poorly absorbed rom the gastrointestinal tract and there ore reach high concentrations in the lumen o the colon.

684 PRINCIPLES OF CHEMOTHERAPY

PFOR-dependent activation P yruva te

Fe rre doxin

Nitroreductase-dependent activation Reduced metronidazole (a ctive )

PFOR Ace tyl CoA

Re duce d fe rre doxin

Metronidazole (ina ctive )

NADP + Nitroreductase NADP H

ADHE Etha nol

Ace ta te

FIGURE 37-7. Fermentation enzymes of anaerobic organisms and mechanisms of metronidazole activation. Anaerobic organisms metabolize pyruvate to acetyl CoA; this conversion is catalyzed by the enzyme pyruvate-ferredoxin oxidoreductase (PFOR). Acetyl CoA is then either hydrolyzed to acetate or oxidized to ethanol by alcohol dehydrogenase E (ADHE). Metronidazole is a prodrug; it contains a nitro group that must be reduced for the drug to become active. Reduced metronidazole is highly effective against anaerobic organisms, probably because of the formation of cytotoxic intermediates that cause DNA, protein, and membrane damage. Two aspects of anaerobic metabolism provide opportunities for selective reduction of the nitro group. First, the reaction catalyzed by PFOR results in the reduction of ferredoxin; reduced ferredoxin can then transfer its electrons to metronidazole, resulting in reduced (active) metronidazole and reoxidized ferredoxin. Second, many anaerobic organisms express nitroreductase enzymes that selectively reduce metronidazole and, in the process, oxidize NADPH to NADP .

Tinidazole

Tinidazole , a second-generation nitroimidazole related to

metronidazole, is also e ective against a number o protozoa and is licensed or the treatment o giardiasis, amebiasis, and vaginal trichomoniasis. Its mechanism o action is unclear but is believed to be similar to that o metronidazole and related to the generation o cytotoxic ree radicals. A particular benef t o tinidazole is that the duration o a therapeutic course o the drug is shorter than that o metronidazole. Tinidazole is also better tolerated than metronidazole, but it is similarly ine ective as a luminicidal agent or the treatment o ameba in ections. Adverse e ects are rare and mild, including gastrointestinal discom ort and the occasional development o a metallic taste. Tinidazole is not recommended or use during the f rst trimester o pregnancy, during breasteeding, and in children less than 3 years o age.

Nitazoxanide

Nitazoxanide is a nitrothiazolyl-salicylamide derivative

structurally related to metronidazole. Nitazoxanide has a broad spectrum o action, including activity against protozoa, anaerobic bacteria, and helminths. It is approved in the United States or use in children with giardiasis and in adults and children with cryptosporidiosis. As a structural analogue o thiamine pyrophosphate, nitazoxanide inhibits the PFOR that converts pyruvate to acetyl CoA in protozoa and anaerobic bacteria (Fig. 37-7). Its mechanism o action against helminths is unclear. A ter oral administration, nitazoxanide is rapidly hydrolyzed to the active metabolite tizoxanide. The active metabolite is excreted in urine, bile, and eces. Nitazoxanide is usually well tolerated with ew reported adverse e ects.

Other Antiprotozoal Agents

Pentamidine can be used to treat early-stage A rican try-

panosomiasis (A rican sleeping sickness), which is caused by Trypanosoma brucei gambiense and certain strains o T. b. rhodesiense . Early-stage trypanosomiasis is def ned as disease that does not involve the central nervous system (CNS).

Pentamidine inhibits DNA, RNA, protein, and phospholipid synthesis. The drug has a high a f nity or DNA in kinetoplasts (a DNA-containing organelle in certain protozoa), and it suppresses kinetoplast replication and unction. Kinetoplastida protozoa include Trypanosoma and Leishmania spp. Pentamidine may also inhibit dihydrofolate reductase . Some strains o Trypanosoma have a high-a f nity uptake system or the drug, contributing to its selectivity. Pentamidine can cause atigue, dizziness, hypotension, pancreatitis, and kidney damage. Pentamidine is now used most commonly as a third- or ourth-line treatment or individuals with Pneumocystis jiroveci (P. carinii) pneumonia (PCP), a common in ection in patients with AIDS. Suramin is another drug used to treat early-stage A rican trypanosomiasis. Suramin interacts with many macromolecules and inhibits numerous enzymes, including those involved in energy metabolism (e.g., glycerol phosphate dehydrogenase). It also inhibits RNA polymerase and thus inter eres with parasite replication. Suramin can cause pruritus, paresthesias, vomiting, and nausea. The biochemical basis or suramin’s relative selectivity or A rican trypanosomiasis is not well understood. Melarsoprol is used as a f rst-line drug in the treatment o late-stage A rican trypanosomiasis (i.e., disease that involves the CNS). Melarsoprol was developed by conjugating the heavy metal chelator dimercaptopropanol to the trivalent arsenic o melarsen oxide. The drug is insoluble in water and is instead dissolved in propylene glycol. Blood trypanosomes lack a unctional tricarboxylic acid cycle and are entirely dependent on glycolysis or ATP production. Melarsoprol inhibits trypanosomal pyruvate kinase, thereby inhibiting glycolysis and decreasing ATP production. A ected trypanosomes quickly lose motility and lyse. Melarsoprol also inhibits the uptake o adenine and adenosine by trypanosomal transporters. Mammalian cells are less permeable to the drug than are trypanosomes, and the drug has some selectivity on this basis. Un ortunately, melarsoprol is still quite toxic to humans (4–6% death rate). Melarsoprol is administered intravenously and can cause severe phlebitis.

C h a p t e r 3 7 Pharmacology of Parasitic Infections

It is also corrosive to plastics, limiting storage and administration options. In addition, 5–10% o individuals with latestage A rican trypanosomiasis develop intense inf ammation o the brain a ter administration o melarsoprol (reactive encephalopathy); this complication is associated with a mortality rate o greater than 50%. Concomitant administration o corticosteroids lessens the likelihood o reactive encephalopathy. Polyneuropathy a ter melarsoprol administration is also common (10%) and can be lessened by concomitant administration o thiamine. Ef ornithine ( -dif uoromethylornithine) is a much less toxic alternative to melarsoprol in the treatment o A rican trypanosomiasis caused by T. b. gambiense (West A rica sleeping sickness). Ef ornithine is highly e ective against both early- and late-stage West A rican sleeping sickness but not against East A rican trypanosomiasis (caused by T. b. rhodesiense). Ef ornithine is a selective and irreversible inhibitor o ornithine decarboxylase and thus o polyamine synthesis. Ornithine decarboxylase converts ornithine to putrescine; this is a rate-limiting step in the synthesis o putrescine and the polyamines spermine and spermidine. Polyamines are involved in nucleic acid synthesis and the regulation o protein synthesis. T. b. gambiense organisms are susceptible to ef ornithine, possibly because o the slow turnover o ornithine decarboxylase in these parasites; T. b. rhodesiense organisms have a higher rate o turnover (as do human cells) and are less sensitive. Ni urtimox is used in the treatment o New World trypanosomiasis (Chagas’ disease), which is caused by Trypanosoma cruzi. The drug undergoes reduction and generates toxic intracellular oxygen radicals in the parasite. It rst orms reduced intermediates such as nitro anion radicals. These radicals can then be reoxidized and, in the process, generate superoxide anions, which react with water to produce cytotoxic hydrogen peroxide. Some parasites, such as trypanosomes, lack catalase and other enzymes capable o degrading hydrogen peroxide. Such parasites are thus sensitive to the toxicity o nitro aromatic drugs. Mammalian cells are protected because o their complement o antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase. Ni urtimox can cause anorexia, vomiting, memory loss, sleep disorders, and seizures. Sodium stibogluconate and meglumine antimonate are used to treat leishmaniasis, which is caused by parasites o the genus Leishmania. These agents contain pentavalent antimony and act by an unknown mechanism. It is postulated that these drugs inhibit the glycolytic pathway and atty acid oxidation, processes that are crucial or intermediary

Th u m b i is a b o y w h o e n jo ys f s h in g in a rive r n e a r h is villa g e in th e De m o cra tic Re p u b lic o Co n g o . At th e a g e o 13, h e e m ig ra te s w ith h is a m ily to th e Un ite d S ta te s . S h o rtly th e re a te r, h e b e g in s to s cra tch h is a rm s a n d le g s vig o ro u s ly. S ix m o n th s la te r, h is m o th e r b rin g s h im to a d e rm a to lo g is t. Phys ica l e xa m in a tio n re ve a ls a m a cu la r a n d p a p u la r ra s h w ith e xco ria tio n s o n th e a rm s a n d le g s , a s w e ll a s a e w s u b cu ta n e o u s n o d u le s . Exa m in a tio n o p e rip h e ra l b lo o d d is clo s e s h ig h -le ve l e o s in o p h ilia .

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metabolism. Pentavalent antimony can also have many nonspeci c e ects, such as modi cation o sul hydryl groups. These drugs can cause bone marrow suppression, a prolonged QT interval, pancreatitis, and rash. Resistance o Leishmania to antimonial agents is being recognized with increasing requency, especially in South Asia. Alternative agents include amphotericin and milte osine . The mechanism o action o milte osine is unknown. It is a synthetic ether phospholipid analogue that is chemically similar to natural phospholipids present in cell membranes. Milte osine has been shown to have antineoplastic, immunomodulatory, and antiprotozoal activity. It is presumed that the cytostatic and cytotoxic e ects o milte osine are caused by inhibition o enzyme systems associated with plasma membranes (such as protein kinase C) and inhibition o phosphatidylcholine biosynthesis. Milte osine may also inhibit platelet activating actor-induced responses and inositol phosphate ormation. The immunomodulatory e ects o milte osine include T-cell activation, inter eron-gamma production in peripheral mononuclear cells, and increased interleukin-2 receptor and HLA-DR expression. The drug received FDA approval in 2014 and can be administered orally to treat patients with leishmaniasis. Use o milte osine may be associated with late clinical relapse o leishmaniasis.

HELMINTHS Helminths are multicellular worms with digestive, excretory, nervous, and reproductive systems. Parasitic helminths can in ect the liver, blood, intestines, and other tissues in human hosts. Clinically signi cant worms can be divided phylogenetically into three classes: nematodes (roundworms), trematodes (f ukes), and cestodes (tapeworms). The presence o a rudimentary nervous system provides a number o possible targets or antihelminthic agents. The physiology o Onchocerca volvulus , which causes onchocerciasis (“river blindness”), provides an example o potential targets or antihelminthic drugs. Although the majority o the ollowing discussion ocuses on the physiology and pharmacology o onchocerciasis, several other antihelminthic agents are also presented.

Physiology o Helminths Humans can become in ected with helminths when they ingest ood or water contaminated with eggs or larvae. In addition, larvae in soil can penetrate human skin, and insects can transmit still other larvae through bites. I humans are the de nitive host, the eggs or larvae develop into adult worms

A n o d u le is e xcis e d a n d e xa m in e d b y a p a th o lo g is t, le a d in g to a d ia g n o s is . Th u m b i b e g in s tre a tm e n t w ith ive rm e ctin b u t re tu rn s th e n e xt d a y e ve ris h a n d e e lin g m o re itchy th a n b e o re .

Questions 9 . What did the pathologist see in the subcutaneous nodule? 1 0 . Why did Thumbi feel worse immediately after treatment with ivermectin?

686 PRINCIPLES OF CHEMOTHERAPY

that can migrate through tissues and enter the sexual stage. During the sexual stage, adult worms release additional eggs or larvae, which can then pass out o the host through the gastrointestinal or urinary tracts. Larvae in humans can also be ingested by insects during a blood meal. In the environment or within vector hosts, eggs or larvae then become inective or humans, and the cycles start over.

Life Cycle of Onchocerca volvulus

Onchocerciasis is one o eight human larial in ections (a speci c type o nematodal worm in ection). In Thumbi’s case, an in ected Simulium spp. blackf y bit and inoculated O. volvulus larvae into his skin in A rica. Adult worms then developed in Thumbi’s subcutaneous tissues. These adult male and emale larial worms came to rest in subcutaneous nodules, in which they mated (Fig. 37-8). Adult worms are large (3–80 cm in length), look like angel-hair pasta, and can live or 10–15 years. The nodules have a characteristic appearance that was recognized by the pathologist. From these nodules (onchocercomata), gravid emales release millions o micro lariae, which migrate reely through the skin and cornea. I ingested by a Simulium f y, additional maturation can occur, and the cycle can continue. The diagnosis o onchocerciasis is usually based on microscopic detection o micro lariae in skin snips, not on pathological examination o excised onchocercomata. Micro lariae are small (200–400 m); as they degenerate and die, they cause local inf ammatory reactions, provoking itching, dermatitis, and, eventually, scarring. When micro lariae die in the cornea, they induce a punctate keratitis that, over years, leads to scarring and blindness. Such ocular involvement made onchocerciasis a leading cause o in ectious blindness in the world (along with trachoma) and is the reason onchocerciasis is also re erred to as “river blindness” (also ref ecting the act that the blackf ies carrying the larvae inhabit areas with f owing streams, such as the one in which Thumbi enjoyed shing). Without treatment, Thumbi could well become one o the thousands o individuals in the world who are currently blind or visually impaired rom onchocerciasis.

S tra tum corne um Epide rmis De rmis

Adult fila ria

S ubcuta ne ous nodule

S ubcuta ne ous s pa ce

Fa t ce lls

Microfila ria (in tis s ue s )

Ivermectin

Neuromuscular Activity

The subcuticular layer o longitudinal muscle in nematodes is inhibited by glutamate and gamma-aminobutyric acid (GABA) and excited by acetylcholine . The motor neurons o invertebrates are unmyelinated, making them more vulnerable to neurotoxins than the myelinated somatic motor neurons o humans. (See Chapter 9, Principles o Nervous System Physiology and Pharmacology, or more in ormation on the human nervous system.) Many antihelminthic agents modulate parasite neuromuscular activity by enhancing inhibitory signaling, antagonizing excitatory signaling (nondepolarizing block), or tonically stimulating excitatory signaling (depolarizing block).

Pharmacology of Antihelminthic Agents Agents That Interrupt Neuromuscular Activity Ivermectin Ivermectin is a semisynthetic macrocyclic lactone that acts

against a broad range o helminths and arthropods and that has been used most extensively to treat and control onchocerciasis. Ivermectin’s exact mechanism o action is unclear, but studies in Caenorhabditis elegans (a soil helminth that is studied extensively in eukaryotic biology as a simple model organism) suggest that the mechanism o action

Corne a l infla mma tion with s cle ros ing ke ra titis

De rma titis

FIGURE 37-8. Life cycle of Onchocerca volvulus. Adult f larial worms mate in subcutaneous nodules in humans, releasing microf lariae that migrate through the skin and subcutaneous tissues and cause dermatitis and pruritus as they die. Microf lariae that die in the cornea induce ocular in ammation, which can lead to corneal scarring and blindness (“river blindness”). Ivermectin, the agent o choice or treating individuals with onchocerciasis, is e ective only against microf lariae; the drug does not kill adult f larial worms.

involves potentiation and/or direct activation o glutamategated chloride channels in nematode plasma membranes. This results in hyperpolarization o neuromuscular cells and causes pharyngeal paralysis. (Note that the glutamate-gated chloride channels in nematodes mediate inhibitory neurotransmission, unlike the excitatory glutamate-gated cation

C h a p t e r 3 7 Pharmacology of Parasitic Infections

channels in humans.) Ivermectin is also thought to a ect GABA inhibitory transmission by potentiating the release o GABA rom presynaptic terminals, directly activating GABA receptors, and/or potentiating the binding o GABA to its receptor. All o these e ects increase GABA-mediated transmission o signals in peripheral nerves, resulting in hyperpolarization. The net e ect is variable, depending on the nematode model system under study, but the ultimate result is blockade of neuromuscular transmission and paralysis of the worm. Pharyngeal paralysis o O. volvulus inhibits nutrient uptake and kills developing larvae (micro lariae). Un ortunately, ivermectin does not kill adult larial worms. It does, however, destroy micro lariae in utero, thereby preventing production and release o new micro lariae rom adult emale worms or at least 6 months. Thus, ivermectin is used to prevent micro laria-mediated ocular damage and to decrease human-to-vector transmission (because micro lariae are inectious to Simulium f ies), but it cannot cure human hosts o O. volvulus in ection. Because the drug is noncurative, it is typically administered to in ected humans every 6–12 months or the li e expectancy o the adult worms (5–10 years). Ivermectin does interact with GABA receptors in vertebrates, but its a nity or invertebrate GABA receptors is about 100- old greater. Cestodes and trematodes lack higha nity ivermectin receptors, which may explain the resistance o these organisms to the drug. GABA receptors in humans are present mainly in the CNS, but because ivermectin does not cross the blood–brain barrier, the drug is generally well tolerated. When the blood–brain barrier is hyperpermeable, as in patients with meningitis, ivermectin can be more toxic and can result in headaches, ataxia, and coma. Adverse e ects o ivermectin are usually attributable to inf ammatory or allergic responses to dying micro lariae (i.e., Mazzotti reaction) and include headaches, dizziness, weakness, rash, pruritus, edema, abdominal pain, hypotension, and ever. This is why Thumbi elt worse the day a ter initiation o treatment. Ivermectin is widely used to treat animals with nematode in ections, and resistance to ivermectin is already recognized in livestock parasites. Although the exact mechanism o resistance is unknown, the P-glycoprotein may be involved. In studies o mice, hypersensitivity to ivermectin results rom disruption o the mdr1a gene, which encodes a P-glycoprotein membrane transporter. Furthermore, analysis o P-glycoprotein cDNA rom Haemonchus contortus (a nematode o veterinary importance) shows 65% homology to Pglycoprotein/multidrug resistance (MDR) protein sequences in mice and humans. P-glycoprotein mRNA expression is higher in ivermectin-selected strains o H. contortus than in unselected strains, and verapamil, which reverses multidrug resistance by blocking P-glycoprotein channels, increases the e cacy o ivermectin. Fortunately, clinically important resistance in humans has not yet been documented. In addition to its use in the treatment o onchocerciasis, ivermectin is used to treat strongyloidiasis and cutaneous larva migrans (both are nematodal in ections) and scabies (an ectoparasitic in estation). Piperazine and Pyrantel Pamoate Piperazine and pyrantel pamoate are antihelminthic agents o

primarily historical interest. They are discussed brief y in the Drug Summary Table.

687

Other Antihelminthic Agents

Albendazole , mebendazole , and thiabendazole inhibit tubulin

polymerization by binding to -tubulin. Evidence suggests that these agents are selective or the nematodal iso orm o -tubulin, thus decreasing host toxicity. Inhibition o tubulin polymerization disrupts nematodal motility and DNA replication (see Chapter 39), leading to degenerative changes in integumental and intestinal cells o helminths and, eventually, causing immobilization and death o the worms. The e ects o the drugs against immotile tissue orms o cestodal larval parasites (e.g., cysticercosis and echinococcosis) are less well understood but may also involve -tubulin binding. In this case, the drugs disrupt the integumental integrity o the protoscolex, a larval structure that eventually becomes the “head” o the adult cestode. Thiabendazole causes signi cant nausea, vomiting, and anorexia at therapeutic doses and is rarely used. Mebendazole and albendazole are better tolerated, and albendazole has the highest oral bioavailability o the three drugs. Praziquantel is the drug o choice or treating adult cestode (tapeworm) and trematode (f uke) in ections. Most importantly, praziquantel is the drug o choice or treating individuals with schistosomiasis, a trematodal in ection that causes considerable morbidity and mortality worldwide. Although the exact mechanism o praziquantel’s action is unknown, it appears to increase parasite membrane permeability to calcium, resulting in contraction and paralysis o the worms. The main adverse e ects o praziquantel include nausea, headache, and abdominal discom ort. Diethylcarbamazine (DEC), a piperazine derivative, is the drug o choice or treating certain larial in ections, including lymphatic lariasis. Its use in the treatment o larial onchocerciasis has been largely supplanted by the use o ivermectin (predominantly because o ivermectin’s tolerability and ease o administration). Unlike ivermectin, however, DEC kills adult larial worms and is thus a curative agent. DEC’s mechanism o action is unknown; current hypotheses include stimulation o innate immune mechanisms, inhibition o microtubule polymerization, and inhibition o arachidonic acid metabolism. DEC is reasonably well tolerated at low doses; its major adverse e ects include anorexia, headache, and nausea. Administration o DEC can, however, precipitate Mazzotti reactions in individuals with heavy micro larial burdens, and such reactions can be atal. Administration o gradually increasing doses o DEC minimizes this possibility. DEC is excreted by the kidneys, and dosing may need to be adjusted in individuals with decreased kidney unction. Antibacterial agents may also have a role in treating certain helminthic in ections. For example, O. volvulus has been ound to contain an obligate symbiont (Wolbachia endobacteria) important in helminth ertility, and the use o doxycycline to treat individuals with onchocerciasis leads to decreased ertility, embryogenesis, and viability o O. volvulus.

CONCLUSION AND FUTURE DIRECTIONS The development o new antiparasitic agents will rely on continued exploitation o molecular and metabolic di erences between parasites and hosts. Recent advances in the

688 PRINCIPLES OF CHEMOTHERAPY

application o molecular biological and genetic techniques to study parasitic eukaryotes, and detailed knowledge o parasite, vector, and host genomes, transcriptomes, and proteomes, should acilitate the development o more selective agents e ective against many parasitic in ections. The development o resistance to antiparasitic agents is o increasing concern, most notably among malarial and leishmanial parasites, and will necessitate both the judicious use o currently available agents and the development o new agents, including antiparasitic vaccines. Despite long-standing e orts to develop e ective treatments or malaria, the disease remains a major global cause o morbidity and mortality, although progress is being made. Development o an e ective malaria vaccine could have a major impact on this global burden. However, the development o an e ective vaccine has been hampered by a number o di f cult scientif c challenges, including the diversity o parasite species and strains, the diversity o parasite li e orms, the intracellular location o the parasites, and the ability o P. falciparum to undergo antigenic variation. The situation has been worsened by the lack o meaning ul economic incentives or vaccine development. Un ortunately, the complexity o the parasites and o their intimate relationship with

in ected hosts suggests that the development o e ective antiparasite vaccines (especially against malaria) will be di f cult.

Suggested Reading Babokhov P, Sanyaolu AO, Oyibo WA, Fagbenro-Beyioku AF, Iriemenam NC. A current analysis o chemotherapy strategies or the treatment o human A rican trypanosomiasis. Pathog Glob Health 2013;107:242–252. (Reviews treatment options and strategies for treating human African trypanosomiasis [“sleeping sickness”].) Dorlo TP, Balasegaram M, Beijnen JH, de Vries PJ. Milte osine: a review o its pharmacology and therapeutic e f cacy in the treatment o leishmaniasis. J Antimicrob Chemother 2012;67:2576–2597. (Reviews use of miltefosine in patients with leishmaniasis.) Fairhurst RM, Nayyar GM, Breman JG, et al. Artemisinin-resistant malaria: research challenges, opportunities, and public health implications. Am J Trop Med Hyg 2012;87:231–241. (Discusses implications of increasing artemisinin resistance among malaria-causing Plasmodium falciparum.) González P, González FA, Ueno K. Ivermectin in human medicine, an overview o the current status o its clinical applications. Curr Pharm Biotechnol 2012;13:1103–1109. (Reviews current uses of this important antiparasitic agent.) Martin C, Gavotte L. The bacteria Wolbachia in f lariae, a biological Russian dolls’ system: new trends in antif larial treatments. Parasite 2010;17:79–89. (Discusses the potential of using drugs that target bacteria [Wolbachia] within parasites to treat patients with parasitic infections).

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C h a p t e r 3 7 Pharmacology o Parasitic In ections 693

Atta c h me nt a nd Atta d e ntry ntt y inh h ib b it ito o rrss Ma ra virocc E n fu viirtide (T-20) Enf 0)

38

Hoss t c e lll

Atta chmee ntt Att a n d e ntry

Io o n c han h a nee l bl blo cke rs Amaa nta A n taa di dine R ima nta dinn e

Unco o a ti ting in g Un c oa te d RNA Unc A orr ne wly s ynthee s iz ize ze d mRNA NA

Pharmacology of Viral Infections NS 3/ 3/4A 4A inhib nh h ibit b it ito o rs

J o n a t h a n Z. Li a n d Do n a ld M . Co e n

Po lyme ras as e in inhi hibito rs Acyclovir Zido Zi dovu vu u di din ne ne S ofo i o ffoss bbuvir INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694–695 Ef vire Efa i nz PHYSIOLOGY OF VIRA AL RE REPLICATIO ON . . . . . . . . . . . . . . . . . . . . . 6994 Viral Life Cyc ycle le . . . . . . . . . . . . . . . . . . . . . . . . 695 NS S 5A iinhibito hibit rs rs PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 69 PH 697 697 L dipa Le di sand vir i Entry Inhibition of Viral Attachment ry . . . . . . . . . . . . . . . . . 698 Maraviroc Inte g ras e inhibito rs Inte r.s. . . . . . . . . . . . . . . . . . . . . . . 698 Enffuvi virt vi rtid rt idee (T id (T-20)) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698 98 R Ra lte lt g gra vir i Inhibition of Viral Uncoating . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698 Inhibition ofo Vi lsGe iinhib E iibit 7000 Pro Pr t aas te nhib nh b ito itssion o rs rs . . . . . . . . . . . . . . . . . . . . . . 70 Inhibition of Viral Genome S a qqu quina quin ina naa vir vir Repl Re plic icat atio ion— n—Po Poly lymerase Inhibitors . . . . . . . . . . . . . . . . . . 702 Ritona Rito Ri R iton tona o nnaaand vir viir Antiherpesvirus Nucleoside Nucleotide Analoguess . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702 Nee Anti-HIV uraminidas i Nucleoside id e inhibito i and hibNucleotide rs rs Analogues . . . . . . . . 705 Z and Nucleotide i i Anti-HBV Nucleoside Analogues . . . . . . . 707 Anti-HCV Nucleoside and Nucleotide Analoguess . . . . . . . 708

INTRODUCTION Viral in ections are among the leading causes o morbidity and mortality worldwide. Although much progress has been made on antiviral drug development, public health measures and prophylactic vaccines remain the primary means by which society controls the spread o viral in ections. The persistence o the acquired immunodef ciency syndrome (AIDS) epidemic makes this pain ully clear. Despite advances in anti-human immunodef ciency virus (HIV) drug therapies, AIDS continues to be a common cause o death, particularly in some A rican nations, where as many as one adult in our is in ected with HIV. This enormous prevalence is largely attributable to ailures in public health measures and the lack o an e ective vaccine against HIV in a setting where anti-HIV drugs are too expensive and the healthcare delivery system is too ragmented. Despite this bleak statistic, the array o drugs available to combat viruses has been instrumental in saving millions o lives each year and in improving the quality o li e or countless others with viral illnesses. This chapter describes the physiology o viral replication and the steps in the viral li e cycle that are targeted by current antiviral medications. Key concepts or the chapter include: (1) viruses replicate intracellularly and utilize host cell machinery; (2) despite this mode o replication, multiple targets have been exploited 694

Hos t ribos om ome

P rot ote in s yynt nthe he s is is

Vira l p rote ins

o s tt-tra P os tra ns la tiona l proce s s ing Nonnucleosid side DNA Polymerase Inhibitors . . . . . . . . . . . . 708 sid Nonnucc le Nonnu leo oside osi id Reversee Tra Transcriptase Inhi hibi ibito tors to ors r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708 088 10 Anti-HCV Nonnucleosi Antiucleo clee oside de RN NA Polymerasee In Inhibitorss . . . 7110 Gee no G nome mee re pli m p lica liica c ti tion on Inhibition t n of o Viral V r Genome G mee Replication—Other pli n Mechanisms c s . . . . . . . . . . . . . . . . . . . . . 710 71 Anti-HCV Anti CV NS5A A Inh Inhibitors rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710 HIV Integrase In Inhi hibi bit bbi ittors orss . . . . . . .As . . . . . .a. nd . . . . . . . . . 710 A. .s.s. e. .mbly Inhi In hiibi biti tion ti on of Viral Maturation r rat . . . . . . . . . . . . . . . . . . . . . . . . . . . 712 ma tura tion t on Inhi hibi biti tion on of Vi Vira rall Re Rele e le lleas aasee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714 Antiviral Drugs w with Unknown M Mechanisms Mechanis echan chaa n off A Action c i . . . . . 71 ctio ct 7166 Docosanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716 Ribavirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Egre s s a nd n716 d rree le a s e Drugss That Modulate the Immunee Syste S System Syst Sy tem . . . . . . . . . . . . . . . 716 CONCLUSION ON N AND A FUTURE DIR DIRECTIONS IRE O . . . . . . . . . . . . . . . . . . 717 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .717

or antiviral drug therapy; and (3) most current antiviral drugs exploit di erences between the structures and unctions o viral and human proteins to achieve selectivity o antiviral action.

PHYSIOLOGY OF VIRAL REPLICATION Viruses replicate by co-opting the host cell’s metabolic machinery. Based on this act, one might think that there would be ewer di erences between viruses and their human hosts to exploit or drug development than between bacteria and humans. However, all viruses encode proteins that are substantially di erent rom their human counterparts. Additionally, certain host proteins are more important or viral replication than they are or human health. In principle, antiviral drugs could target many o these proteins. In practice, however, relatively ew viral proteins and even ewer host proteins have thus ar served as use ul targets or therapy. Nevertheless, it is a testament to the remarkable progress in antiviral drug development that the number o viral proteins that have been exploited or antiviral therapy is greater than the number o bacterial proteins that have been exploited or antibacterial therapy. However, most antiviral drugs are active against only one or a ew viruses while most antibacterial drugs target multiple bacterial species. This di f culty arises because viruses are a heterogeneous group o

C h a p t e r 3 8 Pharmacology o Viral In ections

Th e ye a r is 1993. Mr. M, a 26-ye a r-o ld m a n , co m p la in s to Dr. Ro s e , h is p rim a ry ca re p h ys icia n , o a s o re th ro a t, e ve r, a n d a tig u e o r th e p a s t s e ve ra l w e e ks . On p h ys ica l e xa m in a tio n , Dr. Ro s e n o te s b ila te ra l ce rvica l lym p h a d e n o p a th y, co n s is te n t w ith th e p a tie n t’s “ f u -like s ym p to m s .” Dr. Ro s e th in ks it like ly th a t Mr. M h a s a n in e ctio n , p o s s ib ly a s im p le “ co ld ,” th e “ f u ,” o r s tre p th ro a t. Be ca u s e o Mr. M’s m o n o n u cle o s is -like s ym p to m s , Dr. Ro s e a ls o in clu d e s h u m a n cyto m e g a lo viru s (HCMV), Ep s te in -Ba rr viru s (EBV), toxo p la s m o s is , a n d HIV in h e r d i e re n tia l d ia g n o s is . La b o ra to ry a n tib o d y te s ts o r S tre p to co ccu s , HCMV, EBV, toxo p la s m o s is , a n d HIV in e ctio n a re n e ga tive . Mr. M is co n ce rn e d a b o u t th e p o s s ib ility o HIV in e ctio n a n d th e la ck o tru ly e e ctive th e ra p ie s o r AIDS , a lth o u g h h e d e n ie s a ny u n p ro te cte d s e xu a l a ctivity, in tra ve n o u s (IV) d ru g u s e , o r o th e r p o te n tia l e xp o s u re ris ks . Dr. Ro s e te lls Mr. M th a t h is s ym p to m s w ill like ly re s o lve w ith re s t b u t th a t h e s h o u ld re tu rn o r o llo w-u p w ith in 6 m o n th s . S h e e xp la in s to Mr. M th a t, i h e h a s re ce n tly b e e n in e cte d w ith HIV, h is b o d y w o u ld n o t ye t h a ve p ro d u ce d s u cie n t a n tib o d ie s to b e co m e e vid e n t o n a n a n ti-HIV a n tib o d y te s t. Five ye a rs la te r, Mr. M re tu rn s to Dr. Ro s e’s o ce . He h a s n o t s e e n a ny p hys icia n in th e in te rim a n d n o w p re s e n ts w ith n e w s ym p to m s . Th e re a re m u ltip le o p e n le s io n s o n h is lip s a n d in h is m o u th , a n d h e co n d e s th a t h e h a s s im ila r le s io n s in h is g e n ita l a re a . An ELISA te s t is p o s itive o r a n ti-HIV a n tib o d ie s ,

in ectious agents, whereas most bacteria share a common cell wall structure and distinct DNA replication, transcription, and translation machineries.

Viral Life Cycle Viruses exist as small particles called virions . Virions consist o a nucleic acid genome packaged into a virus-encoded protein shell called a capsid. In some viruses, the capsid is surrounded by an envelope , a lipid bilayer membrane that contains virus-encoded envelope proteins. Viral genomes can consist o DNA or RNA and can be single- or doublestranded. Almost all viruses have the same general li e cycle or replication (Fig. 38-1) with some variations. Figure 38-2 illustrates the specif c li e cycle or HIV, which, as a retrovirus, contains RNA that is copied into DNA. At the start o in ection, the virus attaches to the host cell. This attachment is mediated by proteins on the viral sur ace that bind specif cally to a particular host membrane component. For example, the HIV viral envelope contains the glycoprotein gp120, a transmembrane protein that mediates binding and attachment o the virus to host cells expressing CD4 and chemokine receptors such as CCR5 or CXCR4 (Fig. 38-2). Next, the virion undergoes entry by crossing a host cell membrane

695

a n d a vira l lo a d m e a s u re m e n t s h o w s h ig h le ve ls o HIV RNA in h is b lo o d . Mr. M’s CD4 co u n t is 100 p e r m m 3 (n o rm a l ra n g e , 800–1,200 p e r m m 3 ). Dr. Ro s e im m e d ia te ly p re s crib e s a d ru g re g im e n o zid o vu d in e (AZT), la m ivu d in e (3TC), a n d rito n a vir, e xp la in in g to Mr. M th a t a co m b in a tio n o a n ti-HIV d ru g s is h is b e s t o p tio n o r re d u cin g th e vira l lo a d a n d o re s ta llin g m o re s e rio u s d is e a s e . In a d d itio n , Dr. Ro s e p re s crib e s o ra l va la cyclovir, a p ro d ru g o a cyclovir, to tre a t Mr. M’s o ra l a n d g e n ita l h e rp e s . Ove r th e n e xt 3 ye a rs , Mr. M’s HIV vira l lo a d a lls to u n d e te cta b le le ve ls a n d h is co n d itio n im p rove s . Th e h e rp e s in e ctio n s a re a ls o ke p t in ch e ck. To d a y, Mr. M a p p e a rs in g o o d h e a lth a n d h e ta ke s h is m e d ica tio n s d ilig e n tly, w h ich is e a s ie r n o w w ith a o n ce -a -d a y p ill co n ta in in g e a vire n z, e m tricita b in e , a n d te n o ovir.

Questions 1 . What is acyclovir’s mechanism o action? 2 . Why does acyclovir not ordinarily cause signif cant toxicity in humans, while AZT does? 3 . What are the mechanisms o action o the three antiHIV drugs prescribed by Dr. Rose in 1998? In the oncea-day pill Mr. M is taking today? 4 . Why is combination antiretroviral therapy required to e ectively treat HIV in ections? 5 . What potential adverse e ects could Mr. M experience rom long-term treatment with ritonavir?

into the cytoplasm. In the case o HIV, the process o entry depends on gp41, a viral envelope protein that uses together the membranes o HIV and the target cell. The virion then loses enough o its capsid proteins—the stage o uncoating—that its nucleic acid becomes available or gene expression. (For retroviruses, uncoating does not lead directly to gene expression; instead, it allows reverse transcription o the viral RNA genome into DNA to occur— as described below, this is a step in genome replication.) Viral gene expression entails transcription o the viral genome into mRNA, translation o mRNA into protein on cellular ribosomes, and a variety o processing events including splicing o mRNA precursors and proteolytic cleavage o viral polyproteins into their individual protein units. For many viruses, gene expression begins with transcription. For certain RNA viruses, such as hepatitis C virus (HCV), the f rst step in gene expression is translation o the viral RNA. Many viruses encode proteins that execute or abet certain o these steps in gene expression, and these proteins can serve as drug targets. Genome replication is the next stage o the cycle. This stage requires a supply o ribonucleoside triphosphates or RNA viruses and deoxyribonucleoside triphosphates or DNA viruses. For DNA viruses, the generation o these deoxyribonucleoside triphosphates occurs via two

696 PRINCIPLES OF CHEMOTHERAPY Virus

Re ce ptor

Attachment and entry inhibitors Ma ra viroc Enfuvirtide (T-20)

Atta chme nt a nd e ntry

Ion channel blockers Ama nta dine Rima nta dine

NS3/4A inhibitors

Hos t ce ll

Uncoa ting Uncoa te d RNA or ne wly s ynthe s ize d mRNA

Polymerase inhibitors Acyclovir Zidovudine S ofos buvir Efa vire nz

Hos t ribos ome

P rote in s ynthe s is

Viral prote ins

P os t-tra ns la tiona l proce s s ing

NS5A inhibitors Le dipa s vir Integrase inhibitors Ra lte gra vir Elvite gra vir Dolute gra vir HIV protease inhibitors Da runa vir Ata za na vir Ritona vir

Ge nome re plica tion

As s e mbly a nd ma tura tion

Egre s s a nd re le a s e

Neuraminidase inhibitors Za na mivir Os e lta mivir

FIGURE 38-1. Viral life cycle and pharmacologic intervention. The viral life cycle can be divided into a sequence of stages, each of which is a potential site for pharmacologic intervention. Shown is a generic replication cycle of viruses in cells, alongside which are listed the names of drug classes and examples of individual agents that block each stage. Many of the currently approved antiviral agents are nucleoside analogues or nonnucleoside compounds that target genome replication, typically by inhibiting viral DNA polymerase or reverse transcriptase. Several other drug classes target other stages in the viral life cycle, including attachment and entry, uncoating, gene expression, assembly and maturation, and egress and release. It should be noted that the individual features of viral replication differ for each type of virus, often presenting unique targets for pharmacologic intervention and drug development. See the legend to Figure 33-2 for additional details.

pathways: the salvage pathway, which employs the pharmacologically relevant enzyme thymidine kinase, and the de novo pathway, which includes the enzyme thymidylate kinase. Nucleoside triphosphates are incorporated into new viral genomes by a viral or cellular polymerase (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance, or more detail on nucleotide metabolism). In the case o herpes simplex viruses 1 and 2 (which will be collectively re erred to as HSV), the generation o deoxyribonucleoside triphosphates includes phosphorylation o nucleosides via the salvage pathway by a viral thymidine kinase; a viral DNA polymerase then adds deoxyribonucleoside triphosphates to the growing DNA genome. Exploitation o this two-step process has led to the development o some o the most e ective and sa e antivirals currently available, because differences between human and viral kinases and polymerases allow drugs to take advantage of two different steps in a single pathway. For many viruses, genome replication requires other kinds o proteins. One example is the HCV NS5A protein.

Viral proteins that are synthesized intracellularly assemble with viral genomes within the host cell in a process known as assembly. For a number o viruses, assembly is ollowed by a process known as viral maturation, which is essential or newly ormed virions to become in ectious. This process typically involves cleavage o viral polyproteins by proteases. For some viruses, maturation occurs within the host cell; or others, such as HIV, it occurs outside the host cell. Viruses egress rom the cell either by cell lysis or by budding through the cell membrane. For inf uenza viruses, the newly ormed virions require an additional step o release rom the extracellular sur ace o the host cell membrane. In summary, nearly all viruses replicate via the ollowing stages: attachment, entry, uncoating, gene expression, genome replication, assembly, and egress. Some viruses have additional stages such as maturation and release. The stages o retrovirus in ection occur in a di erent order rom those o most other viruses, and retroviruses have additional steps and stages in their li e cycle. For example, genome replication o HIV includes the additional step o integration, in which the viral genome is incorporated into the host

C h a p t e r 3 8 Pharmacology o Viral In ections 1 Atta chme nt

697

2 Fus ion

HIV

s s RNA gp120 gp41 Protease Ma trix prote in Integrase Core prote in Reverse transcriptase

CD4

Che mokine re ce ptor

3 Re ve rs e tra ns cription DNA Integrase

5 Tra ns cription 4 Inte gra tion RNA (ge nomic a nd mRNA) Protease

6 Tra ns la tion

Integrase Core prote in Reverse transcriptase

7 Virion a s s e mbly a nd budding

8 Ma tura tion (Protease)

FIGURE 38-2. Life cycle of HIV. HIV is a retrovirus that in ects CD4 cells. 1. Virus attachment is dependent on binding interactions between viral gp41 and gp120 proteins and host cell CD4 and certain chemokine receptors. 2. Fusion o the viral membrane (envelope) with the host cell plasma membrane allows the HIVgenome complexed with certain virion proteins to enter the host cell. 3. Uncoating permits the single-stranded RNA (ssRNA) HIVgenome to be copied by reverse transcriptase into double-stranded DNA. 4. The HIVDNA is integrated into the host cell genome in a reaction that depends on HIV-encoded integrase. 5. Gene transcription and post-transcriptional processing by host cell enzymes produce genomic HIV RNA and viral mRNA. 6. The viral mRNA is translated into proteins on host cell ribosomes. 7. The proteins assemble into immature virions that bud rom the host cell membrane. 8. The virions undergo proteolytic cleavage, maturing into ully in ective virions. Currently approved anti-HIV drugs target viral attachment and usion, reverse transcription, integration, and maturation. The development o drug resistance can be signif cantly retarded by using combinations o drugs that target a single stage (e.g., two or more inhibitors o reverse transcription) or more than one stage in the HIV li e cycle (e.g., reverse transcriptase inhibitors and protease inhibitors).

genome (Fig. 38-2). Specif c host and/or viral proteins are involved in each o these stages. Di erences between viral and host proteins at any o these stages can be targeted or antiviral therapy. Di erent viruses have vastly di erent arrays o genes. Some, such as hepatitis B virus (HBV), have compact genomes that encode only coat proteins and a ew proteins used mainly in gene expression and genome replication. Others, such as herpesviruses, encode scores o proteins that per orm many di erent unctions. The viral proteins that most requently have served as targets or antiviral drugs are enzymes involved in genome replication, although multiple other proteins acting at di erent stages in the viral li e cycle serve as targets, too.

PHARMACOLOGIC CLASSES AND AGENTS This section o the chapter reviews the mechanisms o antiviral drugs that target di erent stages o the viral li e cycle. Understanding the mechanisms o antiviral drugs has relied strongly on studies o viruses that are resistant to these drugs. Resistance to an antiviral drug usually implies that the drug acts, at least in part, by inter ering directly with a virusspecif c process rather than by incapacitating a host cell process. In most cases, then, mutations that con er resistance to an antiviral drug a ect the target(s) o that drug and suggest that the drug acts selectively against that target. Thus, mapping drug-resistance mutations to viral genes, along with

698 PRINCIPLES OF CHEMOTHERAPY

demonstrating that the drug interacts with the product o that viral gene in a way that explains drug action, has been a major method by which the drug mechanisms described below have been elucidated.

Inhibition of Viral Attachment and Entry All viruses must in ect cells to replicate. There ore, inhibiting the initial stages o viral attachment and entry provides a conceptual “preventive” measure against in ection. Drugs that act at these stages do not need to enter cells, which can be an advantage. Two anti-HIV drugs, maraviroc and enfuvirtide (T-20), act at these stages. Both drugs have unusual properties or antiviral agents: maraviroc targets a host protein rather than a viral protein, and en uvirtide is a peptide.

Maraviroc

Maraviroc targets the chemokine receptor CCR5, which is a host plasma membrane protein. The development o maraviroc stemmed rom clinical studies o individuals who had been exposed repeatedly to HIV, yet did not develop AIDS. It was ound that some o these individuals have a deletion in the CCR5 gene. Absence o the CCR5 gene product prevents in ection by the strains o HIV that are most requently transmitted between individuals. The deletion also causes increased risk o clinical mani estations rom West Nile disease but otherwise appears to have little negative impact on human health. Drug companies per ormed screens or compounds that could prevent binding o chemokines to CCR5 and chemically modif ed the leading candidate compounds to optimize their pharmacodynamic and pharmacokinetic properties. (Such “target-based screens” had earlier achieved success in the development o anti-HIV nonnucleoside reverse transcriptase inhibitors; see Box 38-1.) Maraviroc, the end result o this process, blocks in ection o HIV strains that use CCR5 or attachment and entry (Fig. 38-3). However, maraviroc is not active against HIV strains that use the CXCR4 receptor. It is approved or use in combination with other anti-HIV drugs in patients who have undetectable levels o CXCR4-using virus (which requires a genotypic or phenotypic diagnostic test o viral tropism).

Enfuvirtide (T-20)

En uvirtide, also known as T-20, is a peptide that is structurally similar to a segment o gp41, the HIV protein that mediates membrane usion. The proposed mechanism or

gp41-mediated membrane usion and T-20 action is illustrated in Figure 38-3. In the native virion, gp41 is held in a con ormation that prevents it rom using membranes or binding T-20. Attachment o HIV to its cellular receptors triggers a con ormational change in gp41 that exposes a segment that can insert into membranes ( usion peptide), a heptad repeat region (HR1), and a second heptad repeat region mimicked by T-20 (HR2). The gp41 then re olds, so that the HR2 segments bind directly to the HR1 segments. This re olding brings the virion envelope and the cell membrane into close proximity, allowing membrane usion to occur (by mechanisms that remain incompletely understood). When T-20 is present, however, the drug binds to the exposed HR1 segments and prevents the re olding process, thereby preventing usion o the HIV envelope with the host cell membrane. En uvirtide is approved or use in combination with other anti-HIV drugs in patients whose HIV in ection has not been controlled by f rst-line anti-HIV medications. En uvirtide is rarely used in clinical practice as it must be administered by twice-daily subcutaneous injections. Local injection site reactions are common and have been associated with bacterial pneumonia and a hypersensitivity reaction. Resistance mutations appear to emerge rapidly in patients who have detectable viremia despite en uvirtide treatment.

Inhibition of Viral Uncoating The adamantanes amantadine and rimantadine (structures in Fig. 38-4) are inhibitors o viral uncoating that are active exclusively against in uenza A virus (and not against in uenza B or C viruses). In uenza viruses (in particular in uenza A viruses) cause 50 million illnesses each year in the United States and tens to hundreds o thousands o hospitalizations. Current vaccines generally are not completely e f cacious and are use ul or only a year at a time due to the rapid evolution o new in uenza strains. Thus, there has long been a considerable need or anti-in uenza drugs. A well-supported model or the mechanism o action o the adamantanes is diagrammed in Figure 38-4. In uenza virions enter cells via receptor-mediated endocytosis and are internalized into endosomes (see Chapter 1, Drug–Receptor Interactions). As endosomes acidi y because o the action o an endosomal proton pump, two events occur. First, the conormation o the viral envelope protein hemagglutinin changes

B O X 3 8 - 1 De ve lo p m e n t o f No n n u cle o s id e Re ve rs e Tra n s crip ta s e In h ib ito rs a n d CCR5 An ta g o n is t The nonnucleoside reverse transcriptase inhibitors (NNRTIs) were discovered by using target-based, high-throughput screening methods. The gene encoding HIV RT was overexpressed in E. coli,i and large amounts o RT were purif ed and used in an RT assay that could be easily automated. Using this assay, many thousands o compounds were screened or the ability to inhibit RT. Candidate compounds were then tested or specif city in a counter-screen by checking their ability to inhibit an unrelated polymerase. The compounds that emerged were chemically modif ed to improve their stability, pharmacokinetics, and toxicity prof le. This process eventually yielded NNRTIs that

are highly specif c, inhibiting HIV-1 RT at low concentrations while not inhibiting even the RT o the closely related virus HIV-2. The CCR5 antagonist maraviroc was also developed using a target-based, high-throughput screen. In this case, the assay was designed to discover lead compounds that prevented the binding o endogenous ligands (chemokines) to CCR5. As with the development o the NNRTIs, the lead compounds were then tested or specif city against CCR5 and chemically modif ed to optimize their potency, antiviral activity, pharmacokinetics, and toxicity prof le. The end result was maraviroc, a selective CCR5 antagonist that is used in combination antiretroviral treatment o adults in ected with CCR5-tropic HIV-1. ■

C h a p t e r 3 8 Pharmacology o Viral In ections

G

CCR5

F

Maraviroc

O

699

F

NH

N N

N N

Maraviroc CCR5 blocke d by ma ra viroc Hos t ce ll pla s ma me mbra ne

A

CD4

Che mokine re ce ptor

Fus ion pe ptide

B

F

Enfuvirtide (T-20)

HR1

gp120

gp41 HR2

gp41

Inte rme dia te

Vira l me mbra ne (e nve lope )

C

D

Tra ppe d inte rme dia te

E

He mifus ion s ta lk

Fus ion pore

FIGURE 38-3. Model for HIVgp41-mediated fusion and maraviroc and enfuvirtide (T-20) action. A. HIVglycoproteins exist in trimeric orm in the viral membrane (envelope). Each gp120 molecule is depicted as a ball attached noncovalently to gp41. B. The binding o gp120 to CD4 and certain chemokine receptors in the host cell plasma membrane causes a con ormational change in gp41 that exposes the usion peptide, heptad-repeat region 1 (HR1) and heptad-repeat region 2 (HR2). The usion peptide inserts into the host cell plasma membrane. C. gp41 undergoes urther con ormational changes, characterized mainly by un olding and re olding o the HR2 repeats. D. Completed re olding o the HR regions creates a hemi usion stalk, in which the outer leaf ets o the viral and host cell membranes are used. E. Formation o a complete usion pore allows viral entry into the host cell. F. En uvirtide (T-20) is a synthetic peptide drug that mimics HR2, binds to HR1, and prevents the HR2–HR1 interaction (dashed arrow). There ore, the drug traps the virus–host cell interaction at the attachment stage, preventing membrane usion and viral entry. G. Maraviroc is a small-molecule antagonist o the CCR5 chemokine receptor; the drug blocks cellular in ection o HIV strains that use CCR5 or attachment and entry (dashed arrow). The structure o maraviroc is shown.

drastically. This con ormational change permits usion o the in uenza virus envelope with the endosome membrane (see the above discussion o HIV-mediated membrane usion). By itsel , this action could liberate viral ribonucleoprotein (including the virion’s RNA genome), but that would not be su f cient to permit its transcription: a second pH-dependent event within the virion is also required. This entails the inux o protons through a proton channel called M2 in the viral envelope, which causes dissociation o the virion matrix protein rom the rest o the ribonucleoprotein. Amantadine and rimantadine inhibit the in ux o protons through M2.

As hydrophobic molecules with a positive charge at one end, these drugs resemble blockers o cellular ion channels (see Chapters 12, Local Anesthetic Pharmacology, and 24, Pharmacology o Cardiac Rhythm), and indeed the adamantanes appear to simply “plug” (physically occlude) the channel. Amantadine can cause light-headedness and di f culty concentrating; these adverse e ects are likely due to its e ects on host ion channels. Indeed, the unintended e ects o amantadine on host channels likely account or this drug’s other therapeutic use—the treatment o Parkinson’s disease (see Chapter 14, Pharmacology o Dopaminergic

700 PRINCIPLES OF CHEMOTHERAPY ADP

Vira l me mbra ne NA

H+

M2 HA bound to s ia lic a cid on ce ll re ce ptor

ATP

CH3 CHNH2

Ma trix prote in

NH2

Inte rna lize d ce ll re ce ptor RNP Endos oma l me mbra ne

Amantadine

Rimantadine

Early endosome

Low pH

Low pH + amantadine or rimantadine

ADP

ADP

Amantadine or rimantadine

H+

H+ Acid-induce d dis s ocia tion of ma trix s tructure

ATP

H+ H+

H+

H+

H+

H+

H+ H+

H+

ATP

H+ H+

Acid-induce d s tructura l cha nge in HA trigge rs me mbra ne fus ion

H+ H+ H+ H+

H+

M2 cha nne l ope ns to pe rmit e ntry of protons

H+

RNP re le a s e d from e ndos ome

RNP not re le a s e d from e ndos ome

H+ M2 cha nne l doe s not ope n

Late endosome

FIGURE 38-4. Uncoating o inf uenza virus and e ect o amantadine and rimantadine. The structures o the adamantanes, amantadine and rimantadine, are shown. In uenza virus enters host cells by receptor-mediated endocytosis (not shown) and is contained within an early endosome. The early endosome contains an H -ATPase that acidif es the endosome by pumping protons rom the cytosol into the endosome. A low pH-dependent con ormational change in the viral envelope hemagglutinin (HA) protein triggers usion o the viral membrane with the endosomal membrane. Fusion alone is not su f cient to cause viral uncoating, however. In addition, protons rom the low-pH endosome must enter the virus through M2, a pH-gated proton channel in the viral envelope that opens in response to acidif cation. The entry o protons through the viral envelope causes dissociation o matrix protein rom the in uenza virus ribonucleoprotein (RNP), releasing RNP and thus the genetic material o the virus into the host cell cytosol. Amantadine and rimantadine block M2 ion channel unction and thereby inhibit acidif cation o the interior o the virion, dissociation o matrix protein, and uncoating. Note that the drug is shown as “plugging” the channel. NA, neuraminidase.

Neurotransmission). Rimantadine is an analogue o amantadine that has a similar antiviral mechanism and ewer adverse e ects compared to amantadine, especially the neurological e ects that can be problematic in the elderly. However, resistance to adamantanes develops rapidly, and resistant viruses retain nearly complete replication capacity (f tness) and pathogenicity. Indeed, adamantanes are no longer recommended or use in the United States due to the high rates o drug resistance, and they have been supplanted by neuraminidase inhibitors (see “Inhibition o Viral Release”).

Inhibition o Viral Gene Expression HCV causes serious liver disease and more deaths in the United States than does HIV. A ter entry o HCV into the cell and uncoating in endosomes, the f rst step in HCV gene

expression is translation o the viral genome. The translation product is a so-called polyprotein that encompasses proteins rom multiple viral genes, and the next, crucial step in HCV gene expression is cleavage o the polyprotein into individual proteins (Fig. 38-5A). Certain o these individual proteins then replicate HCV RNA, producing new genomes to be translated and cleaved. Other viral proteins assemble RNA-containing viral particles that are then released rom the cell. HCV encodes a protease, called NS3/4A, which is essential or several o the cleavages o the polyprotein. Additionally, this enzyme may play a role in counteracting host innate immune responses, particularly those elicited by inter eron alpha. Given the success o drugs targeting the HIV protease, which is required or the maturation step in its virus’ li e cycle (see “Inhibition o Viral Maturation”), there was considerable interest in developing drugs that target the HCV

C h a p t e r 3 8 Pharmacology o Viral In ections

701

A Core

E1

E2

p7

NS 2

NS 3

4A

S tructura l

B

NS 4B

NS 5A

NS 5B

Non-s tructura l

O O

N

S

O H N

NH

O

O

N

N

N H

O

N H

O H N

H N

O O

O O

O

Telaprevir

O

N

N S O

N

S

Simeprevir HN

O O O

O

H

N

H N H

O

O

H N H N

H N

NH2

N O

N

O

O O

NH

O

Boceprevir

N

N

Paritaprevir

FIGURE 38-5. The HCV polyprotein and anti-HCV protease inhibitors. A. The HCV RNA genome is translated into a polyprotein, which is shown as a long rectangle, with the segments corresponding to the indicated individual viral proteins. The polyprotein is then cleaved into the individual proteins by host peptidases (sites o cleavage are indicated by open triangles), the viral NS2 protease (green triangle), and the viral NS3/4A protease (blue triangles). (Modif ed rom Ray SC, Bailey J R, Thomas DL. Hepatitis C virus. In: Knipe DM, Howley PM, Cohen J I, et al, eds. Fields virology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2013:795–824.) B. The structures o telaprevir, boceprevir, simeprevir, and paritaprevir are shown. Telaprevir and boceprevir covalently react with their target via ketoamide groups, while simeprevir and paritaprevir are noncovalent inhibitors with macrocyclic structures. These drugs are approved or use only in combination with other anti-HCV therapies.

NS3/4A protease. In 2011, two such drugs, telaprevir (which is no longer marketed) and boceprevir (Fig. 38-5B), were the f rst agents that act directly against HCV (direct-acting antivirals, DAAs) to be approved by the US Food and Drug Administration (FDA). In 2013 and 2014, respectively, the NS3/4A inhibitors simeprevir and paritaprevir (Fig. 38-5B), which can be administered once a day, were approved. All our drugs were developed by an iterative approach similar to that used to discover inhibitors o the HIV protease (see discussion below and Fig. 38-11), although, among other di erences, telaprevir and boceprevir covalently react with their target via ketoamide groups rather than binding tightly via noncovalent interactions like the anti-HIV drugs. Simeprevir and paritaprevir are noncovalent inhibitors with

macrocyclic structures that increase the a f nity o binding by decreasing entropic e ects. All our drugs inhibit HCV NS3/4A protease with higher potency than they do human proteases. None o the NS3/4A inhibitors are approved or use as monotherapies. Telaprevir, boceprevir, and simeprevir are approved or use in combination with inter eron alpha (modif ed by pegylation to permit less requent dosing) and ribavirin (see below or urther in ormation on these two agents), which was the previous standard o care or HCV therapy. The addition o the protease inhibitors results in a substantially higher rate o “sustained virological response,” which is tantamount to cure o HCV in ection. However, these inter eron-based combinations entail 6 months or more o

702 PRINCIPLES OF CHEMOTHERAPY

treatment and are limited by the development o resistance in some patients, by the requirement or injection o intereron alpha, and by major adverse e ects o inter eron alpha and ribavirin. Relative to telaprevir and boceprevir, simeprevir and paritaprevir represent an advance, in that they can be administered once a day, have a more avorable adverse e ect prof le, and are also approved or use in combination with particular DAAs that inhibit HCV genome replication (see below)—simeprevir with so osbuvir; paritaprevir with ombitasvir and dasabuvir—in a regimen that requires no inter eron alpha and, in many cases, only 3 months o treatment. However, each o the protease inhibitors is approved or only certain genotypes o HCV, and resistance can arise. Indeed, it is strongly recommended that testing or resistance occur prior to use o simeprevir. Paritaprevir is ormulated together with the anti-HIV protease inhibitor ritonavir (see below), which increases paritaprevir serum levels by blocking its hepatic metabolism and is not recommended or use in patients with decompensated liver disease. Depending on the HCV subtype, the DAA combination containing paritaprevir may be co-administered with ribavirin, which has its own toxicities. Newer NS3/4A protease inhibitors are under development.

Inhibition of Viral Genome Replication— Polymerase Inhibitors The vast majority o drugs that inhibit viral genome replication inhibit a polymerase. Most viruses encode their own polymerases. These enzymes are required to replicate the viral genomes and di er in various ways rom human polymerases, making them excellent targets or antiviral drugs. Viruses expressing polymerases that have been success ully targeted to yield drugs approved by the FDA include certain human herpesviruses, HIV, HBV, and HCV. Most o these drugs are so-called nucleoside analogues (Fig. 38-6). Several, as discussed below, are nonnucleoside inhibitors o a polymerase. The latter do not structurally resemble physiologic nucleosides. All nucleoside analogues must be activated by phosphorylation, usually to the triphosphate orm, in order to exert their e ect. In their phosphorylated orm, these agents mimic nucleoside triphosphates, which are the natural substrates o polymerases. Nucleoside analogues inhibit polymerases by competing with the natural triphosphate substrate; these analogues are also typically incorporated into the growing DNA or RNA chain, where they often terminate elongation. Either or both o these eatures—enzyme inhibition and incorporation into DNA or RNA—can be important or antiviral activity. The more e f ciently cellular enzymes phosphorylate the nucleoside analogue, and the more potently the phosphorylated orms inhibit cellular enzymes, the more toxic the nucleoside analogue will be. There ore, selectivity depends on how much more e f ciently viral enzymes phosphorylate the drug than cellular enzymes do, as well as how much more potently and e ectively viral DNA synthesis is inhibited than analogous cellular unctions are. The challenge in designing nucleoside analogues is to make the drug appear enough like a natural nucleoside that it can be activated and its triphosphate can inhibit a viral polymerase, but not so much like a natural nucleoside that it inhibits cellular processes. All nucleoside analogues employ variations on this theme to achieve their respective degrees o selectivity.

Antiherpesvirus Nucleoside and Nucleotide Analogues

Although the diseases caused by herpesviruses are not li ethreatening or most immunocompetent people, some—such as eye in ections and genital herpes, caused by HSV, and shingles, caused by reactivation o varicella zoster virus (VZV)—can be serious nonetheless. For immunocompromised patients such as Mr. M, herpesvirus diseases such as HSV esophagitis and HCMV retinitis or neurologic disease can be devastating or even atal. Herpesviruses also have the property o latency, in which viral genomes reside inside a cell and abundantly express a ew genes (at most), thus avoiding immune surveillance. The viruses can then reactivate long a ter the primary in ection and cause disease. No currently available antiviral drug attacks viruses during latency; rather, all available drugs act only on actively replicating virus. Herpesvirus replication corresponds roughly to the schematic in Figure 38-1. All herpesviruses contain doublestranded DNA encoding a variety o proteins involved in DNA replication. These proteins are categorized in two groups. The f rst group, which includes the viral DNA polymerase , participates directly in DNA replication and is essential or virus replication. The second group participates indirectly, or example, by helping to synthesize the deoxyribonucleoside triphosphates necessary or DNA replication. For some herpesviruses, including HSV and VZV, one o these proteins is a viral thymidine kinase (TK). Some other viruses, including HCMV, do not encode a TK but instead encode a protein kinase that induces the expression o cellular enzymes that synthesize deoxyribonucleoside triphosphates. Proteins in the second group are not essential or virus replication in cell culture or in certain cells in mammalian hosts because cellular enzymes can substitute or their activities. Herpesvirus DNA polymerases and kinases are su f ciently di erent rom their cellular counterparts to enable development o selective antiviral nucleoside analogues. Acyclovir Acyclovir (ACV) is a drug used against HSV and VZV. Acy-

clovir illustrates the undamental mechanisms o nucleoside analogues, and it is the drug that convinced the medical community that antivirals could be sa e and e ective. Acyclovir was discovered in a screen o compounds or activity against HSV replication. It exhibits a high therapeutic index (toxic dose/e ective dose) because o its high selectivity. Accordingly, acyclovir toxicity is generally a clinical problem only when the drug is administered intravenously at high doses or very serious in ections or in the setting o renal insu f ciency. The structure o acyclovir consists o a guanine base attached to a broken (acyclic) and incomplete sugar ring (Fig. 38-6). Despite the lack o a complete sugar ring, HSV and VZV TK can phosphorylate acyclovir much more e f ciently than any mammalian enzyme can. There ore, HSV- and VZV-in ected cells contain much more phosphorylated acyclovir than do unin ected cells; this f nding accounts or much o acyclovir’s antiviral selectivity. Phosphorylation o ACV produces the compound ACV monophosphate. This compound is then converted to ACV diphosphate and ACV triphosphate by cellular enzymes (Fig. 38-7A). ACV triphosphate inhibits the herpesvirus DNA polymerase; moreover, it inhibits viral DNA polymerase more potently than cellular DNA polymerases. In vitro, inhibition o HSV DNA polymerase is a three-step process.

A Na tive nucle os ide s NH2 N HO

O N

N

N

HO

N

O OH

NH

N

O

N

HO

NH2

O

De oxygua nos ine

NH

O

N

O

H2 N

NH2

O

O

N

HO

NH2

O

N

NH2

Va lga nciclovir (prodrug) NH2

N

N

NH

N

N

NH2

O

N

N

OH

Ga nciclovir

O

N

N

O

OH

O N

N

O

H2 N

NH

NH2

Va la cyclovir (prodrug)

Acyclovir

N

NH

N

O

NH

N

O

De oxythymidine

O

HO

N

O

OH

De oxycytidine

O

O

N

N O

OH

N

HO

HO

O

N

B Antihe rpe s virus nucle os ide a nd nucle otide a na logue s

N

NH

N

OH

De oxya de nos ine

O

NH2

OH

NH2 HO

O

OH

N

P

O

O

O

HO

Fa mciclovir (prodrug)

P e nciclovir

O

Cidofovir

C Anti-HIV nucle os ide a nd nucle otide a na logue s O NH HO O

NH2

O

N

HO

N

O

F

N

NH O

NH2

HO

O

N

N

HO

O

S

O

N

S O

O

N3

Zidovudine (AZT)

S ta vudine (d4T)

La mivudine (3TC)

Emtricita bine (FTC) NH2 N

O

NH

N HO

N

O

N

NH

N

O

N

O

HO

N

O

N

N

O

O

NH2

O

O

P

N

N

O

O

O

Dida nos ine (ddI)

Te nofovir dis oproxil

Aba ca vir

D Anti-he pa titis B nucle os ide a nd nucle otide a na logue s NH2 N O HO

P

N

E Anti-RNA virus nucle os ide a na logue s

N

N CH2

N

N

O

O

O NH N

O

N

NH

NH2

HO

N

O

Ade fovir

HO HO

Ente ca vir

O

HO

O P O NH

N O

Te lbivudine

OH

NH

O

NH2 N

O

OH

O

O

N

O

OH

O

F

OH

Riba virin

S ofos buvir

FIGURE 38-6. Antiviral nucleoside and nucleotide analogues. A. The nucleosides used as precursors or DNA synthesis are depicted in their anti con ormations. Each nucleoside consists o a purine (adenine and guanine) or pyrimidine (cytosine and thymidine) base attached to a deoxyribose sugar. These deoxyribonucleosides are phosphorylated in stepwise ashion to the triphosphate orms (not shown) or use in nucleic acid synthesis. B. Except or cido ovir, the antiherpesvirus nucleoside and nucleotide analogues are structural mimics o deoxyguanosine. For example, acyclovir consists o a guanine base attached to an acyclic sugar. Cido ovir, which mimics the deoxyribonucleotide deoxycytidine monophosphate, uses a phosphonate (C–P) bond to mimic the physiologic P–O bond o the native nucleotide. Valacyclovir, amciclovir, and valganciclovir are more orally bioavailable prodrugs o acyclovir, penciclovir, and ganciclovir, respectively. C. Anti-HIVnucleoside and nucleotide analogues mimic a variety o endogenous nucleosides and nucleotides and contain variations not only in the sugar but also in base moieties. For example, AZT is a deoxythymidine mimic that has a 3 -azido group in place o the native 3 -OH. Stavudine and lamivudine also contain modif ed sugar moieties linked to natural base moieties. Teno ovir, which is shown as its prodrug teno ovir disoproxil, is a phosphonate analogue o deoxyadenosine monophosphate. O the analogues that contain modif ed base moieties, didanosine mimics deoxyinosine and is converted to dideoxyadenosine, while emtricitabine contains a uoro-modif ed cytosine and abacavir contains a cyclopropyl-modif ed guanine. D. Telbivudine is an L-stereoisomer o thymidine, ade ovir is a phosphonate analogue o the endogenous nucleotide deoxyadenosine monophosphate, and entecavir is a deoxyguanosine analogue with an unusual moiety substituting or deoxyribose. These three compounds, together with lamivudine and teno ovir (see panel C), are approved or use in the treatment o HBVin ection. E. So osbuvir, which contains uracil linked to a modif ed sugar linked to a phosphoramidite that is modif ed to increase uptake by liver cells, is approved or use against the RNA virus HCV. Ribavirin, which contains a purine mimic attached to ribose, is approved or use against the RNA viruses HCV and RSV.

704 PRINCIPLES OF CHEMOTHERAPY A

O

O N

NH

N HO

N

N

HSV or VZV thymidine kinase

OH

HO

NH2

P

O

NH

N O

N

NH2

O

O Acyclovir monophos pha te

Acyclovir

Cellular kinase

O N

HO

OH P

O

O

OH P O

O

OH P

N

NH

N O

O

N

NH2

Cellular kinase

HO

O

O

OH P O

Acyclovir triphos pha te (pppACV)

O

OH P

NH

N O

N

NH2

O

O Acyclovir diphos pha te

Vira l DNA polyme ra s e pppACV

B

ACV

ACV

pppdG pppdC dC

dC

dG

1

2 Binding of pppACV to vira l DNA polyme ra s e compe te s for binding of pppdG.

dC

dG

dG

3 ACV is incorpora te d into growing DNA cha in, blocking furthe r cha in growth.

Whe n the ne xt de oxynucle os ide triphos pha te binds , vira l DNA polyme ra s e is "froze n."

FIGURE 38-7. Mechanism of action of acyclovir. A. Acyclovir is a nucleoside analogue that is selectively phosphorylated by HSV or VZV thymidine kinase to generate acyclovir monophosphate. Host cellular enzymes then sequentially phosphorylate acyclovir monophosphate to its diphosphate and triphosphate (pppACV) forms. B. Acyclovir triphosphate has a three-step mechanism of inhibition of herpesvirus DNA polymerase in vitro: (1) it acts as a competitive inhibitor of dGTP (pppdG) binding; (2) it acts as a substrate and is base-paired with dC in the template strand to become incorporated into the growing DNA chain, causing chain termination; and (3) it traps the polymerase on the ACV-terminated DNA chain when the next deoxyribonucleoside triphosphate (shown here as dCTP, or pppdC) binds.

In the f rst step, ACV triphosphate competitively inhibits deoxyguanosine triphosphate (dGTP) incorporation (note that high concentrations o dGTP can reverse inhibition at this early step). Next, ACV triphosphate acts as a substrate and is incorporated into the growing DNA chain opposite a C residue. The polymerase translocates to the next position on the template but cannot add a new deoxyribonucleoside triphosphate because there is no 3 -hydroxyl on ACV triphosphate; hence, ACV triphosphate is an obligate chain terminator. Finally, provided that the next deoxyribonucleoside triphosphate is present, the viral polymerase reezes in a “dead-end complex,” leading to apparent inactivation o the

enzyme (Fig. 38-7B). (The mechanism o polymerase “ reezing” is incompletely understood.) Interestingly, cellular DNA polymerase does not undergo inactivation to the dead-end complex. It is not yet known whether the inactivating step is important in vivo or whether ACV incorporation and chain termination alone are su f cient to inhibit viral replication. Acyclovir resistance occurs in requently during treatment o immunocompetent patients with cold sores or genital herpes. It occurs relatively o ten (5–10% o patients), however, during treatment o immunocompetent patients with HSV eye in ections and immunocompromised hosts with various herpetic diseases.

C h a p t e r 3 8 Pharmacology of Viral Infections

Valacyclovir is a prodrug orm o acyclovir that has

705

Valganciclovir is a prodrug orm o ganciclovir that has

approximately f ve old higher oral bioavailability than acyclovir (Fig. 38-6). This compound, which contains an acyclovir structure covalently attached to a valine moiety, is rapidly converted to acyclovir a ter oral administration.

higher oral bioavailability than ganciclovir. Valganciclovir is a valine ester o ganciclovir, making the relationship between valganciclovir and ganciclovir similar to that between valacyclovir and acyclovir (Fig. 38-6).

Famciclovir and Penciclovir Famciclovir (Fig. 38-6) is the diacetyl 6-deoxy analogue o penciclovir, the active orm o the drug. Famciclovir is well

Cidofovir

absorbed orally and subsequently modif ed by an esterase and an oxidase to yield penciclovir. In humans, this results in approximately 70% oral bioavailability. Like acyclovir, penciclovir consists o a guanine linked to an acyclic sugar-like molecule that lacks a 2 position but retains the 3 position and has a CH2 group in place o the ether oxygen. Penciclovir’s mechanism o action is similar to that o acyclovir (Fig. 38-7), with some quantitative di erences. Famciclovir is used in the treatment o HSV in ections and shingles, and penciclovir ointment is used to treat cold sores caused by HSV. Ganciclovir

HCMV is much less sensitive to acyclovir than HSV and VZV are, primarily because much less phosphorylated acyclovir accumulates in HCMV-in ected cells than in HSV- or VZV-in ected cells. Ganciclovir is a nucleoside analogue that was originally synthesized as a derivative o acyclovir with the intention o developing another anti-HSV drug, but it proved too toxic or that indication. It turned out, however, that ganciclovir is much more potent than acyclovir against HCMV, and ganciclovir was the f rst antiviral drug approved or use against HCMV. Like penciclovir, ganciclovir contains a guanine linked to an acyclic sugar-like molecule that lacks a 2 position and retains the 3 CHOH group that is missing in acyclovir (Fig. 38-6), but unlike penciclovir, ganciclovir retains the ether oxygen. Thus, ganciclovir more closely resembles the natural compound, deoxyguanosine, and this resemblance may account or its greater toxicity. (In act, ganciclovir is so toxic that it should be used only or serious in ections.) As mentioned above, HCMV does not encode a homolog o HSV TK (which phosphorylates ganciclovir very e f ciently) but rather encodes a viral protein kinase that induces expression o host cell enzymes. Remarkably, this viral protein kinase, called UL97, directly phosphorylates ganciclovir, leading to a 30- old increase in the amount o phosphorylated ganciclovir in in ected cells compared to unin ected cells. Ganciclovir triphosphate inhibits HCMV DNA polymerase more potently than it does cellular DNA polymerases. Although ganciclovir is not an obligate chain terminator, its incorporation does result in chain termination a ter the next nucleotide is incorporated. The mechanism o chain termination involves an exonuclease activity o the viral DNA polymerase, which rapidly excises any nucleotides that are incorporated therea ter. Thus, as with acyclovir and HSV, ganciclovir is selective against HCMV at two steps: phosphorylation and DNA polymerization. However, the selectivity against HCMV at each step is not as great as the selectivity o acyclovir against HSV; accordingly, ganciclovir is more toxic than acyclovir. Toxicity is most commonly mani ested as bone marrow suppression, especially neutropenia. Ganciclovir resistance due to mutations in either or both the UL97 and DNA polymerase genes is a clinical problem in a substantial raction o patients.

This phosphonate-containing acyclic cytosine analogue represents a twist on the mechanism o action o antiherpesvirus nucleoside analogues. Indeed, cido ovir can be considered a nucleotide rather than a nucleoside analogue. With its phosphonate group, cidofovir mimics deoxycytidine monophosphate (dCMP); thus, in e ect, it is already phosphorylated (Fig. 38-6). There ore, cido ovir does not require viral kinases or its phosphorylation, and, accordingly, it is active against UL97-mutant viruses that are resistant to ganciclovir. As predicted by its structural resemblance to a phosphorylated compound, cido ovir is not orally available and is there ore administered intravenously. Nevertheless, this drug enters cells with reasonable e f ciency. It is urther phosphorylated (twice) by cellular enzymes to yield an analogue o dCTP, which inhibits herpesvirus DNA polymerases more potently than cellular DNA polymerases. Like ganciclovir, cido ovir is not an obligate chain terminator, but it can induce chain termination. Cido ovir is approved or use in the treatment o HCMV retinitis in patients with HIV/AIDS. Cido ovir diphosphate has a long intracellular hal -li e; there ore, its use requires relatively in requent dosing (only once each week or less). Because o its mechanism o renal clearance, cido ovir must be co-administered with probenecid. (Probenecid inhibits a proximal tubule anion transporter and thereby decreases cido ovir excretion.) Nephrotoxicity is a major problem, and great care must be taken in administering this drug. Other Antiherpesvirus Nucleoside Analogues

Several nucleoside analogues with antiherpesvirus activity were developed and approved be ore the development o acyclovir. These agents are more toxic than acyclovir and so are not widely used, but are listed in the Drug Summary Table.

Anti-HIV Nucleoside and Nucleotide Analogues

HIV is a retrovirus. All retroviruses contain an RNA genome within a capsid surrounded by a lipid envelope studded with glycoproteins. The capsid also contains three enzymes that are especially important rom a pharmacologic perspective: reverse transcriptase, integrase, and protease. All three enzymes are essential or HIV replication (Fig. 38-2). Reverse transcriptase (RT) is a DNA polymerase that can copy both DNA and RNA. RT copies the RNA retrovirus genome into double-stranded DNA a ter the virus enters a cell. The viral DNA is then integrated into the host genome through the action o the viral enzyme integrase (see below). Subsequently, cellular RNA polymerase copies the integrated viral DNA back into RNA to make both ull-length genomic viral RNA and the mRNAs that encode the various viral proteins. The structural proteins assemble onto the ulllength genomic RNA, and soon therea ter, the virus buds through the cell membrane and matures into a orm capable o in ecting new cells. The protease cleaves viral proteins during assembly and maturation (see discussion below). Without these cleavages, the viral particles that are ormed remain unctionally immature and nonin ectious.

706 PRINCIPLES OF CHEMOTHERAPY

Similar to herpesviruses, HIV orms latent in ections in humans, and no available antiviral drug attacks HIV during latency. Rather, the available drugs act only on replicating virus. Zidovudine Zidovudine (azidothymidine [AZT]) was the f rst FDA-

approved anti-HIV drug. Although it has largely been supplanted by newer nucleoside analogues with higher e f cacy, lower toxicity, and improved pharmacokinetic properties, AZT illustrates many o the important principles o antiHIV nucleoside analogues. Like the antiherpesvirus nucleoside analogues described above, AZT has an altered sugar moiety. Specif cally, AZT contains a thymine base attached to a sugar in which the normal 3 hydroxyl has been converted to an azido group (Fig. 38-6). Thus, like acyclovir, AZT is an obligate chain terminator. AZT is a substrate or cellular thymidine kinase, which phosphorylates AZT to AZT monophosphate. (Unlike herpesviruses, HIV does not encode its own kinase.) AZT monophosphate is then converted to the diphosphate orm by cellular thymidylate kinase and to the triphosphate orm by cellular nucleoside diphosphate kinase. Thus, unlike acyclovir and ganciclovir, there is no selectivity at the activation step, and phosphorylated AZT accumulates in almost all dividing cells in the body, not just infected cells. The accumulation o phosphorylated AZT in almost all dividing cells largely accounts or the increased toxicity o AZT compared to a drug such as acyclovir. AZT triphosphate targets HIV reverse transcriptase and is a substantially more potent inhibitor o HIV RT than o the human DNA polymerases that have been tested. The mechanism by which AZT inhibits RT is not entirely resolved, but incorporation o AZT triphosphate into the growing DNA chain is clearly important or antiviral activity. Resistance to AZT inexorably appeared in patients when it was used as a monotherapy. High-level resistance was typically associated with the accumulation o several mutations in the gene encoding HIV RT. Like all polymerases, HIV RT catalyzes not only a orward reaction but also a back-reaction in which the two linked phosphates (pyrophosphate), which are cleaved rom the nucleoside triphosphate or drug triphosphate during incorporation, can combine with the newly extended primer template to regenerate the triphosphate and the original primer template. With HIV RT, this “excision” reaction can be supported by ATP as well as by pyrophosphate. Many mutations that cause resistance to AZT and some other nucleoside analogues avor this ATPdependent excision reaction. Thus, AZT can be compared with acyclovir and ganciclovir (Table 38-1). Acyclovir is the most selective o these

drugs because it is highly selective at both the activation (kinase) and inhibition (polymerase) steps. AZT is probably the least selective o the three drugs because it is nonselective at the activation step. Although AZT is relatively selective at the inhibition step, phosphorylated orms o AZT inhibit important cellular enzymes. For example, AZT monophosphate is both a substrate and an inhibitor o cellular thymidylate kinase, which is essential or cellular replication. Ganciclovir is intermediate in selectivity, with modest selectivity at both the activation and inhibition steps. AZT toxicity is a serious clinical problem and led to its being administered in doses lower than those that achieve maximum e f cacy. In particular, AZT causes bone marrow suppression, which is mani ested most commonly as neutropenia and anemia. AZT toxicity appears to be caused not only by the e ects o AZT triphosphate on cellular polymerases but also by the e ects o AZT monophosphate on cellular thymidylate kinase (see above). The limited clinical e ectiveness o AZT and problems with its toxicity and resistance led to the development o other anti-HIV drugs and to the use o combination chemotherapy or HIV (Box 38-2). Other Anti-HIV Nucleoside Analogues with Mechanisms o Resistance Similar to That o AZT

Most anti-HIV nucleoside analogues—other than lamivudine and emtricitabine (see below)—have mechanisms o action and mechanisms o resistance similar to those o AZT (Fig. 38-6 and Drug Summary Table). Most exhibit toxicities that are thought to be due, at least in part, to inhibition o mitochondrial DNA polymerase by drug triphosphates, but these vary rom drug to drug. Several o these drugs can be used at e f cacious doses with much less toxicity than AZT. Teno ovir, which contains a phosphonate like the antiHCMV drug cido ovir, is ormulated as an orally available prodrug, teno ovir disoproxil (Fig. 38-6); the prodrug can be administered just once a day. Teno ovir has been combined with other drugs that have di erent resistance mechanisms, which also can be administered once a day, to provide a much simpler treatment regimen than the original anti-HIV combinations. Lamivudine and Emtricitabine

Two nucleoside analogues—lamivudine (3TC) and emtricitabine (FTC)—di er rom the other anti-HIV nucleoside analogues in their structure and mechanism o resistance. These drugs are L-stereoisomers, not the standard D-stereoisomer o biological nucleosides and the other antiHIV nucleoside analogues, and they contain a sul ur atom in their f ve-membered ring (Fig. 38-6). Like AZT and the other anti-HIV nucleoside analogues, 3TC and FTC are

TABLE 38-1 Selectivity o Action o Antiviral Nucleoside Analogues Is Determined by Specif city o Viral and Cellular Kinases and Polymerases DRUG

KINASE SPECIFICITY

POLYMERASE SPECIFICITY

Acyclovir

Viral TK

Cellular kinases

Viral DNA polymerase

Ganciclovir

Viral UL97

Cellular kinases

Viral DNA polymerase

Zidovudine (AZT)

Cellular TK

Drugs are presented in order o selectivity o action:

, large di erence in specif city;

Viral RT

Cellular DNA polymerase Cellular DNA polymerase

Cellular DNA polymerase

, modest di erence in specif city. TK, thymidine kinase; RT, reverse transcriptase.

C h a p t e r 3 8 Pharmacology o Viral In ections

obligate chain terminators. However, resistance to these compounds is not usually conferred by the same mutations that confer resistance to the other anti-HIV nucleoside analogues. Rather, alteration of a single residue drastically reduces incorporation of these compounds into the growing primer template. As a result, 3TC or FTC have frequently been combined with one of the other anti-HIV nucleoside analogues, since resistance to one compound does not usually result in resistance to the other (Box 38-2). Additionally, 3TC and FTC are biotransformed to relatively weak inhibitors of mitochondrial DNA polymerase. FTC can be administered just once a day and is often used in combination with other once-a-day anti-HIV drugs.

707

Anti-HBV Nucleoside and Nucleotide Analogues

In addition to their use in treating HIV infections, 3TC/FTC and tenofovir are used in patients with chronic HBV infections and evidence of active virus replication. (FTC is not FDA-approved for treatment of HBV but is often used in patients who are co-infected with HIV and HBV.) Three other nucleoside analogues are also approved for use against HBV: these include adefovir, which, like cidofovir and tenofovir, is a nucleoside phosphonate; telbivudine , which is simply L-thymidine; and entecavir, which is an unusual deoxyguanosine analogue (Fig. 38-6). HBV is an unusual DNA virus. Within the HBV virion is a partially double-stranded DNA genome and a viral DNA

B O X 3 8 - 2 Co m b in a tio n An tivira l Th e ra p y in th e Tre a tm e n t o f HIV When AZT was f rst introduced, monotherapy with this drug delayed disease progression in HIV-in ected individuals and prolonged the survival o patients with advanced AIDS. In the late 1980s and early 1990s, this was a major advance in treatment. Since then, however, the drawbacks o AZT as monotherapy have become well recognized. AZT causes considerable toxicity— including anemia, nausea, headache, insomnia, arthralgia, and, rarely, lactic acidosis—and it e ects only a modest (three old to ten old) and transient decrease in the viral load o HIV in plasma. Most patients treated with AZT as monotherapy inexorably progressed to AIDS. AZT-resistant virus could be detected in most o these patients, and it is generally accepted that these AZT-resistant variants contributed to the low long-term e f cacy o AZT monotherapy. Similar problems have been encountered with the use o most other anti-HIV drugs as monotherapy. When 3TC, the NNRTIs, or protease inhibitors were used as single agents, although the initial antiviral e f cacy was greater than that o AZT ( 30- old reduction in the concentration o HIV in plasma), it was still incomplete, and resistance developed even more quickly than it did with AZT. Toxicities, un avorable pharmacokinetic properties, and drug–drug interactions are also signif cant problems with many o the available agents. Because o these drawbacks, combination chemotherapy (i.e., the use o “drug cocktails”; see Chapter 41, Principles o Combination Chemotherapy) has become the standard o care or HIV-in ected individuals. The cocktails are more e f cacious than single agents, inducing larger decreases in the viral load o HIV. Combination chemotherapy also decreases the emergence o resistance, both because virus replication is more e f caciously inhibited and, there ore, the chances or mutations to arise during replication are reduced, and because multiple mutations are required to con er resistance to all the drugs in the cocktail. In theory, combination chemotherapy can permit each drug to be used at lower doses, thereby reducing toxicity. It is now widely accepted that patients diagnosed with HIV in ection should start therapy immediately with combination chemotherapy rather than with a single drug. Indeed, all new anti-HIV drugs are now approved by the FDA or combination use only, and certain

combinations o drugs are combined into single pills. In 2006, the f rst single pill co- ormulated regimen o teno ovir, emtricitabine, and e avirenz was approved or use on a once-a-day basis. Several other one-pill once-daily co- ormulated regimens have been approved since then, and the reduced pill burden has been shown to improve both adherence and clinical outcome. In antibacterial and antineoplastic combination chemotherapy, it is typical that only agents a ecting di erent targets are combined (see Chapter 41). However, in anti-HIV combination chemotherapy, two or even three RT inhibitors (e.g., teno ovir, emtricitabine, and e avirenz) have been combined with evident benef t. One actor accounting or this success could be the incomplete e f cacy o each drug alone; combining these drugs could allow or greater e f cacy. (Because some o these drugs have toxicity prof les that di er rom one another, it is possible to combine these agents without a signif cant increase in overall toxicity.) A second actor is that mutations con erring resistance to one drug do not ordinarily con er resistance to the other drugs. For example, mutants resistant to AZT and most other nucleoside analogues remain sensitive to 3TC or FTC and to NNRTIs. A third possible actor is that mutations con erring resistance to one drug can suppress the e ects o mutations con erring resistance to another drug, although the clinical signif cance o this f nding is controversial. A ourth possible actor—perhaps the most important—is that many resistance mutations decrease the “f tness” o the virus; that is, its ability to replicate in the patient. Thus, under some circumstances, it may even be benef cial to include in a combination therapy regimen a drug to which the virus is resistant in order to maintain selective pressure in avor o that less-f t, drug-resistant virus. In many patients undergoing combination anti-HIV therapy (o ten called highly active antiretroviral therapy or HAART), the concentration o virus in the blood drops below the limit o detection ( ewer than 20-50 copies o HIV RNA/mL in a standard test). However, anti-HIV drugs, like antiherpesvirus drugs, attack only replicating virus and not latent virus, and the best evidence is that HAART will need to be maintained li e-long. Despite this limitation, HAART has been a tremendous success story, saving millions o lives worldwide and preventing countless additional in ections. ■

708 PRINCIPLES OF CHEMOTHERAPY

polymerase that also unctions as an RT. Upon entry into the cell nucleus, this polymerase completes the synthesis o the viral DNA. The resulting DNA does not ordinarily integrate; rather, it serves as an episomal template or transcription by cellular RNA polymerase, which copies it into RNA to make both ull-length genomic RNA and the mRNAs that encode the various viral proteins. Structural proteins, including the viral polymerase, then assemble onto the ull-length genomic RNA. Within the resulting particles, which are still inside the in ected cell, the polymerase copies the RNA into partially double-stranded DNA. Finally, the virus particle buds out o the cell, acquiring a lipid envelope. The triphosphate orms o the f ve di erent nucleoside analogues are potent inhibitors o the HBV polymerase; they become incorporated into the growing DNA chain and cause chain termination (although some are not obligate chain terminators). Drug resistance is an important consideration in the treatment o HBV with these drugs. Resistance to 3TC/FTC and telbivudine occurs relatively rapidly due to mutations similar to those that con er resistance o HIV to 3TC and FTC. Multiple mutations are required or resistance to entecavir, which may contribute to the relatively slow development o resistance to this drug in patients. Interestingly, some o these mutations do not con er resistance per se but rather seem to increase f tness in the presence o the other mutations. Ade ovir, telbivudine, and entecavir are all relatively well tolerated. Mitochondrial toxicity is a risk or all o these drugs. Cases o myopathy and peripheral neuropathy have been reported with telbivudine, and lactic acidosis has been reported with all o the anti-HBV nucleoside analogues.

Anti-HCV Nucleoside and Nucleotide Analogues

The RNA virus HCV encodes an RNA-dependent RNA polymerase. Based on the success o nucleoside analogue inhibitors o viral DNA polymerase, much e ort has gone into developing such inhibitors o HCV RNA polymerase. The f rst o these to be approved is sofosbuvir (Fig. 38-6), which contains uracil. Recall that uracil is the base in uridine, a normal nucleoside precursor o RNA. Despite its activity as an RNA polymerase inhibitor, so osbuvir does not contain a hydroxyl group at the 2 position o the sugar moiety. Instead, that position is modif ed with uorine and a methyl group. Like cido ovir, teno ovir, and ade ovir, so osbuvir contains a phosphate mimic—but in this case, a phosphoramidite rather than a phosphonate—that can be urther phosphorylated by cellular enzymes to a triphosphate mimic that has a long cellular hal -li e. The phosphoramidite is urther modif ed with additional groups that are cleaved o in hepatocytes; i.e., so osbuvir is a prodrug. At concentrations that show little or no cytotoxicity, including mitochondrial toxicity, so osbuvir is highly e f cacious at inhibiting HCV RNA polymerase and genome replication. Although certain mutations in the gene coding or the viral RNA polymerase can con er resistance to so osbuvir, such mutations do not readily arise during treatment with the drug, evidently because they decrease viral f tness. Presumably, the enzyme would need to change its active site or resistance to occur, and such changes would be expected to impair enzyme e f ciency. As the f rst DAA approved or use in an all-oral regimen without inter eron alpha (but with ribavirin) against certain genotypes o HCV, so osbuvir trans ormed HCV treatment. It has subsequently been approved or use without ribavirin in combination with the protease inhibitor

simeprevir (see above) or the NS5A inhibitor ledipasvir (see below) against certain HCV genotypes. In many cases, only a 2- to 3-month course o combination antiviral treatment is needed to cure the disease. So osbuvir is generally well tolerated, and serious adverse reactions have been limited.

Nonnucleoside DNA Polymerase Inhibitors

Nucleoside analogues can inhibit cellular as well as viral enzymes. As a result, e orts have been made to discover compounds with di erent structures that can more selectively target viral enzymes. The f rst such compound to be used clinically was foscarnet (phosphonoformic acid [PFA]; Fig. 38-8). Foscarnet has a relatively broad spectrum o activity in vitro (including against HIV), but clinically, it is used to treat certain serious HSV and HCMV in ections in which therapy with acyclovir or ganciclovir has not succeeded (e.g., because o resistance). Mechanistically, oscarnet di ers rom nucleoside analogues in that it does not require activation by cellular or viral enzymes; rather, oscarnet inhibits viral DNA polymerase directly by mimicking the pyrophosphate product o DNA polymerization. Moreover, a crystal structure suggests that oscarnet occupies the position o two o the phosphates on the incoming deoxyribonucleoside triphosphate, thus stalling the polymerase. Selectivity results rom the increased sensitivity o viral DNA polymerase to oscarnet relative to cellular enzymes. As might be expected o a compound that so closely mimics a natural compound (pyrophosphate), oscarnet’s selectivity is not as high as acyclovir’s; it inhibits cell division at concentrations not much higher than its e ective antiherpesvirus concentration. Major drawbacks to oscarnet use include its lack o oral bioavailability and its poor solubility; renal impairment is its major dose-limiting toxicity. Resistance can also arise.

Nonnucleoside Reverse Transcriptase Inhibitors

The nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz, nevirapine , delavirdine , etravirine , and rilpivirine were developed using the rational approach o target-based, high-throughput screening (Box 38-1 and Fig. 38-8). Indeed, the NNRTIs were among the f rst successes o this now widely used approach. Unlike the nucleoside analogues, these drugs inhibit their target directly, without the need or chemical modif cation. X-ray crystallographic studies have shown that NNRTIs bind near the catalytic site o RT. NNRTIs permit RT to bind a nucleoside triphosphate and primer template but inhibit the joining o the two. The NNRTIs are orally bioavailable, and their adverse e ects (most commonly, rash) are typically less serious than those o oscarnet and most nucleoside analogues. The main limitation o NNRTI use is that resistance develops rapidly; just a single mutation that prevents drug binding is su f cient or high-level resistance with little f tness cost. This limitation requires that these drugs must be used in combination with other anti-HIV drugs (Box 38-2). One NNRTI, efavirenz, was the f rst anti-HIV drug to be taken once a day. In 2006, a single pill combining e avirenz, teno ovir, and FTC was approved by the FDA or once-aday administration. Since then, a co- ormulated single pill containing rilpivirine, teno ovir, and FTC has also become available. While e avirenz-containing treatments are requently associated with neuropsychiatric adverse e ects, the rilpivirine-based regimen is recommended as f rst-line treatment only or HIV-1-in ected individuals with viral RNA load 100,000 copies/mL and CD4 count 200 cells/mm3.

C h a p t e r 3 8 Pharmacology o Viral In ections NH2

N

N

Br

N N

O

O

P

S

OH

HO

N

O

N

O

OH

HN N H

H N

N H

O

O Etravirine

Delavirdine

Foscarnet

N

N

H

C

S

O

C

HN

N

O

N

N

O

N

709

O

N

N

O HN

NH

N

Nevirapine

N

Rilpivirine

Dasabuvir

H

N

O O F3C

O

N

H

H

F

F

N

Cl O N H

O N

O

N

N N O

N

N H

Efavirenz

H

O

H

Ledipasvir

O C[CH3 ]3

O

O N N H

N

N H O

O H3 CO 2 CHN

N

Ombitasvir

NHCO 2 CH3

FIGURE 38-8. Nonnucleoside DNA polymerase and reverse transcriptase inhibitors and NS5A inhibitors. Foscarnet is a pyrophosphate analogue that inhibits viral DNA and RNA polymerases. Foscarnet is approved or the treatment o HSV and HCMV in ections that are resistant to antiherpesvirus nucleoside analogues. The nonnucleoside reverse transcriptase inhibitors (NNRTIs) delavirdine, etravirine, nevirapine, rilpivirine, and e avirenz inhibit HIV-1 reverse transcriptase. The NNRTIs are approved in combination with other antiretroviral drugs or the treatment o HIV-1 in ection. Note that the structures o the NNRTIs are signif cantly di erent rom those o the anti-HIV nucleoside and nucleotide analogues (compare with Fig. 38-6). Dasabuvir inhibits HCV RNA polymerase. The anti-HCVNS5A inhibitors, ledipasvir and ombitasvir, have two old symmetric elements, which permit very tight binding to the dimeric NS5A protein. These compounds are approved or treatment o HCV disease in combination with other anti-HCV drugs.

710 PRINCIPLES OF CHEMOTHERAPY

FIGURE 38-9. Integration of HIVDNA into cellular DNA and effect of anti-HIVintegrase inhibitors. A. Schematic rendering o the action o HIVintegrase. Doublestranded HIVDNA is generated by reverse transcription as a blunt-ended, linear molecule with repeated sequences known as long terminal repeats (LTR) at both ends. The 5 LTR includes the promoter/enhancer or HIVtranscription, and the 3 LTR includes the polyadenylation signal. At the termini o both LTRs are identical sequences o our base pairs. In the f rst step o integration (3 end processing), HIVintegrase removes the two terminal nucleotides rom the 3 strands rom both ends o the viral DNA, resulting in two-base (AC), 5 overhangs. In the second step (strand trans er), integrase creates a staggered cleavage o host DNA and then catalyzes the attack o the 3 OH ends o the viral DNA on phosphodiester bonds in the host DNA, resulting in the ormation o new phosphodiester bonds linking host and viral DNA at both ends o the viral genome. The AC overhang o viral DNA is not joined, and the process also results in single-stranded gaps in the host DNA on each side o the viral genome. This leads to the third step (repair/ligation), in which the AC overhangs are removed and the gaps in host DNA f lled in, creating a short duplication o host sequences on either side o the integrated viral DNA. The integrase inhibitors raltegravir, elvitegravir, and dolutegravir inhibit the strand trans er reaction. B. Structures o raltegravir, elvitegravir, and dolutegravir. C. Molecular mechanism by which raltegravir inhibits HIV integrase. The left panel shows the active site o a retroviral integrase bound to viral DNA in the absence o drug, while the right panel shows the same active site in the presence o raltegravir. The integrase protein is shown in green with alpha helices shown as helical ribbons and beta strands as arrows. Asp and Glu residues that coordinate magnesium ions, the viral DNA, and raltegravir are shown as stick models and the magnesium ions as gray spheres. The 3 -hydroxyl group o viral DNA is adjacent to the magnesium ions in the absence o drug (left panel) but angled away rom the ions when raltegravir is bound (right panel). (Panel C was kindly provided by Peter Cherepanov.)

Many companies have employed a strategy similar to that used to discover the NNRTIs to identi y nonnucleoside inhibitors o HCV RNA polymerase. Dasabuvir was the f rst o these to be approved by the FDA (in 2014) (Fig. 38-8). The binding o dasabuvir to the polymerase is thought to inhibit initiation o HCV RNA synthesis. The drug is highly potent (nM range) against replication o certain HCV genotypes, but resistance can arise rapidly when it is used as monotherapy. Accordingly, dasabuvir is approved or use only in combination with the HCV protease inhibitor paritaprevir (see above) and the NS5A inhibitor ombitasvir (see below). Other inhibitors in this class are expected to enter clinical practice in the near uture.

concentrations (IC50’s) that are in the picomolar or even subpicomolar range in some cases. NS5A inhibitors o ten retain potency against multiple genotypes o HCV and are associated with ew adverse e ects. Resistance to these inhibitors arises rapidly, however, requiring that they be used in combination chemotherapy. The combination o ledipasvir–so osbuvir is generally well tolerated. Ledipasvir and so osbuvir are both substrates o the P-glycoprotein drug transporter; concomitant administration o ledipasvir–so osbuvir with intestinal P-glycoprotein inducers (e.g., ri ampin, phenytoin, tipranavir/ ritonavir) may decrease drug levels and is not recommended (see Chapter 5, Drug Transporters). Ledipasvir–so osbuvir may increase teno ovir serum levels in certain situations and should be administered cautiously to individuals who are coin ected with HCV and HIV.

Inhibition of Viral Genome Replication— Other Mechanisms

HIV Integrase Inhibitors

Anti-HCV Nonnucleoside RNA Polymerase Inhibitors

Anti-HCV NS5A Inhibitors

As a complement to the target-based screens used to discover NNRTIs (Box 38-1) and the nonnucleoside inhibitors o HCV RNA polymerase, many companies have developed cell-based screens to search or novel inhibitors o viral replication. These screens typically assay viral replication by measuring the activity o a oreign gene product (a reporter) that is encoded in the viral genome being assayed. These assays can be automated such that large libraries o chemicals can be screened or activity over relatively short time rames. An advantage o this approach is that one can identi y inhibitors o targets or which there is no biochemical assay. A new drug class—the anti-HCV NS5A inhibitors—is a striking example o the success o this screening approach. The f rst o these inhibitors to be approved by the FDA (in 2014) were ledipasvir (in combination with so osbuvir) and ombitasvir (in combination with paritaprevir and dasabuvir) (Fig. 38-8). NS5A is a somewhat enigmatic viral protein that is essential or viral genome replication. There is evidence that this protein helps the NS5B RNA polymerase synthesize long chains o RNA. NS5A is also thought to alter the host cell environment in order to abet viral RNA synthesis and to have roles at later stages o in ection. The protein is a dimer, and NS5A inhibitors similarly display either rough (ledipasvir) or complete (ombitasvir) two old symmetry. This structure promotes tight binding o the drugs, with each hal o the molecule engaging an NS5A monomer. Additionally, it appears that drug binding to a small raction o NS5A molecules in the in ected cell is su f cient to inhibit HCV replication. These eatures collectively lead to remarkably potent inhibition o HCV genome replication, with 50% inhibitory

Integrase, the enzyme that carries out HIV genome integration, is an essential enzyme or HIV genome replication. Integrase assembles onto sequences at the ends o HIV DNA, cleaves dinucleotides rom each 3 strand, trans ers these strands to target (cellular) DNA, and covalently ligates the HIV DNA to target DNA (Fig. 38-9A). Scientists developed an assay or inhibition o the DNA strand trans er reaction o integrase, and this assay was used to screen or active compounds. Three such compounds have been success ully developed into orally available FDA-approved drugs, raltegravir (the f rst in its class), elvitegravir, and dolutegravir (structures in Fig. 38-9B). The structure o raltegravir or elvitegravir bound to an enzyme that is closely related to HIV integrase and is complexed with viral DNA ends reveals a ascinating mechanism o action o the drugs. Raltegravir and elvitegravir not only bind to amino acid residues o the protein but also bind to magnesium ions chelated by acidic active-site residues (DDE) and to DNA. Interestingly, part o the drug occupies a position such that the 3 -hydroxyl o the viral DNA is angled away rom the active site, thus preventing strand trans er (Fig. 38-9C). All three drugs are approved or use in combination with other anti-HIV drugs in HIV-in ected individuals, including those who have not previously been treated with antiretroviral drugs (treatment-naïve). O note, raltegravir is approved or use in pediatric cases, and elvitegravir is ormulated as a once-a-day pill with FTC, teno ovir, and an inhibitor o the cytochrome P450 enzyme that metabolizes elvitegravir (cobicistat). Dolutegravir has a higher barrier to genetic resistance than raltegravir and elvitegravir do, and dolutegravir remains active against many viruses that are resistant to the other two integrase inhibitors. Dolutegravir is available as a single co- ormulated tablet with abacavir and lamivudine.

C h a p t e r 3 8 Pharmacology of Viral Infections A

HIV DNA

5' LTR

CAGT3' GTCA5'

5'ACTG 3'TGAC

3' LTR

3' e nd proce s s ing

5' LTR

CA3' GTCA5'

5'ACTG 3'AC

3' LTR

Integrase inhibitors Ta rge t (ce llula r) DNA p VWXYZ VWXYZ p

S tra nd tra ns fe r 5'

AC TG VWXYZAC

CAVWXYZ GTC A

5' LTR

3' LTR

5'

Re pa ir/liga tion VWXYZTG VWXYZAC

CAVWXYZ GTVWXYZ

5' LTR

3' LTR

O

B N

O O -K+

N

F

O

Cl

HO

N H N

H N

N

O

N HO

O

O

F

O

Raltegravir

Elvitegravir O

OH F

O

N

H N

N O

H

F

O

Dolutegravir

C Vira l DNA

Vira l DNA

3'-OH 3'-OH E

α4

E D

D β1 Inte gra s e

RAL D

β4 α2

α4

D

β4

β1

α2

Inte gra s e

711

712 PRINCIPLES OF CHEMOTHERAPY

Inhibition o Viral Maturation For many viruses, including HIV, the assembly o proteins and nucleic acids into particles is not su f cient to produce an in ectious virion; rather, an additional step called maturation is required. In most cases (including HIV), these viruses encode proteases that are essential or maturation. The HIV protease cleaves gag and gag-pol polyproteins to yield unctional capsid proteins and viral enzymes.

Lessons learned rom the development o the HIV protease inhibitors were highly valuable in developing the HCV NS3/4A protease inhibitors (see above). The approved and marketed antiviral drugs that target HIV protease—saquinavir, ritonavir, osamprenavir, indinavir, nelf navir, lopinavir, atazanavir, tipranavir, and darunavir (Fig. 38-10)—are success ul examples o rational drug design (Box 38-3 and Fig. 38-11). HO O

O

OH P

N

S

O O

N H O

O

O

H2 N

Fosamprenavir NH2

O S

N

O

HO

H NH

HN

N

O

O

OH

N

N

N

O O

S

NH

Indinavir

O

O N H

O

OH

H N

N

O

H N

N H

N H

OH

N

Lopinavir

O

O

Saquinavir

O N H

H

N

O

H

Darunavir

H N

N H NH2 OH

O O

O

N

H

O

O

H N

HO

S

O

N H

N

N

OH

OH

Ritonavir

H N

O

S

H

H

Nelfinavir N OH

OH

O H3 CO

N H

H N O

O N

N H

H N

OCH3

O

NH S O2

O F3C

Atazanavir

O

N

Tipranavir

FIGURE 38-10. Anti-HIVprotease inhibitors. Shown are the structures o the anti-HIVprotease inhibitors osamprenavir, darunavir, saquinavir, lopinavir, indinavir, ritonavir, nelf navir, atazanavir, and tipranavir. These compounds mimic peptides (peptidomimetics), and all but tipranavir contain peptide bonds.

C h a p t e r 3 8 Pharmacology o Viral In ections

713

A

O Le u As n

H N

O

H N

Ile Le u

N

As n

P rote a s e a tta ck

HO

H N

H N

O OH

Ile

N

Rota tiona l a xis of s ymme try P-3 P-2

P-1 (P he )

P1 (P ro) P2 Mode l of tra ns ition s ta te on s ubs tra te s e que nce

pol S ubs tra te s e que nce

B OH

OH H2 N

Cbz

NH2

Va l

H N

H N

Va l

OH

Cbz Cbz

Val

O

H N

N H

N H

Va l

Cbz

OH

A-74704 P rote a s e IC 50 = 5 nM Antivira l a ctivity < 1 µM

A-74702 P rote a s e IC 50 > 200 µM

O N

N

A-75925 P rote a s e IC 50 < 1 nM Antivira l a ctivity < 1 µM P oor a que ous s olubility

OH

O

H N

N H

H N

N H

O

OH

N

N

O

A-77003 P rote a s e IC 50 < 1 nM Antivira l a ctivity = 0.1 µM Good s olubility P oor ora l bioa va ila bility

O N S

N

O H N

N H O

N H OH

O

S N

Ritona vir P rote a s e IC 50 < 1 nM Antivira l a ctivity = 25 nM Fa ir s olubility Good ora l bioa va ila bility

C

FIGURE 38-11. Steps in the evolution of ritonavir. A. The HIVpol gene product has a phenylalanine (Phe)–proline (Pro) sequence that is unusual as a cleavage site or human proteases. HIV protease cleaves this Phe–Pro bond. The transition state o the protease reaction includes a rotational axis o symmetry. B. Structure-based development o a selective HIVprotease inhibitor began with a compound (A-74702) that contained two phenylalanine analogues and a CHOH moiety between them. This compound, which had weak inhibitory activity, was then modif ed to maximize antiprotease activity while also maximizing antiviral activity, aqueous solubility, and oral bioavailability. The maximization o antiprotease activity was measured as a progressive reduction in IC50, the drug concentration required to cause 50% inhibition o the enzyme. See Box 38-3 or details. C. Structure o ritonavir (space-f lling structure) bound to HIV protease, with beta strands shown as green arrows, alpha helices in purple, and the amino acids that bind ritonavir depicted in stick orm.

714 PRINCIPLES OF CHEMOTHERAPY

B O X 3 8 - 3 De ve lo p m e n t o f Rito n a vir The development o ritonavir is an example o structure-based (“rational”) drug design. Scientists began with a model o the transition state that orms during the cleavage o a substrate by HIV protease (Fig. 38-11). An analogue o the transition state was designed using just one residue on each side o the cleavage site. Knowing that HIV protease is a symmetric dimer, the scientists chose to use the same residue—phenylalanine—on both sides o the cleavage site, with a CHOH group that mimics the transition state as the center o symmetry. This molecule, A-74702, was a very weak inhibitor o HIV protease, but adding symmetric groups at both ends to orm A-74704 (Fig. 38-11, where Val is valine and Cbz is carbobenzyloxy) resulted in a 40,000- old increase in potency (IC50 5 nM). All attempts to modi y A-74704 to improve aqueous solubility also reduced potency, however, so a related potent inhibitor, A-75925, in which the center o symmetry was a C-C bond between two

For several reasons, HIV protease was (and remains) an attractive target or pharmacologic intervention. First, it is essential or HIV replication. Second, a point mutation is su f cient to inactivate the enzyme, suggesting that a small molecule might success ully inhibit activity. Third, the sequences cleaved by HIV protease are conserved and somewhat unusual, suggesting both specif city and a starting point or drug design. Fourth, HIV protease—unlike the human proteases most closely related to it—is a symmetric dimer o two identical subunits, each o which contributes to the active site, again suggesting both specif city and a starting point or drug design. Fi th, the enzyme can be easily overexpressed and assayed, and its crystal structure has been solved. All o these actors increased the likelihood that a drug discovery e ort would be success ul. The HIV protease inhibitor ritonavir provides an example o rational drug design. Ritonavir is a peptidomimetic (i.e., it mimics the structure o a peptide; see Box 38-3 and Fig. 38-11). Its design began with the identif cation o one o the natural substrates o HIV protease, a site or cleaving a longer protein into reverse transcriptase. This site is unusual in that it contains a phenylalanine–proline (Phe–Pro) bond (Fig. 38-11A); mammalian enzymes rarely, i ever, cleave at such a site. To take advantage o the symmetric dimer eature o the HIV protease structure, correspondingly symmetric inhibitors were designed in which the Pro was replaced with a Phe. Moreover, CHOH was used in place o the native C O o the peptide bond in order to mimic the transition state o protease catalysis, which is the catalytic intermediate that binds the enzyme most tightly (Fig. 38-11). The designed inhibitors, unlike the original peptide and the native transition state, cannot be cleaved by the enzyme. How these symmetric inhibitors evolved into ritonavir is discussed in Box 38-3 (also see Fig. 38-11). Although clever design is no guarantee that a drug will be active against a virus by the expected mechanism, the protease inhibitors do act as expected. (Interestingly, darunavir inhibits not only the activity o the protease but also its dimerization.) The compounds are potent in cell culture, albeit o ten less potent against virus replication than against

CHOH groups, became the sca old or urther modif cations. Symmetric changes to both ends o the molecule resulted in a soluble, highly potent inhibitor, A-77003. This compound was not orally bioavailable, however. Further modif cations, which removed a central OH group and altered other moieties at each end o the molecule, resulted in a compound—ritonavir—that was less soluble but had improved antiviral activity and good oral bioavailability. Therapeutically achievable plasma concentrations o ritonavir greatly exceed the concentration required or antiviral activity. In the process o structure-based drug design, successive modi ications to these molecules took advantage o x-ray structures o HIV protease complexed to each inhibitor. By examining these structures, scientists were able to make in ormed guesses about which specif c chemical groups to add or subtract. The result was the therapeutically use ul HIV protease inhibitor ritonavir. ■

enzyme activity in vitro. As expected, HIV-in ected cells exposed to protease inhibitors continue to make viral proteins, but these proteins are not processed e f ciently. Viral particles bud rom the in ected cells, but these particles are immature and nonin ectious. Used in combination with other anti-HIV drugs, protease inhibitors have had a major impact on AIDS therapy (Box 38-2). Currently, ritonavir-boosted atazanavir and darunavir are the two protease inhibitors recommended as components o f rst-line therapy. The main adverse e ects o atazanavir are indirect hyperbilirubinemia and jaundice. Darunavir has a sul onamide moiety and its main adverse e ect is a rash. All protease inhibitors also alter at distribution and cause metabolic abnormalities, which has limited their use. The mechanisms o these adverse e ects remain poorly understood.

Inhibition of Viral Release Rational design has also led to the development o inhibitors o in uenza virus neuraminidases. The rationale or these inhibitors, which block viral release rom the host cell, ollows rom the mechanism o viral attachment and release. In uenza virus attaches to cells via interactions between hemagglutinin, a protein on the viral envelope, and sialic acid moieties, which are present on many cell sur ace glycoproteins. Upon egress o in uenza virus rom cells at the end o a round o replication, the hemagglutinin on nascent virions again binds to the sialic acid moieties, thereby tethering the virions to the cell sur ace and preventing viral release. To overcome this problem, in uenza virus encodes an envelope-bound enzyme, called neuraminidase , which cleaves sialic acid rom the membrane glycoproteins and thereby permits release o the virus. Without neuraminidase, the virus remains tethered and cannot spread to other cells. In 1992, the structure o the neuraminidase–sialic acid complex was solved. The structure showed that sialic acid occupies two o three well- ormed pockets on the enzyme. Based largely on this structure, a new sialic acid analogue was designed to maximize energetically avorable interactions in

C h a p t e r 3 8 Pharmacology o Viral In ections

715

A

HO

B

HO HO

HO

O

O

NH H2 N

O

S ia lic a cid

H2N O

Zanamivir

Glyce rol Ca rboxyla te

Oseltamivir

Hydrophobic group

Glyce rol Ca rboxyla te

Gua nidino

Hydroxyl

Active s ite of ne ura minida s e

O

H N

HN O

S ia lic a cid

O

O

H N

OH

HO

C

COOH

COOH

OH H N

OH

Zanamivir

Ca rboxyla te

Hydrophobic pocke t

GS 4071 (a ctive me ta bolite of prodrug oseltamivir)

FIGURE 38-12. Structure-based design of neuraminidase inhibitors. A. Shown is a model o sialic acid (space-f lling structure) bound to the inf uenza A virus neuraminidase, with the amino acids that bind sialic acid depicted in stick orm. This structure was used to design transition state analogues that bind more tightly to neuraminidase than sialic acid does, resulting in potent inhibitors o the enzyme. B. Structures o sialic acid and the neuraminidase inhibitors zanamivir and oseltamivir. C. Schematic diagram o the active site o inf uenza virus neuraminidase, depicting the binding o sialic acid, zanamivir, and GS4071 to several di erent eatures o the active site. (Oseltamivir is the ethyl ester prodrug o GS4071.)

all three o the potential binding pockets (Fig. 38-12). This compound, now known as zanamivir, inhibits neuraminidase with a Ki o about 0.1 nM. Zanamivir is active against both in uenza A and in uenza B, with potencies o about 30 nM. However, zanamivir has low oral bioavailability and must be administered by inhaler. E orts to improve on zanamivir’s pharmacokinetics resulted in a new drug, oseltamivir (Fig. 38-12), whose oral availability is approximately 75%. Oseltamivir binds well to two o the three binding pockets o the neuraminidase. When taken prophylactically, oseltamivir reduces the number o u cases in susceptible populations (e.g., nursing home residents). Both oseltamivir and zanamivir reduce the duration o u symptoms in most patients who are already in ected with the virus. However, this reduction is only

1 day on average, and even this modest e ect (although it can be quite meaning ul to the “ u” su erer) requires that the drugs be taken within 2 days o the onset o symptoms. Oseltamivir is also used to treat patients who are at high risk or severe in ection (e.g., immunocompromised individuals) or those who already have severe in ection. Concerns about H5N1 avian in uenza (“bird u”) or the 2009 H1N1 (“swine u”) pandemic strain have led to stockpiling o oseltamivir. Although mutations that cause resistance to neuraminidase inhibitors reduce viral f tness and were not detected in treated patients or a number o years, in uenza strains have since evolved with additional mutations that compensate or the loss o f tness. Regardless, the neuraminidase inhibitors represent a triumph o rational drug design.

716 PRINCIPLES OF CHEMOTHERAPY

Antiviral Drugs with Unknown Mechanisms of Action Despite the increasing success o rational drug design, some antiviral agents act by unknown or only partially understood mechanisms.

Docosanol

n-Docosanol is a 22-carbon saturated alcohol with activity

against HSV and certain other enveloped viruses. Although shorter chain saturated alcohols have long been known to inactivate virion in ectivity, but also exhibit cytotoxicity, docosanol has been reported to lack signif cant cytotoxicity. Cell culture studies suggest that docosanol acts, at least in part, between the stage o HSV attachment and the stage o viral protein translation, with some e ects on virus entry at certain doses. Cells must be pretreated with docosanol or hours or an antiviral e ect to become mani est, and there is evidence that, during this time, docosanol is metabolized and incorporated into host cell membranes. However, it is not clear what basis, i any, exists or selectivity o antiviral action; no docosanol-resistant mutants have been reported that could shed light on the drug’s mechanism o action. Docosanol is FDA-approved as an over-the-counter topical treatment or recurrent oral– acial HSV episodes (cold sores), although its clinical e f cacy is controversial, as is the relationship o any such e f cacy to an antiviral e ect.

Ribavirin

Ribavirin exhibits activity against many viruses in vitro. In

patients, however, ribavirin has been approved by the FDA only (1) in aerosol orm (in e ect, topical application to the lungs) or severe respiratory syncytial virus (RSV) in ection and (2) in oral orm, in combination with inter eron alpha, with so osbuvir, or with the DAA combination o paritaprevir, ombitasvir, and dasabuvir or chronic HCV in ection. It has also been used to treat other viral in ections, such as the li e-threatening in ections caused by Lassa ever virus. Structurally, ribavirin di ers rom the other nucleoside analogues in Figure 38-6 in that it has a natural sugar moiety (ribose) attached to a non-natural base-like moiety that most resembles a purine (adenine or guanine). Ribavirin is converted to monophosphate, diphosphate, and triphosphate orms by cellular enzymes. The phosphorylated orms can inhibit various host and viral enzymes, and the triphosphate derivative can also be incorporated into RNA by viral RNA polymerases and induce deleterious mutations. There is also evidence that ribavirin can enhance immune responses. Which o these proposed mechanisms o ribavirin action are relevant or the therapeutic e ect o the drug on viral in ections in humans is not f rmly established. Indeed, or HCV, ribavirin by itsel has little i any e ect on the levels o virus in treated patients. Moreover, ribavirin has important toxicities, including anemia. Regardless, learning more about the mechanisms o ribavirin action may lead to improved antiviral therapies.

Drugs That Modulate the Immune System Three classes o drugs that make explicit use o host immune processes are used to treat viral in ections. These classes include immunization, inter erons, and imiquimod. For background on the immune system, see Chapter 42, Principles o In ammation and the Immune System.

Active and passive immunization inhibit viral in ection by

providing antibodies against virion sur ace proteins; these antibodies then block the attachment and penetration o virions into cells and increase virion clearance. Some antibodies are directly virucidal, causing virions to be destroyed or inactivated be ore the virus can interact with its receptor(s) on target cells. There are, o course, many vaccines that are examples o active immunization against viruses (e.g., measles, mumps, rubella, hepatitis B), and most o these vaccines are used prophylactically. One example o a vaccine used therapeutically is rabies vaccine , which can save the lives o individuals who are already in ected with rabies virus. Examples o passive immunization are the prophylactic use o either pooled human immune globulins with anti-RSV activity or a humanized monoclonal antibody, palivizumab, to prevent RSV in ection in high-risk children. The inter erons and imiquimod make use o the innate immune response (see Chapter 42) and do not directly target viral gene products. Inter erons were f rst recognized as proteins that are produced in response to virus in ection and that can inhibit replication o the same or other viruses. There are two major types o inter erons. Type I interferons include interferon- and interferon- , which are produced by many cell types and interact with the same cell sur ace receptor. Type II interferons include interferon- , which is typically produced by cells o the immune system, especially T cells, and interacts with a di erent receptor. Interaction o inter erons with their receptors induces a series o signaling events that activate and/or induce the expression o proteins that combat viral in ections. One relatively well understood example o such a protein is a protein kinase, called PKR, which is activated by double-stranded RNA. (Double-stranded RNA is o ten produced during viral in ections.) PKR phosphorylates a component o the host translational machinery, thereby turning o protein synthesis and thus the production o virus in in ected cells. Interferon- is used as a therapeutic agent in the treatment o HCV, HBV, condyloma acuminata (which is caused by certain human papillomaviruses [HPVs]), and Kaposi’s sarcoma (which is caused by Kaposi’s sarcoma-associated herpesvirus [KSHV], also known as human herpesvirus 8). Inter eron- is usually administered in a orm that has been modif ed with polyethylene glycol (pegylated) to improve its pharmacokinetic prof le a ter injection. Although the mechanism by which inter erons inhibit the replication o certain viruses is reasonably well understood (e.g., by inducing PKR), the mechanisms by which inter erons act against HCV, HBV, HPVs, and KSHV remain poorly understood. Interestingly, all o these viruses encode proteins that inhibit inter eron action. Understanding the mechanism o this inhibition may aid understanding o the action o inter erons in inhibiting viral replication. This is an active area o investigation. Inter eron- is also used to treat certain relatively rare malignancies, and interferon- is used to treat multiple sclerosis. Again, the mechanisms by which inter erons exert their therapeutic e ects in these clinical settings are poorly understood. For multiple sclerosis, there is evidence that, while boosting innate immune responses, type I inter erons also suppress certain in ammatory responses. Imiquimod is approved or the treatment o certain diseases caused by HPVs. Imiquimod interacts with the Tolllike receptors TLR7 and TLR8 to boost innate immunity,

C h a p t e r 3 8 Pharmacology of Viral Infections

including the secretion o inter erons. Toll-like receptors are membrane proteins that recognize pathogen-associated molecular patterns. Activation o Toll-like receptors induces intracellular signaling events that are important or de ense against pathogens. In the case o imiquimod, it is not clear exactly how this stimulation results in e ective treatment o disease caused by HPV.

CONCLUSION AND FUTURE DIRECTIONS The various stages in the viral li e cycle provide a basis or understanding the mechanisms o action o currently available antiviral drugs and or developing new antiviral therapies. The vast majority o antiviral drugs available today inhibit viruses at the genome replication stage by taking advantage o structural and unctional di erences between viral and host polymerases. In addition, maraviroc and en uvirtide (T-20) inhibit HIV attachment and entry, adamantanes inhibit in uenza A uncoating, protease inhibitors inhibit viral gene expression (HCV) and viral maturation (HIV), and neuraminidase inhibitors inhibit in uenza release. It is important to bear in mind, however, that many o these drugs inhibit only one virus (e.g., HIV), and, in some cases, only one species o that virus (e.g., HIV-1 but not HIV-2). Only a tiny raction o viruses known to cause human disease can be treated e ectively with the antiviral therapies that are currently available. Nevertheless, great strides have been made. At this writing, new antiviral small-molecule drugs are under review at the FDA, and many other small molecules, antibodies, and other modalities, such as oligonucleotides that block viral gene expression by RNA intererence, are under investigation. (An antisense DNA-based oligonucleotide that blocks HCMV replication has been FDA-approved [ omivirsen], but it is no longer marketed and whether it actually acts by blocking viral gene expression has not been established.) In the case o Mr. M, the treatment o HIV with a combination o drugs could reduce viral loads

717

to undetectable levels and delay the progression o AIDS or many years. Although antiviral therapies do not yet represent either prevention or cure or this disease, such therapies have already decreased the morbidity and mortality o HIV/AIDS in millions o individuals, essentially converting HIV in ection rom a death sentence to a manageable chronic disease.

Acknowledgment We thank Robert W. Yeh or his valuable contributions to this chapter in earlier editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy and Peter Cherepanov or Figure 38-9C, illustrating the mechanism o integrase inhibitors.

Suggested Reading Coen DM, Richman DD. Antiviral agents. In: Knipe DM, Howley PM, Cohen JI, et al., eds. Fields virology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2013. (Detailed review o the general and specif c aspects o the mechanisms and uses o antiviral drugs.) Dorr P, Westby M, Dobbs S, et al. Maraviroc (UK-427,857), a potent, orally available, and selective small-molecule inhibitor o chemokine receptor CCR5 with broad-spectrum anti-human immunodef ciency virus type 1 activity. Antimicrob Agents Chemother 2005;49:4721–4732. (Describes the development o an antiviral drug, maraviroc, that acts by blocking a host target.) Hare S, Gupta SS, Valkov E, Engelman A, Cherepanov P. Retroviral intasome assembly and inhibition o strand trans er. Nature 2010;464:232–236. (Presents the crystal structure o a retroviral integrase bound to viral DNA and integrase inhibitors, thereby elucidating mechanisms o integrase action and drug inhibition.) Hay AJ, Wolstenholme AJ, Skehel JJ, Smith MH. The molecular basis o the specif c anti-in uenza inhibition o amantadine. EMBO J 1985;4: 3021–3024. (This classic paper illustrates how viral genetics can be used to identi y a drug target.) Sof a MJ, Bao D, Chang W, et al. Discovery o a -D-2 -deoxy-2 - - uoro2 - -C-methyluridine nucleotide prodrug (PSI-7977) or the treatment o hepatitis C virus. J Med Chem 2010;53:7202–7218. (Describes the iterative process undertaken to discover so osbuvir.) von Itzstein M, Wu WY, Kok GB, et al. Rational design o potent sialidasebased inhibitors o in uenza virus replication. Nature 1993;363:418–423. (Describes the structure-based design o zanamivir.)

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722 PRINCIPLES OF CHEMOTHERAPY

39 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance

C

David A. Ba rb ie a n d David A. Fra n k

I TR IN T ODUCTION OD & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723–724 BIOC BI OCHE EMI MIST S RY OF GE GENO NOME ME SYNTHESIS, STABILITY, AND MAINT NTEN NANCE . . . . . . . . . . . . . . . . . . . . . . . . . 72 7244 Nucl Nu cleoti tidde Syynth thes hesis is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 7244 Purine Ribo Ribonucleotide onucle l otide Synthe Synthesiss . . . . . . . . . . . . . . . . . . . . . 724 Pyrimidine Ribonucleotide Synthesis . . . . . . . . . . . . . . . . . . 724 Ribonucleotide Reduction and Thymidylate Synthesis . . . 725 Nucleic Acid Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725 DNA Repair and Chromosome Maintenance . . . . . . . . . . . . . 725 Mismatch Repairr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727 Base Excision Repair. r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727 Nucleotide Excision Repairr . . . . . . . . . . . . . . . . . . . . . . . . . . 728 Double-Strand Break Repairr . . . . . . . . . . . . . . . . . . . . . . . . . 728 Telomere Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730 Microtubules and Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 732 Inhibitors of Thymidylate Synthase . . . . . . . . . . . . . . . . . . . . . . 733 Inhibitors of Purine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 733

INTRODUCTION Cancer therapy has traditionally been based on the principle that tumor cells are traversing the cell cycle requently and are thus more sensitive than normal cells to inter erence with DNA synthesis and mitosis. Indeed, the antimetabolites , a class o agents that are analogues o endogenous olates, purines, and pyrimidines, and that unction as inhibitors o the enzymes o nucleotide synthesis, were some o the f rst drugs to be tested as chemotherapeutic agents. In the late 1940s, Sidney Farber and colleagues administered the anti olate compound aminopterin to patients with acute leukemia and observed temporary remissions in more than hal o the patients. Because o their rapid growth and division, cancer cells are also thought to be more sensitive than normal cells to the e ect o DNA-damaging agents. Also in the late 1940s, nitrogen mustards —derivatives o agents that had been ound to cause bone marrow suppression through wartime exposures—were

Inhibitor ors rss of Ri Ribo bonu n cleotide de Reduc educta tase se . . . . . . . . . . . . . . . . . . 73 7344 Puri Purine Pu rine ne a nd nd P Pyrimid yrim yrimid idin din inee An Ana A naalog logues guess Th That att Are Incor nc orpo pora r ted into DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 7355 A ents That Directlly Modif Ag difyy DN DNA A St S ructuure . . . . . . . . . . . . . . 735 Alkylating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 7355 Platinum Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 Bleomycinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739 Topoisomerase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Camptothecinss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Anthracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Epipodophyllotoxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Amsacrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741 Microtubule Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741 Inhibitors of Microtubule Polymerization: Vinca Alkaloidss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741 Inhibitors of Microtubule Depolymerization: Taxanes . . . . 741 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 742 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .742

tested in patients with lymphoma and leukemia and shown to induce remissions. These and other f ndings have since led to the development o multiple classes o antineoplastic drugs designed to inter ere with the building blocks o DNA synthesis and mitosis, or to produce DNA damage and chromosomal instability, and thereby to promote cytotoxicity and programmed cell death (apoptosis ). Un ortunately, the therapeutic window o these drugs is narrow because they also a ect normal cells that routinely undergo cell division in tissues such as the gastrointestinal tract and bone marrow. That being said, cancer cells can o ten be induced to undergo apoptosis more readily than normal cells, providing some degree o selectivity. Use o combination chemotherapy with agents rom di erent classes has helped to enhance e f cacy while minimizing overlapping dose-limiting toxicities, but the ability to cure patients with most types o advanced cancer remains limited. In part, this limited e f cacy is due 723

724 PRINCIPLES OF CHEMOTHERAPY

On e d a y, J L, a 23-ye a r-o ld g ra d u a te s tu d e n t w h o h a s h e re to o re b e e n in g o o d h e a lth , n o tice s w h ile s h o w e rin g th a t h e h a s d e ve lo p e d a h a rd lu m p in h is le t te s tis . Co n ce rn e d b y th e f n d in g , J L’s p hys icia n o rd e rs a n u ltra s o u n d e xa m in a tio n , w h ich s h o w s a s o lid m a s s s u g g e s tive o ca n ce r. Th e te s tis is re m ove d s u rg ica lly; p a th o lo g ic re vie w co n f rm s th e d ia g n o s is o te s ticu la r ca n ce r. A ch e s t x-ra y re ve a ls s e ve ra l lu n g n o d u le s , w h ich a re th o u g h t to re p re s e n t m e ta s ta tic s p re a d o th e ca n ce r. J L is tre a te d w ith s e ve ra l cycle s o co m b in a tio n ch e m o th e ra p y, in clu d in g b le o m ycin , e to p o s id e , a n d cis p la tin . Th e lu n g n o d u le s d is a p p e a r co m p le te ly. On e ye a r la te r, J L is a b le to re s u m e h is s tu d ie s , a n d th e re a re n o s ig n s o re cu rre n ce

to the development o multiple resistance mechanisms, including the ailure o tumor cells to undergo apoptosis in response to DNA damage or stress. In addition, it is increasingly apparent that populations o cancer stem cells may have low proli eration rates and other properties that render them resistant to cytotoxic chemotherapy.

BIOCHEMISTRYOF GENOME SYNTHESIS, STABILITY, AND MAINTENANCE The central dogma o molecular biology states that DNA contains all the in ormation necessary to encode cellular macromolecules—specif cally, that DNA is transcribed into RNA, and RNA is then translated into proteins. Antimetabolites inhibit the synthesis o nucleotides, which are the building blocks o both DNA and RNA. Figure 39-1A provides an overview o nucleotide synthesis, and Figure 39-1B shows the steps at which some o the drugs discussed in this chapter inhibit nucleotide metabolism.

Nucleotide Synthesis Nucleotides, the precursors o DNA and RNA, include the purine nucleotides and the pyrimidine nucleotides. Purines and pyrimidines are the bases that are used to determine the chemical code within DNA and RNA. Adenine and guanine are purines; cytosine, thymine, and uracil are pyrimidines. Nucleosides are derivatives o purines and pyrimidines that are conjugated to ribose or deoxyribose. Nucleotides are monophosphate, diphosphate, and triphosphate esters o the corresponding nucleosides. For example, an adenine base covalently linked to a ribose sugar and a diphosphate ester is called adenosine diphosphate (ADP). The various purine and pyrimidine bases, nucleosides, and nucleotides are shown in Table 39-1. Nucleotide synthesis involves three general sets o sequential reactions: (1) synthesis o ribonucleotides, (2) reduction o ribonucleotides to deoxyribonucleotides, and (3) conversion o deoxyuridylate (dUMP) to deoxythymidylate (dTMP) (Fig. 39-2). Ribonucleotide synthesis di ers or purines and pyrimidines; there ore, the synthesis o each class o molecules is discussed individually. All ribonucleotides are reduced to deoxyribonucleotides by a single

o th e ca n ce r. No n e th e le s s , a t e ve ry s u b s e q u e n t o llo w-u p vis it, J L’s p hys icia n a s ks h im w h e th e r h e is d e ve lo p in g s h o rtn e s s o b re a th .

Questions 1 . How did serendipity lead to the discovery o cisplatin, the most e f cacious drug against testicular cancer? 2 . What is the molecular target o each o the drugs in J L’s combination chemotherapy regimen? 3 . Why does J L’s physician inquire about shortness o breath at each ollow-up visit? 4 . By what mechanisms could bleomycin, etoposide, and cisplatin act synergistically against J L’s testicular cancer?

enzyme, ribonucleotide reductase . Deoxyribonucleotides generated rom ribonucleotides and rom dUMP are used or DNA synthesis. Because olate is an essential co actor or the synthesis o purine ribonucleotides and dTMP, olate metabolism is discussed separately (see Chapter 33, Principles o Antimicrobial and Antineoplastic Pharmacology).

Purine Ribonucleotide Synthesis

Adenine and guanine , the purine bases shown in Table 39-1,

are synthesized as components o ribonucleotides ( or RNA synthesis) and deoxyribonucleotides ( or DNA synthesis). Derivatives o adenine and guanine, which include ATP, GTP, cAMP, and cGMP, are also used or energy storage and cell signaling. Purine synthesis begins with the assembly o inosinate (IMP) rom a ribose phosphate, moieties derived rom the amino acids glycine, aspartate, and glutamine, and one-carbon trans ers catalyzed by tetrahydrofolate (THF), as shown in Figure 39-2. Because o the central role o THF in purine synthesis, one important chemotherapeutic strategy is to reduce the amount o THF available to the cell and thereby to inhibit purine synthesis. Figure 39-3 shows the central role o IMP in purine synthesis. IMP can be aminated to AMP or oxidized to GMP. In turn, AMP and GMP can be converted to ATP and GTP, respectively, and then incorporated into RNA, or reduced to dAMP and dGMP, respectively, as described below. Purine bases, nucleosides, and nucleotides are readily interconverted by multiple enzymes within the cell. In one such reaction, the enzyme adenosine deaminase (ADA) catalyzes the irreversible conversion o adenosine or 2 -deoxyadenosine to inosine or 2 -deoxyinosine, respectively. Inhibition o ADA causes the intracellular stores o adenosine and 2 -deoxyadenosine to exceed those o the other purines, ultimately resulting in metabolic e ects that are toxic to the cell (see discussion o pentostatin below).

Pyrimidine Ribonucleotide Synthesis

Pyrimidine ribonucleotides are synthesized according to the metabolic pathway shown in Figure 39-4. The basic pyrimidine ring, orotate, is assembled rom carbamoyl phosphate and aspartate. Orotate then reacts with a ribose phosphate; the decarboxylation product o this reaction yields

C h a p t e r 3 9 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance

725

uridylate (UMP). As with IMP in purine synthesis, UMP has

A

P urine pre curs ors

P yrimidine pre curs ors Fola te

a central role in pyrimidine synthesis. UMP is itsel a nucleotide component o RNA, as well as the common precursor o the RNA and DNA components cytidylate (CMP), deoxycytidylate (dCMP), and deoxythymidylate (dTMP). CTP is ormed by the amination o UTP.

Ribonucleotide Reduction and Thymidylate Synthesis Inos ine monophos pha te (IMP )

P yrimidine s

Ribonucle otide s De oxyribonucle otide s DNA RNA P rote in

B Methotrexate P urine precurs ors

P yrimidine pre curs ors Fola te

6-Mercaptopurine Thioguanine IMP 6-Mercaptopurine Thioguanine

P yrimidine s

Ribonucle otide s Hydroxyurea De oxyribonucle otide s

Fludarabine Cytarabine Cladribine

DNA

RNA

P rote in

FIGURE 39-1. Overview of de novo nucleotide biosynthesis. A. Folate is an essential cofactor in the synthesis of inosine monophosphate (IMP), from which all purine nucleotides are derived. Pyrimidine synthesis does not require folate, although folate is required for the methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) (see Fig. 39-2). Ribonucleotides contain one of the purine or pyrimidine bases linked to ribose phosphate. Subsequent reduction of the ribose at the 2 position produces deoxyribonucleotides. Deoxyribonucleotides are polymerized into DNA, while ribonucleotides are used to form RNA (not shown). The central dogma of molecular biology states that the DNA code determines the sequence of RNA (transcription) and that RNA is then translated into protein. B. Methotrexate inhibits dihydrofolate reductase (DHFR) and thereby prevents the utilization of folate in purine nucleotide and dTMP synthesis. 6-Mercaptopurine and thioguanine inhibit the formation of purine nucleotides. Hydroxyurea inhibits the enzyme that converts ribonucleotides to deoxyribonucleotides. Fludarabine, cytarabine, and cladribine are purine and pyrimidine analogues that inhibit DNA synthesis. 5-Fluorouracil inhibits the enzyme that converts dUMP to dTMP (not shown). Sulfonamides are discussed in Chapter 33.

The ribonucleotides ATP, GTP, UTP, and CTP, which are required or RNA synthesis, are assembled on a DNA template and linked to orm RNA. Alternatively, ribonucleotides can be reduced at the 2 position on ribose to orm the deoxyribonucleotides dATP, dGTP, dUTP, and dCTP. The conversion o ribonucleotides to deoxyribonucleotides is catalyzed by the enzyme ribonucleotide reductase . (In actuality, ribonucleotide reductase uses as substrates the diphosphate orms o the our ribonucleotides to produce dADP, dGDP, dUDP, and dCDP; nucleotides can, however, be readily interconverted among their monophosphate, diphosphate, and triphosphate orms.) Note, in Figures 39-2 through 39-4, that ribonucleotide reductase catalyzes the ormation o the DNA precursors dATP, dGTP, and dCTP. The DNA precursor dTTP is not synthesized directly by ribonucleotide reductase, however. Rather, dUMP must be modif ed to orm dTMP. As demonstrated in Table 39-1, dTMP is the product o dUMP methylation. The methylation o dUMP to dTMP is catalyzed by thymidylate synthase , with methylenetetrahydroolate (MTHF) serving as the donor o the methyl group (Fig. 39-4). As MTHF donates its methyl group, it is oxidized to dihydro olate (DHF). DHF must be reduced to THF by dihydrofolate reductase (DHFR) and then converted to MTHF in order to serve as the co actor or another cycle o dTMP synthesis. Inhibition o DHFR prevents the regeneration o tetrahydro olate and thereby inhibits the conversion o dUMP to dTMP, eventually resulting in an insu f cient cellular level o dTMP or DNA replication.

Nucleic Acid Synthesis Provided that su f cient levels o nucleotides are available, DNA and RNA can be synthesized, and protein synthesis, cell growth, and cell division can occur. Many drugs, including the antimetabolites discussed in this chapter, inhibit both DNA and RNA synthesis. To avoid repetition, a detailed discussion o DNA and RNA synthesis is provided in Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation. For the purposes o this chapter, the reader should be aware that RNA and DNA are formed by polymerization of ribonucleotides and deoxyribonucleotides, respectively. RNA polymers are elongated by the enzyme RNA polymerase , and DNA is elongated by DNA polymerase . Although antimetabolites primarily inhibit the enzymes that mediate nucleotide synthesis, some antimetabolites also inhibit DNA and RNA polymerases (see below).

DNA Repair and Chromosome Maintenance Mutations and other DNA lesions can arise spontaneously or as a result o exposure to DNA-damaging chemical agents or radiation. Several general pathways exist or repair o these lesions, including mismatch repair (MMR) or DNA replication errors, base excision repair (BER) or small base modif cations and single-strand breaks, nucleotide excision repair (NER) or removal o bulky adducts, and

726 PRINCIPLES OF CHEMOTHERAPY

TABLE 39-1 Purine and Pyrimidine Derivatives: Bases, Nucleosides, and Nucleotides Ba s e

Ribonucle os ide

Ribonucle otide

NH2 N NH2 N

N

N

N H

N

N -

O

N

H

Ade nine (A)

H

H

OH

H OH

O

P

O

O

N

N

HO

-

H

H H

H OH

OH

O

P

H

OH

H

O

O

N

O

-

H

H

De oxya de nos ine

Ade nyla te (AMP )

N

N

O

O

H H

NH2

N

N

N

O

-

Ade nos ine

N

N

N

O

De oxyribonucle otide

NH2

NH2

N

HO

De oxyribonucle os ide

H

H

OH

H

H

De oxya de nyla te (dAMP )

Purines O N

O

N

N N H

N

N

HO

NH

N

NH NH2 -

O

NH2

H

H

H

OH

H OH

O

P O

O

N

H

H

H

OH

H OH

H

N

O

O

N H

H

N

O -

O

P

O

OH

H OH

H

Cytidine

Cytos ine (C)

H

H

OH

H OH

N H

O

O

H

Ura cil (U)

H

OH

H OH

P O

H

H

H

OH

H OH

Uridine

OH

H

H

N

O

H

O

P

H

O H

H

H

H

OH

H

H

De oxycytidyla te (dCMP )

De oxcytidine

O

O

NH

NH O

N

O -

O H H

Uridyla te (UMP )

O

P O

H

OH

H

N

O O H

H

H

H

OH

H

O

NH N

NONE

NONE

NH

O

HO

N H

O

H

Purine synthesis

P RP P

Pyrimidine synthesis

Fola te

P RP P

Amino a cids

IMP

Ribonucle otide s

AMP

GMP

UMP

CMP

dUMP

De oxyribonucle otide s dAMP

H

dGMP

dTMP

DNA

dCMP

O

P O

H

De oxythymidine

Thymine (T)

Amino a cids

OH

H

N

O -

O H

H

De oxyuridyla te (dUMP )

De oxyuridine

O

O

O

-

O

NH

O

O

O-

H

OH

N

O -

O

-

H

NH2

N

H

NH2

De oxygua nyla te (dGMP )

HO

O

H

N O

N

O -

O H

H

NH O

N

O

H

O

N

O -

De oxygua nos ine

Cytidyla te (CMP )

HO

NH

H

O

NH

Pyrimidines

OH

P O

HO

O

O

H

NH2

O

O-

H

O

H

NH

N

O

NH2 -

N

O H

N

NH2

N

N

N HO

Gua nyla te (GMP )

HO

N

NH

O

NH2

NH2

NH2

O

-

Gua nos ine

Gua nine (G)

N

NH

N

O

O

O

O

O

O

O

-

H

H

H

OH

H

H

De oxythymidyla te (dTMP )

FIGURE 39-2. Nucleotide synthesis. Purine synthesis (left) begins with the formation of inosine monophosphate (IMP) from amino acids, phosphoribosylpyrophosphate (PRPP), and folate. IMP is aminated to adenylate (AMP) or oxidized to guanylate (GMP). The ribonucleotides AMP and GMP are reduced to form the deoxyribonucleotides deoxyadenosine monophosphate (dAMP) and deoxyguanosine monophosphate (dGMP), respectively. (The conversion of ribonucleotides to deoxyribonucleotides actually takes place at the level of the corresponding diphosphates and triphosphates, e.g., ADP → dADP and ATP → dATP.) Pyrimidine synthesis (right) begins with the formation of orotate from aspartate and carbamoyl phosphate (see Fig. 39-4). Orotate is ribosylated and decarboxylated to uridylate (UMP); amination of UMP yields cytidylate (CMP). (The conversion of UMP to CMP actually takes place at the level of the corresponding triphosphates, i.e., UTP → CTP.) The ribonucleotides UMP and CMP are reduced to form the deoxyribonucleotides deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP). dUMP is converted to deoxythymidine monophosphate (dTMP) in a reaction that depends on folate. At the level of the corresponding triphosphates (not shown), deoxyribonucleotides are incorporated into DNA, and ribonucleotides are incorporated into RNA (not shown). Note the central role of folate as an essential cofactor in the synthesis of purine nucleotides and dTMP.

C h a p t e r 3 9 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 6-Mercaptopurine Thioguanine

GTP

Inosine XMP monophosphate IMP (IMP) dehydrogenase

GMP Ribonucleotide reductase

6-Mercaptopurine

Hydroxyurea

ATP

Ade nylos uccina te

(IMPDH)

727

Hydroxyurea

AMP Ribonucleotide reductase

dGMP

dAMP

dGTP

dATP Fludarabine Cladribine

DNA

DNA

FIGURE 39-3. Details of purine synthesis. Inosine monophosphate, or IMP, occupies a central position in the synthesis of purine nucleotides. IMP is oxidized by IMP dehydrogenase (IMPDH) to xanthylate (XMP), which is converted to guanosine monophosphate (GMP). GMP can be incorporated into DNA or RNA as deoxyguanosine triphosphate (dGTP) or guanosine triphosphate (GTP), respectively. Alternatively, IMP can be aminated to adenosine monophosphate (AMP) through an adenylosuccinate intermediate. AMP can be incorporated into DNA or RNA as deoxyadenosine triphosphate (dATP) or adenosine triphosphate (ATP), respectively. 6-Mercaptopurine and thioguanine inhibit IMPDH and thus interrupt GMP synthesis. 6-Mercaptopurine also inhibits the conversion of IMP to adenylosuccinate and thus interrupts AMP synthesis. Hydroxyurea inhibits ribonucleotide reductase and thus inhibits formation of the deoxyribonucleotides required for DNA synthesis. Fludarabine and cladribine are halogenated adenosine analogues that inhibit DNA synthesis.

As pa rta te

homologous recombination or nonhomologous end-joining Ca rba moyl phos pha te

Orota te P RP P

UMP Ribonucleotide reductase

MTHF THF

DHF

Hydroxyurea

dUMP

Thymidylate synthase

DHFR

UTP

dTMP

CTP Ribonucleotide reductase

dCTP

or double-strand breaks (Fig. 39-5). DNA repair pathways are important not only because they can alter the e f cacy o chemotherapy but also because loss o these pathways requently contributes to tumor development via impairment o genomic integrity and acilitation o mutations in oncogenes and tumor suppressor genes. Telomeres , the repeat sequences that cap the ends o chromosomes, also play an important role in genome stability and prevention o chromosome usions. The enzyme telomerase , which prevents telomere shortening in cancer cells, represents a key component in the process o immortalization and oncogenic trans ormation.

Cytarabine

Mismatch Repair 5-Fluorouracil

DNA

Methotrexate

dTTP

DNA

FIGURE 39-4. Details of pyrimidine synthesis. Aspartate (an amino acid) and carbamoyl phosphate combine to form orotate, which then combines with phosphoribosylpyrophosphate (PRPP) to form uridylate (UMP). UMP occupies a central position in the synthesis of pyrimidine nucleotides. UMP can be sequentially phosphorylated to uridine triphosphate (UTP). UTP is incorporated into RNA (not shown) or aminated to form cytidine triphosphate (CTP). CTP is incorporated into RNA (not shown) or reduced by ribonucleotide reductase to deoxycytidine triphosphate (dCTP), which is incorporated into DNA. Alternatively, UMP can be reduced to deoxyuridylate (dUMP). Thymidylate synthase converts dUMP to deoxythymidylate (dTMP) in a reaction that depends on folate. dTMP is phosphorylated to deoxythymidine triphosphate (dTTP), which is incorporated into DNA. Hydroxyurea inhibits the formation of deoxyribonucleotides and thereby inhibits DNA synthesis. Cytarabine, a cytidine analogue, inhibits the incorporation of dCTP into DNA. 5-Fluorouracil inhibits dTMP synthesis by inhibiting thymidylate synthase. Methotrexate inhibits dihydrofolate reductase (DHFR), the enzyme responsible for regenerating tetrahydrofolate (THF) from DHF. By inhibiting DHF reductase, this drug inhibits the formation of methylenetetrahydrofolate (MTHF), which is the folate compound required for dTMP synthesis.

During DNA replication, errors such as single-base mismatches and insertions or deletions o microsatellite repeat sequences (microsatellite instability) are recognized and repaired by proteins o the mismatch repair (MMR) system. For single-base mismatches, recognition involves a heterodimer between the MSH2 protein and MSH6, while or insertion/deletion loops, MSH2 can also partner with MSH3 (Fig. 39-6). These complexes recruit the proteins MLH1 and PMS2 (as well as MLH3 or insertion/deletion loops), which, in turn, recruit exonucleases and components o the DNA replication machinery or excision and repair o the lesion. Germline mutations in MLH1, PMS2, MSH2, or MSH6 are associated with 70–80% o cases o hereditary nonpolyposis colon cancer. In addition, microsatellite instability, a hallmark o de ective MMR, is observed in 15–25% o sporadic colorectal cancers.

Base Excision Repair

DNA single-strand breaks (SSB), which may be ormed directly by ionizing radiation or indirectly due to enzymatic excision o a modif ed base by a DNA glycosylase, activate the enzyme poly(ADP-ribose) polymerase 1 (PARP1) (Fig. 39-7). At the site o the break, PARP1 trans ers ADP-ribose moieties rom nicotinamide adenine dinucleotide (NAD) to itsel and to a number o other proteins involved in DNA and

728 PRINCIPLES OF CHEMOTHERAPY

Re plica tion e rrors

Oxyge n ra dica ls Ionizing ra dia tion Che mica ls (nitros a mine s ) Che mothe ra pe utic a ge nts (a lkyla ting a ge nts , te mozolomide )

UV ra dia tion Che mica ls (2-AAF, be nzo(a )pyre ne ) Che mothe ra pe utic a ge nts (pla tinum a ge nts )

Ionizing ra dia tion Che mica ls (biofla vonoids , ra diomime tic che mica ls ) Che mothe ra pe utic a ge nts (ble omycin, topois ome ra s e I a nd II inhibitors )

Ba s e pa ir mis ma tche s Ins e rtion/de le tion loops

Aba s ic s ite s Ba s e modifica tions S ingle -s tra nd bre a ks

Bulky a dducts

Double -s tra nd bre a ks

Mis ma tch re pa ir

Ba s e e xcis ion re pa ir

Nucle otide e xcis ion re pa ir

Double -s tra nd bre a k re pa ir

FIGURE 39-5. Mechanisms of DNA damage and repair. Several general pathways mediate repair o DNA lesions in response to DNA damage. Replication errors typically result in base pair mismatches or insertion/deletion loops in regions o microsatellite DNA repeats; these lesions are repaired by the mismatch repair (MMR) pathway. Oxygen radicals, ionizing radiation, and various chemicals and chemotherapeutic agents can cause abasic site ormation, base modif cations, and single-strand breaks, which are repaired by the base excision repair (BER) pathway. Ultraviolet (UV) irradiation and certain DNAmodi ying chemicals and chemotherapeutic agents can cause the ormation o bulky adducts that are excised and repaired by the nucleotide excision repair (NER) pathway. Ionizing radiation, radiomimetic chemicals, bleomycin, and natural (bio avonoids) and chemotherapeutic (camptothecins, anthracyclines, epipodophyllotoxins) topoisomerase inhibitors can induce double-strand DNA breaks that trigger repair by the double-strand break (DSB) repair pathway. 2-AAF, 2-acetylamino uorene.

Single-base mismatch

MS H2

MS H6

A T T G C T T AGG C T AACGC AT C CG

Insertion/deletion loop

MS H2

T A T A T

A T A T A

MS H3/6

AT AT A T AT AT A T AT AT AT A T AT AT

MLH1

P MS 2

MLH1

MS H2

MS H6

MS H2

T A T A T

A T A T A

chromatin metabolism. The covalent addition o negatively charged ADP-ribose oligomers alters the interactions o these proteins with DNA and with other proteins. PARP1 recruits the BER protein XRCC1; together with DNA polymerase and DNA ligase III, XRCC1 acilitates repair o the lesion. PARP1 has also been implicated in the recognition o DNA double-strand breaks (DSB) and in the recruitment o DNAdependent protein kinase in DSB repair (see below), as well as in cell death pathways, modif cation o chromatin structure, transcriptional regulation, and mitotic apparatus unction.

Nucleotide Excision Repair P MS 2/ MLH3 MS H3/6

A T T G C T T AGG C T AACGC AT C CG

AT AT A T AT AT A T AT AT AT A T AT AT

A T T G C T T AGG C T AACG AAT C CG

AT AT AT AT AT AT A T AT AT AT A T AT AT

FIGURE 39-6. Mismatch repair pathway. Replication errors can result in single-base mismatches or insertion/deletion loops in microsatellite repeat regions, the latter as a result o intrastrand complementary base-pairing. Single-base mismatches are recognized by an MSH2/MSH6 heterodimer, and insertion/deletion loops are recognized by an MSH2/MSH3 or MSH2/ MSH6 heterodimer. Additional components o the mismatch repair machinery are then recruited, including MLH1/PMS2 or single-base mismatches or MLH1/PMS2 or MLH1/MLH3 or insertion/deletion loops. Exonucleases and components o the DNA replication machinery are subsequently recruited or excision and repair o the lesions.

In response to the ormation o bulky adducts that distort the DNA double helix, such as those induced by ultraviolet radiation and DNA-damaging chemotherapeutic agents, a complex set o proteins recognizes and initiates repair o the lesion via a process termed nucleotide excision repair (NER). Repair involves local opening o the double helix around the site o the damage, incision o the damaged strand on both sides o the lesion, excision o the oligonucleotide containing the lesion, and, f nally, DNA repair synthesis and ligation. The endonuclease ERCC1 plays an important role in targeted excision o the DNA lesion. The genes involved in nucleotide excision repair were in part identif ed rom study o the clinical syndromes xeroderma pigmentosa and Cockayne syndrome , which are rare photosensitivity disorders that exhibit de ects in NER.

Double-Strand Break Repair

In response to a double-strand break, activation o the ataxia telangiectasia mutated (ATM) kinase results in generation o the phosphorylated histone gamma-H2AX at the site o the break. Together with the protein MDC1, gamma-H2AX recruits to the locus o DNA damage a complex (MRN) containing the proteins Mre11, Rad50, and Nijmegen breakage syndrome gene 1 (NBS1) (Fig. 39-8). The breast and ovarian

C h a p t e r 3 9 Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance PARP 1 His tone AT TGC T AGG C T AACG AAT C CG

S is te r chroma tids DS B

NAD Nicotina mide ADP r ADP r ADP r ADP r

ADP r ADP r ADP r ADP r

His tone modifica tion

ATM H2AX

P

PARP 1 His tone AT TGC T AGG C T AACG AAT C CG MDC1 MRN comple x re cruitme nt ADP r ADP r ADP r ADP r

P

MRE11 NBS 1

RAD50

ADP r ADP r ADP r ADP r

XRCC1 PARP 1 His tone

AT TGC T AGG C T AACG AAT C CG

Nucle a s e -me dia te d re s e ction

A T T G C T T AG G C T AACG AAT C CG

FIGURE 39-7. Base excision repair pathway. The enzyme poly(ADP-ribose) polymerase 1 (PARP1) is recruited to single-strand break sites resulting rom ionizing radiation or base lesion excision. PARP1 poly-ADP ribosylates a variety o targets at the site o injury, including itsel and histones. The proteins modif ed with ADP-ribose (ADPr) oligomers then recruit additional proteins, such as XRCC1, which, in turn, recruit DNA polymerase and DNA ligase III to repair the lesion.

RAD52

BRCA1-P RAD51-BRCA2 RAD54 S tra nd inva s ion

BRCA2 RAD51

DNA s ynthe s is ; bra nch migra tion

DNA polymerase DNA ligase

FIGURE 39-8. Double-strand break repair pathway. The ataxia telangiectasia mutated (ATM) kinase recognizes and binds to double-strand DNA break sites. Upon activation, the ATM kinase marks the site by generating the phosphorylated histone gamma-H2AX. Gamma-H2AX and the protein MDC1 recruit the Mre11/Rad50/Nijmegen breakage syndrome gene 1 (NBS1) complex (MRN) to the site o injury. A ter RAD52 is recruited and nucleases mediate DNA resection, BRCA1 is recruited to the site and phosphorylated by ATM, ATR, and CHK2 kinases. Together with RAD51 and BRCA2, phosphorylated BRCA1 acilitates repair o the double-strand break by homologous recombination (depicted in the f gure) or nonhomologous end-joining (NHEJ ; not shown).

Liga tion; junction re s olution

Accura te ly re pa ire d DNA

ATM ATR, CHK2

BRCA1

729

730 PRINCIPLES OF CHEMOTHERAPY [TTAGGG]n

2-30 kb

3' 5'

[AATCCC]n

cancer susceptibility gene product BRCA1 is also phosphorylated by the kinases ATM, ATR, and CHK2 in response to the double-strand break, and phosphorylated BRCA1, RAD51, and BRCA2 are also recruited to the break site. Subsequent repair is mediated either by homologous recombination, with formation and resolution of a Holliday junction (Fig. 39-8), or by nonhomologous end-joining (NHEJ ), in which DNA-dependent protein kinase and a complex of proteins, including XRCC4, catalyze nucleolytic processes that allow end-joining by DNA ligase IV. The DNA repair effected by homologous recombination is more accurate than that mediated by NHEJ.

Unknown nuclease

s s [TTAGGG]n 3' 5' 50-300 nt TRF1 + othe r fa ctors

Folding

3' 5'

Telomere Biology

TRF2 + othe r fa ctors

S tra nd inva s ion

ds t-loop 5'

ss

3' D loop

FIGURE 39-9. Telomere structure. Human telomeres are 2 to 30 kilobases (kb) in length and consist o the simple sequence repeats TTAGGG. A 3 -terminal 50- to 300-nucleotide (nt) single-stranded overhang is generated by an as yet unidentif ed nuclease. The telomere binding proteins TRF1, TRF2, and other actors acilitate olding and proximal invasion o double-stranded telomeric DNA by the single-stranded overhang to generate a stable t-loop structure. This structure plays an important role in capping and protecting the ends o chromosomes.

Human telomeres consist of the simple repeat sequence TTAGGG. These repeats are shaped, folded, and bound by a complex of proteins to form a unique structure termed a t-loop (Fig. 39-9). In the t-loop structure, a long singlestranded overhang at the 3 end of the DNA invades the proximal double-stranded DNA component; this process is facilitated by TRF1, TRF2, and other protein factors. The t-loop and its associated complex of proteins are thought to play important roles in capping and protecting the chromosome end, as well as protecting telomeres from recognition by the DNA damage checkpoint machinery. Because DNA polymerase is unable to replicate the ends of linear chromosomes completely, telomeres shorten with each division in normal cells. Telomere shortening ultimately results in disruption of the telomeric caps, activation of a DNA damage checkpoint, and a state of cell cycle arrest termed cellular senescence (Fig. 39-10). When cells are able to bypass this checkpoint via inactivation of the tumor suppressor protein p53, which normally regulates

Te lome ra s e a ctiva tion

p53 los s , pRB los s

Ea rly popula tion doubling

La te popula tion doubling

p53 activa tion, pRB a ctiva tion

Cris is

Immorta liza tion

S e ne s ce nce

Te lome re s

Long

Me dium

S hort

S hort

Long

P rolife ra tion

Ye s

Ye s

No

Ye s

Ye s

Ce ll de a th

No

No

No

Ye s

No

FIGURE 39-10. Chromosome maintenance and its relationship to immortalization. As primary cells undergo successive population doublings, telomeres progressively shorten due to the inability o DNA polymerase to replicate the ends o linear chromosomes. Ultimately, a checkpoint is triggered, mediated by the proteins p53 and pRB, which results in a state o growth arrest termed cellular senescence. Senescence can be bypassed by inactivation o p53 and pRB; ultimately, however, the critically short telomeres cause the cells to enter a state termed crisis and to die. Activation o telomerase allows cells to maintain adequate telomere length and divide indef nitely, resulting in immortalization. Notably, exogenous expression o telomerase alone in primary cells is su f cient or these cells to bypass senescence and become immortalized.

C h a p t e r 3 9 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance

cell cycle arrest or apoptosis in response to DNA damage, chromosome usions are observed. It is thought that the progressive shortening o telomeres with age promotes genomic instability and contributes to oncogenesis. However, cells also continue to die under these conditions. Activation o the enzyme telomerase , which is a reverse transcriptase that uses an RNA template to synthesize TTAGGG repeats, allows cells to restore telomere length and divide indef nitely. Telomerase activation is observed in normal germline cells and some stem cell populations and has been shown to maintain the presence o the 3 overhang in normal cells. The immortalization process associated with telomerase activation is also essential or tumor ormation and maintenance. In a minority o tumors, an alternative lengthening o telomeres (ALT) pathway is activated.

S ubunit

β-tubulin

731

24 nm

(GTP a s e a ctivity)

GDP GTP

α-tubulin

GTP GTP

Microtubules and Mitosis Once a cell has replicated its DNA, it is prepared to undergo mitosis. In this process, chromosomes condense and are segregated into two identical daughter cells. The cell cycle transitions rom DNA replication (S phase) to G2 phase and then to mitosis (M phase) are complex and depend on the coordinated action o a number o so-called cyclin-dependent kinases (CDKs ; see Chapter 33). Progression through mitosis is also acilitated by enzymes that include aurora kinases and pololike kinases. Many cancer cells exhibit dysregulation o cell cycle timing and abnormalities in mitosis. Thus, pharmacologic inhibition o these regulatory kinases is an active area o cancer research. Currently, however, the microtubule machinery represents the primary target o agents that act in mitosis. Microtubules are cylindrical, hollow f bers composed o polymers o tubulin, which is a heterodimeric protein consisting o -tubulin and -tubulin subunits (Fig. 39-11). -Tubulin and -tubulin are encoded by separate genes, but they have similar three-dimensional structures. Both - and -tubulin bind GTP; in addition, -tubulin (but not -tubulin) can hydrolyze GTP to GDP. Microtubules originate rom a central microtubule organizing center (the centrosome, which includes two centrioles and associated proteins), where -tubulin (a protein with homology to tubulin and -tubulin) nucleates tubulin polymerization. Nascent microtubules assemble into protof laments, which are longitudinal polymers o tubulin subunits. Each protof lament interacts laterally with two other protof laments to orm a hollow-core tube, 24 nm in diameter, which consists o 13 protof laments arranged concentrically. Because tubulin is a heterodimer, this tube has inherent asymmetry; the end o a microtubule nearest the centrosome is bordered by -tubulin and is called the ( ) (minus) end, while the end o a microtubule extending rom the centrosome is bordered by -tubulin and is called the ( ) (plus) end (Fig. 39-11). Tubulin units are added at di erent rates to the ( ) and ( ) ends; the ( ) end grows (adds tubulin) twice as ast as the ( ) end. Microtubules are not static structures. Rather, they possess an inherent property known as dynamic instability (Fig. 39-12). Tubulin heterodimers add to the end o the microtubule with GTP bound to both -tubulin and -tubulin subunits. As the microtubule grows, the -tubulin o each tubulin heterodimer hydrolyzes its GTP to GDP. The hydrolysis o GTP to GDP introduces a con ormational change in tubulin that destabilizes the microtubule. The exact mechanism o this destabilization is unknown, but it may be related to a

FIGURE 39-11. Microtubule structure. Microtubules are hollow cylindrical tubes that polymerize from tubulin subunits. Each tubulin subunit is a heterodimer composed of -tubulin (shades of purple) and -tubulin (shades of blue). Both -tubulin and -tubulin bind GTP (dark shades of purple and blue); -tubulin hydrolyzes GTP to GDP after the tubulin subunit is added to the end of a microtubule (lighter shades of purple and blue). Microtubules are dynamic structures that grow and shrink lengthwise; the cylindrical tubes are composed of 13 subunits arranged concentrically, resulting in a diameter of 24 nm. Note that microtubules have an inherent structural asymmetry. One end of a microtubule is limited by -tubulin and is referred to as the ( ) (minus) end; the opposite end is limited by -tubulin and is referred to as the ( ) (plus) end.

decrease in the strength o lateral protof lament interactions or an increase in the tendency or protof laments to “curve” away rom the straight microtubule. There ore, microtubule stability is determined by the rate o microtubule polymerization relative to the rate o GTP hydrolysis by -tubulin. I a microtubule polymerizes tubulin aster than -tubulin hydrolyzes GTP to GDP, then, in the steady state, there is a cap o GTP-bound -tubulin at the ( ) end o the microtubule. This GTP cap provides stability to the microtubule structure, allowing urther polymerization o the microtubule. Conversely, i tubulin polymerization proceeds more slowly than the hydrolysis o GTP to GDP by -tubulin, then, in the steady state, the ( ) end o the microtubule is enriched with GDP-bound -tubulin. This GDPbound tubulin con ormation is unstable and causes rapid depolymerization o the microtubule. The ability o microtubules to assemble and disassemble rapidly is important or their many physiologic roles. Pharmacologic agents can disrupt microtubule unction either by preventing the assembly o tubulin into microtubules or by stabilizing existing microtubules (and thereby preventing microtubule disassembly). Microtubules have important physiologic roles in mitosis, intracellular protein tra f cking, vesicular movement, and cell structure and shape. Mitosis is the physiologic role that is targeted pharmacologically; the other physiologic roles, however, predict many o the adverse e ects o drugs that interrupt microtubule unction. Recall that microtubules nucleate rom centrosomes, which consist o centrioles and other associated proteins. In mitosis,

732 PRINCIPLES OF CHEMOTHERAPY

P re e xis ting microtubule

GTP -bound tubulin ca p

A

High conce ntra tions of GTP -bound tubulin

Low conce ntra tions of GTP -bound tubulin

β-tubulin α-tubulin

B

Le ngthe ne d microtubule Ra te of GTP hydrolys is = Ra te of polyme riza tion GTP ca p pre s e rve d

C

Ra te of GTP hydrolys is > Ra te of polyme riza tion GTP ca p s hrinks

D

Los s of GTP ca p Uns ta ble microtubule ; de polyme riza tion

FIGURE 39-12. Dynamic instability of microtubules. A. A preexisting microtubule is characterized by tubulin subunits that have predominantly hydrolyzed the GTP on -tubulin to GDP (light purple and light blue). However, -tubulin subunits that have recently been added to the microtubule have not yet hydrolyzed GTP (dark purple and dark blue). The GTP-bound tubulin subunits form a GTP-bound tubulin cap at the ( ) end of the microtubule. B. In the presence of a high concentration of GTP-bound free tubulin subunits, new GTP-bound tubulin is added to the ( ) end of the microtubule at a rate that equals or exceeds the rate of GTP hydrolysis by -tubulin. Maintenance of a GTP-bound tubulin cap results in a stable microtubule. C. In the presence of a low concentration of GTP-bound free tubulin subunits, new GTP-bound tubulin is added to the ( ) end of the microtubule at a rate less than the rate of GTP hydrolysis by -tubulin. This results in shrinkage of the GTP-bound tubulin cap. D. A microtubule that lacks a GTP-bound tubulin cap is unstable and undergoes depolymerization.

the two centrosomes align at opposite ends o the cell. Microtubules are extremely dynamic during M phase; they grow and shrink during M phase at rates much greater than during other phases o the cell cycle. This increased dynamic instability during M phase allows microtubules to locate and attach to the chromosomes. The microtubules emanating rom each centrosome bind to kinetochores, which are proteins that attach to the centromere o a chromosome. Once the kinetochore o each chromosome is attached to a microtubule, microtubuleassociated proteins act as motors to align the kinetochorebound chromosomes at the equator o the cell (def ned by the midpoint between the two centrosomes). When every chromosome has aligned at the equator, the microtubules shorten, separating a diploid pair o chromosomes into each hal o the cell. Finally, cytokinesis (division o the cytoplasm) occurs, and two daughter cells are ormed. Although many other proteins are involved in the regulation o mitosis, microtubules have a critical role in the process. Disruption o microtubule unction reezes cells in M phase, leading eventually to the activation o programmed cell death (apoptosis).

PHARMACOLOGIC CLASSES AND AGENTS Traditional antineoplastic chemotherapy can be subdivided into several classes o agents. The antimetabolite drugs are compounds that either inhibit the enzymes involved in nucleotide synthesis and metabolism or are incorporated as analogues into DNA and result in chain termination or strand breaks. These drugs act primarily during the S phase o the cell cycle, when cells are undergoing DNA replication. Another broad class o agents, which induce cytotoxicity by modif cation o DNA structure and generation o DNA damage, includes alkylating agents, platinum compounds, bleomycin, and topoisomerase inhibitors. These drugs exert their e ects during multiple phases o the cell cycle. The f nal category o agents inhibits microtubule assembly or depolymerization, disrupting the mitotic spindle and inter ering with mitosis. For a summary o the major classes o chemotherapeutic agents, their cell cycle specif city, and major toxicities, see Table 41-2 (Chapter 41, Principles o Combination Chemotherapy).

C h a p t e r 3 9 Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance

Inhibitors o Thymidylate Synthase Thymidylate (dTMP) is synthesized by the methylation o 2 -deoxyuridylate (dUMP). This reaction, which is catalyzed by thymidylate synthase, requires MTHF as a co actor (Fig. 39-4). 5-Fluorouracil (5-FU; Fig. 39-13) inhibits DNA synthesis, primarily by inter ering with the biosynthesis o thymidylate. 5-FU is f rst converted to 5- uoro-2 -deoxyuridylate (FdUMP) by the same pathways that convert uracil to dUMP. FdUMP then inhibits thymidylate synthase by orming, together with MTHF, a stable, covalent ternary enzyme–substrate–co actor complex. Cells deprived o dTMP or a su f cient period o time undergo so-called thymineless death. 5-FU can also be metabolized to oxuridine triphosphate (FUTP), which can be incorporated into mRNA in place o uridylate and can thereby inter ere with RNA processing. Either inhibition o thymidylate synthase by FdUMP or inter erence with RNA processing by FUTP, or a combination o the two mechanisms, could explain the toxic e ect o 5-FU on cells. However, certain 5-FU congeners that inhibit thymidylate synthase but are not incorporated into RNA show antitumor e f cacy similar to that o 5-FU. This f nding points to thymidylate synthase inhibition as the dominant mechanism o 5-FU action. 5-FU is used as an antineoplastic agent, especially in the treatment o carcinomas o the breast and gastrointestinal tract. 5-FU has also been used in the topical treatment o premalignant keratoses o the skin and o multiple superf cial basal cell carcinomas. Because 5-FU depletes thymidylate rom normal cells as well as cancer cells, this agent is highly toxic and must be used with care. Capecitabine is an orally bioavailable prodrug o 5-FU. It is absorbed across the gastrointestinal mucosa and converted by a series o three enzymatic reactions to 5-FU. Capecitabine is approved or the treatment o metastatic colorectal cancer and as second-line therapy in metastatic breast cancer. Clinical trials have demonstrated that the e f cacy o oral capecitabine is similar to that o intravenous 5-FU. Elucidation o the mechanism o action o 5-FU has led to the use o a 5-FU/ olinic acid (leucovorin) combination as f rst-line chemotherapy or colorectal cancer. Because 5-FU inhibits thymidylate synthase by orming a ternary complex involving the enzyme (thymidylate synthase), substrate (5-FdUMP), and co actor MTHF, it was hypothesized that increasing the levels o MTHF would potentiate the activity o 5-FU. Clinical trials proved this hypothesis to be correct by showing that the e f cacy o the combined regimen is greater than that o 5-FU alone. This is an important example

o the use o mechanistic knowledge to improve the clinical e ectiveness o a drug. Pemetrexed is a olate analogue that, similar to endogenous olate and the dihydro olate reductase (DHFR) inhibitor methotrexate (see Chapter 33), is transported into cells by the reduced olate carrier and polyglutamated by the intracellular enzyme olylpolyglutamate synthase. Polyglutamated pemetrexed is a potent inhibitor o thymidylate synthase and a much weaker inhibitor o DHFR; similar to 5-FU, its cytotoxic e ect is likely due to the induction o “thymineless” cell death. (Note that the 5-FU derivative 5-FdUMP inhibits thymidylate synthase by binding to the dUMP [substrate] site on the enzyme, whereas pemetrexed inhibits thymidylate synthase by binding to the MTHF [co actor] site on the enzyme.) Pemetrexed is approved or the treatment o all subtypes o non-small cell lung cancer except or the squamous subtype, due to lack o e f cacy o the drug in this subtype. Pemetrexed is also used in combination with cisplatin (see below) in the treatment o malignant pleural mesothelioma. To reduce toxicity to normal cells, patients treated with pemetrexed are also given olic acid and vitamin B12 supplementation.

Inhibitors o Purine Metabolism 6-Mercaptopurine (6-MP) and azathioprine (AZA), a prodrug

that is nonenzymatically converted to 6-MP in tissues, are inosine analogues that inhibit interconversions among purine nucleotides (Fig. 39-14). 6-Mercaptopurine contains a sul ur atom in place o the keto group at C-6 o the purine ring. A ter its entry into cells, mercaptopurine is converted by the enzyme

O

O HN

HN

H2 N

N

Ura cil

F

5-Fluorouracil (5-FU)

FIGURE 39-13. Structures o uracil and 5-f uorouracil. Note the structural similarity between uracil and 5- uorouracil (5-FU). Uracil is the base in dUMP, the endogenous substrate or thymidylate synthase (see Fig. 39-4), and 5-FU is metabolized to FdUMP, an irreversible inhibitor o thymidylate synthase.

N

HN H2 N

N H

N

Thioguanine

NO 2

N N

S

S N N H

Azathioprine (prodrug) O

O

N H

Gua nine

N

NH

NH

S

N

HN

N

O

733

N

HN N

N H

6-Mercaptopurine

FIGURE 39-14. Structures o guanine, thioguanine, azathioprine, and 6-mercaptopurine. Thioguanine, azathioprine, and 6-mercaptopurine are structural analogues o purines. Thioguanine resembles guanine and can be ribosylated and phosphorylated in parallel with endogenous nucleotides. The nucleotide orms o thioguanine irreversibly inhibit IMPDH (see Fig. 39-3) and, upon incorporation into DNA, inhibit DNA replication. Azathioprine is a prodrug orm o 6-mercaptopurine; azathioprine reacts with sul hydryl compounds in the liver (e.g., glutathione) to release 6-mercaptopurine. The nucleotide orm o 6-mercaptopurine, 6-thioinosine-5 -monophosphate (T-IMP), inhibits the enzymes that convert IMP to AMP and GMP (see Fig. 39-3). T-IMP also inhibits the f rst committed step in purine nucleotide synthesis.

734 PRINCIPLES OF CHEMOTHERAPY

hypoxanthine-guanine phosphoribosyltrans erase (HGPRT; see

Chapter 49, Integrative In ammation Pharmacology: Gout) to the nucleotide orm, 6-thioinosine-5 -monophosphate (T-IMP). T-IMP is thought to inhibit purine nucleotide synthesis by several mechanisms. First, T-IMP inhibits the enzymes that convert IMP to AMP and GMP, including inosine monophosphate dehydrogenase (IMPDH) (Fig. 39-3). Second, T-IMP (as with AMP and GMP) is a “ eedback” inhibitor o the enzyme that synthesizes phosphoribosylamine, which is the f rst step in purine nucleotide synthesis. Both o these mechanisms lead to marked decreases in the cellular levels o AMP and GMP, which are essential metabolites or DNA synthesis, RNA synthesis, energy storage, cell signaling, and other unctions. 6-MP may also inhibit DNA and RNA synthesis by less well-characterized mechanisms. The major clinical application o 6-MP is in acute lymphoblastic leukemia (ALL), especially in the maintenance phase o a prolonged combination chemotherapy regimen. 6-MP is also active against normal lymphocytes and can be used as an immunosuppressive agent. For unknown reasons, the prodrug AZA is a superior immunosuppressant compared to 6-MP and is typically the drug o choice or this application. AZA is discussed in detail in Chapter 46, Pharmacology o Immunosuppression. Both the e ectiveness and the toxicity o 6-MP are potentiated by allopurinol. Allopurinol inhibits xanthine oxidase, thereby preventing the oxidation o 6-MP to its inactive metabolite 6-thiouric acid. (In act, allopurinol was discovered in an e ort to inhibit the metabolism o 6-MP by xanthine oxidase.) Co-administration o allopurinol and 6-MP allows the dose o 6-MP to be reduced by two-thirds (although toxicity is proportionally increased as well). Allopurinol is o ten used as a single agent to prevent the hyperuricemia that could result rom the destruction o cancer cells by chemotherapeutic agents (tumor lysis syndrome ). The use o allopurinol in the treatment o gout is presented in Chapter 49. Pentostatin (Fig. 39-15) is a selective inhibitor o adenosine deaminase (ADA). The drug is a structural analogue o the intermediate in the reaction catalyzed by ADA and binds to the enzyme with high a f nity. The resulting inhibition o ADA causes an increase in intracellular adenosine and 2 -deoxyadenosine levels. The increased adenosine and 2 -deoxyadenosine have multiple e ects on purine nucleotide metabolism. In particular, 2 -deoxyadenosine irreversibly inhibits S-adenosylhomocysteine hydrolase, and the resulting increase in intracellular S-adenosylhomocysteine is toxic to lymphocytes. This action may account or the e ectiveness o pentostatin against some leukemias and lymphomas. Pentostatin is especially e ective against hairy cell leukemia.

Inhibitors o Ribonucleotide Reductase Hydroxyurea inhibits ribonucleotide reductase by scaveng-

ing a tyrosyl radical at the active site o the enzyme. In the absence o this ree radical, ribonucleotide reductase is unable to convert nucleotides to deoxynucleotides, and DNA synthesis is thereby inhibited. Hydroxyurea is approved or use in the treatment o adult sickle cell disease and certain neoplastic diseases. The mechanism o action o hydroxyurea in the treatment o sickle cell disease may or may not be related to inhibition o ribonucleotide reductase. As an alternative to this mechanism,

NH2

A N N HO

N

OH

N

N NH

O H

H

H

OH

H OH

N HO

N

O

Ade nos ine H

H

H

OH

H

H

Pentostatin (2'-Deoxycoformycin)

B NH2 N

N

NH2 N HO

N

Cl

N

N

O H

H

H

OH

H

N

O H

Cladribine

HO

P OH

N

F

O

O H H

OH

HO H

H

Fludarabine-5'-phosphate

FIGURE 39-15. Structures o adenosine, pentostatin, cladribine, and f udarabine. A. Pentostatin inhibits adenosine deaminase (ADA), the enzyme that converts adenosine and 2 -deoxyadenosine to inosine and 2 -deoxyinosine, respectively. Pentostatin binds to ADA with very high a f nity (Kd 2.5 10 12 M) because it structurally resembles the intermediate (transition state) in this enzymatic reaction. B. Cladribine and udarabine-5 -phosphate are also adenosine analogues. Cladribine is a chlorinated purine analogue that is incorporated into DNA and causes DNA strand breaks. Fludarabine phosphate is a uorinated purine analogue that is incorporated into DNA and RNA; this drug also inhibits DNA polymerase and ribonucleotide reductase.

hydroxyurea has been shown to increase the expression o the etal iso orm o hemoglobin (HbF), which inhibits the polymerization o sickle hemoglobin (HbS) and thereby decreases red blood cell sickling under conditions o hypoxia. Hydroxyurea signif cantly decreases the incidence o pain ul (vaso-occlusive) crisis in patients with sickle cell disease. The mechanism by which hydroxyurea increases HbF production is unknown. The role o hydroxyurea in the treatment o sickle cell disease is discussed urther in Chapter 45, Pharmacology o Hematopoiesis and Immunomodulation. Hydroxyurea is most commonly used in the treatment o myeloproli erative disorders such as polycythemia vera and essential thrombocytosis or or palliative control o blood counts in acute myelogenous leukemia. In myeloproli erative disorders, hydroxyurea can be used as a single agent or in combination with other agents to inhibit the excessive growth o myeloid cells in the bone marrow. The applications o hydroxyurea or these indications have been limited somewhat by concerns that long-term hydroxyurea use may be leukemogenic; there ore, this may be an example o the phenomenon that certain antitumor agents can also cause cancer.

C h a p t e r 3 9 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance

Purine and Pyrimidine Analogues That Are Incorporated into DNA A number o antimetabolites exert their major therapeutic e ect by acting as “rogue” nucleotides. These drugs are substrates or the various pathways o nucleotide metabolism, including ribosylation, ribonucleotide reduction, and nucleoside and nucleotide phosphorylation. The sugar triphosphate orms o these drugs can then be incorporated into DNA. Once incorporated into DNA, these compounds disrupt the structure o DNA, resulting in DNA chain termination, DNA strand breakage, and inhibition o cell growth. Thioguanine is a guanine analogue in which a sul ur atom replaces the oxygen atom at C-6 o the purine ring (Fig. 39-14). As with 6-mercaptopurine, thioguanine is converted by HGPRT to its nucleotide orm, 6-thioguanosine-5 -monophosphate (6-thioGMP). Unlike T-IMP, the nucleotide orm o 6-mercaptopurine, 6-thioGMP is a good substrate or guanylyl kinase, the enzyme that catalyzes the conversion o GMP to GTP. By this mechanism, 6-thioGMP is converted to 6-thioGTP, which is incorporated into DNA. Within the structure o DNA, 6-thioGTP inter eres with RNA transcription and DNA replication, resulting in cell death. 6-ThioGMP also irreversibly inhibits IMPDH and thereby depletes cellular pools o GMP (Fig. 39-3). Thioguanine is used in the treatment o acute myelogenous leukemia. Major adverse e ects o thioguanine include bone marrow suppression and gastrointestinal injury. Fludarabine-5 -phosphate (Fig. 39-15) is a uorinated purine nucleotide analogue that is structurally related to the antiviral agent vidarabine (see Chapter 38, Pharmacology o Viral In ections). The triphosphate orm o udarabine is incorporated into DNA and RNA, causing DNA chain termination. Fludarabine triphosphate also inhibits DNA polymerase and ribonucleotide reductase and thereby decreases nucleotide and nucleic acid synthesis in cells. The relative importance o these actions in mediating the cellular toxicity o the drug remains to be elucidated. Fludarabine5 -phosphate is used in the treatment o lymphoproli erative disorders, especially chronic lymphocytic leukemia (CLL) and low-grade B-cell lymphomas. Cladribine is a chlorinated purine analogue that is structurally related to udarabine-5 -phosphate (Fig. 39-15). Cladribine triphosphate is incorporated into DNA, causing strand breaks. Cladribine also depletes intracellular pools o the essential purine metabolites NAD and ATP. Cladribine is approved or use in the treatment o hairy cell leukemia and has been used experimentally in the treatment o other types o leukemia and lymphoma. Cytarabine (araC) is a cytidine analogue that is metabolized to araCTP (Fig. 39-16). AraCTP competes with CTP or DNA polymerase, and incorporation o araCTP into DNA results in chain termination and cell death (Fig. 39-4). Synergism between cytarabine and cyclophosphamide has been noted, presumably because o the reduced DNA repair caused by cytarabine’s inhibition o DNA polymerase. Cytarabine is used to induce and maintain remission in acute myelogenous leukemia; it is especially e ective or this indication when combined with an anthracycline (see below). 5-Azacytidine is a cytidine analogue. The triphosphate metabolite o 5-azacytidine is incorporated into DNA and RNA (Fig. 39-16). Once incorporated into DNA, azacytidine intereres with cytosine methylation, altering gene expression and promoting cell di erentiation. Azacytidine and its 2 -deoxy

NH2

NH2

N N

N

N

O

N

HO

O

HO

O H

735

O

H

H

OH

H OH

H

Cytidine

H

H

OH

H OH

5-Azacytidine NH2 N N

O

HO O H H

OH

HO H

H

Cytosine arabinoside (Cytarabine, araC)

FIGURE 39-16. Structures of cytidine, cytarabine, and 5-azacytidine. Cytarabine and 5-azacytidine are both analogues of the nucleoside cytidine. Cytarabine has an arabinose sugar in place of ribose (note the chirality of the hydroxyl group highlighted in blue). The incorporation of cytarabine triphosphate (araCTP) into DNA inhibits further nucleic acid synthesis, because the replacement of 2 -deoxyribose by arabinose interrupts strand elongation. 5-Azacytidine has an azide group (highlighted in blue) within the pyrimidine ring; this drug is incorporated into nucleic acids and interferes with the methylation of cytosine bases.

derivative decitabine (5-aza-2 -deoxycytidine) are approved or the treatment o myelodysplastic disease. Gemcitabine is a uorinated cytidine analogue in which the hydrogen atoms on the 2 carbon o deoxycytidine are replaced by uorine atoms. The diphosphate orm o gemcitabine inhibits ribonucleotide reductase; the triphosphate orm o gemcitabine is incorporated into DNA, inter ering with DNA replication and resulting in cell death. Gemcitabine is active in several solid tumors, including pancreatic, breast, bladder, and non-small cell lung cancer, and is also used in regimens or hematologic malignancies such as Hodgkin’s disease.

Agents That Directly Modify DNA Structure Alkylating Agents

The advent o modern chemotherapy dates to the 1940s, when highly reactive alkylating agents were f rst noted to induce remissions in otherwise untreatable malignancies. The clinical use o these agents was sparked by observations that nitrogen mustards, derivatives o wartime agents that caused dramatic suppression o hematopoietic cells, could have therapeutic utility in blood-derived malignancies such as leukemias and lymphomas. Soon therea ter, it was suggested that alkylating agents could also be use ul in treating epithelial tumors, mesenchymal tumors, carcinomas, and sarcomas; in

736 PRINCIPLES OF CHEMOTHERAPY

act, alkylating agents are commonly used against all o these diseases today. Alkylating agents—such as cyclophosphamide , bendamustine , mechlorethamine , melphalan, chlorambucil, and thiotepa —are electrophilic molecules that are attacked by nucleophilic sites on DNA, resulting in the covalent attachment o an alkyl group to the nucleophilic site. Depending on the particular agent, alkylation can take place on nitrogen or oxygen atoms o the base, the phosphate backbone, or a DNAassociated protein. The N-7 and O-6 atoms o guanine bases are particularly susceptible to alkylation. Alkylating agents typically have two strong leaving groups (Fig. 39-17). This structure con ers the ability to bis -alkylate (per orm two alkylating reactions), enabling the agent to cross-link the DNA molecule either to itsel —by linking two guanine residues, or example—or to proteins. Bis-alkylation (cross-linking) seems to be the major mechanism o cytotoxicity (Fig. 39-18A). Alkylation o guanine residues can also result in cleavage o the guanine imidazole ring, abnormal base-pairing between the alkylated guanine and thymine, or depurination (i.e., excision o the guanine residue) (Fig. 39-18B–D). Ring cleavage disrupts the molecular structure o DNA; anomalous DNA base-pairing causes miscoding and mutation; and depurination leads to scission o the sugar–phosphate DNA backbone. Importantly, the mutations caused by these processes can increase the risk o developing new cancers. Although all nitrogen mustards are relatively reactive, the individual agents vary in the speed with which they react with nucleophiles; this act has signif cant impact on their clinical use. Highly unstable compounds, such as mechlorethamine, cannot be administered orally because such agents alkylate target molecules within seconds to minutes. Because o this high reactivity, these molecules are power ul vesicants (causing blisters) and can severely damage skin and so t tissue i they leak out o blood vessels. The rapid reactivity o alkylating agents can be exploited by in using the drug directly into the site o a tumor. For example, thiotepa can be instilled into the bladder to treat superf cial bladder cancers. In contrast to mechlorethamine and thiotepa, chlorambucil and melphalan are much less reactive and can be administered orally. Cyclophosphamide is particularly use ul because it is a nonreactive prodrug that requires activation by the hepatic cytochrome P450 system; this agent can be administered either orally or intravenously (Fig. 39-19). Nitrosoureas , such as bendamustine and BCNU (carmustine ; Fig. 39-17), target DNA in much the same way as do Cl H O N P

O Cl

N

N

O O

Cl Cyclophosphamide

N H

Cl

N

BCNU (Carmustine, a nitrosourea)

FIGURE 39-17. Structures of cyclophosphamide and BCNU. Cyclophosphamide and BCNU (carmustine) each have two chloride leaving groups (blue). The presence of two leaving groups allows these alkylating agents to bis-alkylate and thereby cross-link macromolecules such as DNA. The ability to cross-link DNA is crucial to the DNA damage caused by these agents.

cyclophosphamide and other alkylating agents. Like cyclophosphamide, these compounds require bioactivation. Unlike most alkylating agents, however, nitrosoureas also attach carbamoyl groups to their DNA-associated targets. It is not clear whether carbamoylation contributes signif cantly to the activity o nitrosoureas. Regardless o the mechanism, however, bendamustine treatment results in signif cantly greater survival compared with traditional cyclophosphamide-based regimens when combined with rituximab (see Chapter 46) or indolent B-cell non-Hodgkin’s lymphomas (NHL). Some alkylating agents are better than others at targeting specif c tumors. For example, nitrosoureas are use ul in the treatment o brain tumors, because their high lipid solubility enables them to cross the blood–brain barrier. Similarly, the alkylating antibiotic mitomycin targets hypoxic tumor cells, such as those at the center o a solid tumor, because it requires bioreductive activation, which occurs more readily in low-oxygen environments. Several nonclassical alkylating agents also deserve mention as clinically use ul drugs. The f rst is dacarbazine , a synthetic molecule that is a component o a potentially curative combination chemotherapy regimen or Hodgkin’s disease. Dacarbazine also has some activity in treating melanoma and sarcomas. Procarbazine is an orally active drug that is used against Hodgkin’s disease. A metabolite o procarbazine unctions as a monoamine oxidase inhibitor, and toxicity related to this activity— such as tyramine sensitivity, hypotension, and dry mouth—can occur. Temozolomide , an oral alkylating agent, is an imidazotetrazine derivative o dacarbazine. Temozolomide is widely used in the treatment o gliomas and o glioblastoma multiorme in particular. Its action is synergistic with radiation, and it enhances survival in glioblastoma when used in combination with radiotherapy or this disease. Finally, altretamine is use ul or treating re ractory ovarian cancer. Although it is structurally related to alkylating agents o the triethylenemelamine class (such as thiotepa), whether the mechanism o action o this drug involves DNA alkylation remains controversial. Through natural selection, tumor cells can develop resistance to a single alkylating agent as well as cross-resistance to other drugs in the same class. Several mechanisms or resistance have been reported. Highly reactive drugs can be deactivated by intracellular nucleophiles such as glutathione . Alternatively, cells can become resistant by reducing uptake o the drug or accelerating DNA repair. One enzyme, O6-methylguanine-DNA methyltransferase (MGMT), prevents permanent DNA damage by removing alkyl adducts to the O6 position o guanine be ore DNA cross-links are ormed. Increased expression o this enzyme in neoplastic cells is associated with resistance to alkylating agents. Conversely, MGMT gene silencing predicts clinical benef t rom temozolomide in glioblastoma. Alkylating agent toxicity is dose-dependent and can be severe. As a rule, adverse e ects result rom damage to DNA in normal cells. Three cell types are pre erentially a ected by alkylating agents. First, toxicity typically mani ests in rapidly proli erating tissues, such as bone marrow, gastrointestinal and genitourinary tract epithelium, and hair ollicles. This results in myelosuppression, gastrointestinal distress, and alopecia (hair loss). Second, organ-specif c toxicity can result rom low activity o a DNA damage repair pathway in that tissue. Third, a tissue can be pre erentially a ected because the toxic compound accumulates in that tissue; or example, acrolein (a byproduct o the activation o cyclophosphamide

C h a p t e r 3 9 Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance

737

O N Cl

Cl

N

N

NH N

NH2

DNA cha in Gua nine

Mechlorethamine

Cl

OH

N N N

N N

NH2

DNA cha in OH N H2 N

N

OH

N N

N

N

N

DNA cha in

DNA cha in

Alkyla te d gua nine

A

D

Cl

OH

N N

N

B N

C

N

N

NH2

N NH2 Gua nine e xcis ion from DNA

Cros s -linke d DNA

O Cl

Cl

OH

N N HN

N

N

NH2

DNA cha in Ring cle a va ge

N

H H

N

N

O

O

N

DNA cha in

N N

DNA cha in

N O

N

H

H

Abnorma l ba s e pa iring (a lkyla te d gua nine hydroge n-bonde d to thymine )

FIGURE 39-18. Biochemical outcomes of guanine alkylation. In reactions such as those exemplif ed here with mechlorethamine, guanine alkylation can cause several types o DNA damage. The nitrogen o mechlorethamine per orms a nucleophilic attack on one o its own -carbons, resulting in an unstable intermediate that is highly electrophilic (not shown). The nucleophilic N-7 o guanine reacts with this unstable intermediate, resulting in an alkylated guanine. Four potential outcomes can result rom this initial alkylation, all o which cause structural damage to DNA. A. The process o alkylation can be repeated, with a second guanine acting as a nucleophile. The resulting cross-linking o DNA appears to be a major mechanism by which alkylating agents damage DNA. B. Cleavage o the imidazole ring disrupts the structure o the guanine base. C. The alkylated guanine can hydrogen-bond to thymine rather than cytosine, leading to a mutation in the DNA. D. Excision o the alkylated guanine residue results in a depurinated DNA strand.

or its analogue ifosfamide ) can produce hemorrhagic cystitis because of accumulation and concentration in the bladder (Fig. 39-19). This toxicity can be treated by using the sulfhydryl-containing molecule mesna , which is also concentrated in the urine and rapidly inactivates the acrolein. The immune response requires rapid proliferation of lymphocytes; this makes lymphocytes especially vulnerable to damage by alkylating agents. Thus, in addition to their anticancer activity, alkylating agents such as cyclophosphamide are also effective at immunosuppression. This “toxicity” has been put to clinical use: administered at doses lower than those used for antineoplastic therapy, alkylating agents are used to treat autoimmune diseases and organ rejection (see Chapter 46).

One approach to limiting toxicity has been to develop alkylating agents that accumulate preferentially inside tumor cells. An example of one such agent is melphalan, or phenylalanine mustard; this agent was designed to target melanoma cells, which accumulate phenylalanine for the biosynthesis of melanin. Another example is estramustine , in which the mustard component is conjugated to estrogen; this agent was designed to target breast cancer cells that express the estrogen receptor. Interestingly, neither melphalan nor estramustine works as intended, although they both have clinical utility; through mechanisms that are still poorly understood, melphalan is active against multiple myeloma, and estramustine is used to treat prostate cancer.

738 PRINCIPLES OF CHEMOTHERAPY Cl H O N P

N

O Cl

Cyclophosphamide (prodrug, ina ctive ) Liver cytochrome P450 oxidase Cl H O N P

HO

Cl

O

N

H O N P

O

N

O Cl

Cl

4-Hydroxycyclophos pha mide (a ctive )

4-Ke tocyclophos pha mide (ina ctive )

O

O H2 N

H

P O

Cl

N

O H

O

H2 N

P OH

Cl

N

+ Cl

Cl

Aldophos pha mide (a ctive )

Acrole in (cytotoxic)

P hos phora mide mus ta rd (cytotoxic)

Aldehyde oxidase O H2 N

HO O

P O

Cl

N

Cl

Ca rboxyphos pha mide (ina ctive )

FIGURE 39-19. Activation and metabolism of cyclophosphamide. Cyclophosphamide is a prodrug that must be oxidized by P450 enzymes in the liver in order to become pharmacologically active. Hydroxylation converts cyclophosphamide to 4-hydroxycyclophosphamide; this active metabolite can be further oxidized to the inactive metabolite 4-ketocyclophosphamide or undergo ring cleavage to the active metabolite aldophosphamide. Aldophosphamide can be oxidized by aldehyde oxidase to the inactive metabolite carboxyphosphamide or be converted to the highly toxic metabolites acrolein and phosphoramide mustard. Accumulation of acrolein in the bladder can cause hemorrhagic cystitis; this adverse effect of cyclophosphamide can be ameliorated by co-administration of mesna, a sulfhydryl compound that inactivates the acrolein (not shown).

Platinum Compounds

The introduction o cisplatin (cis-diamminedichloroplatinum [II]) into clinical use in the 1970s trans ormed previously intractable tumors, such as testicular cancer, into curable ones. As with the alkylating agents, the anticancer properties o cisplatin were discovered by a chance observation. While studying the e ects o electricity on bacteria, investigators ound that a product o the platinum electrode was inhibiting DNA synthesis in the microbes. The compound

was purif ed and ound to be cisplatin, which consists o a platinum atom bound to two amines and two chlorines in the cis con ormation. This serendipitous f nding led to the clinical use o cisplatin, which is now the most active drug in the treatment o testicular cancer (see the case o JL). As an antitumor agent, cisplatin is thought to act similarly to bis-alkylating agents (i.e., alkylating agents with two leaving groups) by targeting nucleophilic centers in guanine (N-7 and O-6), adenine (N-1 and N-3), and cytosine (N-3). The cis con ormation o cisplatin (Fig. 39-20) allows the drug to orm intrastrand cross-links between adjacent guanine residues, resulting in DNA damage (Fig. 39-21B). This structural eature is critical to the action o cisplatin; the trans isomer, although capable o binding covalently to DNA, has little antitumor activity. Tumor cells can develop resistance to cisplatin by enhancing the repair o DNA lesions, decreasing drug uptake, or enhancing drug inactivation via up-regulated synthesis o nucleophiles such as glutathione. As JL’s case demonstrates, cisplatin is e f cacious in the treatment o genitourinary cancers, including cancers o the testis, bladder, and ovary. Cisplatin and the related compound carboplatin (Fig. 39-20) are also among the most e f cacious drugs used against lung cancer. As with many chemotherapeutic agents, the rationale or the e f cacy o cisplatin and carboplatin in the treatment o certain tumor types over others is not clear. Cisplatin can be administered intravenously, but it can also be e ective when exposed directly to tumor cells. One example is in treating ovarian cancer, which spreads along the inner lining o the peritoneal cavity. For this application, cisplatin is in used directly into the peritoneal cavity to achieve high local concentrations o the drug while decreasing systemic toxicity. JL’s oncologist considered cisplatin toxicities care ully in determining the dose o this drug to administer and the other agents that would be included in the combination chemotherapy regimen. Because the dose-limiting toxicities o cisplatin, bleomycin, and etoposide di er rom one another, each o these drugs could be used at the maximum-tolerated dose (see Chapter 41). For cisplatin, the dose-limiting toxicity is nephrotoxicity. Gastrointestinal symptoms such as nausea and vomiting are also common; this is o concern because dehydration due to protracted vomiting can exacerbate cisplatin-induced kidney damage and lead to irreversible renal ailure. Neurotoxicity, primarily mani ested as paresthesias o the hands and eet and hearing loss, also occurs

O H3 N H3 N

Pt

Cl Cl

Cisplatin

H3 N H3 N

Pt

O O

O Carboplatin

FIGURE 39-20. Structures of cisplatin and carboplatin. Cisplatin and carboplatin are coordinated complexes of platinum (Pt). The cis structure of these molecules (i.e., the presence of the two leaving groups on the same side of the molecule, rather than on opposite corners) provides them with the ability to cross-link adjacent guanines on the same DNA strand (intrastrand cross-link) or, much less frequently, on opposite DNA strands (interstrand cross-link). Similar compounds with trans conformations cannot effectively cross-link adjacent guanines.

C h a p t e r 3 9 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance A

739

B

C

FIGURE 39-21. Interactions of bleomycin, platinum compounds, and anthracyclines with DNA. A. Bleomycin (highlighted in orange) binds to the DNA double helix and thereby exposes nucleotides in DNA to the iron (II) atom (large red ball) that is complexed to bleomycin. In the presence of molecular oxygen, the iron–bleomycin complex causes single-strand and double-strand breaks in DNA by a free radical mechanism. B. Platinum complexes (highlighted in orange) cross-link N-7 atoms on adjacent guanine residues, forming intrastrand DNA cross-links. C. Daunorubicin, an anthracycline (highlighted in orange), intercalates into DNA structure (see expanded view on right) and thereby prevents the strand passage and religation steps that are part of the catalytic cycle of type II topoisomerase (see Fig. 34-4). Anthracyclines may also damage DNA by a free radical mechanism.

requently. Thiol-containing compounds, such as amifostine , can ameliorate cisplatin nephrotoxicity without diminishing its antitumor e ects. Carboplatin, a cisplatin analogue that is less nephrotoxic, has replaced cisplatin in many chemotherapy regimens. Oxaliplatin, a third platinum compound, has activity in the treatment o colorectal cancer and other gastrointestinal malignancies. Like cisplatin, oxaliplatin causes cumulative neurotoxicity; oxaliplatin also induces a unique acute neurotoxicity that is exacerbated by exposure to cold temperatures.

Bleomycin

The bleomycins , a amily o natural glycopeptides synthesized by a species o Streptomyces, have prominent cytotoxic activity. A mixture o several o these glycopeptides, di ering only in side chains, is used clinically (Fig. 39-21A). Bleomycin binds DNA and chelates iron (II), leading to the ormation o ree radicals that cause single- and double-strand DNA breaks. As with many chemotherapeutic agents, multidrugresistance mechanisms, such as increased drug e f ux rom tumor cells, can reduce tumor susceptibility to bleomycin.

740 PRINCIPLES OF CHEMOTHERAPY

In chelating iron, bleomycin orms a heme-like ring. It is believed that the chelated complex abstracts a hydrogen radical rom the 4 position o a nearby pyrimidine residue (thymine or cytosine). The unstable intermediate decomposes in the presence o oxygen to produce an abstracted pyrimidine and a ree phosphodiester at one or both DNA strands (Fig. 39-21A). Relative to other DNA-damaging agents, bleomycin causes less myelosuppressive toxicity. Because o its reactivity with oxygen, however, bleomycin can cause pulmonary f brosis , the drug’s most problematic and dose-limiting toxicity. The e ects o bleomycin on pulmonary unction are cumulative and irreversible. There ore, this agent’s use is largely restricted to potentially curative combination chemotherapy regimens or testicular cancer and Hodgkin’s disease. In JL’s case, it was concern regarding pulmonary toxicity that led his physician to monitor his lung unction closely throughout therapy and to inquire about shortness o breath on each visit. Worsening lung unction would have required adjustments in JL’s therapy.

Topoisomerase Inhibitors Several chemotherapeutic agents damage DNA by exploiting the natural nuclease/ligase unction o topoisomerases. The basic physiology o this process is discussed in Chapter 34. The antineoplastic camptothecins , anthracyclines , epipodophyllotoxins , and amsacrine act in this manner. These compounds inter ere with the proper unction o topoisomerases and cause cellular topoisomerases to participate in DNA destruction.

Camptothecins

The camptothecins are semisynthetic molecules derived rom alkaloid extracts o Camptotheca plants. Camptothecins target topoisomerase I, causing DNA strand damage. Topoisomerase I modulates supercoiling by complexing with DNA and nicking one o its two strands (see Fig. 34-3). The camptothecins act by stabilizing this nicked DNA complex and preventing topoisomerase I rom religating the strand break. Other replication enzymes then bind to the camptothecin–DNA–topoisomerase complex, converting the single-strand DNA lesion to a double-strand break. Neoplastic cells are o ten unable to repair the resulting damage. Two camptothecin derivatives have clinical utility— irinotecan and topotecan. Irinotecan was initially introduced or the treatment o advanced colon cancer, although it may also be e ective in treating other tumor types. It is a water-soluble prodrug that is cleaved by the enzyme carboxylesterase to release the lipophilic metabolite SN-38. Although SN-38 is approximately 1,000- old more active than irinotecan in inhibiting topoisomerase I, it is more highly protein-bound than irinotecan and has a much shorter hal -li e in vivo. Thus, the relative contribution o SN-38 to the anticancer e ects o irinotecan is unclear. Irinotecan use is limited by severe gastrointestinal toxicity, leading to potentially li e-threatening diarrhea. As with many other chemotherapeutic agents, irinotecan also causes dosedependent bone marrow suppression. SN-38 is metabolized by UDP-glucuronosyltrans erase (UGT) A1, and patients with abnormalities in this enzyme (Gilbert’s syndrome) are highly susceptible to irinotecan toxicity. This f nding supports the hypothesis that SN-38 is an important contributor to the e ects o irinotecan.

Topotecan is used in the treatment o metastatic ovarian cancer, small cell lung cancer, and other neoplasms. Specif cally, this agent shows e ectiveness in treating ovarian neoplasms that are resistant to cisplatin, which are otherwise di f cult to treat e ectively.

Anthracyclines

Anthracyclines, natural antitumor antibiotics isolated rom a species o the ungus Streptomyces, are among the most clinically use ul cytotoxic cancer chemotherapeutic agents. Although several mechanisms appear to be involved in their activity, the ability o the anthracyclines to damage DNA most likely results rom their intercalation into DNA (Fig. 39-21C). This intercalation inter eres with the action o topoisomerase II, resulting in DNA lesions such as strand scission and, ultimately, in cell death (see Fig. 34-4). Like many other antineoplastic agents, anthracyclines cause myelosuppression and alopecia. Anthracyclines are excreted in bile, and their dose must be reduced in patients with liver dys unction. These agents are major components o chemotherapy regimens or a variety o malignancies, particularly hematologic cancers (such as leukemias and lymphomas) and breast cancer. Daunorubicin is an anthracycline that is commonly used to treat acute myeloid leukemia and acute lymphocytic leukemia. The best known drug in this group, doxorubicin (hydroxydaunorubicin or Adriamycin® ), remains a key component o regimens used to treat lymphoma and breast cancer, although it has a higher risk o gastrointestinal toxicity than daunorubicin and can be associated with heart ailure . It is thought that doxorubicin acilitates the excessive production o ree radicals in the myocardium and thereby damages cardiac cell membranes. Cardiotoxicity is related to both the peak plasma concentration and the cumulative dose o doxorubicin. Cardiotoxicity can be reduced by coadministration o dexrazoxane , which is thought to inhibit ree radical ormation by chelating intracellular iron and preventing iron-mediated ree radical generation. Liposomal doxorubicin (Doxil® ) is a specif c ormulation that improves tumor delivery by enhancing penetration o drug into the cell membrane. In addition, liposomal doxorubicin can limit cardiotoxicity by reducing membrane lipid peroxidation and ree radical generation.

Epipodophyllotoxins

Like anthracyclines, epipodophyllotoxins appear to act primarily by inhibiting topoisomerase II-mediated religation o double-strand DNA breaks (see Fig. 34-4). The antineoplastic agents etoposide (VP-16) and teniposide (VM-26) are semisynthetic derivatives o a compound isolated rom the plant Podophyllum. These drugs bind topoisomerase II and DNA, trapping the complex in its cleavable state. Tumor cells o ten develop resistance to etoposide by increasing their expression o P-glycoprotein. This protein normally serves as an e ux pump to rid the cell o toxic molecules such as natural metabolic side-products, but it can also remove chemotherapeutic agents derived rom natural products be ore those agents have exerted their cytotoxic e ect. Etoposide is use ul or treating testicular cancer, lung cancer, and leukemia, while both etoposide and teniposide are used to treat various lymphomas. Bone marrow suppression is the chie toxicity o the two epipodophyllotoxins in clinical use.

C h a p t e r 3 9 Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance

Combining drugs that damage DNA directly, such as cisplatin and bleomycin, with drugs that inhibit topoisomerase II, such as etoposide, can have power ul synergistic anticancer e ects. This synergy may relate to the role o topoisomerases in repairing DNA damage or to the combined ability o these drug classes to induce su f cient DNA damage to trigger apoptosis. In practice, drugs o these classes are co-administered in many success ul antineoplastic regimens. As JL’s case demonstrates, the combination o etoposide, bleomycin, and cisplatin can cure most cases o metastatic testicular cancer.

741

Vinca alkaloids Excha nge a ble GTP binding s ite

V

Taxanes

β-tubulin

T

None xcha nge a ble GTP binding s ite

C

Amsacrine

Amsacrine is another example o a chemotherapeutic agent

that acts primarily by inhibiting topoisomerase II-mediated religation o double-strand DNA breaks. This compound targets DNA by intercalating between base pairs, distorting the double helix, producing DNA–protein cross-links, and creating both single- and double-strand DNA lesions. Its clinical use is generally restricted to the treatment o recurrent leukemia and ovarian cancer.

Microtubule Inhibitors Microtubules depend on dynamic instability or physiologic unctioning. Without the ability to change length quickly, microtubules can do little other than lend structural support to a quiescent cell. Although microtubules play important roles in many aspects o cellular physiology, drugs that inhibit microtubule unction are pre erentially toxic to M-phase cells. Vinca alkaloids inhibit microtubule polymerization, while taxanes inhibit microtubule depolymerization. Two other inhibitors o microtubule polymerization, griseo ulvin and colchicine, are discussed in Chapter 36, Pharmacology o Fungal In ections and Chapter 49, respectively.

α-tubulin

FIGURE 39-22. Tubulin binding sites of microtubule-inhibiting drugs. The tubulin heterodimer is composed o -tubulin (purple) and -tubulin (blue). -Tubulin and -tubulin both bind GTP. The GTP on -tubulin is not hydrolyzed; or this reason, the GTP binding site on -tubulin is re erred to as the nonexchangeable GTP binding site. -Tubulin hydrolyzes GTP to GDP; or this reason, the GTP binding site on -tubulin is re erred to as the exchangeable GTP binding site. The two major classes o antineoplastic microtubule inhibitors bind to distinct sites on the tubulin heterodimer. Vinca alkaloids, which inhibit microtubule polymerization, bind to a site on -tubulin located near the exchangeable GTP binding site (V). Vinca alkaloids bind pre erentially at the ( ) end o microtubules and thereby inhibit the addition o new tubulin subunits to the microtubule. Taxanes, which stabilize polymerized microtubules, bind to a di erent site on -tubulin (T). Taxanes may stabilize either the interactions between tubulin subunits or the shape o microtubule protof laments. Colchicine binds to a site located at the inter ace between -tubulin and -tubulin (C). Colchicine is not used in cancer chemotherapy but is used in the treatment o gout (see Chapter 49).

Inhibitors of Microtubule Polymerization: Vinca Alkaloids

The vinca alkaloids vinblastine and vincristine are natural products originally isolated rom the periwinkle plant, Vinca rosea. Vinca alkaloids bind to -tubulin on a portion o the molecule that overlaps with the GTP-binding domain (Fig. 39-22). The binding o vinca alkaloids to -tubulin at the ( ) end o microtubules inhibits tubulin polymerization and thereby prevents microtubule extension. Because microtubules must constantly add tubulin to maintain stability (i.e., they must retain a GTP-bound tubulin cap), inhibition o tubulin addition eventually leads to the depolymerization o existing microtubules (Fig. 39-12). Vinblastine is used to treat certain lymphomas and, as part o a multidrug regimen (with cisplatin and bleomycin), to treat metastatic testicular cancer. Pharmacologic doses o the drug cause nausea and vomiting. Myelosuppression is the dose-limiting adverse e ect o vinblastine. Vincristine plays an important role in the chemotherapy o pediatric leukemias. It is also a component o chemotherapy regimens used to treat Hodgkin’s disease and some non-Hodgkin’s lymphomas. Pharmacologic doses o vincristine cause nausea and vomiting. Vincristine causes some myelosuppression, but not to the same degree as vinblastine. Peripheral neuropathy is usually the dose-limiting adverse e ect o vincristine; this toxicity may result rom inhibition o the microtubule tra f cking unction in long peripheral nerves that extend rom the spinal cord to the extremities.

Eribulin is an analogue o a natural product rom the

Halichondria genus o marine sponges. It binds to the ( ) end o microtubules and inhibits microtubule dynamics. Eribulin was approved in 2010 or use in the treatment o metastatic breast cancer.

Inhibitors of Microtubule Depolymerization: Taxanes

The taxanes, which include paclitaxel, docetaxel, and cabazitaxel, are natural products originally derived rom the bark o the western yew tree. Taxanes bind to the -tubulin subunit o microtubules at a site distinct rom the vinca alkaloid binding site (Fig. 39-22). Paclitaxel has been shown to bind to the inside o microtubules. Unlike the vinca alkaloids, taxanes promote microtubule polymerization and inhibit depolymerization. Stabilization o the microtubules in a polymerized state arrests cells in mitosis and eventually leads to programmed cell death (apoptosis). Two leading hypotheses have been proposed or the apparent microtubule-stabilizing properties o taxanes. First, taxanes could strengthen the lateral interactions between microtubule protof laments. Increased lateral interactions would decrease the tendency or protof laments to “peel away” rom the microtubule cylinder. Second, taxanes could straighten individual protof laments. Once -tubulin hydrolyzes GTP to GDP, protof laments have a tendency to “curl,” which produces a strain on the integrity o the microtubule cylinder. By straightening protof laments, taxanes could

742 PRINCIPLES OF CHEMOTHERAPY

reduce the tendency or the protof laments to separate rom the intact microtubule. In vivo, both o these mechanisms may be important or taxane-mediated stabilization o microtubules; alternative mechanisms are also possible. Paclitaxel is used as an antineoplastic agent in the treatment o many solid tumors, especially breast, ovarian, and non-small cell lung cancer. Paclitaxel has several important adverse e ects. An acute hypersensitivity reaction occurs commonly in response to paclitaxel, or more likely to the vehicle in which paclitaxel is solubilized; this e ect can be obviated by administration o dexamethasone (a glucocorticoid receptor agonist) and a histamine H1 receptor antagonist be ore treatment with paclitaxel. Many patients experience myalgias and myelosuppression rom paclitaxel, and high doses o the drug can cause pulmonary toxicity. Peripheral neuropathy, typically mani esting as a “stocking and glove” sensory def cit in the extremities, can limit the cumulative amount o drug that can be administered sa ely. Abraxane ® is an albumin-bound orm o paclitaxel with a mean particle size o 130 nanometers. The albumin-bound paclitaxel nanoparticles do not cause a hypersensitivity reaction, do not require premedication, and cause less myelosuppression than traditional, solvent-based paclitaxel. Abraxane® is approved or the treatment o metastatic breast cancer and, in combination with gemcitabine, or the f rstline treatment o pancreatic cancer. Docetaxel is most commonly used in the treatment o breast cancer, non-small cell lung cancer, and prostate cancer. As with paclitaxel, docetaxel causes an acute hypersensitivity reaction that can be obviated by preadministration o glucocorticoids. Docetaxel occasionally exhibits the drug-specif c adverse e ect o uid retention, which likely arises rom increased capillary permeability. Docetaxel does not cause neuropathy as requently as paclitaxel does. The myelosuppression associated with docetaxel is pro ound, however, and is usually dose-limiting. Cabazitaxel is a third taxane that has recently been approved or the treatment o hormone-re ractory prostate cancer a ter ailure o docetaxel-based treatment. Treatment with cabazitaxel is also associated with signif cant neutropenia.

CONCLUSION AND FUTURE DIRECTIONS The antineoplastic agents described in this chapter exert their e ects on the genome by preventing e f cient DNA replication, inducing DNA damage, and inter ering with mitosis. Because many normal cells as well as cancer cells are transiting through the cell cycle, these agents are associated with multiple dose-limiting toxicities. In addition, although cancer cells are susceptible to DNA damage, in some instances, mutations in key checkpoint proteins such as p53 prevent the apoptosis that would otherwise be induced by these agents. Novel approaches are being developed to target DNA damage more specif cally. For example, it has been shown that mice def cient in PARP1 are able to overcome the deect in single-strand break repair by converting single-strand

breaks to double-strand breaks and then repairing the DNA by the DSB repair pathway. Furthermore, normal human cells treated in culture with PARP1 inhibitors are capable o undergoing normal cell division, although these cells do mani est increased susceptibility to DNA damage as a consequence o de ective single-strand break repair. In contrast, cells def cient in BRCA1 or BRCA2, which are involved in DSB repair, are killed in response to treatment with PARP1 inhibitors; compared to normal cells, BRCA1 or BRCA2 cells are up to 1,000- old more sensitive to the action o PARP1 inhibitors. Presumably, the BRCA1 and BRCA2 cells are more sensitive due to impairment o both singlestrand break and DSB repair pathways, resulting in lethal accumulation o DNA damage. Based on these f ndings, PARP1 inhibitors are currently in clinical trials or the treatment o BRCA-def cient breast cancer and ovarian cancer, and may be e ective in other tumors in which the DNA damage response is compromised. The investigational PARP1 inhibitor olaparib has had mixed results in general but may show particular activity in tumors with BRCA mutations. The observation that telomerase is expressed in most cancer cells and is key to the process o immortalization highlights this enzyme as an important target in uture cancer therapy. Although telomerase is expressed to some degree in stem cells and normally cycling cells, most normal cells lack telomerase expression. There ore, the dependency o tumor cells on the immortalized state could provide telomerase inhibitors with a avorable therapeutic index. However, e ective agents have yet to be discovered, and one concern is that multiple cell divisions may be required or telomere length to shorten to a level that is critical or cell survival. Combinations o telomerase inhibitors with traditional cytotoxic agents or newer molecularly targeted therapies could yield synergistic e ects. Such strategies, as well as those described in Chapter 40, Pharmacology o Cancer: Signal Transduction, will help to advance cancer therapy by moving beyond general cytotoxic approaches and ocusing treatment instead on the molecular abnormalities responsible or driving oncogenesis.

Suggested Reading Bishr M, Saad F. Overview o the latest treatments or castration-resistant prostate cancer. Nat Rev Urol 2013;10:522–528. (Discusses recent advances in the treatment of prostate cancer, including a review of cabazitaxel.) Brody LC. Treating cancer by targeting a weakness. N Engl J Med 2005;353:949–950. (Discusses advances in targeted cancer therapy.) Gazdar A. DNA repair and survival in lung cancer. N Engl J Med 2007;356:771–773. (Discusses DNA repair pathway status in relationship to survival and chemotherapy responsiveness.) O’Sullivan Coyne G, Chen A, Kummar S. Delivering on the promise: poly ADP ribose polymerase inhibition as targeted anticancer therapy. Curr Opin Oncol 2015;27:475–481. (Reviews the development and clinical activity of PARP inhibitors as anticancer drugs.) Peltomaki P. Role o DNA mismatch repair de ects in the pathogenesis o human cancer. J Clin Oncol 2003;21:1174–1179. (Reviews the pathophysiology of DNA repair mechanisms.) Van der Jagt R. Bendamustine or indolent non-Hodgkin lymphoma in the ront-line or relapsed setting: a review o pharmacokinetics and clinical trial outcomes. Exp Rev Hematol 2013;6:525–537. (Reviews the pharmacology of bendamustine.)

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C h a p t e r 3 9 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 749

Tr ns Tra me mbraa ne doma maa in

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Pharmacology of Cancer: Signal Transduction B

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Tra nss me m bra mb b ra ne d. .oma in. . . . . . . . . . . . . 7750 INT IN TRODUCTION & CASE . . . . . . . . . doma . . . . .in 750–751 75 500– ––775511 BIOCHEMISTRY OF INTERCELLULA AR AN AND D INTRACELLULAR SIIGN GNAL AL TRANSDUCTION . . . . . . . . . . . .Ty . .r 7500 Tyr Grow owth th Fac acto c to tors rs a nd Growth Factor Receptors . . . . . . . . . . . . 75 750 50 0 Intracellular Signal Transducttion ionIna Pa Path ways natctive ctiv ey prote in in . . . . . . 751 Prot Pr otea easo some me Str truc uctu ture re a nd d Function 7522 tyr ty yro ros oss ine inee kina kin inaa s e. . . . . . . 75 Angiogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756 ((e.g., g , J AK2) AK2) PHAR PH ARM MACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 757 Growth Factor Receptor and Signal Transduction Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . 757 EGF Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . 758 ALK/ROS1 Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759 BCR-Abl/C-KIT/PDGFR Inhibition n . . . . . . . . . . . . . . . . . . . . . 759

INTRODUCTION Traditional antineoplastic therapy has consisted o agents directed against DNA replication and cell division. These drugs exhibit some degree o selectivity against cancer cells, which tend to have a higher growth raction and, in some cases, an increased susceptibility to DNA damage compared to normal cells. However, the therapeutic window o these drugs is narrow, resulting in toxicity to normal stem cells and in hematologic and gastrointestinal adverse e ects. With the impressive advances in basic tumor cell biology over the last several decades and the identif cation o numerous oncogenes and tumor suppressor genes, the potential exists or development o agents that are targeted more specif cally at the molecular circuitry responsible or the dysregulated proli eration o cancer cells. An early example o such a drug was the selective estrogen receptor modulator tamoxifen (see Chapter 30, Pharmacology o Reproduction). Tamoxi en is still one o the most active agents in the treatment o hormone receptor-positive breast cancer, with a relatively modest adverse e ect prof le. Subsequently, the remarkable success o imatinib mesylate in the treatment o chronic myelogenous leukemia has suggested that, in some cases, tumor cells are dependent on oncogenes such as BCR-Abl or their survival. Like tamoxi en, imatinib remains in wide use today. This chapter highlights the basic principles and agents o targeted cancer therapy, detailing recent advances and directions or the uture. 750

BT BT TK K Inhibitors In nnhhibi ibbiito itor it to oorrss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760 ors 7 FLT3 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776 FL 7600 .y.r. . . . . . . . . T . .y.r. . . . . . . . . . 776 7600 J AK2 AT yr y2 IInhibitors . . . . . . . . . . .P. .Ty Tyr Ty Tyr Tyr P RAS RAS/M AS/M /MAP Kinase Pathway ayy IInnh nhibi hibition hibi ibitionn . . . . . . . . . . . . . . . . . . 760 7 PI3K/A / KT T/m mTO TOR R In Inhhibi hibito tors rs . . . . . . . . . . . . . . . . . . . . . . . . . . . 761 7 Tyros T yr yro yros o s ine i kina kin ki s e Proteasome t Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761 7 a c cti ctivity tivi ti i vity it i ty y Angiogenesis Inhib hibit itor orss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762 7 Anti-VEGF and Anti-VEGFR A Antibodi Antibodies ntib ntibod nti nt d s Tyrr . . . . . . . . . . . . 762 Tyr Ty ribood P VEGFR Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762 Thalidomide and Lenalidomidee . . . . . . . . . . . . .Intra Intra ce llula lllu u la r3 Tumor-Specif c Monoclonal Antibodies . . . . . . . ta rge t pro 763iin prote ote CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 764 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .764

BIOCHEMISTRY OF INTERCELLULAR AND INTRACELLULAR SIGNAL TRANSDUCTION Growth Factors and Growth Factor Receptors External signals stimulate cell growth and proli eration via the interaction o growth actors with specif c cell sur ace receptors. Growth actor receptors typically contain an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic domain that has either intrinsic tyrosine kinase activity or an associated protein tyrosine kinase (Fig. 40-1). In general, binding o the growth actor ligand results in receptor oligomerization, a con ormational change in the cytoplasmic domain o the receptor, and tyrosine kinase activation. Intracellular targets are subsequently phosphorylated, propagating a signal that culminates in progression through the cell cycle and cellular proli eration. One example o a receptor tyrosine kinase is the epidermal growth factor receptor (EGFR), which possesses intrinsic tyrosine kinase activity and is a member o the broader ErbB amily o proteins, including EGFR (ErbB1), HER2/neu (ErbB2), ErbB3, and ErbB4. Binding o epidermal growth actor (EGF) or trans orming growth actor- (TGF- ) to EGFR results in receptor homodimerization and propagation o a growth signal. In addition, heterodimerization between

C h a p t e r 4 0 Pharmacology o Cancer: Signal Transduction

MW is a 65-year-old wom an with m etastatic non-sm all cell lung cancer. She has ne ver s m oked, a nd her prim ary tum or is an aden ocarcino m a with b ron chio alveo lar eatu re s. He r o n co lo g is t co n sident with carb op latin and pem etrexed b ut ers treatm en also sends genetic testing o her tum or or m utations in the ep id erm al growth actor receptor (EGFR) an d or genetic rearrangem ents o anaplastic lym phom a kinase (ALK), ROS1, and RET. Her tum or is ound to harbor an activating m utation in the EGFR kinase dom ain at codon 858, resulting in a substitution o arginine or leucine (L858R). MW is there ore treated with the oral EGFR tyrosine kinase inhibitor (TKI) erlotinib. She develops a skin rash and diarrhea but otherwise tolerates this m edication well. Restaging com puted tom ography scans are per orm ed 2 m onths a ter starting treatm ent with erlotinib. These scans reveal a dram atic reduction

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FIGURE 40-1. Structure and function of growth factor receptors. A. Growth actor receptors exemplif ed by the epidermal growth actor (EGF) receptor contain an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic domain with intrinsic tyrosine kinase activity. Binding o ligand results in receptor homodimerization (or heterodimerization with other amily members), triggering activation o the tyrosine kinase, autophosphorylation o the receptor on tyrosine (Tyr ) residues, and phosphorylation o intracellular target proteins. B. Growth actor receptors exemplif ed by the type I cytokine receptors (such as the erythropoietin [EPO] receptor) lack intrinsic tyrosine kinase activity. Instead, the receptors are associated with intracellular protein tyrosine kinases such as JAK2. Upon ligand-induced receptor dimerization, the associated kinase is activated and autophosphorylated on tyrosine residues, resulting in recruitment and phosphorylation o intracellular target proteins.

751

in MW’s tum or burden, and a ter 6 m onths, there is no residual evidence o cancer. Un ortunately, MW subsequently develops recurrence o her disease. A repeat biopsy reveals that the tum or now also shows am plif cation o the MET receptor tyrosine kinase. She decides to participate in a clinical trial o a MET inhibitor or treatm ent o her recurrent non-sm all cell lung cancer.

Questions 1 . How does signaling through EGFR promote cell growth and survival? 2 . By what mechanism does erlotinib inhibit EGFR and inhibit cancer cell growth? 3 . How does amplif cation o MET expression lead to tumor recurrence despite treatment with erlotinib? 4 . What is the most common mechanism o EGFR TKI resistance, and how might this be overcome?

amily members can occur, yielding urther diversity in the signal that is transduced. ErbB receptors are expressed on epithelial cells and are o ten activated or overexpressed in a variety o carcinomas (e.g., EGFR in non-small cell lung cancer and HER2/neu in breast cancer). Other examples o receptor tyrosine kinases include the insulin-like growth actor receptor 1 (IGF1R), plateletderived growth actor receptor (PDGFR), f broblast growth actor receptor (FGFR), C-KIT, Bruton’s tyrosine kinase (BTK), FMS-like tyrosine kinase (FLT3), anaplastic lymphoma kinase (ALK), ROS1, RET, and MET. Signaling through these receptors activates the growth o certain hematopoietic and mesenchymal tissues, and dysregulation o these receptors is requently observed in specif c myeloproli erative disorders, leukemias, sarcomas, and epithelial cancers (Table 40-1). Other hematopoietic receptors rely on interaction with an associated cytoplasmic tyrosine kinase or transduction o a growth signal. For example, type I cytokine receptors such as the erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), and G-CSF receptor (GCSFR) orm specif cally oriented homodimers upon binding o ligand, resulting in activation o the associated tyrosine kinase JAK2, which leads to urther signaling and ultimately to cell growth. Activating mutations o the receptors themselves (e.g., EpoR) have been implicated in conditions such as congenital polycythemia. A common activating mutation in JAK2, resulting in the conversion o valine to phenylalanine at position 617 (V617F), has been ound in a majority o patients with the myeloproli erative disorder polycythemia vera and in a signif cant raction o patients with essential thrombocythemia and myeloid metaplasia with myelof brosis.

Intracellular Signal Transduction Pathways Activation o a growth actor receptor initiates the transduction o a series o intracellular signals, culminating in events such as cell cycle entry, promotion o protein translation and cell growth, and enhanced cell survival. Two broad categories o pathways activated by receptor tyrosine kinases are the RAS-MAP kinase pathway and the phosphatidylinositol3-kinase (PI3K)-AKT pathway (Fig. 40-2).

752 PRINCIPLES OF CHEMOTHERAPY

TABLE 40-1 Receptor Tyrosine Kinases Associated with Cancer RECEPTOR TYROSINE KINASE

MALIGNANCY OR MYELOPROLIFERATIVE DISORDER

EGFR (ErbB1)

Non-small cell lung cancer Head and neck cancer Colon cancer Pancreatic cancer Glioblastoma

HER2/neu (ErbB2)

Breast cancer Ovarian cancer Head and neck cancer

PDGFR

Hypereosinophilic syndrome Mast cell disease Dermatof brosarcoma protuberans Gastrointestinal stromal tumor (GIST)

FGFR3

Multiple myeloma Bladder cancer

C-KIT

Gastrointestinal stromal tumor (GIST) Systemic mastocytosis

FLT3

Acute myelogenous leukemia

RET

Multiple endocrine neoplasia type 2 Familial medullary thyroid carcinoma

MET

Hepatocellular carcinoma Melanoma Glioblastoma Epithelial malignancies

The Kirsten ras gene was initially identif ed as a retroviral oncogene in rats and subsequently ound to have several human homologues, including K-ras, H-ras, and N-ras. The protein (RAS) encoded by ras is targeted to the plasma membrane by the arnesyltrans erase-mediated addition o a hydrophobic arnesyl group to its COOH-terminus; this targeting brings RAS into close proximity to activated receptor tyrosine kinases. Intracellular nonreceptor tyrosine kinases such as ABL and SRC, also originally identif ed as oncogene products, can activate signaling through RAS as well (Fig. 40-2A). Upon activation by binding to GTP, RAS triggers a series o phosphorylation events mediated by the kinases RAF, MEK, and ERK (MAP kinase), the targets o which include transcription actors that promote activation o genes involved in proli eration. For example, activation o cyclin D transcription results in cyclin D expression and binding to its catalytic partners, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) (Fig. 40-3). These complexes initiate phosphorylation o the retinoblastoma protein (pRB), thereby li ting pRB’s repression o the transcription actor E2F. E2F mediates the expression o components o the DNA replication machinery and enzymes involved in nucleotide synthesis. Thus, phosphorylation o pRB by cyclin D–CDK4/6 and subsequent activation o other cyclin–CDK complexes (such as cyclin E–CDK2) result in the transition rom G1 to S phase and progression through the cell cycle. While such signaling cascades might seem unnecessarily complicated, they allow or the integration o diverse extracellular and

intracellular signals, the opportunity or multiple points o eedback control, and the tight regulation o critical events such as cellular proli eration. A second key intracellular signaling pathway is controlled by the lipid kinase PI3K. Stimulation o receptors or growth actors such as insulin or insulin-like growth actors (IGFs) commonly leads to activation o PI3K via an associated insulin receptor substrate protein (IRS). ErbB amily members can also activate this pathway via phospholipase C- (PLC- ), and RAS can also promote signaling through this pathway (Fig. 40-2B). Activation o PI3K results in generation o phosphatidylinositol-3,4,5-trisphosphate (PIP3) rom plasma membrane phospholipids, activation o phosphoinositide dependent kinase-1 (PDK-1) via translocation to the cell membrane, and phosphorylation o AKT by PDK-1. This pathway is negatively regulated by the lipid phosphatase PTEN, which degrades PIP3. Downstream e ects o AKT activation include promotion o translation and cell growth by the mammalian target o rapamycin (mTOR). In addition, phosphorylation o the orkhead amily o transcription actors (FOXO) by AKT results in their exclusion rom the nucleus, preventing expression o genes involved in cell cycle arrest, stress resistance, and apoptosis. Thus, a net e ect o activating the PI3K-AKT pathway is the promotion o cell survival. Activating mutations in the catalytic subunit o PI3K (PI3KCA) and inactivating mutations in PTEN are requently observed in a variety o malignancies, including breast cancer, colon cancer, prostate cancer, and glioblastoma. In addition to activating the MAP kinase and PI3K pathways, RAS also activates the RAL signaling pathway (Fig. 40-2B). RAL signaling remains incompletely understood, but this pathway activates specif c kinases involved in innate immunity and vesicle tra f cking. RAL signaling is also essential or RAS-mediated tumorigenesis. Signaling via type I cytokine receptors is associated with activation o the J AK-STAT pathway (Fig. 40-2C). Receptor dimerization activates JAKs, or Janus kinases, via transphosphorylation, allowing the recruitment o STAT proteins through their SH2 domains. STATs are a amily o proteins that shuttle rom the cytoplasm to the nucleus to regulate transcription directly. Phosphorylation o STAT proteins by JAKs leads to the ormation o SH2 domain-mediated STAT homo- or heterodimers that translocate to the nucleus and regulate transcription. Growth actor receptors such as EGFR, as well as intracellular tyrosine kinases such as SRC, can also signal through activation o STATs.

Proteasome Structure and Function Key cellular processes such as cell cycle progression and apoptosis are also regulated at the post-translational level by protein degradation. One o the major systems involved in this control is the ubiquitin–proteasome pathway, which comprises three enzymes that target specif c proteins or ubiquitin conjugation and degradation by the proteasome (Fig. 40-4A). Ubiquitin is a 9-kDa protein that derives its name rom its widespread distribution in tissues and its conservation across eukaryotes. The f rst enzyme involved in the process, E1, uses ATP to activate ubiquitin. The second enzyme in the cascade, E2, is a ubiquitin-conjugating enzyme that transiently carries ubiquitin and acts in conjunction with the third enzyme, the ubiquitin ligase E3, to orm a polyubiquitin chain that is trans erred to the target protein on an internal lysine residue.

C h a p t e r 4 0 Pharmacology o Cancer: Signal Transduction A

Cetuximab (anti-ErbB1) Trastuzumab (anti-ErbB2)

EGFR

B IGF1

EGF

IGF1R

P P Tyr

Farnesyltransferase inhibitors ABL

P

EGFR

P Tyr

Gefitinib Erlotinib

Tyr P

753

PI3K inhibitors

PI3K

PTEN

PIP3

Tyr P

RAS

S RC

RAS

RAL

Imatinib, dasatinib, bosutinib, nilotinib

RAF

Sorafenib, vemurafenib, dabrafenib

MEK

TBK1 PDK

Trametinib

NF-ΚB pa thwa y AKT inhibitors

MAP K

Everolimus, temsirolimus

AKT

mTOR

FOXO Nucle us

MAP K MYC J UN

Nucle us

FOS FOXO Ge ne tra ns cription

Myc/ J un/Fos

Tra ns cription of ge ne s ne e de d for a poptos is a nd ce ll cycle a rre s t

S ynthe s is of prote ins ne e de d for ce ll growth a nd ce ll cycle progre s s ion

FIGURE 40-2. Intracellular signaling pathways. A. The RAS-MAP kinase pathway is actiC EP O

EGF

EP OR

EGFR

P Tyr

Tyr P

P Tyr

Tyr P

J AK2 Ruxolitinib P

S TAT S TAT P

S RC

Nucle us

Ge ne tra ns cription P

S TAT S TAT P

vated by multiple growth actor receptors (here exemplif ed by the EGF receptor, EGFR) as well as several intracellular tyrosine kinases such as SRC and ABL. RAS is recruited to the plasma membrane by arnesylation and activated by binding to GTP. Activated RAS stimulates a sequence o phosphorylation events mediated by RAF, MEK, and ERK (MAP) kinases. Activated MAP kinase (MAPK) translocates to the nucleus and activates proteins such as MYC, J UN, and FOS that promote the transcription o genes involved in cell cycle progression. Cetuximab and trastuzumab act as antagonists at the EGF receptor (ErbB1) and HER2 receptor (ErbB2), respectively. Gef tinib and erlotinib inhibit the receptor tyrosine kinase. Farnesyltrans erase inhibitors prevent RAS activation. Imatinib, dasatinib, bosutinib, and nilotinib inhibit ABLkinase; sora enib, vemura enib, and dabra enib inhibit RAF kinase; and trametinib inhibits MEKkinase. B. The PI3 kinase (PI3K) pathway is activated by a number o growth actor receptors (here exemplif ed by the insulin-like growth actor receptor 1 [IGF1R] and the epidermal growth actor receptor [EGFR]). Activated PI3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP3), which activates phosphoinositide dependent kinase-1 (PDK). In turn, PDKphosphorylates AKT. PTEN is an endogenous inhibitor o AKT activation. Phosphorylated AKT transduces multiple downstream signals, including activation o the mammalian target o rapamycin (mTOR) and inhibition o the FOXO amily o transcription actors. mTOR activation promotes the synthesis o proteins required or cell growth and cell cycle progression. Because the FOXO amily o transcription actors activates the expression o genes involved in cell cycle arrest, stress resistance, and apoptosis, inhibition o FOXO promotes cell proli eration and resistance to apoptosis. RAS activates not only the MAP kinase pathway diagrammed in panel A but also the PI3K pathway diagrammed in panel B and the NF- B pathway diagrammed in Figure 40-5B. Activation o the NF- B pathway by RAS involves direct activation o RAL, which activates the serine/threonine kinase TBK1. Everolimus and temsirolimus inhibit mTOR, and inhibitors o PI3K and AKT are in development. C. The STAT pathway is activated by SRC and by a number o growth actor receptors (here exemplif ed by the erythropoietin receptor [EPOR], which signals to STAT proteins through J AK2 kinase, and by the EGF receptor [EGFR], which signals to STAT proteins indirectly). Phosphorylation o STAT induces SH2 domain-mediated homodimerization, and phosphorylated STAT homodimers translocate to the nucleus and activate transcription. Ruxolitinib inhibits J AK2; this agent is used to treat myelof brosis. Ruxolitinib and other J AK2 inhibitors are under evaluation or the treatment o polycythemia vera and essential thrombocythemia, which o ten share a common activating mutation in J AK2 (V617F).

754 PRINCIPLES OF CHEMOTHERAPY

MAP K

Cyclin D

p16

CDK4/6

Cyclin D CDK4/6

P P

RB

P

P

RB

S pha s e ge ne s

E2F

S pha s e ge ne s

E2F

G1 phase

S phase

FIGURE 40-3. Regulation of the G1–S cell cycle transition. Activation o MAP kinase results in increased expression o the D-type cyclins. Cyclin D binds to its catalytic partners cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), which phosphorylate the retinoblastoma protein (RB). Phosphorylation o RB releases its transcriptional repression o S-phase genes, allowing the transcription actor E2F to activate the transcription o genes needed or entry into S phase. These genes include cyclin E as well as DNA polymerase and the enzymes involved in nucleotide synthesis. Cyclin E binds to its catalytic partner CDK2, which urther phosphorylates RB, creating a positive eedback loop that drives cells into S phase (not shown). The CDK2/ CDK4/CDK6 system is counterbalanced by cyclin-dependent kinase inhibitors (CDKIs) such as p16, which inhibits CDK4/6, and p21 and p27, which inhibit CDK2 (not shown).

B

E1 is nonspecif c, and there are a number o di erent E2 ubiquitin-conjugating enzymes with a limited degree o specif city. The E3 ubiquitin ligase component is largely responsible or target protein specif city. The RING amily o E3 ligases contains a characteristic RING f nger domain with conserved histidine and cysteine residues complexed with two central Zn2 ions. RING E3 ligases can be subdivided into single-subunit E3 ligases and multisubunit complexes such as the Skp1-Cullin-Fbox protein amily (SCF) E3 ligases. In the latter complexes, the RING f nger component, Rbx, is distinct rom the specif city component, the F-box protein, which is so named because o a characteristic moti f rst identif ed in cyclin F. Once proteins are selectively ubiquitinated, they are targeted or degradation by the 26S proteasome, which is a cylindrical particle present in both cytoplasm and nucleus. The core 20S subunit is the catalytic component with multiple proteolytic sites, while the 19S regulatory component mediates binding to ubiquitin-conjugated proteins and has multiple ATPases involved in substrate un olding and delivery to the central 20S chamber. Substrates are cleaved progressively, with one protein being completely degraded be ore the next protein enters. Short peptide segments, on average 6 to 10 amino acids in length, are extruded and subsequently hydrolyzed to individual amino acids in the cytosol. Regulation o protein degradation occurs largely at the level o the E3 ubiquitin ligase and governs key aspects o cell cycle control, apoptosis, and other important cellular processes (Fig. 40-4B). For example, CBL is a single-subunit RING E3 ubiquitin ligase that targets phosphorylated EGFR amily members or degradation. In addition, both cyclins Single-subunit RING E3 ligases

Multisubunit RING E3 ligases

Ub Ub

Ub

A S te p 1

S te p 2

Ub

Ub

Ub

E1

Ta rge t

E2

Ub

Ub

Ub

Ub

Skp1

Ub

Ub

PPi + AMP

Ub

Ub

Ub

S te p 3

Ub

E3

Ta rge t

Rbx

Ta rge t

F-box

Ub

Ta rge t

Cullin

E3 Single-subunit ligase ATP +

E1

E2

Ta rge t

CBL MDM2

Ub

Ub

Ubiquitin

EGFR p53

F-box protein ligase S kp2 Fbw7 βTrCP

Target p27, FOXO Cyclin E β -ca te nin, Iκ Bα

Bortezomib

26S prote a s ome

SCF-like ligase

Ub Ub

Ta rge t prote in fra gme nts

Target

Target

Ana pha s e promoting comple x

Cyclin B

VHL

HIF-1 α

FIGURE 40-4. The ubiquitin–proteasome pathway. A. Ubiquitin (Ub) is activated by ATP-dependent conjugation to E1, the f rst enzyme in the pathway. Activated ubiquitin is then passed rom the active-site cysteine o E1 to the active-site cysteine o the ubiquitin-conjugating enzyme E2, which unctions coordinately with the ubiquitin ligase E3 to attach ubiquitin to protein targets. Polyubiquitination o target proteins results in their recognition by the 26S proteasome, which consists o a 19S outer regulatory subunit and a 20S internal core chamber. The proteasome mediates proteolytic degradation o the target protein into short peptide ragments. Bortezomib is a proteasome inhibitor that has been approved or use in multiple myeloma and is under investigation or use in other malignancies. B. The RING amily o E3 ubiquitin ligases consists o single-subunit enzymes (left) and multisubunit protein complexes (right). Single-subunit ligases include CBL, which targets EGFR or degradation, and MDM2, which targets p53 or degradation. Multisubunit RING E3 ligase complexes include SCF and SCF-like amily members, which are named or their Skp1, Cullin, and F-box protein subunits. The F-box protein component mediates target protein specif city; or example, Skp2 targets p27 and FOXO or degradation, Fbw7 targets cyclin E or degradation, and TrCP targets -catenin and I B or degradation. SCF-like ligase complexes include the anaphase-promoting complex, which targets cyclin B or degradation, and VHL, which targets hypoxia-inducible actor-1 (HIF-1 ) or degradation.

C h a p t e r 4 0 Pharmacology of Cancer: Signal Transduction

and cyclin-dependent kinase inhibitors are major targets or ubiquitin-mediated proteasomal degradation. The anaphasepromoting complex is a multisubunit RING-containing E3 ligase that is activated by phosphorylation late in mitosis, triggering degradation o cyclin B and progression through mitosis. Regulation o the G1–S cell cycle transition is in part mediated by the cyclin-dependent kinase inhibitor p27, which inhibits cyclin E–CDK2 and cyclin A–CDK2 complexes. Degradation o p27 is regulated by another SCF E3 ligase, which binds p27 via its F-box specif city component Skp2. Thus, overexpression o Skp2, which is ound in a number o tumor types, can promote cell cycle progression by degrading p27. Degradation o FOXO by Skp2 is a second mechanism by which overexpression o Skp2 may promote tumorigenesis. Yet another SCF E3 ligase complex regulates cyclin E activity by targeting it or degradation via the F-box protein Fbw7. Loss o Fbw7 has been implicated in tumor progression due to high levels o cyclin E. Another example o an E3 ligase with a critical role in the regulation o apoptosis and cell cycle regulation is MDM2, A

755

a single-subunit RING E3 ligase that targets p53 or degradation. Activation o MDM2 is linked to impairment o apoptosis and promotion o tumorigenesis via loss o p53. MDM2 is inhibited by the p14ARF protein, which shares the same genomic locus as the CDK4/6 inhibitor p16. Disruption o this locus, which is one o the most common events in cancer, leads ultimately to both p53 and pRB inactivation. Other key cellular pathways regulated by ubiquitinmediated proteasomal degradation include the Wnt signaling pathway and the nuclear actor-kappa B (NF B) pathway. Both pathways are targeted by the common F-box protein TrCP, which recognizes phosphorylated substrates (Fig. 40-5). Activation o Wnt signaling prevents phosphorylation o -catenin, which allows it to escape recognition by TrCP and ubiquitin ligation by SCF E3 ligase. Unphosphorylated -catenin then translocates to the nucleus with its partners TCF/LEF and activates transcription o genes such as myc and cyclin D1. This pathway is also regulated by the adenomatous polyposis coli (APC) gene, which orms

Wnt Frizzle d

Abs e nce of Wnt

AP C comple x (a ctive )

β-ca te nin

β-ca te nin

βTrCP comple x

P

Dis he ve le d

P

AP C comple x (ina ctive )

β-ca te nin

Ub Ub Ub Ub Ub

β-ca te nin

P

Nucle us

26S prote a s ome

β-ca te nin TCF/ LEF

Tra ns cription of ge ne s promoting ce ll cycle progre s s ion

Ub Ub

β-ca te nin fra gme nts

FIGURE 40-5. Wnt signaling and NF B pathways. A. In the absence of Wnt signaling, -catenin is phosphorylated by the adenomatous polyposis coli (APC) protein complex. Phosphorylated -catenin is recognized by TrCP and thereby targeted for ubiquitin-mediated proteasomal degradation. Activation of Wnt signaling inhibits APC function, allowing -catenin to accumulate and translocate to the nucleus. In the nucleus, -catenin complexes with its partners TCF/LEF and activates the transcription of genes promoting cell cycle progression. Hereditary or acquired loss of APC allows accumulation of -catenin, contributing to oncogenesis in colon cancer. (continued)

756 PRINCIPLES OF CHEMOTHERAPY B

Absence of stimuli

IκB kina s e (ina ctive )

IκB

Multiple stimuli

IκB kina s e (a ctive )

NFκB

Ina ctive comple x

IκB

IκB

NFκB

P

βTrCP comple x

Ub Ub Ub Ub Ub

IκB

P

Nucle us

26S prote a s ome

NFκB

Tra ns cription of ge ne s involve d in prolife ra tion a nd infla mma tion

Ub Ub

IκB fra gme nts

FIGURE 40-5. (continued) B. Similarly, the I B protein is targeted or ubiquitin-mediated proteasomal degradation as a result o phosphorylation by I B kinase and recognition by TrCP. In the absence o stimuli, I B binds to and inhibits NF B. In the presence o stimuli, proteasomal degradation o I B allows NF B to translocate to the nucleus and activate the transcription o genes involved in proli eration and inf ammation.

part o the complex that promotes phosphorylation and subsequent destruction o -catenin. Loss o APC in colorectal cells prevents phosphorylation o -catenin, leading to -catenin accumulation and to promotion o cancer. The F-box protein TrCP also regulates signaling through NF B, which is inhibited by its association with the inhibitor o NF B (I B). Phosphorylation o I B by a amily o I B kinases (IKKs) allows TrCP to bind to I B and activate its proteasome-mediated degradation. The release o I B inhibition allows NF B to translocate to the nucleus and activate transcription o genes involved in in ammation, proli eration, and survival. Specif c IKKs may be aberrantly activated in cancer cells and thereby generate an environment avoring tumor cell survival.

receptors, VEGFR1 (also known as Flt-1), VEGFR2 (Flk-1/ KDR), and VEGFR3 (Flt-4). The VEGF receptors are receptor tyrosine kinases. Neuropilins (NRP-1 and -2) are coreceptors that lack an intracellular signaling domain and enhance the binding o ligand to VEGFR1 and VEGFR2. VEGFR1 and VEGFR2 are expressed on the vascular endothelium

TABLE 40-2 Vascular Endothelial Growth Factor Receptors RECEPTOR TISSUE EXPRESSION CORECEPTORS

LIGANDS

VEGFR1

Vascular endothelium Hematopoietic cells Smooth muscle cells Osteoclasts

Neuropilin-1 Neuropilin-2

VEGF-A VEGF-B PlGF-1 PlGF-2

VEGFR2

Vascular endothelium Neuronal cells

Neuropilin-1 Neuropilin-2

VEGF-A VEGF-E

VEGFR3

Vascular endothelium Lymphatic endothelium Monocytes and macrophages

None

VEGF-C VEGF-D

Angiogenesis Solid tumors require development o a neovasculature in order to sustain growth and survive conditions o hypoxia. Tumor angiogenesis is a complex process involving a number o di erent pro- and antiangiogenic actors. The vascular endothelial growth factor (VEGF) amily o proteins and receptors has emerged as a key regulator o this process. The VEGF amily consists o seven ligands, including VEGF-A, -B, -C, -D, and -E and placenta growth actor (PlGF)-1 and -2 (Table 40-2). These ligands have varying a f nities or the major VEGF

VEGFR, vascular endothelial growth actor receptor; PlGF, placenta growth actor.

C h a p t e r 4 0 Pharmacology of Cancer: Signal Transduction

and play key roles in angiogenic signaling, while signaling through VEGFR3 appears to play a major role in lymphangiogenesis (i.e., development o new lymphatic vessels). VEGFR2, which appears to be the major proangiogenic receptor targeted by VEGF-A, signals via both a RAF/MAP kinase pathway to promote proli eration o endothelial cells and a PI3K/AKT pathway to promote endothelial cell survival. VEGF also potently induces vascular permeability, utilizing similar signaling pathways both to promote the ormation o transendothelial vesicular organelles and to open interendothelial junctions. Invasion and migration o endothelial cells is promoted by activation o matrix metalloproteinases and serine proteases and by reorganization o intracellular actin. Activation o VEGF is mediated by stimuli such as hypoxia, by cytokines and growth actors, and by a variety o oncogenes and tumor suppressor genes. Regulation o the response to hypoxia is mediated by von Hippel-Lindau protein (VHL), a component o an SCF-like RING E3 ubiquitin ligase complex that targets hypoxia-inducible factor-1 (HIF-1 ) or degradation (Fig. 40-6). Loss o VHL is the def ning event in the inherited VHL syndrome and is a requent f nding in sporadic clear cell renal carcinoma. Under normoxic conditions, HIF-1 undergoes oxygendependent hydroxylation, which allows VHL binding and subsequent ubiquitin-mediated degradation. Under hypoxic conditions, HIF-1 is not hydroxylated and VHL is unable

to bind it. Native HIF-1 is thus allowed to translocate to the nucleus and pair with its binding partner HIF-1 to activate transcription o hypoxia-inducible genes such as VEGF, PDGF- , and TGF- . In this manner, angiogenesis is stimulated by hypoxic conditions or by inappropriate activation o HIF-1 due to loss o VHL expression in tumors. Cytokines such as IL-1 and IL-6, as well as prostaglandin products o COX-2 activation, can also stimulate VEGF production. Signaling via EGFR amily members, PDGFR, and the insulin-like growth actor-1 receptor (IGF-1R) have also been shown to induce VEGF expression. Finally, activation o oncogenes such as RAS, SRC, and BCR-Abl and inactivation o tumor suppressor genes such as p53 and PTEN can result in VEGF production and thus promote angiogenesis and tumor maintenance.

PHARMACOLOGIC CLASSES AND AGENTS Growth Factor Receptor and Signal Transduction Antagonists The identif cation o specif c pathways that are dysregulated in certain tumors a ords the potential to target key components o these pathways in a more selective manner.

Normal or high O2

Low O2

HIF-1α P HD

VHL comple x

HIF-1α P HD

O2

HIF-1α

757

OH

Ub Ub Ub Ub Ub

HIF-1α

OH

Nucle us

26S prote a s ome HIF-1α HIF-1β

Tra ns cription of VEGF, P DGF-β, TGF-α, EP O ge ne s

Ub Ub

HIF-1α fra gme nts

FIGURE 40-6. Regulation of the response to hypoxia. Left panel: Under normal or high oxygen conditions, hypoxia-inducible factor-1 (HIF-1 ) is hydroxylated (in an oxygen-dependent reaction) by the prolyl hydroxylase PHD. Hydroxylated HIF-1 is recognized by VHLand thereby targeted for ubiquitin-mediated proteasomal degradation. Right panel: PHD is inactive under low oxygen conditions, allowing HIF-1 to accumulate and translocate to the nucleus. In the nucleus, HIF-1 complexes with HIF-1 and activates the transcription of hypoxia-inducible genes such as VEGF, PDGF- , TGF- , and erythropoietin (EPO).

758 PRINCIPLES OF CHEMOTHERAPY

While the growth actor and signal transduction pathways described above are active during normal cell physiology, some tumors may become dependent on one pathway in particular or their growth and survival. Conversely, in normal cells, the redundancy o signaling pathways allows or compensation, as exemplif ed by the observation that inactivation o the EGFR gene in the mouse causes minimal de ects. Thus, the therapeutic window o the newer targeted agents tends to be wider than that o traditional cytotoxic chemotherapy, with a di erent spectrum o adverse e ects.

EGF Receptor Antagonists Gef tinib, Erlotinib, and A atinib

The expression o EGFR on epithelial cells and its amplif cation and/or activation in a signif cant raction o non-small cell lung cancers (NSCLCs) led to the development and testing o small-molecule EGFR tyrosine kinase inhibitors (TKIs) in patients with advanced NSCLC. The f rst o these agents to be tested was gef tinib. Gef tinib is an orally bioavailable drug that competes with ATP or binding to the cytoplasmic tyrosine kinase domain o EGFR and thereby acts as a reversible inhibitor o EGFR tyrosine kinase activity (Fig. 40-2A). In patients with metastatic NSCLC who had received multiple prior chemotherapy regimens, response rates to gef tinib were on the order o 10% in studies conducted in the United States and 20% in trials conducted in Japan and Europe. During the course o these studies, it was noted that patients who tended to respond were emale, nonsmokers, Asian, and with adenocarcinoma tumor histology. Given the dramatic responses in some cases, sequencing o the EGFR gene was carried out in tumors rom these patients. Common activating mutations were ound in the kinase domain o EGFR, including L858R and in- rame deletions spanning positions 746 and 753. These mutations enhance tyrosine kinase activity in response to EGF and increase sensitivity to gef tinib. Signals generated by these mutant EGFRs activate MAP kinase, AKT, and STAT pathways, leading to promotion o cell survival. Recent studies have shown that screening lung cancer patients or EGFR mutations can identi y specif c patients who are likely to benef t rom treatment with EGFR inhibitors, even prior to the initiation o standard chemotherapy. Erlotinib is an oral small-molecule EGFR TKI similar to gef tinib (Fig. 40-2A). Both drugs yielded similar results in phase II studies, with similar adverse e ects, including skin rash and diarrhea. Pivotal randomized phase III studies, however, demonstrated a statistically signif cant survival benef t or erlotinib but not or gef tinib (in patients who were not stratif ed by EGFR mutation status). Thus, erlotinib was approved by the US Food and Drug Administration (FDA) or treatment o metastatic NSCLC, while gef tinib is still utilized in Europe and Asia; both drugs are now directed toward patients with activating EGFR mutations. The second-generation EGFR TKI a atinib, which binds covalently to EGFR and is thus an irreversible inhibitor, was recently approved by the FDA specif cally or patients with lung cancer and EGFR mutations. Patients who initially respond to erlotinib or gef tinib but subsequently develop resistance most o ten carry a single secondary mutation, T790M, within the EGFR kinase domain. EGF receptors carrying both activating mutations and the T790M secondary mutation exhibit reduced sensitivity to inhibition by erlotinib and gef tinib. Despite their covalent cross-linking to EGFR, second-generation

irreversible inhibitors such as a atinib have ailed to overcome resistance generated by the T790M mutation, in part due to toxicity associated with inhibition o the wild-type kinase. Novel third-generation EGFR TKIs that are also irreversible yet bind selectively to the T790M mutant protein are entering the clinic and showing promising activity or the large group o patients who develop acquired resistance to erlotinib or gef tinib. Amplif cation o MET is another mechanism o acquired resistance to EGFR inhibition. MET is a receptor tyrosine kinase that is normally under the control o the ligand hepatocyte growth actor (HGF). MET amplif cation allows cancer cells to re-activate downstream survival pathways that are blocked by EGFR inhibition. MET inhibitors are currently in clinical development; these agents could potentially be used in combination with EGFR antagonists to combat acquired drug resistance. Erlotinib has shown less impressive activity in a wide variety o other epithelial malignancies in which EGFR is overexpressed, including colon cancer, pancreatic cancer, and head and neck cancer. EGFR is requently amplif ed, mutated, or overexpressed in patients with glioblastoma, but response rates o only 10–20% are seen with EGFR antagonists, similar to the response rates in patients with advanced NSCLC. A constitutively active EGFR genomic deletion variant, EGFRvIII, has been identif ed in a signif cant raction o patients with glioblastoma. Because this mutant receptor relies on PI3K/AKT signaling, it was hypothesized that loss o PTEN might impair response to EGFR inhibitors in this setting by activating AKT independently (Fig. 40-2B). Indeed, co-expression o EGFRvIII and PTEN in glioblastoma correlates with response to erlotinib. Cetuximab, Panitumumab, Trastuzumab, and Lapatinib

Strategies to target signaling by EGFR amily members have also included the development o monoclonal antibodies that bind with high a f nity to the extracellular ligand-binding domain o the receptor. One example is cetuximab, a chimeric mouse/human IgG1 monoclonal antibody that binds EGFR (ErbB1) with high specif city and an a f nity greater than that o the physiologic ligands EGF or TGF- (Fig. 40-2A). When administered in combination with irinotecan, cetuximab improves response rates in EGFR-expressing colorectal cancers. Panitumumab is a humanized IgG2 monoclonal antibody directed against EGFR that has activity in a subset o patients with colorectal cancer. The principal adverse e ects o cetuximab and panitumumab are similar to those o the small-molecule EGFR antagonists, including skin rash and diarrhea. Interestingly, the development o skin rash in response to cetuximab correlates with tumor response, perhaps re ecting the degree o EGFR blockade by cetuximab. As a single agent, cetuximab enhances the e f cacy o radiation therapy in locally advanced head and neck cancer, improving locoregional control and overall survival compared to radiation therapy alone. Less dramatic e ects have been seen in NSCLC, in which EGFR mutations do not predict responsiveness to cetuximab. KRAS activating mutations are associated with resistance to EGFR inhibitors. This is likely due to the act that constitutive activation o RAS can bypass the upstream blockade in EGFR signaling (Fig. 40-2A). To help predict responsiveness to EGFR inhibitor therapy, routine testing o KRAS mutational status is now per ormed in tumors such as lung cancer and colon cancer.

C h a p t e r 4 0 Pharmacology of Cancer: Signal Transduction

Trastuzumab, another chimeric mouse/human IgG monoclo-

nal antibody, is directed against ErbB2 (HER2) (Fig. 40-2A). Approximately 25–30% o breast cancers are associated with amplif cation and overexpression o HER2/neu; these cancers also display more aggressive behavior. HER2 amplif es the signal generated by other ErbB amily members via the ormation o heterodimers. Trastuzumab down-regulates HER2 and thereby disrupts this signaling. In vivo, trastuzumab also appears to induce antibody-dependent cellular cytotoxicity and inhibit angiogenesis. Trastuzumab has signif cant activity in breast cancers with high levels o HER2 amplif cation. In addition to the intrinsic activity o trastuzumab in the advanced and metastatic breast cancer settings, treatment o HER2-amplif ed breast cancers with trastuzumab in the adjuvant setting (i.e., a ter surgical resection o the tumor) enhances the e f cacy o chemotherapy and reduces rates o cancer recurrence by 50%. The principal adverse e ect o trastuzumab is cardiotoxicity, particularly when used in combination with anthracyclines. Trastuzumab does not cross the blood–brain barrier, and thus CNS relapse o breast cancer may occur. Lapatinib, a small-molecule dual EGFR/HER2 inhibitor, has also been approved or the treatment o metastatic breast cancer with HER2 overexpression. Lapatinib crosses the blood–brain barrier and shows activity against brain metastases. Trastuzumab emtansine (T-DM1) is an antibody– drug conjugate that links trastuzumab to the cytotoxic agent mertansine and specif cally delivers this toxin to HER2expressing cells. T-DM1 has shown signif cant activity in trastuzumab-re ractory breast cancer.

ALK/ROS1 Inhibitors Crizotinib and Ceritinib

The success o erlotinib in treating EGFR-mutant lung cancers encouraged investigators to study mutations in other growth actor receptors in patients with cancer. It was discovered that a subset o smokers with adenocarcinoma harbor rearrangements in the ALK receptor tyrosine kinase. Based on this f nding, crizotinib, initially identif ed as a MET and ALK TKI, was repurposed or this population o lung cancer patients and ound to exhibit remarkable activity, leading to its approval by the FDA. As with EGFR TKIs, however, treatment with crizotinib leads to acquired resistance through a variety o mechanisms. Recently, the second-generation ALK TKI ceritinib was approved by the FDA based on its ability to overcome crizotinib resistance, and additional inhibitors and drug combination strategies are being developed. Lung adenocarcinomas in light smokers continue to be subdivided urther into oncogene-dependent genotypes, such as those characterized by ROS1 and RET rearrangements. Crizotinib also inhibits ROS1 and has activity in the subgroup o lung cancers with ROS1 rearrangements. Several multitargeted kinase inhibitors such as sunitinib and cabozantinib are being evaluated or RET-rearranged lung cancers.

BCR-Abl/C-KIT/PDGFR Inhibition Imatinib Imatinib is a small-molecule tyrosine kinase inhibitor that

was initially developed as a 2-phenylaminopyrimidine derivative specif c or PDGFR. Imatinib was subsequently ound to be a potent inhibitor o ABL kinases, including the BCR-Abl usion protein generated as a result o the t(9;22)

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chromosomal translocation (Philadelphia chromosome) ound in chronic myelogenous leukemia (CML) (Fig. 40-2A). Imatinib was also ound to inhibit the receptor tyrosine kinase C-KIT. Imatinib is the canonical example of a targeted therapeutic agent, because BCR-Abl is uniquely expressed by leukemic cells and is essential for their survival. Initial in vitro studies demonstrated that imatinib potently and specif cally inhibits the growth o cells expressing BCRAbl. Subsequent evaluation o an oral ormulation in mice demonstrated suppression o growth o human BCR-Ablpositive tumors with minimal adverse e ects. Early studies o imatinib in patients with chronic-phase CML yielded impressive results, with normalization o blood counts (a hematologic response) in 95% o patients and signif cant reduction in cells expressing the Philadelphia chromosome (a cytogenetic response) in 41% o patients. In a phase III study, imatinib was superior to standard treatment with inter eron and cytarabine in patients with chronic-phase CML, with a hematologic response rate o 95% and a complete cytogenetic response in 76% o patients. Treatment o accelerated or blast-phase CML with imatinib is less e ective but is associated with some responses. Imatinib is relatively well tolerated; its principal adverse e ects are myelosuppression, superf cial edema, nausea, muscle cramps, skin rash, and diarrhea. Mutation o C-KIT, the receptor or stem cell actor (SCF), is ound requently in gastrointestinal stromal tumor (GIST) and in the myeloproli erative disorder systemic mastocytosis . In GIST, mutations and in- rame deletions o C-KIT are typically ound in the juxtamembrane domain, resulting in constitutive activation o the tyrosine kinase in the absence o ligand. In contrast, in systemic mastocytosis, the characteristic C-KIT-activating mutation D816V is within the tyrosine kinase domain itsel . Imatinib has shown signif cant activity in advanced gastrointestinal stromal tumor, but it has proven largely ine ective in the treatment o systemic mastocytosis. Indeed, biochemical studies show that the drug is not e ective at targeting C-KIT kinases with the D816V mutation. Both idiopathic hypereosinophilic syndrome and a variant o systemic mastocytosis with eosinophilia are characterized by expression o the FIPL1-PDGFRA usion protein. This protein, which is generated by an interstitial chromosomal deletion, causes constitutive signaling through PDGFRA. Inhibition o PDGFRA by imatinib has been a success ul therapeutic approach in both conditions. Dasatinib, Bosutinib, and Nilotinib

Most patients with CML experience durable remissions with imatinib treatment. However, a raction o patients show evidence o the BCR-Abl transcript when sensitive tests such as reverse transcriptase polymerase chain reaction (RT-PCR) are used or detection, even in cases in which a complete cytogenetic response is observed. Moreover, some patients with CML develop acquired resistance to imatinib, occasionally due to amplif cation o BCR-Abl but more commonly due to acquisition o resistance mutations. Crystallographic studies show that imatinib targets the ATP-binding site o ABL when the activation loop o the kinase is closed, thereby stabilizing the protein in an inactive con ormation (see Fig. 1-2). A minority o resistance mutations inter ere directly with imatinib binding, while most resistance mutations a ect the ability o ABL to adopt the closed con ormation to which imatinib binds.

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A second class o tyrosine kinase inhibitors, the SRC– ABL inhibitors, bind to the ATP-binding site in ABL irrespective o the con ormational status o the activation loop. One o these drugs, dasatinib, is more potent than imatinib and has signif cantly greater e f cacy than imatinib against wild-type BCR-Abl. Dasatinib also inhibits the activity o most clinically relevant imatinib-resistant BCRAbl iso orms, with the exception o the T315I mutation (Fig. 40-2A). Bosutinib is another SRC–ABL inhibitor that inhibits most BCR-Abl resistance alleles, with the exception o T315I and V299L. Investigators have used a structure-based approach to improve the e f cacy o imatinib, substituting alternative binding groups or the N-methylpiperazine group. This approach has resulted in the development o nilotinib. The a f nity o nilotinib or wild-type BCR-Abl is signif cantly higher than that o imatinib, and nilotinib inhibits most imatinibresistant mutants with the exception o T315I (Fig. 40-2A). Dasatinib, bosutinib, and nilotinib have shown signif cant activity in patients with CML who have developed resistance to imatinib and are approved by the FDA or this setting. Another potent and broad-spectrum inhibitor, ponatinib, which inhibits VEGFR and FGFR amily members in addition to ABL, SRC, and other kinases, has shown activity even in the resistant T315I population but is associated with increased vascular toxicity.

BTK Inhibitors

Ibrutinib is a covalent and relatively selective inhibitor o

BTK, which is expressed in B cells and promotes B-cell receptor signaling and lymphocyte survival. Ibrutinib has shown remarkable activity in re ractory B-cell malignancies such as mantle cell lymphoma and was also recently approved by the FDA or the treatment o chronic lymphocytic leukemia (CLL). Ibrutinib is currently under evaluation or the treatment o a variety o other B-cell malignancies, including di use large B-cell lymphoma and plasma cell dyscrasias such as multiple myeloma and Waldenstrom’s macroglobulinemia.

FLT3 Inhibitors

One o the most common mutations in acute myelogenous leukemia (AML), occurring in approximately 25–30% o patients, involves internal tandem duplication within the juxtamembrane domain o the FLT3 receptor tyrosine kinase. This mutation results in ligand-independent dimerization and activation o signaling via the RAS/MAPK and STAT pathways. Several FLT3 inhibitors have been developed and demonstrate anti-leukemia cell activity in vitro. Sorafenib, a multitargeted kinase inhibitor developed against RAF but also with activity against VEGFR (see below) and FLT3, has shown signif cant activity in patients with relapsed/ re ractory AML carrying FLT3 mutations.

J AK2 Inhibitors

Although BCR-Abl was discovered decades ago to be the pathophysiologically important lesion in CML, the genetic basis o the other major myeloproliferative disorders (polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelof brosis) remained obscure. It is now apparent that a common activating mutation in JAK2 (V617F) underlies the aberrant signaling and proli eration in most cases, although how one mutation leads to

this spectrum o disorders remains unclear (see Fig. 40-2C). The V617F mutation is located in the pseudokinase domain o JAK2, and disruption o this autoinhibitory region leads to unchecked activity o the kinase. In vitro, selective JAK2 inhibitors cause cells containing the JAK2 V617F mutation to be growth inhibited and undergo apoptosis; in animal models, JAK2 inhibitors demonstrate therapeutic e f cacy against JAK2(V617F)-induced hematologic disease. Ruxolitinib is the f rst JAK2 inhibitor to be approved by the FDA, based on its activity in myelof brosis. Ruxolitinib is also under evaluation or the treatment o polycythemia vera and essential thrombocythemia, as are several other JAK2 inhibitors, such as momelotinib, that have entered late-stage clinical trials. These and other JAK inhibitors are also being evaluated or use in the treatment o a variety o solid tumors that aberrantly activate cytokine signaling pathways.

RAS/MAP Kinase Pathway Inhibition

Oncogenic mutation o ras is one o the most common events in malignancy, occurring in approximately 30% o human cancers. K-ras mutations are requently observed in non-small cell lung cancer, colorectal cancer, and pancreatic carcinoma, while H-ras mutations are ound in kidney, bladder, and thyroid cancers, and N-ras mutations occur in melanoma, hepatocellular carcinoma, and hematologic malignancies. However, despite the requency o these mutations, inhibition o RAS has thus ar been di f cult to achieve and has yielded minimal clinical success. Most e orts have ocused on targeting arnesylation o RAS and inhibiting downstream e ectors. Farnesylation o RAS is essential or its association with the plasma membrane and its subsequent activation. Several arnesyltrans erase inhibitors (FTIs) have been developed that inhibit RAS arnesylation (Fig. 40-2A). While these inhibitors demonstrate activity against RAS in vitro, some RAS mutants exhibit resistance. Moreover, there are many other targets o arnesylation that could be inhibited by FTIs, and such inhibition is likely responsible or the cytotoxic e ects o these drugs. FTIs that have been tested clinically include tipifarnib and lonafarnib. Tipi arnib has demonstrated activity in relapsed/re ractory AML, although responses appear to be independent o ras mutations. Clinical testing o FTIs in solid tumors has not yet met with success. Immediately downstream o RAS is the serine/threonine kinase RAF, which phosphorylates MEK, which in turn phosphorylates MAP kinase, leading to transcription actor activation (Fig. 40-2A). There are three RAF amily members—A-RAF, B-RAF, and C-RAF. Activating mutations in B-RAF have been ound in a signif cant raction o malignant melanomas and are also observed at a lower requency in lung, colorectal, ovarian, and thyroid cancers. Sorafenib was initially designed as a C-RAF inhibitor, but it also inhibits B-RAF, among other kinases. Sora enib has shown signif cant activity against melanoma cell lines that contain activating B-RAF mutations but has been less impressive clinically. Vemurafenib is a more potent and selective inhibitor o B-RAF that was designed to target the common B-RAF V600E mutation observed in melanoma. Vemura enib treatment o patients with B-RAF(V600E)-mutant melanoma has been associated with signif cant responses and has led to approval o the drug by the FDA, although resistance invariably develops. Dabrafenib is another potent B-RAF inhibitor with activity in this population o melanoma patients.

C h a p t e r 4 0 Pharmacology of Cancer: Signal Transduction

There are two MEK homologues directly downstream o RAF: MEK1 and MEK2. Both o these homologues have dual serine/threonine kinase activity, phosphorylating and activating ERK1 and ERK2. Trametinib is a highly potent inhibitor o both MEK1 and MEK2 (Fig. 40-2A). Based on its activity in B-RAF-mutant melanoma, trametinib has been approved by the FDA or this indication. “Vertical inhibition” (or sequential inhibition) o the RAF-MEK-ERK pathway by combining dabra enib and trametinib results in urther response and delayed resistance in patients with B-RAF-mutant melanoma. These inhibitors are also being explored or use in the subset o lung cancers that harbor B-RAF mutations. MEK inhibitors are also being explored as a downstream method o targeted therapy or KRAS-driven cancers. However, clinical-stage MEK inhibitors such as trametinib and selumetinib have shown limited single-agent activity in KRAS-mutated lung and pancreatic cancers, likely due to the act that KRAS engages multiple other pathways in parallel. MEK inhibitors are currently being combined with traditional chemotherapy as one strategy to enhance activity. Another approach involves combining MEK inhibitors with targeted inhibitors against downstream signaling components o the RAS/PI3K/AKT/mTOR or RAS/RAL/cytokine pathways. Clinical trials evaluating these combination therapy strategies or KRAS-driven lung and gastrointestinal cancers are underway.

PI3K/AKT/mTOR Inhibitors

Signaling via the PI3K/AKT pathway leads to downstream activation o the mammalian target o rapamycin (mTOR) (Fig. 40-2B). mTOR is a serine/threonine kinase that regulates multiple cellular unctions, including cell growth and proli eration, via activation o protein synthesis. mTOR regulation is accomplished in part by activation o the 40S ribosomal protein S6 kinase (p70S6k) and inactivation o the 4E-binding protein (4E-BP1), which regulates translation o certain mRNAs. Dysregulated mTOR activity is seen in a wide variety o malignancies in which the PI3K pathway is activated or PTEN is lost. In addition, hamartoma syndromes such as tuberous sclerosis result in activation o mTOR. The tuberous sclerosis protein complex (TSC1/2) acts as an intermediary between AKT and mTOR: native TSC1/2 inhibits mTOR, and activation o AKT results in phosphorylation o TSC1/2 and subsequent de-repression o mTOR. TOR was originally identif ed in a screen or mutations in yeast that con erred resistance to rapamycin, and mTOR was subsequently discovered as its mammalian homologue. Rapamycin (also known as sirolimus ) binds to FKBP12, a member o the FK506-binding protein amily, and the rapamycin–FKBP12 complex binds to mTOR and inhibits its activity. In addition to its immunosuppressive properties, rapamycin promotes cell cycle inhibition, apoptosis, and angiogenesis inhibition by blocking translation o downstream targets o mTOR such as cyclin D1, c-MYC, the antiapoptotic protein BAD, and HIF-1 . A number o rapamycin derivatives are currently undergoing clinical testing in a wide variety o malignancies, including temsirolimus and everolimus . Both are soluble ester analogues o rapamycin that demonstrate dose-dependent inhibition o tumor cell growth in vitro. Temsirolimus is approved or treatment o renal cell carcinoma and has shown activity in breast cancer and mantle cell non-Hodgkin’s lymphoma.

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Everolimus is approved or treatment o renal cell carcinoma, breast cancer, and pancreatic cancer. Toxicities include skin rash, mucositis, thrombocytopenia, and leukopenia. It is likely that particular subsets o patients will benef t rom mTOR inhibitors, and uture clinical trials will be designed accordingly. For example, in renal cell carcinoma, activation o HIF-1 due to loss o VHL expression has been shown to sensitize cells to mTOR inhibition and may explain the clinical activity o temsirolimus in a subset o patients. Studies o patients with bladder cancer who show a dramatic response to temsirolimus therapy have led to the identif cation o activating mutations in mTOR, which may, in uture studies, identi y a population o patients predicted to benef t rom this drug. Everolimus treatment is e ective in patients with subependymal giant cell astrocytomas due to TSC loss, leading to approval o the drug or this indication. Furthermore, because particular pathways such as estrogen receptor or EGFR signaling are dependent on PI3K/AKT/ mTOR signaling (Fig. 40-2B), combination therapies using estrogen receptor or EGFR antagonists and mTOR inhibitors are being explored. A variety o PI3K and AKT inhibitors are in clinical development. In addition, because rapamycin derivatives inhibit only a portion o mTOR unction, and result in eedback activation o AKT signaling, second-generation mTOR inhibitors have been developed and are in clinical trials. These agents are competitive inhibitors o mTOR that bind to the active site o the enzyme. Moreover, since eedback activation o PI3K/AKT signaling typically involves engagement o IGF1R, strategies to target this receptor directly are also undergoing clinical testing.

Proteasome Inhibitors In light o the importance o ubiquitin-mediated proteasomal degradation in regulating the cell cycle, apoptosis, and other processes involved in neoplastic trans ormation, proteasome inhibitors have been tested in vitro and in vivo or antitumor e ects. The small molecule bortezomib, a dipeptide with a linked boronate moiety, targets with high a f nity and specif city an active-site N-terminal threonine residue within the 20S catalytic subunit o the proteasome (Fig. 40-4A). Bortezomib induces growth inhibition and apoptosis o tumor cells with relatively ew toxic e ects on normal cells. Clinically, the e ects o bortezomib are reversible, requiring intravenous dosing on a twice-weekly schedule. Bortezomib has demonstrated considerable e f cacy in clinical trials involving patients with multiple myeloma. Principal adverse e ects include neuropathy, thrombocytopenia, and neutropenia. Given its relatively modest adverse e ect prof le, bortezomib has also been incorporated into combination regimens or primary therapy o multiple myeloma, with some o the highest response rates seen to date in this disease. In addition, bortezomib is being tested alone and in combination with standard chemotherapy in a wide variety o other malignancies. Several mechanisms have been proposed to explain the e f cacy o bortezomib in multiple myeloma. One mechanism involves inhibition o NF B through stabilization o I B (Fig. 40-5B). Since NF B activates the transcription o genes promoting cell proli eration and blocking apoptosis in response to in ammation and other stimuli, antagonism o these actions by bortezomib would be expected to lead to

762 PRINCIPLES OF CHEMOTHERAPY

growth inhibition and apoptosis. A second proposed mechanism involves accumulation o mis olded proteins, leading to cell death. Like the plasma cells rom which they arise, multiple myeloma cells synthesize large amounts o immunoglobulin. The proteasome may play an important role in degrading mis olded proteins in these cells, and inhibition o proteasome unction by bortezomib could be lethal in this setting. It has also been proposed that bortezomib can cause stabilization o CDK inhibitors and o p53. Indeed, mutation o p53 is associated with resistance to bortezomib. A second mechanism o bortezomib resistance involves increased expression o heat shock protein-27 (HSP-27), and studies designed at inhibiting heat shock proteins are underway both to overcome bortezomib resistance and to enhance its e f cacy. Carf lzomib is a second-generation proteasome inhibitor that exhibits stronger and irreversible binding to the proteasome and can overcome bortezomib resistance. This drug and other second-generation proteasome inhibitors in development may urther advance the treatment o multiple myeloma and other cancers.

Angiogenesis Inhibitors Recognition o the primary role o VEGF and its receptors in the regulation o angiogenesis has led to strategies to block VEGF unction as a means o disrupting tumor vasculature. The most success ul approaches to date have included the development o neutralizing antibodies against VEGF or VEGFR and small-molecule inhibitors o the VEGFR tyrosine kinase domain.

Anti-VEGF and Anti-VEGFR Antibodies

Bevacizumab is a recombinant humanized mouse IgG1

monoclonal antibody directed against VEGF-A, one o the major proangiogenic VEGF amily members (Table 40-2). In mouse models, blocking VEGF with a monoclonal antibody inhibits angiogenesis and growth o human tumor xenogra ts. Early clinical studies were designed to test the e f cacy o bevacizumab in metastatic renal cell carcinoma, because most o these cancers overexpress VEGF as a result o loss o VHL expression and consequent HIF-1 activation. Incorporation o bevacizumab into standard chemotherapy regimens has yielded success in several tumor types. Addition o bevacizumab to chemotherapy or metastatic colon cancer has shown signif cant improvements in response rates and in survival. Improvement in survival has also resulted rom the addition o bevacizumab to carboplatin and paclitaxel or the treatment o metastatic NSCLC, although patients with cerebral metastasis, squamous cell tumor histology, and central tumors were excluded rom these studies because intratumoral bleeding could lead to potentially atal cerebral hemorrhage or severe hemoptysis. Signif cant activity has also been seen in metastatic renal cell carcinoma and glioblastoma, and bevacizumab is also approved or use in these settings. The potentiation o cytotoxic chemotherapy by bevacizumab and its modest activity as a single agent suggest that its mechanism o action may not be as simple as induction o tumor hypoxia and starvation o nutrients. Activation o VEGFR signaling increases vascular permeability, resulting in high interstitial uid pressures in tumors. This high interstitial uid pressure is postulated to prevent optimal delivery o chemotherapy to the tumor. Indeed, inhibition o VEGF with bevacizumab has been shown to decrease vascular

permeability, reduce interstitial uid pressure, and improve drug delivery to tumors. Adverse e ects o bevacizumab include proteinuria, hypertension, risk o thrombosis or bleeding, risk o gastrointestinal per oration, and impairment o wound healing. Ramucirumab, a recombinant IgG1 monoclonal antibody that binds VEGFR-2, shares a similar adverse e ect prof le, consistent with on-target toxicity related to inhibition o this signaling axis. Ramucirumab has recently been approved by the FDA or the treatment o metastatic gastric cancer and gastroesophageal junction adenocarcinoma.

VEGFR Inhibitors

Other strategies to inhibit VEGF signaling have included the development o small-molecule inhibitors o VEGFR tyrosine kinase activity. The small-molecule inhibitors o VEGFR are o special interest because these agents inhibit multiple receptor tyrosine kinases (Table 40-3). For example, vandetanib inhibits VEGFR-1, VEGFR-2, VEGFR-3, RET, and EGFR. RET is the oncogene that predisposes to multiple endocrine neoplasia (MEN) type 2 disease and medullary thyroid cancer, and vandetanib has been approved by the FDA or the latter indication. Cabozantinib, an inhibitor o VEGFR, C-KIT, RET, FLT3, and MET, among other kinases, is also e ective in the treatment o metastatic medullary thyroid cancer. The treatment o clear cell renal cell carcinoma provides another example o how the broad activity o these agents could be utilized. Loss o VHL expression and activation o HIF-1 result in expression o VEGF, PDGF- , and TGFin a substantial raction o these tumors, and inhibition o VEGF alone with bevacizumab has yielded only modest benef t in patients with metastatic renal carcinoma. More signif cant activity has been seen with the receptor tyrosine kinase inhibitors sunitinib, which inhibits VEGFRs, PDGFR, C-KIT, RET, and FLT3, and sora enib, which inhibits not only B-RAF but also VEGFRs, PDGFR, C-KIT, RET, and FLT3. Pazopanib and axitinib are two other multitargeted VEGFR TKIs that have activity in this setting. Given the

TABLE 40-3 Examples o Vascular Endothelial Growth Factor Receptor Inhibitors VEGFR TYROSINE KINASE INHIBITORS

TARGETS

Sunitinib

VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, C-KIT, RET, FLT3, CSF-1R

Sora enib

VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, C-KIT, RET, FLT3, B-RAF

Pazopanib

VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, C-KIT, FGFR-1, FGFR-3, Itk, Lck, c-Fms

Axitinib

VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, C-KIT

Cabozantinib

VEGFR-1, VEGFR-2, VEGFR-3, C-KIT, RET, FLT3, MET

Vandetanib

VEGFR-1, VEGFR-2, VEGFR-3, RET, EGFR

VEGFR, vascular endothelial growth actor receptor; PDGFR, platelet-derived growth actor receptor; FLT3, FMS-like tyrosine kinase; FGFR, f broblast growth actor receptor; Itk, interleukin-2 receptor inducible T-cell kinase; EGFR, epidermal growth actor receptor.

C h a p t e r 4 0 Pharmacology of Cancer: Signal Transduction

re ractory nature o renal cell carcinoma to traditional chemotherapy, the development and use o these new agents, based on a deeper understanding o the tumor cell biology, represents a major advance in the treatment o this tumor. Sunitinib, sora enib, and other multitargeted VEGFR inhibitors also have activity in a variety o other solid tumors. For example, sunitinib is e ective in the treatment o GISTs that are re ractory to imatinib, and sora enib is utilized or therapy o hepatocellular carcinoma. Regora enib, another broad-spectrum inhibitor o VEGFR and other receptor tyrosine kinases, is approved or use in metastatic colorectal cancer.

Thalidomide and Lenalidomide

Thalidomide is a synthetic glutamic acid derivative that was

ound to have sedative and antiemetic properties and was marketed outside the United States during the mid-1950s as a treatment or morning sickness in pregnant women. Tragically, thalidomide was discovered to be teratogenic, causing severe developmental de ormities including stunted limb development (phocomelia). Thalidomide was subsequently shown to have immunomodulatory properties, inhibiting the synthesis o TNF- and demonstrating e f cacy in the treatment o erythema nodosum leprosum (ENL). In addition, it was hypothesized that the abnormal limb development caused by thalidomide was due to antiangiogenic properties, and indeed, it has since been shown that thalidomide inhibits basic f broblast growth actor (bFGF)-induced angiogenesis. Thalidomide has also been shown to costimulate T cells. Given its combination o properties, thalidomide is now termed an immunomodulatory drug (IMiD). Because increased microvascular density in the bone marrow is associated with poor outcomes in multiple myeloma, thalidomide was initially tested in patients with advanced disease and ound to have signif cant clinical activity. Currently, the combination o thalidomide and dexamethasone is a standard f rst-line regimen or patients with multiple myeloma. Principal adverse e ects include risk o thrombosis, neuropathy, constipation, and somnolence. Although thalidomide was initially evaluated or the treatment o multiple myeloma due to its immunomodulatory and antiangiogenic properties, the precise mechanism o its anticancer activity has remained unclear. Lenalidomide is a synthetic second-generation IMiD analogue o thalidomide. While maintaining the antiangiogenic activity o thalidomide, lenalidomide also exhibits enhanced inhibition o TNF- and costimulation o T cells as well as direct antitumor activity with induction o apoptosis. Lenalidomide shows activity even in thalidomide-re ractory multiple myeloma and, when combined with bortezomib and dexamethasone, yields very high response rates in the primary treatment o multiple myeloma. The incidence o thrombosis with lenalidomide is markedly reduced compared to that with thalidomide, and lenalidomide causes less neuropathy, constipation, and somnolence as well. Lenalidomide also shows signif cant activity in the treatment o myelodysplastic syndromes, principally in patients with a deletion o the long arm o chromosome 5 (del 5q) or with normal cytogenetics. The main adverse e ects o lenalidomide are myelosuppression and thrombocytopenia. The molecular mechanism o lenalidomide action was recently identif ed. Lenalidomide binds to cereblon, a multisubunit E3 ubiquitin ligase (Fig. 40-4). The binding

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o lenalidomide to cereblon directs its activity to the B-cell transcription actors Ikaros amily zinc f nger proteins 1 and 3 (IKZF1 and IKZF3), resulting in their proteasomal degradation. This discovery not only illuminates the particular e f cacy o this class o drugs in multiple myeloma but may also lead to the development o third-generation inhibitors with even more specif c activity and therapeutic benef t in myeloma or other diseases.

Tumor-Specif c Monoclonal Antibodies Most hematologic malignancies express specif c cell surace markers that have been used to subclassi y the malignancies by immunohistochemistry and ow cytometry. The development o chimeric monoclonal antibodies against several o these antigens has provided the opportunity or targeted antibody therapy in a number o these disorders (Table 54-1). Although the mechanism o action o monoclonal antibodies is incompletely understood, it is likely related to the induction o antibody-dependent cell-mediated cytotoxicity and apoptosis. For example, B-cell lymphomas characteristically express the CD20 cell sur ace antigen, which is normally ound almost exclusively on mature B cells. The anti-CD20 IgG1 monoclonal antibody rituximab has demonstrated signif cant single-agent activity and enhancement o the e ects o chemotherapy in B-cell non-Hodgkin’s lymphoma (NHL) and is now routinely used in the therapy o this disorder. Principal adverse e ects include immunosuppression due to the targeting o normal mature B cells and hypersensitivity reactions related to the chimeric nature o the antibody. O atumumab is another humanized anti-CD20 antibody that binds CD20 with higher a f nity than rituximab and with a slower dissociation rate; this agent has shown e f cacy in re ractory CLL. Alemtuzumab is a humanized monoclonal antibody directed against the pan-leukocyte antigen CD52. This agent has been used in the treatment o CLL and as a component o conditioning regimens or stem cell transplantation. Because alemtuzumab induces lysis o both T-cell and B-cell populations, its principal adverse e ect is signif cant immunosuppression, including increased risk or Pneumocystis jiroveci pneumonia and or ungal, cytomegalovirus, and herpesvirus in ections. There ore, prophylaxis or opportunistic in ections is required. Conjugation o radioactive isotopes to anti-CD20 antibodies, such as iodine-131 (131I) tositumomab and yttrium-90 (90Y) ibritumomab tiuxetan, has allowed targeted radioimmunotherapy o B-cell NHL. Ibritumomab in particular has been incorporated into treatment regimens or patients with re ractory disease. In addition to the a orementioned trastuzumab emtansine, two urther examples o antibody–toxin conjugates are denileukin di titox and gemtuzumab ozogamicin. Denileukin di titox, a recombinant usion protein composed o ragments o diphtheria toxin and human IL-2, targets the CD25 component o the IL-2 receptor and has demonstrated activity in T-cell NHL. Gemtuzumab ozogamicin is a conjugate between the antitumor antibiotic calicheamicin and a monoclonal antibody directed against CD33, which is expressed on the sur ace o leukemic blasts in more than 80% o patients with AML.

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CONCLUSION AND FUTURE DIRECTIONS Elucidation o the molecular and biochemical circuitry that regulates normal cell proli eration and identif cation o the key mutations that promote oncogenesis have provided the ability to target specif c pathways that are dysregulated in tumors. The success o imatinib in the treatment o CML demonstrates that cancers can become dependent on oncogenes such as BCR-Abl, requiring oncoprotein signaling or continued proli eration and survival. Although inhibitors o receptor tyrosine kinases and intracellular kinases have a higher therapeutic index than traditional antineoplastic therapies and have had some success in certain tumors, responses are neither durable nor complete in many cases. The identif cation o subsets o tumors in which specif c pathways are activated, such as the EFGR mutation in NSCLC, will guide therapy and improve response rates. Oncogenic microarray signatures and correlations between specif c mutations and sensitivity to targeted agents will acilitate the design o clinical trials ocusing on subsets o patients with the highest likelihood o response. E f cacy will also be improved with second- and third-generation drugs that have higher selectivity or targets and the ability to overcome resistance mutations. It is clear, however, that multiple actors contribute to tumor development, including downstream mutations in pathways regulating cell cycle progression, apoptosis, proteasomal degradation, and angiogenesis. The biology o these processes and o tumor cell invasion and acquisition o metastatic potential will likely provide novel targets or directed therapy. As with combination chemotherapy, the success ul targeted therapies o the uture will likely involve inhibition o multiple pathways using a combination o agents directed at the de ects ound in individual tumors. Furthermore, systematic approaches involving RNA inter erence and other genetic

or chemical screens may identi y previously unanticipated vulnerabilities associated with specif c cancer genotypes, a concept derived rom “synthetic lethal” screening in yeast. The improved selectivity inherent in such strategies will likely give them a superior therapeutic index compared to traditional combination antineoplastic chemotherapy and will hope ully be met with greater clinical success.

Suggested Reading Bartlett JB, Dredge K, Dalgleish AG. The evolution o thalidomide and its IMiD derivatives as anticancer agents. Nat Rev Cancer 2004;4:314–322. (Historic and scientif c overview o thalidomide and its derivatives.) Hanahan D, Weinberg RA. Hallmarks o cancer: the next generation. Cell 2011;144:646–674. (Seminal overview o the characteristic genetic changes leading to oncogenesis.) Kaelin WG Jr. The concept o synthetic lethality in the context o anticancer therapy. Nat Rev Cancer 2005;5:689–698. (Novel approaches to cancer genotype-guided drug development.) Krause DS, van Etten RA. Tyrosine kinases as targets or cancer therapy. N Engl J Med 2005;353:172–187. (Overview o tyrosine kinase inhibition in cancer therapy.) Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012;149:274–293. (Reviews the role o the mTOR pathway in health, disease, and aging, with a ocus on treatments targeting this pathway.) Lu G, Middleton RE, Sun H, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction o Ikaros proteins. Science 2014;343:305–309. (Demonstrates the molecular mechanism o action o lenalidomide.) Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 2005;23:4776–4789. (Biochemical details o ubiquitin pathways.) Shaw AT, Hsu PP, Awad MM, Engelman JA. Tyrosine kinase gene rearrangements in epithelial malignancies. Nat Rev Cancer 2013;13:772–787. (Reviews the etiology, pathogenesis, clinical eatures, and targeted treatments or epithelial cancers with ALK, ROS1, and RET mutations.) Tan CS, Gilligan D, Pacey S. Treatment approaches or EGFR-inhibitorresistant patients with non-small-cell lung cancer. Lancet Oncol 2015;16: e447–459. (Overview o EGFR pathways and treatments in non-small cell lung cancer.)

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C h a p t e r 4 0 Pharmacology o Cancer: Signal Transduction 769

41

A0 Ann ta gonis tic

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Principles of Combination Chemotherapy M

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Q u e n t in J . Ba c a , Do n a ld M . Co e n , a n d David E. Go la n S ynn e rg is ticc INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770–771 INT 0Y. . . . . . . . . . . . . . . . 770 ANTIMICROBIAL COMBINA ATION THER RAP APY 0 M nimum Inhibitory Connc e nt Mi ntra rati tion on and Minimum Bact Ba cter eric icid ic iddall Con oncentrration . . . . . . . . . . . . . . . . . . . . . . . . . . 770 Types of Drug Interactionns—S Syner ynergy gy, Ad Addi d tivity y, andd An an Anta tago goni nism sm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772 7772 Examples of Antimicrobial Comb mbin inat atio ionn Th Thee rapy. . . . . . . . . . 773 Tube Tu berc rcul ulos osis iss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773 Synergistic Combinations. . . . . . . . . . . . . . . . . . . . . . . . . . . . 774 Co-administration of Penicillins with -Lactamase Inhibitorss . . . . . . . . . . . . . . . . . . . . . . . . . . . 775 Polymicrobial and Life-Threatening Infectionss . . . . . . . . . 775 Unfavorable Drug Combinations . . . . . . . . . . . . . . . . . . . . . . . . 775

INTRODUCTION Many in ections and some cancers can be successully treated with single-drug therapies. Such regimens o ten ail, however, when pathogens or tumors develop resistance to chemotherapeutic agents, when multiple pathogens with di erent drug susceptibilities are simultaneously present, or when the dose o the therapeutic agent is limited by toxicity. Under these circumstances, combination chemotherapy may o er decisive advantages. The drugs in a multidrug regimen can interact synergistically to enhance the antimicrobial or antineoplastic e ectiveness o the combination and can decrease the likelihood that resistance will emerge. Combinations are requently used when treatment must be initiated be ore the def nitive identif cation o the pathogen, and synergistic combinations can be used to reduce toxicity when the individual drugs in the combination have low therapeutic indices. Although combination chemotherapy opens new avenues or the expedient elimination o a pathogen or tumor, it also introduces the potential or multiple adverse e ects and drug interactions. The goal o any combination drug regimen should be to e f ciently remove the o ending pathogen or tumor without incurring unacceptable host toxicity.

770

ANTIVIRAL COMBINATION MBINATION THERAPY THER ERAP AP PY . . . . . . . . . . . . . . . . . . . . . 775 7766 ANTINEOPLASTIC OPLASTIC COMBINA COMBINATION NC CHEMO HEMOTHERAPY. . . . . . . . 77 General Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . B ..0 . . 77 7766 Rationale for Combinatio ion Ch Chem mot othe hera rapy py . . . . . . . . . . . . . . . . 779 Exam Ex ampl p es of Antineoplastic Combination Chemotherapy . . . 779 Hodg Ho Hodgkin’s dgkin s Diseasee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779 Testicular Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780 Treatment of Refractory or Recurrent Disease . . . . . . . . . . . . 780 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 781 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .781

MIIC of MIC of D Drug ru rug ug gB

ANTIMICROBIALCOMBINATION THERAPY In the treatment o microbial in ections, drug combinations are used (1) to prevent the emergence o drug resistance, (2) to enhance the activity (e f cacy) o the drug therapy against a specif c in ection (synergy), (3) to reduce toxicity to the host, (4) to treat multiple simultaneous in ections (sometimes called polymicrobial infections), and (5) to treat a li e-threatening in ection empirically be ore the microorganism causing the in ection has been identif ed. Because microbes are genetically distant rom humans, antimicrobial drug combinations can target several di erent molecules that are specif c to the microbe(s), potentially without a concomitant increase in adverse e ects. In contrast, antineoplastic drug combinations are o ten limited by adverse e ects (see below). The ollowing section provides a conceptual ramework or the di erent types o antimicrobial drug interactions and discusses specif c examples o antimicrobial combination therapy.

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Antimicrobial agents with activity against a pathogenic bacterial, protozoal, or ungal microorganism can be

C h a p t e r 4 1 Principles o Combination Chemotherapy 771

Mr. M is a 27-ye a r-o ld m a n fro m ru ra l Ha iti w h o p re s e n ts to a clin ic w ith a ch ro n ic co u g h . He ca n n o t a ffo rd tre a tm e n t a t a p riva te clin ic, s o h e g o e s to a d ru g s to re a n d a s ks th e p h a rm a cis t fo r a p p ro p ria te m e d ica tio n . Th e p h a rm a cis t th in ks th a t Mr. M co u ld h a ve tu b e rcu lo s is , a n d h e s e lls Mr. M a 2-w e e k s u p p ly o f is o n ia zid a n d rifa m p in . Mr. M ta ke s b o th d ru g s fo r a co u p le o f d a ys , b u t th e y m a ke h im n a u s e a te d , a n d h e d e cid e s to ta ke ju s t th e is o n ia zid fo r 2 w e e ks . His s ym p to m s re s o lve . Th re e m o n th s la te r, Mr. M’s co u g h re tu rn s . Th is tim e , h e n o tice s b lo o d in h is s p u tu m a n d h e h a s n ig h t s w e a ts . He ta ke s th e re m a in d e r o f th e 2-w e e k s u p p ly o f rifa m p in a n d e xp e rie n ce s a b rie f lu ll in h is s ym p to m s . With in a fe w d a ys , h o w e ve r, h is co u g h , b lo o d y s p u tu m , a n d n ig h t s w e a ts re tu rn . Be ca u s e h e d o e s n o t h a ve e n o u g h m o n e y to b u y a d d itio n a l d ru g s , h e tra ve ls to th e n e a re s t g ove rn m e n t h o s p ita l to s e e k fre e ca re a n d m e d ica tio n s . Th e g ove rn m e n t d o cto r ta ke s th re e s p u tu m s a m p le s , a ll o f w h ich a re p o s itive fo r a cid -fa s t b a cilli. Th e d o cto r a ls o s e n d s s p u tu m to th e la b o ra to ry fo r cu ltu re , b u t s in ce Myco b a cte riu m tu b e rcu lo s is (th e ca u s a tive a g e n t o f

characterized by the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) or the drug–pathogen pair. The MIC is def ned as the lowest concentration o drug that inhibits growth o a culture o the microorganism a ter 18–24 hours o incubation in vitro. The MBC is def ned as the lowest concentration o drug that kills 99.9% o a culture o the microorganism a ter 18–24 hours o incubation in vitro. In general, the MBC is higher than the MIC. Comparisons between the MICs or MBCs and the clinically achievable concentrations o antimicrobial drugs allow these drugs to be grouped broadly into two categories: cidal and static (Table 41-1; also see Chapter 33, Principles o Antimicrobial and Antineoplastic Pharmacology). An antimicrobial agent is static (e.g., bacteriostatic, ungistatic) i its MIC is within the therapeutic range o the drug but

TABLE 41-1 Examples of Bactericidal and Bacteriostatic Antibiotics BACTERICIDAL ANTIBIOTICS BACTERIOSTATIC ANTIBIOTICS

CONCENTRATIONDEPENDENT

Chloramphenicol Clindamycin Ethambutol Macrolides Sul onamides Tetracyclines Trimethoprim

Aminoglycosides Bacitracin Quinolones

TIME-DEPENDENT -Lactams Isoniazid Metronidazole Pyrazinamide Ri ampin Vancomycin

tu b e rcu lo s is ) is s lo w-g ro w in g , s h e a ls o s ta rts Mr. M o n a m u ltid ru g re g im e n co n s is tin g o f is o n ia zid , rifa m p in , p yra zin a m id e , a n d e th a m b u to l fo r 2 m o n th s , fo llo w e d b y is o n ia zid a n d rifa m p in fo r 4 m o n th s . Several weeks later, the culture reveals that Mr. M’s tu b e rcu lo s is is n o t s u s ce p tib le to e ith e r is o n ia zid o r rifam pin. Mr. M’s doctor is now s eeking a new recom m e nda tio n for trea tm e nt.

Questions 1 . Why did the government doctor prescribe our di erent drugs or Mr. M? 2 . How is resistance trans erred rom one generation o tubercle bacilli to the next? How does this resistancetrans er mechanism compare to the mechanism by which penicillin resistance is trans erred? 3 . Why were Mr. M’s initial e orts at treatment unsuccess ul? What treatment strategy could have been employed to avoid Mr. M’s treatment ailure? 4 . Does Mr. M have multidrug-resistant tuberculosis (MDR-TB)? Should he stay on the our-drug regimen that includes isoniazid and ri ampin? I not, how should his treatment be modif ed?

its MBC is not. The agent is cidal (e.g., bactericidal, ungicidal) i its MBC is within the therapeutic range o the drug. Note that the MIC and MBC re er to a specif c drug– microbe pair under a specif c set o conditions. Many drugs with activity against an organism are static in one growth medium but cidal in another or are cidal at su f ciently high concentrations in vitro. Furthermore, or any particular drug, the MIC and MBC may di er rom one microbe to the next. Indeed, a drug may be static against one organism and cidal against another. As an operational def nition, we can state that, at therapeutic concentrations, cidal drugs kill the microorganism, while static drugs merely arrest microbial growth. In this def nition, the therapeutic concentration re ers to plasma drug levels that are su f cient or pharmacologic activity (here, killing or arresting the growth o the microorganism) without unacceptable toxicity to the patient. For example, most inhibitors o bacterial cell wall synthesis are bactericidal, whereas most inhibitors o bacterial protein synthesis are bacteriostatic (see Chapter 34, Pharmacology o Bacterial In ections: DNA Replication, Transcription, and Translation, and Chapter 35, Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis). As noted in Chapter 33, an important distinction between static and cidal drugs lies in their clinical applications. In general, the successful use of static drugs to treat infections requires an intact host immune system. This is because static drugs do not kill microorganisms but only prevent them rom multiplying. Accordingly, such drugs rely on the host’s immune and in ammatory mechanisms to e ect clearance o organisms rom the body. These drugs are more e f cacious when initiated early in the course o an in ection (i.e., when the in ectious burden is lower). Furthermore, removal o a

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concentration is higher than the minimum bactericidal concentration (MBC). Thus, the overriding consideration or the clinical use o such agents is not the absolute drug concentration that is achieved, but the duration o time during which the drug concentration remains in the therapeutic range (which is def ned as [drug] MBC). In contrast, concentration-dependent bactericidal agents exhibit a rate o killing that increases with drug concentration or [drug] MBC. For such agents, a single very large dose may be su f cient to eliminate the in ection.

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Time FIGURE 41-1. Comparison of the effects of bacteriostatic and bactericidal drugs on bacterial growth kinetics in vitro. In the absence o drug, bacteria grow with exponential (f rst-order) kinetics. A bactericidal drug kills the target organism, as demonstrated by the time-dependent decrease in the number o live bacteria. A bacteriostatic drug prevents microbial growth without killing the bacteria. Removal o a bacteriostatic drug is ollowed by an exponential increase in bacterial number as the previously inhibited bacteria resume growth. Bacteriostatic drugs eradicate in ections by limiting the growth o the in ecting organism or a long enough period o time to allow the host immune system to kill the bacteria.

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static drug be ore the immune system has completely cleared the in ection can result in resumption o microbial growth and reappearance o in ection (Fig. 41-1). According to their mechanism o cell killing, bactericidal agents can be urther characterized as time-dependent or concentration-dependent (Fig. 41-2). Time-dependent bactericidal agents exhibit a constant rate o killing that is independent o drug concentration, provided that the drug

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Drug conce ntra tion FIGURE 41-2. Relationship between rate of microbial killing and drug concentration for time-dependent and concentration-dependent bactericidal drugs. Time-dependent bactericidal agents exhibit a constant rate o microbial killing at concentrations o drug greater than the minimum bactericidal concentration (MBC) (solid line). In contrast, concentration-dependent bactericidal agents exhibit increased killing with increasing drug concentration (dashed line). Note that the e f cacy o concentration-dependent bactericidal agents eventually plateaus because the e ective concentration o the drug becomes limited by the rate o drug di usion to its molecular target.

Types of Drug Interactions—Synergy, Additivity, and Antagonism The discussion has thus ar considered the general properties o drugs used as single agents to treat a microbial in ection. When such drugs are used in combination with other agents, these e ects can be modif ed (either enhanced or diminished). In act, some drugs that have little or no activity against an organism when used as single agents can show high activity when used in combination with another agent. One example o this concept involves the treatment o Enterococcus faecalis, a Gram-positive organism that exhibits little susceptibility to aminoglycosides . Recall that aminoglycosides are thought to kill bacteria by inducing misreading o the genetic code and translation o de ective proteins, which cause urther cellular damage (see Chapter 34). In the case o E. faecalis, aminoglycosides are unable to penetrate the organism’s thick cell wall to reach their target, the 30S ribosomal subunit. However, when used in combination with a cell wall synthesis inhibitor such as vancomycin or a -lactam antibiotic, aminoglycosides are able to reach the bacterial ribosomes and e ectively kill the bacteria (see Chapter 35). The potentiating e ect o the cell wall synthesis inhibitor on the activity o the aminoglycoside is an example o the important pharmacologic concept o synergy. From this example, one could ask whether combining two drugs with individual activity against a particular microbe always results in a more e f cacious drug combination. Surprisingly, or many combinations, this turns out not to be the case. In act, when two drugs with activity against the same pathogen are combined, the drugs can interact to diminish the e f cacy o the combination relative to each alone (antagonism). Alternatively, the drugs may not interact, and the e ect o the combination is simply the sum o the e ects o each drug used individually (additivity). The interaction between two antimicrobial drugs is o ten quantif ed by selecting a particular endpoint (e.g., inhibition o bacterial growth) and then measuring the e ect o various combinations o the two drugs that reach this endpoint. When such data are plotted, additional in ormation can be obtained (Fig. 41-3). The x- and y-intercepts correspond to the MICs o each o the two drugs, and the concavity o the curve indicates the nature o the interaction between the two drugs—concave-up is synergistic; concave-down is antagonistic; linear is additive. The ollowing discussion provides a mathematical rationale or these relationships. Suppose that Drugs A and B inhibit a particular enzyme required or bacterial growth. In this case, the ratio [A]/ MICA would represent the raction o bacterial growth inhibition that can be attributed to the presence o Drug A. This is known as the fractional inhibitory concentration of A (FICA). Similarly, FICB [B]/MICB is the raction

C h a p t e r 4 1 Principles o Combination Chemotherapy 773

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MIC of Drug B FIGURE 41-3. Quantif cation o additive, synergistic, and antagonistic drug interactions. Drug combinations can exhibit additive, synergistic, or antagonistic e ects. The nature o this interaction can be depicted graphically by observing the e ect o each drug on the other’s minimum inhibitory concentration (MIC). I two drugs have an additive interaction, then the addition o increasing amounts o Drug B to Drug A results in a linear decrease in the MIC o Drug A; in this case, each o the two drugs can be thought o as interchangeable. I two drugs have a synergistic interaction, then the addition o Drug B to Drug A results in a signif cantly lower MIC or Drug A (i.e., there is an increase in the potency o Drug A). I two drugs have an antagonistic interaction, then the addition o Drug B to Drug A does not signif cantly lower the MIC o Drug A; in some cases (not shown), higher doses o each drug must be administered to achieve the same e ect as that o each drug used alone. A0 and B0 are the MICs o Drugs A and B, respectively, when used as single agents.

o growth inhibition that can be attributed to Drug B. Now, suppose that the concentration o A is decreased by a small amount, d[A]. To compensate or this loss o growth inhid[A]/MICA), the concentration o B must bition (dFICA be increased by an amount d[B]. For additive drugs, the ratio d[A]/d[B] (which is the same as the slope o the curve in Fig. 41-3) is a constant because one unit o A has exactly the same activity as (MICA/MICB) units o B. For example, A and B could bind to independent sites on the enzyme (i.e., each drug has no e ect on the binding o the other drug). In contrast, i A and B are synergistic, then the amount o B (d[B]) required to compensate or a decrease in A ( d[A]) depends on the amount o A that is already present. Because o the potentiating e ect o Drug A on Drug B, d[B] is smaller or higher [A] (i.e., d2[A]/d[B]2 0, which corresponds to the concave-up curve in Fig. 41-3). For example, the binding o A could induce a con ormational change in the enzyme that enhances the binding o B. By extension, A and B are antagonistic i the amount o B required to compensate or a small decrease in the concentration o A is larger or higher [A] (i.e., d2[A]/d[B]2 0, which corresponds to the concave-down curve in Fig. 41-3). For example, the binding o A could induce a con ormational change in the enzyme that reduces the binding o B. Because o its intuitiveness and simplicity, the mathematical model described above is o ten used to def ne synergy, additivity, and antagonism. However, the experimental determination and quantitative analysis o the e ects o

multiple drugs are complex topics that are beyond the scope o this text. The interested reader is re erred to the work o TC Chou (1984, 2006) or an extensive treatment o this subject. Several generalizations can be made concerning the nature o drug interactions between di erent classes o antimicrobial agents. First, many bacteriostatic drugs (e.g., tetracycline , erythromycin, chloramphenicol) antagonize the action o bactericidal drugs (e.g., vancomycin, penicillin) by inhibiting cell growth and/or preventing the cellular processes that are required or cidal drugs to act (described below in more detail). Second, two bactericidal drugs usually act synergistically in combination. One notable exception to the latter generalization is that ri ampin, a bactericidal inhibitor o RNA polymerase, antagonizes other bactericidal drugs by inhibiting cell growth. Finally, the interactions between two bacteriostatic drugs are o ten additive but cannot be predicted in all cases.

Examples o Antimicrobial Combination Therapy Tuberculosis

The treatment o tuberculosis illustrates one o the principal reasons that combinations o drugs are used—to suppress the emergence o resistance. In the course o this illness, tuberculous bacilli (also called mycobacteria) are inhaled and phagocytosed by alveolar macrophages, in which the bacilli multiply within intracellular vacuoles. A predominantly T-cell–mediated lymphocytic response is elicited, and the macrophages and helper T cells orm large granulomas that wall o the in ected sites. Activated macrophages are usually able to keep the in ection under control by killing the multiplying bacilli but are un ortunately unable to eradicate the in ection completely. Tissue damage is caused by the release o neutral proteases and reactive oxygen intermediates rom activated macrophages, with the end result that central necrosis occurs in the tuberculous cavities in the lungs. Inside each o these cavities, as many as 108 to 109 living bacilli may be held in check by macrophages and helper T cells. Success ul cure o tuberculosis in ections typically requires the use o combinations o drugs with antimycobacterial activity. Commonly used drugs include isoniazid, ri ampin, pyrazinamide , and ethambutol (see Chapter 35). As illustrated in the case o Mr. M, a standard regimen could consist o 2 months o isoniazid, ri ampin, pyrazinamide, and ethambutol, ollowed by 4 months o isoniazid and ri ampin. Kanamycin and other second-line drugs are sometimes substituted or one or two drugs in this regimen i resistance develops. Isoniazid and ri ampin are o ten the pre erred drugs in such regimens because o their ability to kill intracellular as well as extracellular mycobacteria. As noted in Chapter 35, resistance to antimycobacterial drugs develops primarily through chromosomal mutations, and the requency o resistance to any one o the drugs is about 1 in 106 bacteria. These mutations are passed on to daughter cells when the bacteria replicate, leading to establishment o a drug-resistant population. Chapter 35 discusses the implications o the act that a tuberculous cavity contains 108 to 109 bacteria, while the requency o mutants resistant to a single drug is about 1 in 106. On average, then, 100 bacteria will already be resistant to each drug in any single lesion, even be ore that drug is administered. Moreover,

774 PRINCIPLES OF CHEMOTHERAPY

treatment with only one drug would result in selection or bacilli that are resistant to that drug. In the case o Mr. M, his initial 2 weeks o isoniazid treatment likely killed all the isoniazid-susceptible bacilli in his cavity. This accounts or the cessation in his symptoms a ter 2 weeks o treatment. However, the 100 or so isoniazid-resistant bacilli that were selected or by Mr. M’s use o monotherapy remained alive and multiplied. I he had taken ri ampin as well as isoniazid, only 1 in 1012 bacilli would likely have been resistant to both drugs, and he might have eradicated the in ection. Over the 3 months during which Mr. M stopped taking isoniazid, the isoniazid-resistant bacilli remaining in his lungs multiplied, leading to a relapse in his symptoms. He then began taking ri ampin. O the 108 to 109 isoniazidresistant bacilli in each lesion, again there was a 1 in 106 probability that a bacillus had mutated to acquire ri ampin resistance. By taking ri ampin or 2 weeks, he killed all the ri ampin-susceptible bacilli but selected or ri ampinresistant organisms. He was there ore le t with bacilli that were both isoniazid-resistant and ri ampin-resistant—the phenotype o multidrug-resistant tuberculosis (MDR-TB). Mr. M will require a new drug regimen or treatment o MDR-TB. Ideally, the regimen should be constructed using drugs that have been shown to be e ective in susceptibility tests. Also, at least initially, drugs that are part o his current treatment plan should be avoided (i.e., pyrazinamide and ethambutol), because his bacilli may have developed resistance to these agents. Isoniazid and ri ampin should certainly be avoided. Treatment or MDR-TB should begin with at least our new drugs to which Mr. M’s TB isolate has proven susceptibility. Such regimens usually include daily dosing o a parenteral aminoglycoside (kanamycin or amikacin) or peptide antibiotic (capreomycin), together with a uoroquinolone (levof oxacin or moxif oxacin), or at least 4–6 months. Three to f ve oral drugs should be co-administered with the aminoglycoside and uoroquinolone or 18–24 months a ter the sputum culture converts to negative. Ethionamide and clo azimine are second-line drugs that could be included in the regimen. Newer drugs that could be included are linezolid, bedaquiline , and delamanid. Note that, as a whole, the second-line regimen will be signif cantly more toxic than the f rst-line regimen. I bacilli isolated rom Mr. M’s sputum prove to be susceptible to pyrazinamide and/or ethambutol, these agents could replace some o the more toxic drugs. Considering all the issues discussed above, MDR-TB is to be avoided at all costs. Patients with drug-susceptible tuberculosis require access to combination therapy as well as help in adhering to the combination therapy to avoid the emergence o drug-resistant bacilli. This rationale is the basis or DOTS (Directly Observed Therapy Short Course), the World Health Organization (WHO)-recommended strategy or tuberculosis treatment. DOTS is a public health program that has f ve components: (1) political commitment and resources or TB control, (2) the use o sputum-smear microscopy or accurate diagnosis o TB in ection, (3) a standardized 6- to 8-month treatment that is directly observed by a community health worker or at least the f rst 2 months, (4) a regular and uninterrupted supply o medicines, and (5) standardized recording and reporting o each patient’s treatment and progress to central authorities. When used in cases o drugsusceptible TB, DOTS has a remarkable cure rate and can prevent the development o resistance. As noted above, treatment o MDR-TB requires therapy that is more intensive,

more invasive, more toxic, and o longer duration than the standard DOTS regimen. The importance o controlling MDR-TB is highlighted by the growing prevalence o extensively drug-resistant TB (XDR-TB), which had been observed in all regions o the world by 2006. XDR-TB has extremely limited treatment options; by def nition, clinical isolates display the phenotype o MDR-TB (resistance to isoniazid and ri ampin) as well as resistance to uoroquinolones and to at least one o the three commonly used parenteral anti-TB drugs (capreomycin, kanamycin, and amikacin). Because the prevalence o XDRTB is generally higher in resource-poor countries and in populations that are co-in ected with human immunodef ciency virus (HIV), world health organizations have recently emphasized the need or a coordinated global response to limit the spread o XDR-TB. Treatment o XDR-TB requently involves the use o up to f ve drugs, guided by drug susceptibility testing.

Synergistic Combinations

A second reason or using a combination drug regimen is to take advantage o the synergy between the actions o the two drugs. This consideration is especially important in the setting o in ections that are not readily handled by the immune de enses o immunocompromised patients. In the immunocompetent patient, bacteriostatic and bactericidal drugs are o ten equally e f cacious in eliminating an in ection. Bactericidal drugs are strongly pre erred, however, in the setting o immunocompromised patients (e.g., HIV/AIDS patients, immunosuppressed transplant patients, and neutropenic cancer patients), endovascular in ection (e.g., bacterial endocarditis), or meningitis. The reason or using bactericidal combinations in the immunocompromised patient should be obvious—the host does not have su f cient numbers o unctioning lymphocytes and/or neutrophils to eliminate even a nondividing bacterial population. In the case o endocarditis, the reason is not so straight orward. In this case, although there is not a def ciency in the absolute number o leukocytes, the phagocytes are unable to e f ciently penetrate the thick “vegetation”—composed o a meshwork o f brin, platelets, and bacterial products—that surrounds the bacteria. Bactericidal drug combinations are sometimes indicated or meningitis to maximize the probability o overcoming the poor opsonization o bacteria by antibody and complement in the immunologically privileged site o the meninges, especially i the responsible organism is not known (see Chapter 9, Principles o Nervous System Physiology and Pharmacology). One example o antibacterial synergy involves the use o a penicillin and an aminoglycoside to treat a common cause o subacute bacterial endocarditis, Streptococcus viridans. As described above, the mechanism o synergy relies on the penicillin inhibiting cell wall biosynthesis, which allows the aminoglycoside to penetrate the thick peptidoglycan layer o this Gram-positive organism. Two other commonly used synergistic combinations include (1) the anti ungal combination o amphotericin B and f ucytosine and (2) the antibacterial and antiprotozoal combination o a sul onamide and trimethoprim or pyrimethamine . These classic examples illustrate two basic mechanisms whereby one drug can potentiate the activity o another. It is thought that, analogous to the action o penicillins, which enhance the uptake o aminoglycosides by Gram-positive

C h a p t e r 4 1 Principles of Combination Chemotherapy 775

bacteria, amphotericin B enhances ucytosine uptake by ungal cells by damaging ergosterol-rich ungal cell membranes (see Chapter 36, Pharmacology o Fungal In ections). Only a ter penetrating the ungal membrane can ucytosine be converted into its active orm (5- uorouracil, which is converted to 5-FdUMP, an irreversible inhibitor o thymidylate synthase) by a ungal-specif c deaminase. Amphotericin B has a low therapeutic index when used as a single agent (primarily as a consequence o its nephrotoxicity), but its synergistic e ect in combination with ucytosine allows a reduction in the dose o amphotericin B required to treat a systemic ungal in ection such as cryptococcal meningitis (with a corresponding reduction in toxicity). Sul amethoxazole and trimethoprim are commonly used in combination in the treatment o Pneumocystis jiroveci pneumonia, an opportunistic in ection requently encountered in patients with AIDS, as well as many urinary tract in ections caused by Gram-negative enteric organisms. An analogous combination, sul adoxine and pyrimethamine , is used in the treatment o malaria, toxoplasmosis, and other protozoal inections. These combinations illustrate a second mechanism whereby drugs can exert a synergistic e ect. The mechanism o synergy is based on the sul a drug’s inhibition o production o dihydro olate, which ordinarily competes with the second drug or binding to its target, dihydro olate reductase (see Chapter 33). The product o this enzyme, tetrahydro olate, is a required substrate or purine biosynthesis and or many one-carbon trans er reactions and is thus necessary or microbial DNA replication and cell division (see Fig. 33-7).

Co-administration of Penicillins with -Lactamase Inhibitors

The combination o a -lactam antibiotic and a -lactamase inhibitor (e.g., clavulanic acid, sulbactam, tazobactam) illustrates a mechanism o drug interaction that is not technically synergistic (because the -lactamase inhibitor has no antibacterial activity on its own) but that shares a unctional similarity with the drug combinations discussed above. Clavulanic acid is an inhibitor o -lactamase, an enzyme used by many -lactam-resistant Gram-positive and Gramnegative bacteria to inactivate penicillins (see Chapter 35). By preventing the hydrolysis and inactivation o penicillins, clavulanic acid (and other -lactamase inhibitors) greatly increases the potency o penicillins (and other -lactams) against bacteria that express -lactamase. This combination has been e ective in the treatment o in ections due to penicillin-resistant Streptococcus pneumoniae, which is a common cause o otitis media in in ants. Such organisms have typically acquired resistance to penicillins through a plasmid-encoded -lactamase.

Polymicrobial and Life-Threatening Infections

Combinations o antimicrobial drugs are used not only to prevent the emergence o resistance and to act synergistically against a specif c, known pathogen but also to treat polymicrobial in ections and in ections in which treatment must be initiated be ore the microbe causing the in ection is identif ed. Consider, or example, the case o a ruptured appendix or colonic diverticulum that has leaked bacteria into the peritoneal cavity and ormed an intra-abdominal abscess. Such an abscess is likely to contain a wide spectrum o microorganisms— much too broad to be targeted e ectively by a single antibiotic. A ter draining the abscess, treatment with a combination o

antibacterial agents such as a f uoroquinolone or -lactam—to kill aerobic Gram-negative Enterobacteriaceae (e.g., Escherichia coli)—and clindamycin or metronidazole —to kill anaerobes (e.g., Bacteroides fragilis; see Chapter 37, Pharmacology o Parasitic In ections)—o ten results in clearance o the in ection. (Note that it may sometimes be necessary to treat with antagonistic drug combinations in order to cover the spectrum o microorganisms that are likely to be present.) In cases where presumptive treatment is indicated be ore the causative microorganism is identif ed, body uids such as blood, sputum, urine, and cerebrospinal uid (CSF) should be submitted or culture be ore initiating therapy. A combination o drugs with activity against the microbes that are most likely to be involved in the in ection (or that could result in the most serious outcome) is then administered until a positive bacteriologic identif cation is made and drug susceptibility results are obtained. At that point, it may be possible to discontinue unnecessary drugs and implement specif c monotherapy.

Un avorable Drug Combinations As mentioned above, antagonistic drug combinations can sometimes be used in combination chemotherapy regimens, although this situation is to be avoided i possible. Antagonism is most commonly observed when static drugs are used in combination with cidal drugs. For example, tetracyclines are bacteriostatic antimicrobials that antagonize the bactericidal activity o penicillins (see Chapter 34). Recall that the bactericidal activity o penicillins depends on cell growth. By inhibiting the transpeptidation reaction involved in bacterial cell wall cross-linking, the penicillins create an imbalance between cell wall synthesis and autolysin-mediated cell wall degradation. I the bacterial cell continues to grow, this leads to spheroplast ormation and eventually to osmotic lysis. A protein synthesis inhibitor such as tetracycline, which arrests cell growth, would there ore antagonize the e ect o a -lactam. Similarly, imidazoles and triazoles are ungistatic agents that antagonize the ungicidal activity o amphotericin B (see Chapter 36). The mechanism o antagonism can be appreciated by noting that amphotericin B acts by binding ergosterol and orming pores in the ungal membrane, whereas imidazoles and triazoles inhibit a microsomal cytochrome P450-dependent enzyme, 14 -sterol demethylase, which is involved in ergosterol biosynthesis. Thus, the imidazoles and triazoles oppose the action o amphotericin B by decreasing the concentration o the target or the latter drug. Despite these considerations, static and cidal antimicrobial drugs are sometimes used clinically in combination when no good alternatives exist. In such cases, it may be necessary to increase the dose o one or both drugs to compensate or the antagonistic drug–drug interaction. The resulting increase in the therapeutic drug concentration(s) can increase the likelihood o adverse e ects.

ANTIVIRAL COMBINATION THERAPY For certain viruses, no drug provides long-term suppressive benef t when used as a single agent. This phenomenon, which is due largely to the development o drug resistance, has been most studied with HIV. The viral li e cycle is central to understanding the reason that monotherapy or HIV ails to suppress long-term viral replication (see Chapter 38, Pharmacology o Viral

776 PRINCIPLES OF CHEMOTHERAPY

In ections; Fig. 38-2). A ter virus attachment, entry, and uncoating, the viral enzyme reverse transcriptase (RT) synthesizes double-stranded DNA rom the single-stranded viral RNA genome. The DNA is then integrated into the host cell genome and transcribed over and over using the host cell’s transcription machinery. These complete genomic transcripts are eventually packaged into virions that in ect new cells. However, HIV RT is relatively un aith ul, so replication error rates are quite high. In addition, transcription o the integrated DNA into RNA is also error prone. As a result, on average, every new HIV particle contains one mutation relative to its parental virus. The resulting error rate is not so high as to be intolerable to the virus, but it is su f ciently high that, a ter repeated cycles o in ection, reverse transcription, and transcription, a substantial number o viruses encode altered targets o anti-HIV therapy and thereby acquire resistance, even prior to treatment. In the setting o high mutation rates, combination antiviral therapy is benef cial. Combinations o RT inhibitors (as f rst shown or zidovudine and lamivudine) are more e ective than one RT inhibitor alone, in part because resistance to one nucleoside analogue does not necessarily con er resistance to another. The current standard o care or treatment o HIV in ection is “triple therapy.” Triple therapy can use many combinations— or example, two nucleoside analogue RT inhibitors and a nonnucleoside reverse transcriptase inhibitor (NNRTI), two nucleoside analogues and a protease inhibitor, or two nucleoside analogues and an integrase inhibitor. Clinical trials have shown that such combinations are able to reduce viral RNA plasma levels below the limit o detection (typically, 50 copies/mL). At such low levels o viral replication, the probability o resistance emerging to any one o the drugs is greatly reduced. Thus, or example, it has been shown that combinations remain e ective or much longer periods o time than does any single agent. Although combination ormulations o common anti-HIV therapies have reduced “pill burden,” simplif ed treatment, and increased adherence, the adverse e ects o some combinations can result in reduced adherence. Despite these concerns, data rom randomized clinical trials now provide conclusive evidence or the use o combination antiretroviral therapy regardless o the stage o disease. There ore, treatment guidelines in the United States as well as those developed by WHO now recommend universal treatment o all patients with HIV in ection, regardless o CD4 T-cell counts. Combination chemotherapy has also become common or treatment o other viruses that require prolonged therapy and have high mutation rates, such as hepatitis B virus (HBV) and hepatitis C virus (HCV). For HIV, HBV, and HCV, an important strategy in designing combination regimens is to include drugs that are less likely to select or resistant mutants because the mutants that are selected are also less f t.

ANTINEOPLASTIC COMBINATION CHEMOTHERAPY Several intrinsic di f culties are aced in the administration o antineoplastic chemotherapy. Cancer cells can be thought o as “altered sel ” cells that maintain similarities to normal, noncancerous cells, o ten making it di f cult to target the cancer cells selectively. Also, many cancer chemotherapeutic

agents have serious adverse e ects that limit their dose and requency o administration. Despite these hurdles, combination chemotherapy has led to remarkable advances in the treatment o cancer, including the examples o Hodgkin’s disease and testicular cancer discussed at the end o this section. Table 41-2 provides an overview o the major antineoplastic drug classes that are currently available, including their mechanisms o action, cell cycle specif cities, major resistance mechanisms, and dose-limiting toxicities. Note that all these drug classes have been discussed in previous chapters; the ollowing discussion integrates relevant in ormation about the individual drugs in a clinical context.

General Considerations To appreciate the challenges that must be aced in treating cancer with drug therapies, it is use ul to examine the current model or oncogenic trans ormation. Normal somatic cells undergo di erentiation as they mature rom a small regenerating stem cell population. Because cells lose the ability to divide as they progress along their di erentiation pathway, malignancies tend to arise in populations o immature or undi erentiated cells. At the molecular level, the process o malignant trans ormation involves multiple steps, including the loss o tumor suppressor gene products (e.g., p53 and Rb) and the activation o proto-oncogenes (e.g., RAS and c-MYC) through processes such as somatic mutation, DNA translocation, and gene amplif cation. Acquired alterations in genes that regulate the progression o cells through the cell cycle con er a growth advantage on malignant cells, which proli erate in the absence o normal growth regulatory signals. Some o the most aggressive trans ormed cells multiply at a rate o about two divisions a day. At this rate, a single such cell could give rise to a clinically detectable mass o 1 g (109 cells) in just 15 days, and a tumor burden o 1 kg (1012 cells), which is o ten incompatible with li e, could be achieved in 20 days. Fortunately, oncogenesis usually occurs much more slowly than this—a act that supports the concept o screening or many types o cancer (e.g., cervical, prostate, and colon). A malignant cell can give rise to a small colony o cells (106 cells) rather quickly, but urther growth is held in check by the limited availability o oxygen and nutrients. Because oxygen can di use passively in tissues over a distance o only 2–3 mm, cells in the center o the growing tumor mass become hypoxic and enter the G0 (resting) phase. Accordingly, the percentage o cells that are actively dividing (i.e., the growth raction o the tumor) decreases as tumor size increases. Moreover, the continued proli eration o cells at the tumor margins causes a urther decrease in the pO2 in the center o the tumor, and hypoxic tumor cells begin to die (central necrosis). The tumor continues to grow, albeit at a slower rate, because the rate o cell division at the margins exceeds the rate o central necrosis. At some point, hypoxic tumor cells can express or induce the stromal expression o angiogenic actors (e.g., vascular endothelial growth actor [VEGF]) that induce vascularization and thus oxygenation o the tumor. Vascularization can be accompanied by a sudden increase in the growth raction as cells are pulled out o G0 phase and into the cell cycle. Because a single malignant cell can expand clonally to give rise to a tumor, it is thought that every malignant cell must be destroyed to e ect a cure o the cancer. This hypothesis, together with the “log-kill” hypothesis or tumor cell killing

C h a p t e r 4 1 Principles o Combination Chemotherapy 777

TABLE 41-2 Classes o Cancer Chemotherapeutic Agents with Selected Examples DRUG CLASS Alkylating agents Cyclophosphamide Platinum complexes Cisplatin Antimetabolites Folic acid metabolism Methotrexate Purine analogues Mercaptopurine Pyrimidine analogues Fluorouracil Substituted urea Hydroxyurea Natural products Bleomycin Camptothecins Camptothecin Anthracyclines Doxorubicin

Epipodophyllotoxins Etoposide Vinca alkaloids Vincristine Taxanes Paclitaxel Di erentiating agents Tretinoin

Endogenous pathway modif ers Hormone modulators Prednisone Tamoxi en Anastrozole Flutamide Leuprolide Immunomodulatory drugs Inter eronInterleukin-2

MECHANISM OF ACTION

MAJ OR RESISTANCE MECHANISM

DOSE-LIMITING TOXICITY

Cross-link DNA, RNA, protein (Cell cycle nonspecif c)

↑ DNA repair, ↓ drug uptake, ↑ drug inactivation

Bone marrow

DNA intrastrand cross-links (G-G) (Cell cycle nonspecif c)

↑ DNA repair, ↓ drug uptake, ↑ drug inactivation

Kidney

Disrupt nucleotide synthesis, utilization, incorporation (Cell cycle S-phase specif c)

↓ drug uptake, ↓ drug activation, ↑ drug inactivation, ↑ or altered target enzyme, salvage pathway

Bone marrow

Inhibits ribonucleotide reductase (Cell cycle S-phase specif c)

↑ DNA repair, ↓ drug uptake, ↑ drug inactivation

Bone marrow

DNA strand scission (Cell cycle G2-phase specif c) Inhibit topoisomerase I (Cell cycle S-phase specif c) DNA intercalation, inhibit topoisomerase II, lipid peroxidation (Cell cycle G2-phase specif c) Inhibit topoisomerase II (Cell cycle S/G2-phase specif c) Disrupt microtubule assembly (Cell cycle M-phase specif c) Disrupt microtubule disassembly (Cell cycle M-phase specif c)

↑ DNA repair?, ↓ drug uptake?, ↑ drug inactivation?, ↑ drug e ux? ↑ drug e ux?

Pulmonary f brosis

↑ drug e ux

Bone marrow, cardiotoxicity

↑ drug e ux ↑ drug e ux

Bone marrow, GI toxicity (diarrhea) Bone marrow, neuropathy

↑ drug e ux

Bone marrow (mild)

Retinoic acid receptor agonist (Induce di erentiation o cancer cells)

PML-RAR-

Glucocorticoid receptor agonist

Loss o hormone sensitivity (↑ or altered target receptor) Loss o estrogen-dependent growth

Cushingoid syndrome

Loss o estrogen-dependent growth Loss o androgen-dependent growth Loss o androgen-dependent growth

Osteoporosis Hepatotoxicity Osteoporosis

Estrogen receptor antagonist/ modulator Aromatase inhibitor Androgen receptor antagonist GnRH receptor agonist

usion gene mutation

Inter eron receptor agonist, specif c mechanism unknown IL-2 receptor agonist (stimulates T- and B-cell proli eration and di erentiation)

Bone marrow

Retinoic acid syndrome

Endometrial cancer, thrombosis

Bone marrow, neurotoxicity, cardiotoxicity Hypotension, pulmonary edema

(See Table 54-2 or more examples) Targeted delivery o compounds or proteins Toxin conjugates Denileukin di titox Small-molecule conjugates Gemtuzumab ozogamicin

Radiotherapy conjugates Iodine-131 tositumomab

Delivers diphtheria toxin to cells expressing IL-2 receptor

Reduction in receptor expression

Severe edema, u-like systemic symptoms

Delivers calicheamicin to myeloid leukemia cells expressing CD33

Reduction in receptor expression

Hepatotoxicity, in usion reactions

Delivers radioactive iodine to cells expressing CD20

Reduction in receptor expression

Hypersensitivity reactions, bone marrow

(See Table 54-5 or more examples)

continues

778 PRINCIPLES OF CHEMOTHERAPY

TABLE 41-2 Classes of Cancer Chemotherapeutic Agents with Selected Examples continued DRUG CLASS Growth factor receptor and signal transduction antagonists Cetuximab

Trastuzumab

BCR-Abl/C-KIT/PDGFR inhibitors

MECHANISM OF ACTION

MAJ OR RESISTANCE MECHANISM

DOSE-LIMITING TOXICITY

Binds to and inhibits epidermal growth actor receptor (EGFR) Binds to ErbB2 (HER2/neu) cell sur ace receptor and controls cancer cell growth Inhibit protein tyrosine kinase domain

EGFR mutation

Skin, GI toxicity (diarrhea)

Signaling pathway modulation, binding site disruption

Cardiotoxicity

Mutation in target enzyme (e.g., BCR-Abl mutation)

Skin, GI (diarrhea), uid retention

Inhibits protein degradation by proteasome

p53 mutation, ↑ HSP-27 expression

Neurotoxicity, bone marrow

Binds to and neutralizes vascular endothelial growth actor (VEGF)

Multiple adaptive and/or intrinsic mechanisms to evade VEGF dependence o tumor angiogenesis

Kidney (proteinuria), hypertension

(See Chapter 40 and Table 54-4 or more examples) Proteasome inhibitors Bortezomib Angiogenesis inhibitors Bevacizumab

(see Chapter 33), suggests that multiple cycles of chemotherapy must be administered at the highest tolerable doses and the most frequent tolerable intervals to achieve a cure. Antineoplastic chemotherapy usually ollows f rst-order kinetics (i.e., a constant fraction o tumor cells is killed with each cycle o chemotherapy). These kinetics o tumor cell killing are unlike the time-dependent killing characteristic o many antimicrobial drugs, which ollows zero-order kinetics (i.e., a f xed number o microbes is killed per unit time). Adding to the di f culty o curative cancer treatment is the phenomenon o tumor progression, in which a clonally derived population o malignant cells becomes heterogeneous through the accumulation o multiple genetic and epigenetic alterations. When subjected to immune surveillance or the administration o an antineoplastic agent, subclones o the tumor with relatively nonantigenic or drug-resistant phenotypes are selected. Mutations that con er drug resistance are o particular concern, because many trans ormed cells, having lost the ability to repair DNA damage, are characterized by genomic instability. Thus, deletions, gene amplif cations, translocations, and point mutations are not in requent events and can result in antineoplastic drug resistance through any o the mechanisms shown in Table 41-3. With the possible exception o some recently developed classes o antineoplastic therapies directed against molecular targets that are selectively expressed by a malignant clone o cells (e.g., a monoclonal antibody directed against a tumor cell antigen or an enzyme inhibitor directed against a mutated signal transduction molecule; see Chapter 1, Drug–Receptor Interactions; Chapter 40, Pharmacology o Cancer: Signal Transduction; and Chapter 54, Protein Therapeutics), antineoplastic chemotherapy has ocused on di erences in the cell cycle between rapidly dividing and normal cells. Some o these agents act by inducing DNA damage and subsequent apoptosis in all phases o the cell cycle, whereas others act selectively in one phase o the cell cycle (see Chapter 33, especially Fig. 33-4). Un ortunately, such drugs are also associated with signif cant

TABLE 41-3 Mechanisms of Tumor Resistance to Cytotoxic Chemotherapeutic Agents MECHANISM OF TUMOR RESISTANCE EXAMPLES Pharmacokinetic Mechanisms Insu f cient accumulation o drug Insu f cient uptake o drug E ux o drug rom tumor cell (MDR phenotype) Un avorable metabolism o drug or prodrug Insu f cient activation o prodrug Increased inactivation o drug Cytidine deaminase overexpression Alkaline phosphatase overexpression

Methotrexate Vinca alkaloids, etoposides, doxorubicin 5-FU, 6-MP, Ara-C, 6-TG Ara-C 6-TG, 6-MP

Pharmacodynamic Mechanisms Overexpression, alteration, or loss o target molecule* Dihydro olate reductase

Methotrexate

Decreased concentration o co actor

5-FU

Increased concentration o competing molecule

Ara-C metabolite (dCTP)

Increased repair o drug-induced lesions in DNA, proteins, or lipids (membranes)

Alkylating agents

Increased utilization o alternate pathways

Antimetabolites

Loss o drug-induced apoptosis

Most antineoplastics

*Due to DNA mutation, amplif cation, deletion, or epigenetic change; altered transcription or post-transcriptional processing; altered translation or posttranslational modif cation; or altered target stability.

C h a p t e r 4 1 Principles of Combination Chemotherapy 779

toxicity, especially in tissues that normally have a high rate o cell turnover (e.g., bone marrow, hair ollicles, intestinal epithelia). Accordingly, neutropenia, thrombocytopenia, anemia, alopecia, nausea, and oral and intestinal ulcerations are common adverse e ects o many cytotoxic antineoplastic agents. Although many rapidly growing lymphomas and leukemias seem to melt away with antineoplastic chemotherapy, more indolent solid tumors must o ten be treated with adjuvant (i.e., chemotherapy-enhancing) radiation therapy and/or surgery. By the time these tumors come to clinical attention, they may be large and may have metastasized. In such cases, surgical removal o the primary tumor is o ten ollowed by radiation therapy and/or systemic chemotherapy, using agents that penetrate the various tissues that could be sites o metastatic disease (e.g., brain, liver). In summary, cancer therapy must eliminate every malignant cell rom the body, making high doses o chemotherapeutic agents desirable. (In practice, immune mechanisms may be able to clear small numbers o remaining cancer cells i these cells are su f ciently immunogenic. Monoclonal antibody therapies that enhance the immunogenicity o cancer cells have recently been approved; these “immune checkpoint inhibitors” block immune co-regulatory pathways such as B7/CTLA-4 and PD-1/PD-L1.) However, the toxicity o these relatively nonselective agents limits their achievable doses. Moreover, resistance to these drugs can develop. Finally, because these agents target mainly rapidly dividing cells, antineoplastic drugs are less e ective against large solid tumors with low growth ractions. Each o these considerations points to the need or combination drug regimens to treat cancer. The basic pharmacologic principles or such regimens are discussed below.

Rationale or Combination Chemotherapy Combination antineoplastic drug regimens typically include agents that act on di erent molecular targets, at di erent phases o the cell cycle, and with di erent dose-limiting toxicities (Table 41-2). This strategy targets asynchronously dividing tumor cells, reduces the emergence o drug resistance, and allows each drug to be given at its highest tolerable dose, thereby maximizing e f cacy without excessive toxicity. Recent advances in supportive therapy have also increased the maximum tolerated doses or many cytotoxic antineoplastic agents. For example, the routine use o antiemetics, autologous bone marrow transplantation, hematopoietic growth actors (e.g., GM-CSF, G-CSF, erythropoietin), and prophylactic broad-spectrum antibiotics has reduced the complications o myelosuppressive chemotherapy regimens. Similarly, allopurinol treatment to prevent the hyperuricemia that could result rom widespread release and metabolism o purines rom necrotic tumor cells (i.e., tumor lysis syndrome ) has reduced the morbidity associated with high doses o systemic chemotherapy (see Chapter 49, Integrative In ammation Pharmacology: Gout). Finally, so-called leucovorin rescue a ter high-dose methotrexate administration selectively spares nonmalignant cells rom death associated with tetrahydro olate depletion (see Chapter 33). Unlike the treatment o bacterial and viral in ections, cytotoxic chemotherapy or cancer o ten employs an intermittent dosing strategy. The main rationale or this strategy is to avoid unacceptable toxicity to normal cells and tissues, or example, by allowing time or bone marrow recovery. Intermittent dosing may also have the advantage o

“pulling” some nondividing cells out o the G0 phase o the cell cycle and making them more susceptible to subsequent cycles o chemotherapy. The latter rationale has prompted the use o adjuvant radiation therapy and the inclusion o cell cycle nonspecif c drugs in certain combination chemotherapy regimens; both o these strategies have been noted to increase the growth ractions o tumors in some studies. Despite these considerations, continuous delivery o cytotoxic chemotherapeutic agents is occasionally benef cial in treating slowly cycling tumors (e.g., multiple myeloma) or in cases where bolus in usion o drug is associated with signif cantly higher toxicity (e.g., anthracyclines ). Many o the growth actor receptor and signal transduction antagonists used in cancer chemotherapy are less toxic than the older cytotoxic antineoplastic agents. The reduced toxicity o these antagonists permits their use in daily dosing regimens, both as single agents and in combination with cytotoxic chemotherapy. One example o such a combination is lapatinib (an inhibitor o EGFR and HER2) and capecitabine (a prodrug orm o uorouracil) in the treatment o HER2-positive metastatic breast cancer (see Chapter 40). Finally, some antineoplastic drug combinations take advantage o known synergies. A clinically important example is the interaction between 5-f uorouracil (5-FU) and methotrexate . These drugs are used in combination in the treatment o many adenocarcinomas, including breast, colon, and prostate cancers. Both drugs are S-phase specif c and have common dose-limiting toxicities (bone marrow and intestinal mucosal damage), so their use in combination may seem surprising (see Chapter 33 and Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance). The mechanism o synergy appears to involve the ability o methotrexate to enhance activation o 5-FU. Recall that methotrexate inhibits purine biosynthesis and that 5-FU is metabolized by cellular salvage pathways that ultimately convert the drug into the active orm 5-FdUMP. The f rst step in the activation o 5-FU requires 5-phosphoribosyl 1-pyrophosphate (PRPP) and is catalyzed by the enzyme phosphoribosyl trans erase: 5-FU PRPP → 5-FUMP PPi. Methotrexate increases cellular levels o PRPP, likely due to decreased PRPP utilization in purine synthesis pathways. The elevated PRPP levels avor the conversion o 5-FU to 5-FUMP, which is ultimately converted to 5-FdUMP by the action o ribonucleotide reductase and other enzymes.

Examples o Antineoplastic Combination Chemotherapy Hodgkin’s Disease

The treatment o Hodgkin’s disease (HD) illustrates the rational use o cytotoxic antineoplastic drug combinations. In this disease, there is clonal proli eration o Reed-Sternberg (RS) cells within a dense, reactive in ammatory cell background. HD originates in a single lymph node and progresses in a contiguous ashion involving adjacent lymphoid tissues. The RS cell is the neoplastic cell; this cell seems to be o B-cell origin, making the disease a true lymphoma. Pathologic subtypes o classical HD, def ned on the basis o RS cell morphology and the pattern o surrounding reactive in ammatory changes, include nodular sclerosing, mixed cellularity, lymphocyte-rich, and lymphocyte-depleted HD. Patients typically present with lymphadenopathy (cervical, supraclavicular, axillary, or inguinal) and/or systemic

780 PRINCIPLES OF CHEMOTHERAPY

TABLE 41-4 The Cotswold/Ann Arbor Staging System for Hodgkin’s Disease (HD)* STAGE

DESCRIPTION

SUBCLASSIFICATION

I

Involvement o a single lymph node region or lymphatic organ (e.g., thymus)

IA: No systemic symptoms IB: Systemic symptoms (e.g., ever, night sweats, weight loss) IE: Involvement o one area o a single organ outside the lymph system

II

Involvement o two or more lymph node regions on the same side o the diaphragm

IIA: No systemic symptoms IIB: Systemic symptoms IIE: Extranodal contiguous extension rom one lymph node region into a nearby organ

III

Involvement o nodal regions on both sides o the diaphragm

IIIA: No systemic symptoms IIIB: Systemic symptoms IIIS: Splenic involvement IIIE: Extranodal contiguous extension

IV

Di use or disseminated disease involving one or more extralymphatic organs (e.g., liver, bone marrow, lungs, cerebrospinal uid)

IVA: No systemic symptoms IVB: Systemic symptoms

*Note: Bulky disease or massive disease (denoted by adding the letter X to the stage) is sometimes used to describe HD tumor masses that measure at least one-third o the diameter o the chest or that are at least 10 cm in diameter in non-chest sites.

symptoms including ever, malaise, pruritus, night sweats, and weight loss. The stage o the disease determines treatment; patients with early-stage disease (stages I and II) receive radiation therapy with or without chemotherapy, and patients with advanced-stage disease (stages III and IV) require combination chemotherapy (Table 41-4). Be ore the introduction o alkylating agents in the mid1960s, single-agent chemotherapy or advanced HD resulted in a median survival o 1 year. With the development o MOPP (mechlorethamine , vincristine [oncovin], procarbazine , and prednisone ), the f rst success ul antineoplastic drug combination or HD, hal o these patients were cured o their disease. Treatment remained limited by signif cant toxicity, however, including early gastrointestinal and neurological complications as well as late sterility and secondary malignancies (myelodysplastic syndrome, acute nonlymphocytic leukemia, and non-Hodgkin’s lymphoma). Further investigation led to the development o the ABVD (doxorubicin [adriamycin], bleomycin, vinblastine , and dacarbazine ) combination, which is less toxic and more e ective than MOPP. ABVD is the current standard o care or early-stage HD and a pre erred treatment option or advanced-stage HD; trials o novel combination therapies are also underway.

The rationale or the ABVD drug combination comes rom the knowledge that it combines both cell cycle selective and nonselective agents as well as drugs with di erent doselimiting toxicities. Compared to MOPP, ABVD is associated with signif cantly ewer hematological and gonadal complications and secondary malignancies.

Testicular Cancer

Principles o antineoplastic combination chemotherapy are also exemplif ed in the treatment o testicular cancer. This tumor arises rom the spermatogenic epithelium o the testis and is usually detected as a testicular mass on physical examination. The tumor metastasizes through lymphatic channels to pelvic and periaortic lymph nodes be ore disseminating widely through hematogenous routes. Treatment o local disease (without evidence o metastasis) involves surgical removal o the a ected testis with or without pelvic radiation. Advanced disease requires systemic treatment with combination chemotherapy. One standard-o -care regimen is BEP (Fig. 41-4). O the three drugs commonly used in this regimen (bleomycin, etoposide , and cisplatin), cisplatin is a cell cycle nonspecif c drug that may draw nondividing tumor cells into the actively cycling pool, where they are susceptible to the synergistic action o the cell cycle specif c agents bleomycin and etoposide. The drugs in this combination have di erent molecular targets, act on di erent phases o the cell cycle, and have di erent dose-limiting toxicities. Intermittent dosing allows each a ected organ (lung, bone marrow, and kidney, respectively) time to recover between cycles. Administered a ter surgical removal o the primary tumor, such a regimen usually results in a cure.

Treatment of Refractory or Recurrent Disease Despite the act that combination chemotherapy has resulted in vastly improved survival or some cancers, many cancers become re ractory to standard combination chemotherapy. I a standard chemotherapy regimen ails, options include experimental drug therapies, palliative care, or novel drugs approved or use a ter treatment ailure. Many patients choose to enroll in experimental clinical trials. This decision may be Drug Ble omycin

Da y 2

Etopos ide

Da y 1–5

Cis pla tin

Da y 1–5

1

Da y 9

8

Da y 16

15

22

Time (da ys ) FIGURE 41-4. The bleomycin-etoposide-platinum (BEP) combination chemotherapy regimen for testicular cancer. The BEP regimen used to treat testicular cancer consists o a combination o bleomycin, etoposide, and a platinum compound. Cisplatin, a platinum compound o ten used in this regimen, is a cell cycle nonspecif c agent; this drug may draw nondividing cells into the cell cycle, where they can be killed by the G2-phase-specif c agent bleomycin and the S/G2-phasespecif c agent etoposide. The intermittent dosing schedule limits drug toxicity and allows time or the bone marrow to recover rom drug-induced myelosuppression. The 3-week cycle shown is typically administered our times in succession (12 weeks total).

C h a p t e r 4 1 Principles of Combination Chemotherapy 781

based on the hope that an investigational agent could prove e f cacious but with the understanding that the true benef t may be realized only by uture patients. Palliative and hospice care are alternatives to continued drug treatment in cases o advanced metastatic disease. An increasing number o agents with novel mechanisms o action are becoming available or disease that is otherwise re ractory to treatment. Many o these agents selectively target tumor-specif c antigens and signal transduction pathways, as discussed in Chapters 40 and 54. Other agents are being designed and tested to target recently discovered pathways that allow tumors to alter cellular energetics and evade immune destruction (see above). Optimizing combinations o these and other antineoplastic agents or e f cacy and sa ety will be an important challenge or the uture.

CONCLUSION AND FUTURE DIRECTIONS The principles o combination chemotherapy highlight the importance o combination drug treatment in a variety o clinical situations. The use o drug combinations has greatly enhanced the e ectiveness o treatment o both in ectious and neoplastic diseases. The advantages o ered by multidrug regimens over individual drug therapy (monotherapy) include increased antimicrobial, antiviral, and antineoplastic e f cacy; decreased overall drug resistance; decreased host toxicity; and broader coverage o suspected pathogenic organisms. These advantages are illustrated in the rational use o drug combinations to treat in ections with Mycobacterium tuberculosis and HIV, as well as neoplastic disorders such as Hodgkin’s disease and testicular cancer. Treatment o multidrug-resistant microorganisms such as MDR-TB and MDR-HIV remains a special challenge, as does treatment o genetically heterogeneous cancers with low growth ractions such as lung, colon, breast, and prostate cancers. Continued ref nement o combination chemotherapy regimens will rely on increased understanding o molecular targets and metabolic pathways used by microorganisms and cancer cells.

Acknowledgment We thank Shreya Kangovi and Gia Landry or initial dra ts o the case o Mr. M and the discussion in the chapter related to his case. We thank Ryan L. Albritton or his valuable contributions to this chapter in the First and Second Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy and Daniel Kuritzkes or help ul comments.

Suggested Reading Canellos GP, Anderson JR, Propert KJ, et al. Chemotherapy o advanced Hodgkin’s disease with MOPP, ABVD, or MOPP alternating with ABVD. N Engl J Med 1992;327:1478–1484. (ABVD remains a standard o care or advanced Hodgkin’s disease.) Chou TC. Theoretical basis, experimental design, and computerized simulation o synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621–681. (Detailed analysis o models or synergistic, antagonistic, and additive drug combinations.) Chou TC, Talalay P. Quantitative analysis o dose-e ect relationships: the combined e ects o multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55. (Detailed analysis o models or synergistic, antagonistic, and additive drug combinations.) Dancey JE, Chen HX. Strategies or optimizing combinations o molecular targeted anticancer agents. Nat Rev Drug Discov 2006;5:649–659. (Discusses principles or determining combinations o antineoplastic agents that could be most promising to test in preclinical and clinical trials.) Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trottie A III, eds. AJCC cancer staging manual. 7th ed. New York: Springer; 2010:607–611. (Lists staging criteria or human cancers.) Gunthard HF, Aberg JA, Eron JJ, et al. Antiretroviral treatment o adult HIV in ection: 2014 recommendations o the International Antiviral Society— USA Panel. JAMA 2014;312:410–425. (Reviews combination therapies recommended or treatment o HIV.) Hanahan D, Weinberg RA. Hallmarks o cancer: the next generation. Cell 2011;144:646–674. (Reviews hallmarks o cancer and discusses therapeutic targeting o pathways required or tumor growth and progression.) Harvey RJ. Synergism in the olate pathway. Rev In ect Dis 1982;4:255–260. (Describes kinetics o synergism between trimethoprim and the sul onamides.) Luo J, Solimini NL, Elledge SJ. Principles o cancer therapy: oncogene and non-oncogene addiction. Cell 2009;136:823–837. (Reviews antineoplastic therapies targeting the hallmarks o cancer and proposes principles or developing new antineoplastic therapies and combinations.) Momtaz P, Postow MA. Immunologic checkpoints in cancer therapy: ocus on the programmed death-1 (PD-1) receptor pathway. Pharmgenomics Pers Med 2014;7:357–365. (Reviews pathways o immune evasion in cancer and therapeutic rationale or development o immune checkpoint inhibitors that block CTLA-4 and PD-1 pathways.) Paltiel AD, Walensky RP, Schackman BR, et al. Expanded HIV screening in the United States: e ect on clinical outcomes, HIV transmission, and costs. Ann Intern Med 2006;145:797–806. (Compares benef ts, risks, and costs o screening or HIV.) Panel on Antiretroviral Guidelines or Adults and Adolescents. Guidelines or the use o antiretroviral agents in HIV-1-in ected adults and adolescents. http://aidsin o.nih.gov/contentf les/lvguidelines/adultandadolescentgl.pd . (Reviews combination therapies recommended or treatment o HIV.) World Health Organization. Guidelines or the programmatic management o drug-resistant tuberculosis: 2011 update. Geneva, Switzerland: World Health Organization; 2011. http://www.ncbi.nlm.nih.gov/books /NBK148644/. (Evaluates evidence and recommends treatment regimens or multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis.) Zumla A, Raviglione M, Ha ner R, von Reyn CF. Tuberculosis. N Engl J Med 2013;368:745–755. (Reviews current recommendations or treatment and status o selected trials o treatment or latent tuberculosis in ection, drug-sensitive active tuberculosis, and drug-resistant active tuberculosis.)

VI Principles o Inf ammation and Immune Pharmacology

Anti An tig g e n re c o g n itio n

T c e ll re s po p o ns ns e Cyto Cy t kine re ce pto torr

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TCR

42

m t s c

o

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a

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MHC M C

Principles o Inf ammation and the Immune System

N

N

o

CD4

A

A

Re s ting g AP C

Na ivve T ce c ll

MHC MH

No re s po pons ns e

TCR

u m

s

t s o

o

o c

c

c h

h

t

t

t

t

i

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W

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n

Eryn L. Roye r a n d Ap ril W. Arm s t ro n g

INTRODUCTION DUCTION CTION T ON & CA TI CASE ASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783–7 AS ASE 7883– 783– 83– 3–784 –7 784 B7 CD28 CD28 C D4SYSTE OVER OV ERVI VIIEW OF THE IMMUN IMMUNE MM CD4 TEM M . . . . . . . . . . . . . . . . . . . . . 784 Inna In nate te Imm m unity . . . . . . . . 784 Acti Ac Activa Activ tiv ti va te te d . . . . . . . . . . . . . . . . . . . . . . . . . . .Activa Activ d Antigen-Presenti ting Cel ells lss . . . . . . . . . . . . . . . . . . . . . . . . . . .te .e785 AP C ytoki Cy to tResponse o kine kin s e . . . . . . .T. .ce Activation ofCthe Innate Immune . . .ll78 7866 Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 7866 Majo Ma jorr Hi Hist stoc ocom ompa pati tibi bili lity ty Complex l x . . . . . . . . . . . . . . . . . . . . 786 Immune Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787 Humoral and Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . 787 Tolerance and Costimulation . . . . . . . . . . . . . . . . . . . . . . . . . 788 CHEMICAL MEDIATORS OF INFLAMMATION . . . . . . . . . . . . . . . 790 790 Histamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790 Complement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790

INTRODUCTION Inf ammation and the immune system are closely intertwined. Inf ammation is composed o a complex web o responses to tissue injury and in ection, characterized by the classic signs o rubor (redness), calor (heat), tumor (swelling), dolor (pain), and functio laesa (loss o unction). The immune system includes the cells and soluble actors, such as antibodies and complement proteins, which mediate the inf ammatory response; these cells and actors both eliminate the inciting inf ammatory stimulus and initiate immunologic memory. A normal inf ammatory response is an acute process that resolves a ter removal o the inciting stimulus. Diseases o inf ammation and immunity can occur due to inappropriate inf ammation or when the normal inf ammatory response progresses to chronic inf ammation, either because o a longterm inappropriate response to a stimulus (e.g., allergies) or because the o ending agent is not removed (e.g., chronic in ection, transplantation, and autoimmunity). Two pharmacologic strategies are used to target the pathophysiology o immune diseases. The rst involves modi cation o the signaling mediators o the inf ammatory process or suppression o components o the immune system. This is the rationale or drugs that modulate eicosanoid pathways (Chapter 43, Pharmacology o Eicosanoids), histamine (Chapter 44, Histamine Pharmacology), and cells o the immune system (Chapter 45, Pharmacology o Hematopoiesis

Eicosaa nooid oiiddss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7790 90 90 Cytokines ess . . . . . . . . . . . . . . .IL-2R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791 1 Other Agents enn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791 91 7791 91 THE INFLAMMATORY T R RESPONSE. . . . . . . . . . . . . . . . . . . . . . . . . 79 IL-2 IL L2 Dila Di lati tion on of Vesselss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791 791 1 Recr Re cruitment of Cells. . . . . . . . . . . . . . . . . . . . . . . . . T ce ll 911e ra tio l pro prolife olife olif tionn Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. .nd . .e re n. d. . di diff d. . ffe ff r791 e nnti tiia tion Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 7911 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792 CHRONIC INFLAMMATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 792 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .793

and Immunomodulation, and Chapter 46, Pharmacology o Immunosuppression). This approach is still in its in ancy, because it depends on understanding the molecular events in the relevant pathway, but it promises to yield multiple new drugs in the oreseeable uture. The second pharmacologic approach, used in diseases such as peptic ulcer disease (Chapter 47, Integrative Inf ammation Pharmacology: Peptic Ulcer Disease), asthma (Chapter 48, Integrative Inf ammation Pharmacology: Asthma), and gout (Chapter 49, Integrative Inf ammation Pharmacology: Gout), involves modi cation o the underlying pathophysiologic stimulus, thus removing the impetus or inf ammation. The di erence between these two approaches is, at times, indistinct and will continue to overlap as the pathophysiology o chronic inf ammatory disease is better understood at the molecular level. This chapter provides su cient background on the physiology o inf ammation and the immune system to understand the subsequent chapters in this section o the textbook. The treatment is necessarily brie , with an emphasis on pharmacologically relevant targets o the inf ammatory response. The chapter is organized in our parts. First, a general overview o the immune system is presented. Second, the molecular signals that mediate cellular communication and inf ammation are introduced. Third, the immune and inf ammatory cells and signaling molecules are discussed in the context o an integrated inf ammatory response. Fourth, chronic 783

784 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Ma rk is s tre s s e d —h e h a s to ta ke th e US Me d ica l Lice n s in g Exa m in a tio n (US MLE) in 2 w e e ks , a n d h e h a s b a re ly b e g u n to s tu d y. Th ro w in g a s id e a n y p re te n s e o f a b a la n ce d life s tyle , Ma rk tra ve ls to th e m icro b io lo g y la b la te o n e n ig h t to re vie w te ch n iq u e s fo r p e rfo rm in g a Gra m s ta in . Wh ile a p p lyin g th e g e n tia n vio le t co m p o n e n t o f th e Gra m s ta in , Ma rk cu ts h is th u m b o n th e e d g e o f th e m icro s co p e s lid e . Th in kin g h e la cks th e tim e to cle a n h is th u m b p ro p e rly, Ma rk co n tin u e s to s tu d y fu rio u s ly. Ove r th e n e xt 5 h o u rs , Ma rk’s th u m b b e co m e s p ro g re s s ive ly s w o lle n , wa rm , re d , a n d te n d e r. Ma rk re ta in s fo cu s a n d co n tin u e s to s tu d y th ro u g h th e e ve n in g . By th a t n ig h t, h o w e ve r, h e d e ve lo p s a fe ve r a n d in cre a s e d s w e llin g in th e th u m b . By th e th ird d a y, p u s b u ild s u p a t th e s ite . By th e fo u rth d a y, h o w e ve r, Ma rk’s b o d y s e e m s to h a ve

inf ammation, a pathologic state that is o ten associated with autoimmunity, is presented. For a more comprehensive presentation o this rapidly changing subject, see “Suggested Reading” at the end o this chapter.

OVERVIEW OF THE IMMUNE SYSTEM The undamental role o the immune system is to distinguish sel rom nonsel . “Nonsel ” can be an in ectious organism, a transplanted organ, or an endogenous tissue that is mistaken or something oreign. Because protection against in ection is the classic role o the immune system, the terms in ection and in ectious agent are generally used to denote the inciting stimulus or an immune response. The immune system can be stimulated to react against any nonsel agent. Skin and other barrier tissues orm the rst line o deense against any in ection. (In the introductory case, Mark’s in ection occurred only a ter he cut his skin.) Once an o ending agent penetrates these barriers, the immune system mounts a response. The immune response consists o innate and adaptive responses. Innate responses are stereotyped reactions to a stimulus (e.g., release o histamine, phagocytosis o a bacterium). In some cases, innate responses are su cient to neutralize the o ending agent. Cells o the innate immune system, especially antigen-presenting cells, can also process the o ending agent into small ragments; this processing is necessary or activation o the adaptive immune system. Adaptive responses are neutralizing reactions that are speci c to the o ending agent (e.g., antibodies, cytotoxic T cells). In general, then, the innate immune system recognizes nonsel and activates the response to an o ending nonsel agent; the adaptive immune system generates a response that specif cally neutralizes or kills that agent. Many di erent cell types are involved in the immune system, and these cell types interact in a complex web o signaling and communication to create the overall response. The cells o the immune system derive rom two types o pluripotent cells in the bone marrow: myeloid stem cells and lymphoid stem cells. The lymphoid stem cell is sometimes called the common lymphoid stem cell because it gives rise to

g o tte n th e b e tte r o f th e o ffe n d in g a g e n t. Th e s w e llin g d e cre a s e s , th e s ite lo s e s its d is tin ctive a n g ry re d a p p e a ra n ce , a n d h is fe ve r a b ru p tly s u b s id e s . Re lie ve d th a t h e h a s n o t b e co m e a ca s u a lty o f h is o w n p ro cra s tin a tio n , Ma rk co n tin u e s s tu d yin g a n d p e rfo rm s w e ll o n h is e xa m , n o t le a s t b e ca u s e h is w o u n d h a s p rovid e d h im w ith fu n d a m e n ta l in s ig h ts in to im m u n o lo g y.

Questions 1 . Which substance likely caused Mark’s immune system to activate in response to bacteria in his wound? 2 . Which mediators accounted or Mark’s ever? 3 . What changes in the vasculature accounted or the immediate swelling o Mark’s thumb? 4 . Which chemical signals mediated the inf ammatory response in Mark’s thumb?

both B cells and T cells. In general, myeloid stem cells give rise to precursor cells o the innate immune system, whereas lymphoid stem cells generate precursor cells o the adaptive immune system; there are some exceptions. Figure 42-1 depicts the myeloid and lymphoid stem cells and the mature cell types into which the precursor cells di erentiate. The derivation o these cell types is also discussed in Chapter 45. A help ul conceptual ramework is to envision the innate immune system as the immunologic memory o a species, which is invariant over the li etime o an individual and generally the same among individuals o the species. In contrast, the adaptive immune system establishes the immunologic memory o an individual over his or her li etime, depending on that individual’s exposure to pathogens, vaccines, or other immunologic stimuli. Adaptive immunity, there ore, is relatively unique to each individual.

Innate Immunity Cells o the innate immune system are the rst responders to an o ending agent that has penetrated the skin or another barrier (Table 42-1). Innate immune cells per orm three important tasks. First, these cells de end against bacterial and parasitic in ections, either by neutralizing the in ectious agent with secreted cytotoxic proteins or by phagocytosis (engul ng) o the bacterium or parasite. Second, phagocytosis o the o ending agent initiates proteolytic digestion o microbial macromolecules to ragments (antigens) that are then displayed, together with major histocompatibility complex (MHC) class II proteins, on the sur ace o antigenpresenting cells. In turn, these antigen-presenting cells, which include macrophages and dendritic cells, activate cells o the adaptive immune system. Third, innate immune cells secrete numerous cytokines (see below) that urther ampli y the immune response. The major cell types o the innate immune system include granulocytes (neutrophils, eosinophils, and basophils), mast cells , and antigen-presenting cells (macrophages and dendritic cells). Some immunologists consider natural killer (NK) cells, NK T cells, and T cells to have innate immune roles; the biology o these cell types is beyond the scope o this text.

C h a p t e r 4 2 Principles o Inf ammation and the Immune System 785

Bone marrow

P luripote nt he ma topoie tic s te m ce ll

Triline a ge mye loid s te m ce ll

Lymphoid s te m ce ll

Me ga ka ryocyte

Blood and tissues Gra nulocyte s

Lymphocyte y s

B ce ll

T ce ll

Ne utrophil

Eos inophil

Ba s ophil

Effe ctor ce lls

P la s ma ce ll

Activa te d T ce ll

Erythrobla s t

Blood Ma s t ce ll

Monocyte / ma cropha ge

P re curs or

Monocyte

P la te le ts

Erythrocyte

Tis s ue ce lls

Ma s t ce ll

Ma cropha ge

FIGURE 42-1. Development of cells of the immune system. All hematopoietic cells develop rom the pluripotent hematopoietic stem cell. This cell gives rise to the lymphoid stem cell and the trilineage myeloid stem cell. The lymphoid stem cell and its progenitor cells (not shown) give rise to mature lymphocytes (B cells and T cells), the cells that mediate adaptive immune responses. When exposed to speci c antigens, B cells di erentiate into antibody-producing plasma cells, and T cells adopt an activated phenotype. The myeloid stem cell and its progenitor cells, including megakaryocytes, erythroblasts, and myeloid precursors (not shown), proli erate and di erentiate into mature neutrophils, eosinophils, basophils, mast cells, monocytes, platelets, and erythrocytes. In the tissues, monocytes di erentiate into macrophages or dendritic cells, and mast cell precursors di erentiate into mast cells. (See Fig. 45-1 or more details about the di erentiation o cell lineages in the bone marrow.)

Granulocyte is a descriptive term based on the appearance o the cytoplasmic granules within these cells. Neutrophils , the most abundant cell type o the innate immune system and the “ rst responders” in inf ammation, are phagocytic cells primarily responsible or de ense against bacterial inection. These cells envelop invading bacteria in phagocytic vesicles and destroy the bacteria within these vesicles using enzymes such as myeloperoxidase. Eosinophils are circulating granulocytes primarily involved in de ense against parasitic in ections. Because parasites are o ten too large to engul , eosinophils attach to a parasite’s exterior and secrete cytotoxic substances directly on the parasite. Both basophils (circulating) and mast cells (tissue-resident) bind IgE antibody, display this IgE on the cell sur ace, and maintain histamine-containing granules that are released when exogenous antigen binds to and cross-links the IgE. Basophils and mast cells are important in allergic responses. Eosinophils and

basophils are so named because they exhibit eosinophilic and basophilic patterns, respectively, when stained with Wright-Giemsa stain.

Antigen-Presenting Cells

Antigen-presenting cells (APCs) process the macromolecules (especially proteins) o an invading agent to display the processed ragments on the sur ace o the APC. In this orm, the ragments serve as molecular ngerprints used by cells o the adaptive immune system to recognize the invading agent. APCs are important initiators o immune responses because, in addition to displaying nonsel antigens to T cells (see below), they provide the costimulatory signals that are necessary or T-cell activation. The concept o costimulation, in which two separate signals are required to initiate an immune response to a stimulus, is discussed below.

786 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

TABLE 42-1 Cells of the Immune System CELL TYPE

FUNCTION

Innate Immunity Macrophage

Tissue-resident cell, derived rom monocyte Phagocytoses cellular and oreign debris Involved in chronic inf ammation Antigen-presenting cell

Dendritic cell

Transports and presents antigen to T cells in lymph nodes Antigen-presenting cell

Neutrophil

Phagocytoses and kills invading pathogens, especially bacteria

Eosinophil

De ends against parasites

Basophil/mast cell

Release histamine, leukotrienes, and other mediators a ter exposure to antigen

Adaptive Immunity Cytotoxic T cell (TC)

E ector o cellular adaptive immunity

Helper T cell (TH)

Controls adaptive immune responses

B cell

Synthesizes and secretes antibody Antigen-presenting cell

In contrast, adaptive immune cells mount a aster and more intense response upon reexposure to the in ectious agent. The pattern recognition unction o innate immune cells is mediated by multiple mechanisms. One important mechanism involves Toll-like receptors (TLRs). TLRs are transmembrane proteins that bind to shared microbial components such as LPS expressed by Gram-negative bacteria, mannans expressed by ungi, and double-stranded RNA expressed by viral pathogens. Ten TLRs are expressed in humans, and each has a characteristic immune cell distribution and set o pathogen-associated ligands. For example, TLR4 is expressed by antigen-presenting cells and it binds to LPS. Binding o TLRs to their ligands activates an intracellular signaling cascade that converges on the expression o proinf ammatory cytokines, leading to urther immune cell recruitment and activation o the inf ammatory response. A undamental role o innate immunity is to provide an immediate “alarm” that recruits elements o adaptive immunity. This alarm indicates that a molecular structure associated with a pathogen has been detected and serves as an early warning system that the adaptive immune system should initiate an immune response against the pathogen-associated antigens encountered in the context o a TLR agonist or another innate immune signal. Several pharmacologic agents are being investigated as modulators o TLR signaling. Imiquimod, discussed in Chapter 38, Pharmacology o Viral In ections, may unction as a TLR agonist.

Adaptive Immunity Monocytes that exit the bloodstream and take up residence in the tissues can di erentiate into macrophages . As “pro essional APCs,” macrophages process and present antigenic ragments o an invading pathogen or recognition by T cells. The ability o macrophages to envelop and destroy pathogens is enhanced by other components o the immune system, including antibodies and complement (which mediate opsonization) and cytokines (which enhance killing ability). In addition, macrophages produce cytokines such as TNF- that modi y immune responses. Dendritic cells are the most important APCs or the initiation o adaptive immune responses. In the nonlymphoid tissues, dendritic cells engul and process oreign antigens. Dendritic cells then migrate to lymphoid tissues, where they present these cognate antigens to T cells via speci c molecular interactions.

Activation of the Innate Immune Response

Innate immune cells respond to common determinants that are present on many invading agents ( or example, lipopolysaccharide [LPS] in the outer membrane o Gram-negative bacteria). In this role, innate immune cells use pattern recognition to phagocytose a class o in ectious agents rather than a speci c in ectious agent. In contrast, adaptive immune cells, as discussed below, mount a speci c response to the three-dimensional con ormation o a particular antigen, re erred to as an epitope . From a teleological perspective, innate immunity provides a broad gating unction, attempting to counteract harm ul e ects o oreign invaders in a rapid manner and to determine whether an in ectious agent should be urther attacked by adaptive immunity, while adaptive immunity provides a specialized response that is speci c to the particular invading in ectious agent. In an individual, innate immune cells respond in the same way and to the same extent to repeated in ections with the same agent.

The main eatures o the adaptive immune system, speci city to oreign antigens and tolerance to sel -antigens, rely on two principles. First, there must be a mechanism to generate a speci c response to a oreign antigen. Second, adaptive immune cells must be able to distinguish native (sel ) cells and soluble actors rom oreign (nonsel ) cells and soluble actors. The rst property is provided by major histocompatibility complex (MHC) proteins and by somatic gene recombination in T cells and B cells, whereas the second property is provided by signals rom the innate immune system, by regulated immune cell development, and by costimulation.

Major Histocompatibility Complex

MHC proteins are transmembrane proteins that bind and display on their sur ace proteolytically degraded protein ragments and, in some cases, glycolipid antigens. There are two classes o MHC proteins: MHC class I and MHC class II. MHC class I proteins primarily display ragments o cytosolic proteins (Fig. 42-2). All nucleated cells express MHC class I proteins; the repertoire o protein ragments displayed by MHC class I proteins on a cell provides a ngerprint or all the proteins expressed within that cell. I a cell is expressing a recognizable pattern o proteins, then it will not be attacked by the immune system. However, i oreign (e.g., viral) proteins are being generated within the cell, then proteolytic ragments o those viral proteins will be displayed on MHC class I proteins at the sur ace o the cell, and the immune system will recognize that cell as virally in ected. Antigens presented by MHC class I proteins are recognized by T cells bearing the cell sur ace protein CD8. (The designation “CD” stands or cluster of differentiation or cluster designation and is a system or naming an ever-growing list o cell-associated antigens. Each antigen must be de ned by at least two di erent monoclonal antibodies in order to earn the CD designation. CD antigens now number in the hundreds and are present on leukocytes and other cell types.)

C h a p t e r 4 2 Principles o Inf ammation and the Immune System 787 A MHC cla s s I

P rote in fra gme nt

CD8 binding s ite

MHC cla s s I prote in β2 microglobulin

P rote in fra gme nts Cytopla s mic prote in

S e cre tory prote in

Endopla s mic re ticulum Nucleated cell

B MHC cla s s II

CD4 binding s ite

P rote in fra gme nt MHC cla s s II prote in

P rote in Endocytos is

De gra da tion

P rote in fra gme nts

MHC class II proteins display protein ragments derived rom endocytic vesicles. In contrast to class I proteins, which are expressed on all nucleated cells, MHC class II proteins are expressed mostly on antigen-presenting cells (e.g., macrophages and dendritic cells), although some other cell types can be induced to express MHC class II proteins. Endocytic vesicles contain antigenic protein ragments derived rom inectious agents a ter phagocytosis and proteolytic processing o those agents. There ore, the protein ragments expressed on MHC class II proteins generally identi y extracellular oreign agents (e.g., bacteria). As discussed below, T cells bearing the cell sur ace protein CD4 recognize antigens presented by MHC class II proteins. In the process, these T cells stimulate the antigen-presenting cells to produce soluble actors called cytokines and chemokines, which, in turn, aid the T cells in responding to the antigen. In general, then, protein fragments bound to MHC class I identify infected cells, whereas fragments bound to MHC class II identify infectious agents. However, because o the phenomenon o cross-presentation, some proteins generated in the cytosol can be presented by MHC class II to CD4 T cells, and some phagocytosed antigens can be presented by MHC class I to CD8 T cells.

Immune Diversity

While MHC proteins provide a mechanism or distinguishing in ected cells and in ectious agents rom unin ected cells, somatic gene recombination and other processes or generating diversity provide a mechanism or generating a specif c response to an in ection. By recombination, immunoglobulin and T-cell receptor genes semi-randomly create millions o modular three-dimensional protein structures, re erred to as variable regions. Recombined variable regions may undergo somatic hypermutation to create additional diversity that, in the aggregate, can recognize almost any structure. This is the primary mechanism by which the immune system generates an astounding diversity o immune responses.

Humoral and Cellular Immunity

Antigen-presenting cell

FIGURE 42-2. Class I and class II major histocompatibility complex proteins. A. Arepresentative raction o cytoplasmic proteins are proteolytically degraded in the cytosol, and the protein ragments are transported to the endoplasmic reticulum (ER). A raction o secretory proteins are degraded directly in the ER. MHCclass I protein, in association with 2 microglobulin, binds a ragment o the degraded cytoplasmic or secretory protein in the ER. The MHC class I:protein ragment complex is transported to the cell sur ace, where it serves as a ngerprint or the diversity o proteins expressed by that cell. The CD8 binding site on MHC class I ensures that the class I protein:antigen complex interacts only with cytotoxic T cells, which express CD8. All nucleated human cells express MHC class I proteins. B. Antigen-presenting cells phagocytose and degrade bacteria and other oreign agents, generating protein ragments that bind to MHC class II protein in the ER. The MHC class II:protein ragment complex is transported to the cell sur ace, where it serves to display all the potentially nonsel antigens that have been ingested by that cell. The CD4 binding site on MHC class II ensures that the class II protein:antigen complex interacts only with helper T cells, which express CD4. Pro essional antigen-presenting cells (B cells, macrophages, and dendritic cells) are usually the only cell types that express MHC class II proteins, but other cells can be induced to express class II proteins and present antigens under some circumstances.

Adaptive immunity is generally divided into humoral immunity and cellular immunity. In the basic (simplif ed) model o the immune system, the primary cells mediating humoral immunity are B cells, and those mediating cellular immunity are T cells (Table 42-1). The humoral response involves the production o antibodies specif c or an antigen. Mature B cells are characterized by CD19 and CD20 expression, and they can di erentiate into plasma cells. Upon antigen stimulation, plasma cells secrete antibodies against extracellular in ectious agents such as bacteria. In contrast, the cellular response involves activation and clonal expansion o T cells that recognize a specif c antigen. Some T cells recognize in ected cells and then lyse those cells using cytotoxic proteins called perforins and granzymes . Cellular immune responses are there ore e ective against many intracellular in ectious agents such as viruses. In addition to their role in cellular immunity, T cells control the extent o immune responses. Each T cell evolves so that it is activated by only one specif c MHC:antigen complex. All T cells express an MHC:antigen-specif c T-cell receptor (TCR). T cells are divided into cytotoxic T cells (TC) and helper T cells (TH) based on the type o coreceptor expressed and the unction imparted by that coreceptor (Fig. 42-3).

788 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY A Cytotoxic T ce ll T ce ll re ce ptor

MHC cla s s I prote in

β2 microglobulin

Antige n

CD8

Cytotoxic T ce ll

Virus -infe cte d ce ll

B He lpe r T ce ll MHC cla s s II prote in T ce ll re ce ptor Antige n

MHC:antigen complex with su ciently high a nity that the TH cell is activated by the antigen-presenting cell. In addition to initiating and strengthening the immune response, TH cells control the type o immune response by producing one or another set o cytokines. TH cells can be generally divided into TH1, TH2, and TH17 subtypes based on the cytokines produced by the cells. TH1 cells characteristically produce IFN- and IL-2, and these cytokines inf uence the development o cell-mediated immune responses o both CD8 TC cells and other CD4 TH cells. IL-2 interacts with CD25, a receptor expressed on activated T cells, to mediate the early steps in T cell activation. TH2 cells characteristically produce IL-4, IL-5, and IL-10, and these cytokines enhance antibody production by B cells. The TH2 cell subtype is more o ten associated with autoimmunity (see Chapter 46). TH17 cells characteristically produce IL-17, IL-21, and IL-22. IL-17 iso orms recruit neutrophils and ampli y the immune response. TH17 cells are generated rom CD4 cells when these cells are stimulated by IL-23. Drugs that block the maturation or growth o TH17 cells are available or clinical use in the treatment o certain autoimmune diseases.

Tolerance and Costimulation IL-2R IL-2

He lpe r T ce ll

CD28

B7

CD4

Antige n-pre s e nting ce ll

FIGURE 42-3. Activation of cytotoxic and helper T cells. T cells mediate and regulate the cellular immune response. A. Cytotoxic T cells (TC) are the primary mediators o cellular immunity. These cells express T-cell receptors (TCR) and CD8. The TCR identif es nonsel antigens bound to MHC proteins, and CD8 ensures that TC cells interact only with cells expressing MHC class I proteins. In the example shown, the interaction o a TC cell with the MHC class I protein o a virus-in ected cell leads to activation o the TC cell and subsequent killing o the virus-in ected cell. B. Helper T cells (TH) are the primary regulators o cellular immunity. These cells express TCR and CD4. CD4 binds to MHC class II proteins on antigen-presenting cells (APCs); this interaction ensures that TH cells interact only with cells expressing MHC class II proteins. An additional degree o specif city is provided by the interaction o CD28 on TH cells with proteins o the B7 amily on APCs; this “costimulatory signal” is required or TH cell activation. In the example shown, the interaction o a TH cell with the MHC class II and B7 proteins o an antigen-presenting cell leads to activation o the TH cell. The activated TH cell secretes IL-2 and expresses the IL-2 receptor (IL-2R); this autocrine pathway stimulates urther TH cell proli eration and activation. IL-2 and other cytokines secreted by the TH cell activate not only TH cells but also TC cells and B cells.

TC cells are the mediators o cellular adaptive immunity.

These cells express the CD8 coreceptor, which recognizes a constant (i.e., antigen-independent) domain on MHC class I proteins. This coreceptor unction allows the antigen-speci c TCR on TC cells to bind a speci c class I MHC:antigen complex with su ciently high a nity that the TC cell is activated by the cell expressing the class I MHC:antigen complex. Speci c activation o the TC cell initiates a chain o events, including the secretion o membrane-penetrating per orins and apoptosis-inducing granzymes, which result in the death o the cell displaying the oreign antigen. TH cells are primarily the regulators o adaptive immunity. TH cells are identi ed by their expression o the CD4 coreceptor, which recognizes an antigen-independent domain on MHC class II proteins. This coreceptor unction allows the antigen-speci c TCR on TH cells to bind a speci c class II

Diversity in the variable regions o immunoglobulins and T-cell receptors creates the potential or some o these molecules to recognize and attack native proteins, a circumstance termed autoimmunity. There are two primary mechanisms or avoiding autoimmunity. The rst is clonal deletion, in which T cells die during development when they express high-a nity receptors that recognize sel -antigen. In a second process re erred to as tolerance or anergy, cells o the immune system undergo a care ully regulated series o steps during development to ensure that mature immune cells do not recognize native proteins. Costimulation—the requirement or multiple simultaneous signals to initiate an immune response—ensures that stimulation o a single immune receptor does not activate a damaging immune reaction. Signal 1 provides speci city, while signal 2 is permissive, ensuring that an inf ammatory response is appropriate. Regulation o costimulatory molecules is a mechanism whereby the innate immune system regulates the extent o an immune response. I antigen is presented without a coincident costimulatory signal (i.e., without innate immune activation), then anergy may result, whereby a cell becomes unreactive and will not respond to urther antigenic stimuli. Drugs that induce anergy could be therapeutically attractive because such agents could allow long-term acceptance o an organ gra t or limit the extent o an autoimmune disease. For T cells, signal 1 is mediated by the MHC:antigen:TCR interaction. Signal 2 is mediated predominantly by the interaction o CD28 on T cells with B7-1 (also called CD80) or B7-2 (CD86) on activated antigen-presenting cells (Fig. 42-4). Resting T cells present CD28, which can bind either B7-1 or B7-2. B7-1 and B7-2 are not normally present on antigen-presenting cells, but their expression is increased by the innate immune system during an immune response to a pathogen. The lack o expression o B7 molecules in the absence o an innate immune response may help to limit inappropriate adaptive immune responses. When a T cell receives both signal 1 and signal 2, the T cell is activated, IL-2 is expressed, and clonal expansion o TH cells speci c or that oreign epitope occurs. Activated T cells eventually down-regulate CD28 expression and up-regulate

C h a p t e r 4 2 Principles o Inf ammation and the Immune System 789

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FIGURE 42-4. Costimulation in the T-cell activation pathway. Two signals are required or activation o a T-cell response to antigen. A. I an antigenpresenting cell (APC) presents an antigen to a T cell in the absence o an appropriate costimulatory signal, the T cell does not respond and may become anergic. B. I an APC presents both the antigen and a costimulatory molecule such as B7, the T cell proli erates and di erentiates in response to the antigenic stimulus. Cytokines secreted by the activated APC augment T cell activation.

CTLA-4 expression. CTLA-4, like CD28, binds B7-1 and B7-2 but with much higher a f nity than CD28. In contrast to the activating CD28 signal, interaction o CTLA-4 with B7-1 or B7-2 inhibits T cell proli eration. This appears to be a physiologic mechanism or sel -limitation o the immune response. Other inhibitory immune checkpoint signals are discussed in Chapter 46. CD40 ligand (CD40L) is another mediator o costimulation. Activated T cells express CD40L (CD154). CD40 is expressed on antigen-presenting cells, including B cells and macrophages

A

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(Fig. 42-5). Interaction o TH cell CD40L with B cell CD40 promotes B cell activation, isotype switching, clonal expansion, and a f nity maturation. Interaction o TH cell CD40L with macrophage CD40 promotes macrophage expression o B7-1 and B7-2. These molecules, as mentioned above, are crucial or costimulation o T cells. This pathway thus provides a positive eedback mechanism whereby activated T cells can promote urther expansion o activated T cells. In addition, the increased expression o B7-1 and B7-2 on macrophages is important or promoting CD8 TC cell activation.

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FIGURE 42-5. Costimulation and the CD40–CD40L interaction. A. An antigen-presenting cell (APC) presents MHC class II-bound antigen to a CD4 T cell. T-cell recognition o antigen initiates an intracellular signaling cascade that leads to expression o CD40 ligand (CD40L) at the T-cell sur ace. B. CD40Lon the activated T cell binds to CD40 on the sur ace o the APC. Activation o CD40 generates an intracellular signaling cascade that leads to expression o B7 on the APC sur ace. C. Enhanced T-cell proli eration and di erentiation are promoted by costimulation o the T cell by the MHC class II:antigen complex (which binds to the T-cell receptor), CD40 (which binds to T-cell CD40L), and B7 (which binds to T-cell CD28). Cytokines secreted by the activated APC augment T-cell proli eration and di erentiation.

790 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Because the CD40–CD40L interaction promotes multiple costimulation pathways, it has been hypothesized that blockade o CD40L could produce tolerance. Preliminary studies have demonstrated that blockade o CD40L with anti-CD40L antibody can indeed produce tolerance and long-term gra t survival in animal models o organ transplantation. Increasing experimental evidence suggests that peripheral tolerance is maintained by a subset o T cells re erred to as regulatory T cells (Treg). These cells, the best characterized o which are CD4 CD25 , elaborate inhibitory cytokines in response to sel -antigens and thereby limit the immune response to these antigens. Pharmacologic induction o Treg cells may have applications in transplantation and autoimmune diseases, including type 1 diabetes.

CHEMICAL MEDIATORS OF INFLAMMATION The discussion to this point has ocused on the cells o the immune system and their roles in mounting an immune response. Equally important are the molecular mediators o immune cell activity. The ollowing discussion highlights endogenous molecules that regulate the inf ammatory process. (Note that signaling pathways or immune cells are discussed mainly in Chapter 46, although there is some overlap among the endogenous mediators o inf ammation and immunity, especially among the cytokines.) The list o mediators is long (Table 42-2), and essentially all o these signaling systems

TABLE 42-2 Chemical Mediators o the Inf ammatory Response RESPONSE

MEDIATORS

Vasodilation

Histamine C3a, C5a (complement components) Prostaglandins (PG) PGI2, PGE1, PGE2, PGD2 Nitric oxide (NO) Bradykinin Plasmin

Increased vascular permeability

Histamine C3a, C5a Leukotrienes (LT), especially LTC4, LTD4, LTE4 Bradykinin Platelet-activating factor Substance P Calcitonin gene-related peptide (CGRP)

Chemotaxis and leukocyte activation

C3a, C5a LTB4, lipoxins (LX) LXA4, LXB4 Platelet-activating factor Bacterial products

Tissue damage

NO Oxygen radicals Neutrophil and macrophage lysosomal products

Fever

PGE2, PGI2, LTB4, LXA4, LXB4 Interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF)

Pain

PGE2, PGI2, LTB4 Bradykinin Substance P CGRP

have been explored as potential pharmacologic targets. Only those mediators most crucial to inf ammation and those or which therapies already exist are discussed in detail here.

Histamine Histamine , one o the initiators o the inf ammatory response,

is constitutively synthesized and stored in the granules o mast cells and basophils. These cells migrate through tissue on a continual basis. Any injury, rom physical trauma to microbial invasion, stimulates mast cells to release histamine into the interstitium. Histamine is re erred to as a vasoactive amine because its inf ammatory e ects occur mainly on the vasculature: histamine release stimulates dilation o arterioles and postcapillary venules, constriction o veins, and contraction o endothelial cells. These e ects are responsible or the early changes in hemodynamics and vascular permeability discussed below. Several classes o pharmacologic agents modi y histamine signaling; these agents are discussed in Chapter 44.

Complement Complement is a system o serine proteases that is one o

the rst innate mechanisms to be activated in response to injury. The complement system can be activated by antigen–antibody interactions (the classical pathway), by direct interactions with oreign sur aces (the alternative pathway), or by interactions with certain complex carbohydrates (the lectin pathway). In each pathway, a series o proteolytic reactions converts a complement precursor protein, re erred to by the letter “C” ollowed by a number ( or example, C3), into its active orm(s), indicated by the letter “a” or “b” ( or example, C3a and C3b; in this case, both orms are active). The general scheme o this pathway is analogous to that o the coagulation cascade (see Chapter 23, Pharmacology o Hemostasis and Thrombosis), in which precursor proteins are proteolytically cleaved to active products that contribute to the actions o the cascade. A ter activation, complement triggers inf ammatory responses by two mechanisms. First, several cleavage products o the complement cascade are potent stimulators o inf ammation. For example, C3b is an important opsonin, and C3a and C5a mediate leukocyte chemotaxis. Second, the nal step in complement activation is the assembly o the membrane attack complex. This complex o complement proteins produces large pores in the outer membrane o Gramnegative bacteria, leading to lysis o the bacteria. A large number o complement regulatory proteins, both soluble and on the cell sur ace, care ully govern and localize complement activation to the site o inf ammation. Pharmacologic inhibitors o complement activation are used to lessen the tissue injury associated with inappropriate inf ammatory responses ( or example, in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome).

Eicosanoids Eicosanoids are metabolites o arachidonic acid, a atty acid

component o phospholipids in the inner leaf et o the plasma membrane o many cell types. Inf ammatory mediators such as cytokines and complement stimulate the enzymatic release o arachidonic acid rom the plasma membrane. Multiple biochemical reactions ensue, resulting in the ormation o prostaglandins, leukotrienes, and other eicosanoids.

C h a p t e r 4 2 Principles o Inf ammation and the Immune System 791

Notably, certain arachidonic acid derivatives are proinf ammatory, whereas others serve to limit inf ammation. This underscores the act that acute inf ammation is a sel -limited process and that the process o pathogen destruction is intimately tied to the process o tissue repair. Chapter 43 is devoted to an in-depth discussion o eicosanoid physiology, pathophysiology, and pharmacology.

Cytokines Cytokines are proteins that act in a paracrine manner to regulate leukocyte activity. Interleukins and tumor necrosis actor (TNF) amily members are cytokines secreted primarily by

cells o the hematopoietic lineage. Interleukin-1 (IL-1) and tumor necrosis actor- (TNF- ) are among the key cytokines elaborated in the acute inf ammatory response; these cytokines were two o the mediators responsible or Mark’s ever. Another member o the TNF amily is the B lymphocyte stimulator (BLyS), which promotes B cell survival and di erentiation. Located on T cells and B cells, TNF receptors contain membrane glycoprotein CD30, which is important in cellular proli eration and survival and serves as a pharmacologic target (see Chapter 46). Chemokines are a subset o cytokines that promote immune cell tra cking, transmigration, and localization to sites o inf ammation. For example, macrophage chemoattractant protein-1 (MCP-1) promotes monocyte transmigration and activation. Other notable cytokines include the hematopoietic growth actors granulocyte-monocyte colony-stimulating actor (GMCSF) and granulocyte colony-stimulating actor (G-CSF) (see Chapter 45). Because cytokines a ect the proli eration and unction o cells that mediate innate and adaptive immune responses, selective inhibition or stimulation o cytokine action has the potential to modulate immune and inf ammatory responses. Pharmacologic uses or cytokine and anticytokine therapies are discussed in Chapter 45 and Chapter 46, respectively.

Other Agents As noted in Table 42-2, other signaling molecules also coordinate the inf ammatory response. These include kinins, platelet-activating actor, nitric oxide, oxygen radicals, and other leukocyte and bacterial products released during phagocytosis. Although pharmacologic agents have been developed to modulate each o these pathways, there are, as yet, no approved anti-inf ammatory drugs that speci cally interrupt the action o these mediators.

THE INFLAMMATORY RESPONSE The cells and soluble mediators o the immune system interact with one another to generate the inf ammatory response , which typically occurs in our phases. First, the vasculature around a site o injury reacts to recruit cells o the immune system. Second, circulating immune cells migrate rom these vessels into the injured tissues, and the mechanisms o innate and adaptive immunity (see above) serve to neutralize and remove the inciting stimulus. Next, the process o repair and tissue healing ensues and the acute inf ammatory process is terminated. I the process o acute inf ammation is not halted but continues to smolder, chronic inf ammation can occur.

Dilation o Vessels Within hours o being cut, Mark’s thumb begins to exhibit the ve classic signs o inf ammation presented in the introduction. Initially, these signs and symptoms result rom alterations in vascular hemodynamics at the site o injury. Injury to a tissue causes the release o inf ammatory mediators (discussed earlier) that dilate arterioles and postcapillary venules; in turn, vasodilation leads to increased blood f ow to the site o injury, causing the clinical signs o redness and warmth. Inf ammatory mediators also cause contraction o vascular endothelial cells, leading to increased capillary permeability and the development o an exudate (i.e., interstitial f uid with a high protein content); in turn, the exudate causes the clinical mani estation o swelling. Pain develops due to both increased tissue pressure and the action o various inf ammatory mediators.

Recruitment o Cells Increased vascular permeability also allows cells in the blood to enter the interstitium. Cellular migration out o the blood is not random; rather, leukocyte recruitment is orchestrated to optimize clearing o the in ection and local repair o the injured tissue (Fig. 42-6). At the onset o an inf ammatory response, the endothelial cells at the site o injury are activated to express adhesion molecules that bind speci c receptors expressed by leukocytes. For example, intercellular adhesion molecules (ICAMs ) and vascular cell adhesion molecules (VCAMs ) expressed by the vascular endothelium bind integrins expressed on the cell sur ace o leukocytes. This interaction causes the leukocytes, which normally roll along the sur ace o the endothelium by means o loose, transient binding interactions, to adhere tightly to the activated endothelium at the site o injury. The adherent leukocytes then bind other endothelial cell receptors that promote transmigration (diapedesis) o the leukocytes rom the vasculature into the interstitium. Speci city o the immune response is achieved according to the pattern o adhesion molecules expressed by the activated endothelium and by the various types o leukocytes; or example, neutrophils dominate the early inf ammatory response, while monocytes predominate a ter 24 hours.

Chemotaxis Once the cells o the immune system cross the endothelial barrier, they migrate through the interstitium to the speci c site o injury or in ection. Immune cell targeting is accomplished by the process o chemotaxis , or chemical signaling. Inf ammatory mediators released at the site o injury, such as N- ormyl peptides derived rom bacterial proteins or endogenous mediators such as C5a and leukotriene B4 (LTB4), create a chemical gradient to which the leukocytes respond, allowing them to crawl pre erentially toward the site o the inf ammatory reaction.

Phagocytosis Upon their arrival at the site o injury or in ection, neutrophils, macrophages, and other cells o the immune system are ready to per orm their duties. However, these cells require one urther stimulus to activate their killing machinery. Foreign substances must be coated by an opsonin be ore they can be ingested (phagocytosed) by leukocytes. Opsonins are

792 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY A Norma l

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FIGURE 42-6. Overview o the inf ammatory response. A. Leukocytes circulating in the blood interact with selectins expressed on the sur ace o vascular endothelial cells. In the absence o inf ammation, the interaction between leukocytes and endothelial cells is weak, and leukocytes either f ow past or roll along the endothelium. Neutrophil rolling is mediated by the interaction between endothelial cell E-selectin and neutrophil sialyl-Lewis x (s-Le x). B. During the inf ammatory response, endothelial cells up-regulate their expression o intercellular adhesion molecules (ICAMs). ICAM expression increases the potential or strong binding interactions between leukocytes and the activated endothelial cells. For example, ICAM-1 on endothelial cells binds tightly to LFA-1 on neutrophils. The enhanced cell–cell interaction leads to tethering (rolling, activation, and rm adhesion) o leukocytes to endothelial cell sur aces and initiates the process o leukocyte diapedesis and transmigration rom the vascular space into extravascular tissues. Leukocytes migrate through injured tissue in response to chemokines such as IL-8, which are inf ammatory mediators released by injured cells and by other immune cells that have already reached the site o injury.

molecular adaptors that coat oreign sur aces and signal leukocytes that a particle should be attacked. The major opsonins consist o complement, immunoglobulins (antibodies), and collectins (plasma proteins that bind to certain microbial carbohydrates). The interaction o a phagocytic cell with an opsonized particle initiates engul ment and destruction o the o ending agent. This step is also a crucial point o interaction between innate and adaptive immunity. Antigenpresenting cells process engul ed particles and present their antigens to B cells and T cells, which then react to the antigens. In the introductory case, Mark’s cut presumably allowed bacteria to penetrate his skin barrier, leading to in ection. The presence o these bacteria initiated an inf ammatory response that included phagocytosis o bacteria by APCs, presentation o bacterial antigens to TH cells, activation and expansion o TH cells, TH cell activation o urther APC-mediated phagocytosis, and synthesis and secretion o antibodies speci c or the bacteria.

Resolution Tissue repair and reestablishment o homeostasis are the nal events in the acute inf ammatory response. The same mediators that activate inf ammation also initiate a cascade o tissue repair; this process is mediated by the release o growth actors and cytokines, including epidermal growth actor (EGF), platelet-derived growth actor (PDGF), basic broblast growth actor-2 (bFGF-2), trans orming growth actor- 1 (TGF- 1), IL-1, and TNF- . These actors act as mitogens or endothelial cells and broblasts and ultimately stimulate healing and scar ormation through angiogenesis ( ormation o new blood vessels) and the ormation o granulation tissue. In the introductory case, the granulation tissue and eventual scar will be the only record o Mark’s acute inf ammatory event. O note, angiogenesis can be a pathologic state when it is associated with abnormal blood vessel growth or tumor growth, and pharmacologic inhibitors o angiogenesis are currently being used to treat

age-related macular degeneration (where abnormal blood vessels obscure vision) and as antineoplastic agents (see Chapter 40, Pharmacology o Cancer: Signal Transduction).

CHRONIC INFLAMMATION Chronic inf ammation is a pathologic state characterized by the continued and inappropriate response o the immune system to an inf ammatory stimulus. Chronic inf ammation accounts or the symptoms o many autoimmune diseases and may be an important cause o organ transplant rejection. In contrast to the acute inf ammatory response, which is dominated by neutrophils, one o the hallmarks o chronic inf ammation is the predominance o macrophages. Activated macrophages secrete collagenases and growth actors in addition to inf ammatory mediators such as proteases and eicosanoids. These secreted products initiate and maintain a cycle o tissue injury and repair, leading to tissue remodeling. Over time, chronic inf ammation can cause relentless tissue destruction. Promising treatments or chronic inf ammation could include cytokine inhibitors that neutralize mediators o the signaling cascades that perpetuate chronic inf ammation. These agents are discussed in Chapter 46.

CONCLUSION AND FUTURE DIRECTIONS The immune system intricately regulates the response to tissue injury and in ection. A complete review o immunology is beyond the scope o this text; instead, the discussion in this chapter presents a broad overview and highlights elements o immunity that may be addressed pharmacologically. Innate immune mechanisms respond to patterned elements shared among a class o in ectious agents, such as bacterial lipopolysaccharide or viral RNA. The innate immune system also processes these agents and presents them to lymphocytes, thereby activating the adaptive immune system.

C h a p t e r 4 2 Principles o Inf ammation and the Immune System 793

The adaptive immune system develops a response speci c to an in ectious agent or inf ammatory stimulus. As part o the inf ammatory response, the adaptive immune response also includes mechanisms that mediate tolerance to distinguish sel rom nonsel ; dysregulation o these mechanisms may lead to chronic inf ammation and autoimmune disease. Many anti-inf ammatory drugs act to deplete populations o innate or adaptive immune cells; this concept is discussed in more detail in Chapter 46. The chemical mediators o the inf ammatory response—including histamine, complement, eicosanoids, and cytokines—are also major targets o current pharmacologic therapies. Macromolecular drugs are playing an increasingly important role in modulation o these chemical mediators; or example, anticytokine antibodies that inhibit tumor necrosis actor- have been developed or the treatment o rheumatoid arthritis, psoriatic arthritis, and inf ammatory bowel disease. A second approach to the modulation o inf ammatory responses is to target the intracellular signaling cascades responsible or the initiation o immune responses. Cyclosporine, discussed in Chapter 46, is an example o such a drug. As the number o agents available or treatment o immune disorders grows, it will also be important to determine whether

macromolecular agents and small-molecule signaling inhibitors can be used in combination to target multiple steps in inf ammation.

Suggested Reading Dinarello CA. Anti-inf ammatory agents: present and uture. Cell 2010; 140:935–950. (A signaling-oriented overview o targets or development o new anti-in ammatory agents.) Ibelgau ts H. COPE: cytokines & cells online path nder encyclopedia. http://www.copewithcytokines.de/cope.cgi. (Website that describes all known actions o cytokines.) Iwasaki A, Medzhitov R. Control o adaptive immunity by the innate immune system. Nat Immunol 2015;16:343–353. (Advances in understanding interactions between the innate and adaptive immune systems.) Littman DR, Rudensky AY. Th17 and regulatory T cells in mediating and restraining inf ammation. Cell 2010;140:845–858. (Discusses advances in T cell subsets and regulatory T cell biology.) Matesanz-Isabel J, Sintes J, Llinas L, de Salort J, Lázaro A, Engel P. New B-cell CD molecules. Immunol Lett 2011;134:104–112. (An updated classif cation o molecules with the CD designation.) Murphy KM, Travers P, Walport M. Janeway’s immunobiology. 8th ed. New York: Garland Publishing; 2011. (A general immunology textbook.) Pier GB, Lyczak JB, Wetzler L. Immunology, in ection and immunity. Washington, DC: ASM Press; 2004. (A detailed text with a ocus on immunologic mechanisms.)

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Thromb Thr hroo mbo mbox b oxxa nee

P GF F

2α INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 794–795 EN . . . . . . . . . . . . . . . . 808 PHAR PH ARMA MACOLOGIC CLASSES AND AGENTS P GH G 2 s y yn yntha t s e re ducta s eGEN Th b Thro mboxane TP ) 4 rrus, Phoospholipase Ph p Inhibitors . (ute . . . rus .us, . . .lung) . . . . . . . . . . . . . . . . . . . . . . . 808 antag nis ts SANO g oEICOSA PHYSIOLOGY OF NOID OID D MET TAB ABOL OLIS ISM M . . . . . . . .(pla . . te . .le. ts. 794 Cyclooxy xyge gena e nase Inhibitors . . . . . . . . . . . . . . . . . .HO . . . . . . . . . . . 808 Generationn o Ar Arachidonic Acid and Omega-3 Traditional tive ve Inh nhib ibit itors:: NSAIDs . . . . . . . . . . . . 8088 Fatt tty Acids ds. . . . . . . . . . . . . . . . . . . . .COOH . . . . . . . . . . . . . . . . . . . . 795 P GE E 2 n Nonselecti O COOH is ome oAcetaminophen ra s e i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810 81 Cyclooxygenase nase Pathway nas Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795 (ma cropha roph ge s, ma Selection s t ce lls ) o of the Appropr O p iaa te NSAID D . . . . . . . . . . . . . . . . . . 810 Prostaglandinss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798 OH COX-2 Inhibitors n . . . . . . . . . . . . . . . . . . . . . . . . .HO . . . . . . . . . . . 81 811 Thro hromb mbbox oxan a e a ndd Prostacyclin an n . . . . . . . . . . . . . . . . . . . . . . 798 OH TxA Prostano noid id d R ecep ec epto tor r Mimetics Mi . . . . . . . . . . . . . . . . . . . . . . . . . 812 Lipoxygenase Pathway Li y . . . . .2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 799 P GF 22α O Thr Th r omboxane Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 Leukotrienes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7999 N None nzymaProtectins, tii t tic Lee ukotriene uk kotri trien enee Inhi Inhibit hibi biti tion ion onCOOH 1 C OOH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 Lipoxins, Resolvins, and Marres esin inss . . . . . . . . . . 802 hhydroly yydroly d lys iis Lipoxygenase oxygenaa se Inhibition Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 812 FP2 Epoxyg ygenas asee Pa Path thwa way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802 5-Lipoxygenase Li ygenase Activating Acti Protein (FLAP) Isoprostanes . . . . . OH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802 HO S mo o th mus cle c o ntrac tio n OH Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Br . . o nc ho c o ns 813 Bro tricc tio n tr Metabolic Inactivation tion o Local Eicosan ti Eicosanoids n . . . . . . . . . . . . . 802 P GE 2 Synthesis Inhibitors . . . . . . . . . . . . . Ute Leukotriene . . .rine . . . .c.o.nntrac 813 tio n Integrated Inf ammation mmation on Schema . . . . . . . COOH . . . . . . . . . . . . . . . . 802 Leukotriene ko ne Receptor Antagonists . . . . . . . . . . . . . . . . . . . . 813 PATHOPHYSIOLOGY OF EICOSANOIDS . . . . . . . . . . . . . . . . . . . . 806 Lipoxins, Aspirin-Triggered Lipoxins, Resolvins/Protectins/ Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807 Maresins, and Lipoxin-Stable Analogues . . . . . . . . . . . . . . 813 Inf ammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 807 Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 813 Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .814 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808 Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808

INTRODUCTION Autacoids are substances that are rapidly synthesized in

response to specif c stimuli, act quickly in the local environment, and remain active or only a short time be ore degradation. Eicosanoids represent a chemically diverse amily o autacoids that are mostly derived rom arachidonic acid. Research on eicosanoids has def ned their vital roles in in ammatory, neoplastic, and cardiovascular physiology and pathophysiology. Numerous pharmacologic interventions in eicosanoid pathways—including the nonsteroidal antiin ammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, leukotriene inhibitors, and others—are use ul in the clinical management o in ammation, pain, and ever. Given the diverse bioactivities o eicosanoids, uture research in eicosanoid physiology and pharmacology may lead to the development o new therapeutics or the in ammatory and autoimmune bases o asthma, glomerulonephritis, cancer, wound healing, cardiovascular diseases, and other clinical conditions.

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PHYSIOLOGY OF EICOSANOID METABOLISM Eicosanoids are centrally involved in a number o metabolic pathways that exhibit diverse roles in in ammation and cellular signaling. The vast majority o these pathways center on reactions involving the metabolism o arachidonic acid (Fig. 43-1). The ollowing section considers the biochemical steps leading to arachidonic acid generation and then discusses the cyclooxygenase, lipoxygenase, epoxygenase, and isoprostane pathways o arachidonic acid metabolism. The word eicosanoid arises rom the Greek root meaning twenty, and the term classically re ers to unbranched 20-carbon molecules derived rom arachidonic acid oxygenation. The term eicosanoid also applies broadly to various other molecules— such as resolvins, protectins, and maresins—that are derived rom docosahexaenoic acid, a 22-carbon precursor. The term docosanoids is sometimes also used to describe these 22-carbon structures.

C h a p t e r 4 3 Pharmacology of Eicosanoids

Ms . G, a 44-ye a r-o ld Na tive Am e rica n e m a le , vis its h e r p hys icia n b e ca u s e o jo in t p a in a n d p ro g re s s ive a tig u e . He r h is to ry re ve a ls g e n e ra l jo in t s ti n e s s a n d p a in o r th e p a s t 3 w e e ks . Th e p a in is w o rs t in th t e m e ta ca rp o p h a la n g e a l a n d p roxim a l in te rp h a la n ge a l jo in ts a n d is e s p e cia lly p ro m in e n t in th e e a rly m o rn in g . Ms . G is a d vis e d to ta ke ib u p ro e n a s n e e d e d , a n d th is m e d ica tio n p rovid e s re lie o h e r p a in o r s o m e tim e . S ix m o n th s la te r, Ms . G h a s co n tin u e d to ta ke ib u p ro e n d a ily an d s h e n o te s in d ig e s tio n a n d n e w vo m itin g o “ co ee g ro u n d s ” -like m a te ria l. He r p hys icia n re co m m e n d s ce s s a tio n o ib u p ro e n a n d a n u p p e r ga s tro in te s tin a l e n d o s co p ic e xa m in a tio n , w h ich re ve a ls ga s tric m u co s a l e ro s io n a n d h e m o rrh a g e . He r p hys icia n is a ls o co n ce rn e d a b o u t in te rva l p ro g re s s io n in Ms . G’s jo in t s ti n e s s a n d p a in a n d re e rs h e r to a rh e u m a to lo g y clin ic. S h e re p o rts to th e rh e u m a to lo g is t th a t h e r p a in h a s p ro g re s s e d to in clu d e b o th e e t, b o th h a n d s a n d w ris ts , b o th e lb o w s , s o m e ce rvica l ve rte b ra e , a n d th e le t s h o u ld e r. Ove r th e p a s t e w m o n th s , s h e h a s h o u rs o m o rn in g p a in , h a s n o te d d i f cu lty w ith b a s ic h o u s e h o ld ta s ks , a n d h a s a vo id e d p hys ica l a ctivity. On e xa m in a tio n , th e m e ta ca rp o p h a la n g e a l a n d p roxim a l in te rp h a la n g e a l jo in ts o b o th h a n d s a re o u n d to b e s w o lle n , te n d e r, a n d wa rm ; Ms . G a ls o h a s ch a ra cte ris tic u ln a r d e via tio n o h e r f n g e rs a n d “ s wa n -n e ck” d e o rm itie s .

Generation of Arachidonic Acid and Omega-3 Fatty Acids Arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid) is

the common precursor to the vast majority o eicosanoids (Fig. 43-1). Arachidonic acid must be biosynthesized rom the essential atty acid precursor linoleic acid (all-cis-9,12octadecadienoic acid), which humans can obtain only rom dietary sources. Eicosapentaenoic acid (all-cis-5,8,11,14,17eicosapentaenoic acid; EPA) and docosahexaenoic acid (allcis-4,7,10,13,16,19-docosahexaenoic acid; DHA) are the precursors to resolvins, protectins, and maresins. Humans can obtain EPA and DHA either rom dietary sources or rom biotrans ormation o the essential atty acid precursor -linolenic acid (all-cis-9,12,15-octadecatrienoic acid). -Linolenic acid, EPA, and DHA are also called omega-3 fatty acids because they contain a double bond between the third and ourth carbons rom the terminal ( ) end o the molecule. Within cells, arachidonic acid does not exist as a ree atty acid but rather is esterif ed to the sn2 position o membrane phospholipids, predominantly phosphatidylcholine and phosphatidylethanolamine. Arachidonic acid is released rom cellular phospholipids by the enzyme phospholipase A2 (Fig. 43-1), which hydrolyzes the acyl ester bond. This important reaction, which represents the f rst step in the arachidonic acid cascade, is the overall rate-determining step in the generation o eicosanoids.

795

S kin n o d u le s a re a p p a re n t o n th e e xte n s o r s u r a ce o b o th o re a rm s . La b o ra to ry te s ts s h o w a n e le va te d e ryth ro cyte s e d im e n ta tio n ra te (ES R), lo w h e m o g lo b in , a n d p o s itive rh e u m a to id a cto r. Syn o via l u id a s p ira te is n o ta b le o r le u ko cyto s is . Ha n d ra d io g ra p h s s h o w e ro s io n a n d o s te o p e n ia . Be ca u s e th e s ym p to m s , e xa m in a tio n , la b o ra to ry s tu d ie s , a n d ra d io g ra p h s a re co n s is te n t w ith a d ia g n o s is o m o d e ra te ly to s e ve re ly a ctive rh e u m a to id a rth ritis , Ms . G is in itia lly s ta rte d o n a co u rs e o ce le coxib (a COX-2 s e le ctive in h ib ito r), e ta n e rce p t (a TNF- a n ta g o n is t), a n d p re d n is o n e (a g lu co co rtico id ). Ove r th e s u b s e q u e n t m o n th s , th e jo in t p a in , s w e llin g , a n d te n d e rn e s s d e cre a s e n o tice a b ly. J o in t u n ctio n in th e h a n d s is re s to re d , a n d Ms . G is a b le to re s u m e s o m e p hys ica l a ctivity.

Questions 1 . Which eicosanoid mediators may be causing Ms. G’s joint pains? 2 . By what mechanism do glucocorticoids such as prednisone affect eicosanoid levels and/or bioactivity? 3 . By what mechanism did ibuprofen cause Ms. G’s gastric erosion and hemorrhage? 4 . What are the potential concerns regarding long-term use of celecoxib? 5 . By what mechanism does etanercept affect eicosanoid levels and/or bioactivity?

There are membrane-bound and soluble iso orms o phospholipase A2, classif ed as secretory (sPLA2) and cytoplasmic (cPLA2), respectively. The numerous phospholipase A2 iso orms are di erentiated based on molecular weight, pH sensitivity, regulation and inhibition characteristics, calcium requirements, and substrate specif city. The existence o multiple iso orms allows or calibrated regulation o the enzyme in di erent tissues to achieve selective biological responses. Phospholipase A2 iso orms relevant to in ammation are stimulated by cytokines (such as TNF- , GM-CSF, and IFN- ) and growth actors (such as epidermal growth actor [EGF] and the MAP kinase–protein kinase C [MAPK– PKC] cascade). Although glucocorticoids were once thought to inhibit phospholipase A2 directly, it has now been shown that this action is mediated by inducing the synthesis o lipocortins, a amily o phospholipase A2-regulatory proteins. One o the lipocortins, annexin 1, mediates some o the antiin ammatory actions o glucocorticoids (see below).

Cyclooxygenase Pathway Unesterif ed intracellular arachidonic acid is converted by cyclooxygenase , lipoxygenase , or the cytochrome-containing epoxygenase enzymes; the specif c enzyme dictates the particular class o local eicosanoids that are generated. The cyclooxygenase pathway leads to the ormation o prostaglandins , prostacyclin, and thromboxanes ; the lipoxygenase pathways lead to leukotrienes and lipoxins ; and the

796 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY Cle a va ge s ite

O R

R = choline , e tha nola mine , or inos itol

O

P O-

O

O

O

TABLE 43-1 Comparison of COX-1 and COX-2

Arachidona te Acyl

P hos pholipids

PROPERTY

COX-1

COX-2

Expression

Constitutive

Inducible; not normally present in most tissues Constitutive in parts o nervous system

Tissue location

Ubiquitous expression

Inf amed and activated tissues

Cellular localization

Endoplasmic reticulum (ER)

ER and nuclear membrane

Substrate selectivity

Arachidonic acid, eicosapentaenoic acids

Arachidonic acid, -linolenate, -linolenate, linoleate, eicosapentaenoic acids

Role

Protection and maintenance unctions

Proinf ammatory and mitogenic unctions

Induction

Generally no induction hCG can up-regulate COX-1 in amnion

Induced by LPS, TNF- , IL-1, IL-2, EGF, IFNmRNA rises 20- to 80- old upon induction Regulated over 1–3 hours

Inhibition

Pharmacologic: NSAIDs (low-dose aspirin)

In vivo: Anti-inf ammatory glucocorticoids, IL-1 , IL-4, IL-10, IL-13 Pharmacologic: NSAIDs, COX-2 selective inhibitors

Phospholipase A2

COOH Cytochrome P450 epoxygenases

None nzyma tic Is opros ta ne s

Ara chidonic a cid Lipoxyge na s e pa thwa ys

Lipoxins Le ukotrie ne s

Cyclooxyge na s e pa thwa ys

Epoxye icos a te tra e noic a cids (EETs )

P ros ta gla ndins P ros ta cyclin Thromboxa ne

FIGURE 43-1. Overview of the role of arachidonic acid in eicosanoid pathways. Phospholipase A2 acts on phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) to release arachidonic acid. Phospholipase A2 cleaves the ester bond (marked by the arrow at “cleavage site”) to release arachidonic acid. Unesteri ed arachidonic acid is then used as substrate or the cyclooxygenase, lipoxygenase, and epoxygenase pathways. The cyclooxygenase pathways produce prostaglandins, prostacyclin, and thromboxane. The lipoxygenase pathways produce leukotrienes and lipoxins. The epoxygenase pathway produces epoxyeicosatetraenoic acids (EETs). Nonenzymatic peroxidation o arachidonic acid generates isoprostanes.

epoxygenase pathways lead to epoxyeicosatetraenoic acids (Fig. 43-1). Cyclooxygenases (also known as prostaglandin H synthases) are glycosylated, homodimeric, membrane-bound, heme-containing enzymes that are ubiquitous in animal cells rom invertebrates to humans. Two cyclooxygenase iso orms, denoted COX-1 and COX-2, are ound in humans. Although COX-1 and COX-2 share 60% sequence homology and near-superimposable three-dimensional structures, the genes are located on di erent chromosomes, and the enzymes di er in cellular, genetic, physiologic, pathologic, and pharmacologic prof les (Tables 43-1 and 43-2). Each cyclooxygenase enzyme catalyzes two sequential reactions. The f rst reaction, the cyclooxygenase step, is the oxygendependent cyclization o arachidonic acid to prostaglandin G2 (PGG2); the second reaction, the peroxidase step, is the reduction o PGG2 to PGH2 (Fig. 43-2). As a result o di erences in cellular localization, regulatory prof le, tissue expression, and substrate requirements, COX-1 and COX-2 ultimately produce di erent sets o eicosanoid products that are involved in di erent pathways and unctions. Constitutively expressed COX-1 is believed to unction in physiologic, or “housekeeping,” activities such as vascular homeostasis, maintenance o renal and gastrointestinal blood ow, renal unction, intestinal mucosal proli eration, platelet unction, and antithrombogenesis. A number o “as-needed,” or specialized, unctions are attributed to the products o the inducible COX-2 enzyme, including roles in in ammation, ever, pain, transduction o pain ul stimuli in the spinal cord, mitogenesis (particularly in the gastrointestinal epithelium),

COX, cyclooxygenase; EGF, epidermal growth actor; hCG, human chorionic gonadotropin; IFN, inter eron; IL, interleukin; LPS, lipopolysaccharide; NSAID, nonsteroidal anti-inf ammatory drug; TNF, tumor necrosis actor.

renal adaptation to stresses, deposition o trabecular bone, ovulation, placentation, and uterine contractions o labor. The role o constitutive COX-2 expression in areas o the nervous system, such as the hippocampus, hypothalamus, and amygdala, remains to be elucidated.

TABLE 43-2 Major Adverse Effects of Nonselective COX Inhibitors and COX-2 Selective Inhibitors ADVERSE EFFECT Gastric ulceration

NONSELECTIVE COX INHIBITORS (NSAIDs)

COX-2 SELECTIVE INHIBITORS / *

Inhibit platelet unction Inhibit labor induction Impair renal unction Hypersensitivity reaction

?

*The gastrointestinal toxicity o COX-2 selective inhibitors may be less than that o nonselective COX inhibitors, but there is still some incidence o toxicity.

C h a p t e r 4 3 Pharmacology o Eicosanoids

797

COOH

Vasodilation Inhibits platelet aggregation

Smooth muscle contraction Inhibits platelet aggregation

Ara chidonic a cid

IP DP COOH

HO

COX-1 and COX-2: cyclooxyge na s e a ctivity

O

NSAIDs, COX-2 inhibitors

COOH

O

HO

OH

P GD2

OH

O

P GI2

COOH

O OOH

Hydrolys is O

Prostacyclin synthase (e ndothe lium)

COOH

PGD2 isomerase (bra in, ma s t ce lls )

P GG 2

HO

COX-1 and COX-2: pe roxida s e a ctivity HO

OH

Ina ctive

6-ke to-P GF 1α O

COOH

O

Vasoconstriction Platelet activation Thromboxane antagonists

OH

Thromboxane synthase (pla te le ts )

TP

PGF2α reductase (ute rus , lung)

P GH2

HO

COOH

O O

PGE2 isomerase (ma cropha ge s , ma s t ce lls )

COOH

OH

HO

TxA2

P GF 2α

O

None nzyma tic hydrolys is

COOH

FP

OH

HO

O OH

P GE 2

TxB2

EP1 Ina ctive

Smooth muscle contraction Bronchoconstriction Uterine contraction

OH

COOH HO

OH

EP4 EP2

EP3

Vasodilation Hyperalgesia Fever Diuresis Immunomodulation

FIGURE 43-2. Prostaglandin biosynthesis, function, and pharmacologic inhibition. The biosynthetic pathways rom arachidonic acid to prostaglandins, prostacyclin, and thromboxane are depicted. Tissue-speci c enzyme expression determines the tissues in which the various PGH2-derived products are biosynthesized. NSAIDs and COX-2 inhibitors are the most important classes o drugs that modulate prostaglandin production. COX, cyclooxygenase; PG, prostaglandin; Tx, thromboxane; DP, PGD2 receptor; EP, PGE2 receptor; FP, PGF2 receptor; IP, PGI2 receptor; TP, TxA2 receptor; NSAID, nonsteroidal antiinf ammatory drug. Note that DP, EP, FP, IP, and TP are all G protein-coupled receptors.

798 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

COOH

OH P ros ta noid ba ckbone

FIGURE 43-3. Prostanoid structure. The prototypical prostanoid structure is a 20-carbon carboxylic acid with a cyclopentane ring and a 15-hydroxyl group. All prostaglandins, thromboxanes, and prostacyclins are based on this common core structure.

Protein kinetic studies suggest that there may be a third unctional cyclooxygenase iso orm. The putative COX-3 iso orm may be a product o the same gene as COX-1 but with di erent protein characteristics, possibly because o alternative mRNA splicing or post-translational modif cation.

Prostaglandins

Prostaglandins represent a large amily o structurally similar compounds that have potent and specif c biological actions. The name o the amily derives rom their initial identif cation in the genitourinary system o male sheep. Prostaglandins all share a chemical structure, called a prostanoid, consisting o a 20-carbon carboxylic acid characterized by a cyclopentane ring and a 15-hydroxyl group (Fig. 43-3). Prostaglandins are divided into three major subseries: PG1, PG2, and PG3. The subscript numeral indicates the number o double bonds present in the molecule. The PG2 series is the most biologically prevalent in humans because these are the direct derivatives o arachidonic acid, which is an eicosatetraenoic acid (C20:4, with the notation indicating the number o carbon atoms and the number o double bonds in the atty acid). The PG1 series derive rom the arachidonic

acid precursor dihomo- -linolenic acid (DHGLA), an eicosatrienoic acid, while the PG3 series derive rom an eicosapentaenoic acid (EPA, C20:5). (As alluded to earlier, protectins, D-series resolvins, and maresins derive rom docosahexaenoic acid, DHA, C22:6.) Prostaglandin PGH2 represents the critical juncture downstream o cyclooxygenase (Fig. 43-2), because it is the immediate precursor to PGD2, PGE2, PGF2 , thromboxane A2 (TxA2), and prostacyclin (PGI2). The distribution o these eicosanoids in various tissues is determined by the expression pattern o the di erent downstream enzymes o prostaglandin synthesis (i.e., PG synthases) (Fig. 43-2). The prostaglandins are important in many physiologic processes, many o which are not directly related to in ammation. These unctions are highlighted in Table 43-3. Note the especially important housekeeping unctions o PGE2, broadly re erred to as cytoprotective roles, in which organs such as gastric mucosa and renal parenchyma are shielded rom the e ects o ischemia by PGE2-mediated vasodilation and overall regulation o blood ow. PGE2 is also involved in in ammatory cell activation, and PGE2 that is biosynthesized by COX-2 and PGE2 synthase in cells near the hypothalamus appears to have a role in ever.

Thromboxane and Prostacyclin

Platelets express high levels o the enzyme thromboxane synthase but do not contain prostacyclin synthase. Thereore, TxA2 is the chief eicosanoid product of platelets. TxA2 has a hal -li e o only 10–20 seconds be ore it is nonenzymatically hydrolyzed to inactive TxB2. TxA2, which signals via a seven transmembrane G protein-coupled receptor (GPCR) Gq mechanism, is a potent vasoconstrictor as well as a promoter o platelet adhesion and aggregation. In contrast, the vascular endothelium lacks thromboxane synthase but expresses prostacyclin synthase. There ore, PGI2 is the primary eicosanoid product of the vascular endothelium.

TABLE 43-3 Prostaglandin Products, Synthesis, Receptors, and Functions TISSUES EXPRESSING SYNTHETIC ENZYME

RECEPTOR TYPE AND SIGNALING MECHANISM

PROSTAGLANDIN

SYNTHETIC ENZYME

FUNCTIONS

PGD2

PGD2 isomerase

Mast cells Neurons

DP Gs

Bronchoconstriction (asthma) Sleep control unctions Alzheimer’s disease

PGE2

PGE2 isomerase

Many tissues, including macrophages and mast cells

EP1 Gq EP2 Gs EP3 Gi EP4 Gs Other

Potentiation o responses to pain ul stimuli Vasodilation Bronchoconstriction Cytoprotective: modulates gastric mucosal acid secretion, mucus, and blood f ow Inf ammatory cell activation Fever Mucus production Possibly erectile unction

PGF2

PGF2 reductase

Vascular smooth muscle Uterine smooth muscle Bronchial smooth muscle

FP Gq

Vascular tone Reproductive physiology (aborti acient) Bronchoconstriction

The prostanoid receptors are all G protein-coupled receptors. DP, prostaglandin (PG) D2 receptor; EP, PGE2 receptor; FP, PGF2 receptor

C h a p t e r 4 3 Pharmacology of Eicosanoids

PGI2, which signals via Gs, unctions as a vasodilator, venodilator, and inhibitor o platelet aggregation. In other words, PGI2 is the physiologic antagonist o TxA2. The vasodilatory actions o PGI2, like those o PGE2, also con er cytoprotective properties. The local balance between TxA2 and PGI2 levels contributes to regulation o arterial resistance (and thus blood pressure) and to thrombogenesis. Imbalances can lead to hypertension, ischemia, thrombosis, coagulopathy, myocardial in arction, and stroke. In certain populations o the northern latitudes (including Inuit, Greenland, Irish, and Danish populations), the incidence o heart disease, stroke, and thromboembolic disorders is less than in other populations. The diet o these northern peoples is richer in f sh oils and, as a result, contains relatively larger amounts o marine oils (including EPA and DHA). Analogous to the conversion o arachidonic acid into TxA2 and PGI2, EPA is converted into TxA3 and PGI3. Importantly, the vasoconstricting and platelet aggregating e ects o TxA3 are relatively weak. As a result, the thromboxane–prostacyclin balance could be tipped toward vasodilation, platelet inhibition, and antithrombogenesis. This is one possible contributor to the observation that these northern populations have a lower incidence o heart disease and is cited as one rationale or increasing dietary f sh consumption. Novel marine oil-derived mediators that possess potent anti-in ammatory and pro-resolving actions have also recently been discovered (see “Lipoxins, Resolvins, Protectins, and Maresins” section).

Lipoxygenase Pathway Besides the cyclooxygenase pathway, the other major metabolic ate o arachidonic acid is the lipoxygenase pathway, which leads to the ormation o leukotrienes and lipoxins. Lipoxygenases are enzymes that catalyze the insertion o molecular oxygen into arachidonic acid, using non-heme iron to generate specif c hydroperoxides. Three lipoxygenases, 5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase (5-LOX, etc.), are the major LOX iso orms ound in humans (Table 43-4). The lipoxygenases are numbered based on the

799

carbon position in arachidonic acid on which they catalyze the insertion o molecular oxygen. The immediate products o lipoxygenase reactions are hydroperoxyeicosatetraenoic acids (HPETEs ). HPETEs can be reduced to the corresponding hydroxyeicosatetraenoic acids (HETEs ) by glutathione peroxidase-dependent enzymes. 5-HPETE ormed by 5-LOX is the direct precursor to leukotriene A4 (LTA4), which itsel is the precursor to all bioactive leukotrienes (Fig. 43-4). Lipoxygenases are also involved in converting 15-HETE and LTA4 to lipoxins (Fig. 43-5). 5-LOX requires translocation to the nuclear membrane or enzymatic activity. The protein 5-lipoxygenase-activating protein (FLAP) helps 5-LOX translocate to the nuclear membrane, orm an active enzyme complex, and accept the arachidonic acid substrate rom phospholipase A2.

Leukotrienes

Leukotriene biosynthesis begins with the 5-LOX-mediated conversion o 5-HPETE to leukotriene A4 (LTA4). Thereore, 5-LOX catalyzes the f rst two steps in leukotriene biosynthesis (Fig. 43-4). It is not known whether 5-HPETE di uses out o the 5-LOX catalytic site between these steps or remains bound to the same 5-LOX enzyme or both reactions. LTA4 is next converted to either LTB4 or LTC4. The enzyme LTA4 hydrolase converts LTA4 to LTB4 in neutrophils and erythrocytes. The conversion o LTA4 to LTC4 occurs in mast cells, basophils, eosinophils, and macrophages by the addition o a -glutamylcysteinylglycine tripeptide (glutathione). LTC4, LTD4, LTE4, and LTF4, which represent the cysteinyl leukotrienes , are interconverted by removal o amino acid portions o the -glutamylcysteinylglycine tripeptide (Fig. 43-4). LTB4 acts via two G protein-coupled receptors, BLT1 and BLT2. Binding o LTB4 to BLT1, which is expressed mainly in tissues involved in host de ense and in ammation (leukocytes, thymus, spleen), leads to proin ammatory sequelae such as neutrophil chemotaxis, aggregation, and transmigration across epithelium and endothelium. LTB4 up-regulates neutrophil lysosomal unction and generates reactive oxygen

TABLE 43-4 Tissue Expression of Lipoxygenases and Products of Lipoxygenase Action LIPOXYGENASE

TISSUE EXPRESSION

PRODUCTS

PATHWAYS

NOTES

5-LOX

Neutrophils Macrophages Mast cells Eosinophils

5-HPETE/5-HETE LTA4 Epoxytetraene "

Leukotrienes/lipoxins Lipoxins Lipoxins/aspirin-triggered lipoxins

Requires FLAP for activity

Platelets Megakaryocytes (tumors) Skin Macrophages GI system Brain

12-HPETE/12-HETE Epoxytetraene " " " "

Lipoxins " " " " "

Macrophages Monocytes Airway epithelium

15-HPETE/15-HETE Epoxytetraene "

Lipoxins " "

12-LOX Platelet type Epidermal type Leukocyte type

15-LOX

FLAP, 5-lipoxygenase activating protein; GI, gastrointestinal; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene.

800 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY S timuli

Ca 2+

PLA2

COOH

Ara chidonic a cid

5-Lipoxygenase

Zileuton, FLAP inhibitors OOH COOH

5-HP ETE

5-Lipoxygenase

Zileuton, FLAP inhibitors O

COOH

LTA4 LTA4 hydrolase

H2 O

LTC4 synthase

Gluta thione

OH COOH

OH

OH

S

COOH

H N

HN H2 N

LTB4

O

COOH

O

COOH

γ-Glutamyl transpeptidase

BLT1

LTC 4

Carboxypeptidase A OH

OH

COO H COOH

Ma jor s ource : Ne utrophils (BLT1 ) Actions : Activa tion of ne utrophils - Ma rgina tion - Migra tion - De gra nula tion - S upe roxide a nion ge ne ra tion - Eicos a noid s ynthe s is

S S H N

H2 N

OH

HN

COOH

H2 N

O

LTD4

O

O

COOH

LTF 4

P la s ma e xuda tion

γ-Glutamyl transpeptidase

Dipeptidase OH

COOH S OH

H2N O

LTE 4 Ma jor s ource : Ma s t ce lls , ba s ophils , e os inophils Actions :

Bronchocons triction Va s ocons triction De cre a s e d corona ry blood flow De cre a s e d ca rdia c contra ctility P la s ma e xuda tion

Zafirlukast Montelukast Cys LT1

FIGURE 43-4. Leukotriene biosynthesis, function, and pharmacologic inhibition. The biosynthetic pathways rom arachidonic acid to the leukotrienes are shown. Zileuton and 5-lipoxygenase activating protein (FLAP) inhibitors prevent the conversion o arachidonic acid to 5-HPETE and LTA4; zileuton has been used in the chronic management o asthma. Zaf rlukast and montelukast are antagonists at CysLT1, the receptor or all cysteinyl leukotrienes (mainly LTC4 and LTD4); these drugs are used in the chronic management o asthma. The cysteinyl leukotrienes also interact with CysLT2 (not shown). BLT1 and BLT2 are LTB4-related G protein-coupled receptors; BLT1 is the major LTB4 receptor. BLT2 (not shown) is the G protein-coupled receptor or 12-HHT, a cyclooxygenase product (see text or details).

C h a p t e r 4 3 Pharmacology o Eicosanoids

801

COOH

Ara chidonic a cid 15-Lipoxygenase 5-Lipoxygenase Peroxidase

OOH COOH

COOH

5-HP ETE

OH 15-HETE 5-Lipoxygenase

5-Lipoxygenase

OOH O

COOH

COOH

LTA4

OH 5-Hydrope roxy, 15-hydroxye icos a te tra e noic a cid

5-Lipoxygenase

15-Lipoxygenase

O

COOH

OH Epoxyte tra e ne Hydrolys is

Hydrolys is

OH

OH

COOH

COOH

OH

OH OH

OH LXA4

FP R2/ALX

LXB4

FIGURE 43-5. Lipoxin biosynthesis. Two main routes lead to biosynthesis o the lipoxins. In each pathway, sequential lipoxygenase reactions are required to generate epoxytetraene, which then undergoes hydrolysis to yield the lipoxins. Left pathway: Arachidonic acid is converted to 15-HETE by sequential activity o 15-lipoxygenase and peroxidase. 15-HETE is converted by 5-lipoxygenase to the chemical intermediate 5-hydroperoxy, 15-hydroxyeicosatetraenoic acid, and 5-lipoxygenase acts on this intermediate to orm an epoxytetraene. Right pathway: Arachidonic acid is converted to 5-HPETE by 5-lipoxygenase, and 5-HPETE is converted to LTA4 by urther action o 5-lipoxygenase. LTA4 is converted to epoxytetraene by 15-lipoxygenase. Common pathway: Epoxytetraene is hydrolyzed to the active lipoxins LXA4 and LXB4. The lipoxins have both anti-inf ammatory and pro-resolving roles, are counterregulators o leukotriene action, and regulate many cytokines and growth actors. LXA4 is a selective agonist at the G protein-coupled receptor FPR2/ALX.

802 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

species (ROS), enhances cytokine production, and potentiates the actions o natural killer (NK) cells. BLT2 has been ound to bind the COX product 12-HHT (12-hydroxy-5,8,10heptadecatrienoic acid) and evoke chemotaxis o leukocytes and may play a role in tumorigenesis. The cysteinyl leukotrienes (LTC4 and LTD4) bind to CysLT1 receptors to cause vasoconstriction, bronchospasm, and increased vascular permeability. Cysteinyl leukotrienes are responsible or the hyperreactivity to stimuli and the airway and vascular smooth muscle contraction that occur in asthmatic, allergic, and hypersensitivity processes. Together, both arms o the leukotriene pathways (i.e., LTB4 and LTC4/ LTD4) play key roles in psoriasis, asthma, arthritis, and various in ammatory responses. They are also key mediators in vascular disease and are likely to be important in atherosclerosis and obesity.

Lipoxins, Resolvins, Protectins, and Maresins

Lipoxins (lipoxygenase interaction products) are derivatives o arachidonic acid containing our conjugated double bonds and three hydroxyl groups. The two main lipoxins, LXA4 and LXB4 (Fig. 43-5), balance the proin ammatory actions o leukotrienes and cytokines and are thus important in coordinating resolution o in ammation. At sites o in ammation, there is typically an inverse relationship between the amounts o lipoxin and leukotriene present. This observation has led to the suggestion that lipoxins may act as counterregulatory signals or negative regulators o leukotriene action. LXA4 receptors are present on neutrophils and in the lung, spleen, and blood vessels. Lipoxins blunt neutrophil chemotaxis, adhesion, and transmigration through endothelium (by decreasing P-selectin expression), limit eosinophil recruitment, stimulate vasodilation (by inducing synthesis o PGI2 and PGE2), inhibit LTC4- and LTD4-stimulated vasoconstriction, inhibit LTB4 in ammatory e ects, and inhibit the unction o NK cells. Lipoxins stimulate the uptake and clearance o apoptotic neutrophils by macrophages and thereby mediate resolution o the in ammatory response. Because lipoxin production appears to be important in the resolution o in ammation, an imbalance in lipoxin– leukotriene homeostasis may be a key actor in the pathogenesis o in ammatory disease. For example, it is possible that Ms. G’s chronic joint in ammation involves an imbalance in the relative amounts o leukotrienes and lipoxins in her a ected joints. Metabolomic approaches have identif ed endogenous amilies o omega-3-derived mediators, called resolvins, protectins, and maresins, that control both the magnitude and duration o in ammation (Fig. 43-6). Together with lipoxins, they constitute a chemical genus o specialized pro-resolving mediators (SPM). Resolvins, protectins, and maresins are biosynthesized rom essential omega-3 atty acid precursors, especially EPA and DHA. The mapping o these endogenous in ammationresolution circuits provides new avenues to probe the molecular bases o many widely occurring in ammatory diseases. Each o the resolvins, protectins, and maresins possesses multiple potent and stereoselective actions in human cells and in animal disease models. In general, these specialized local mediators limit neutrophil recruitment to sites o inammation and stimulate macrophages to take up and remove apoptotic cells rom the in ammatory site. Resolvins

and protectins are produced not only in in ammatory sites but also in bone marrow and brain, where they also appear to possess potent local mediator actions. Importantly, identif cation o unctional SPM biosynthesized during in ammation-resolution indicates that resolution is an active process, which is a paradigm shi t rom the prior belie that the dampening o acute in ammation is a passive event in vivo. De ective or def cient resolution mechanisms may underlie some chronic in ammatory diseases and suggest the potential or resolution pharmacology. In the uture, avenues to control in ammation may be complemented by novel therapeutics that stimulate key endogenous mechanisms o in ammation resolution.

Epoxygenase Pathway Microsomal cytochrome P450 epoxygenases oxygenate arachidonic acid, resulting in the ormation o epoxyeicosatetraenoic acid (EET) and hydroxyacid derivatives (Fig. 43-1). The epoxygenase pathway is important in tissues that do not express COX or LOX, such as certain cells o the kidney. Epoxygenation o arachidonic acid produces our di erent EETs, depending on which double bond in arachidonic acid is modif ed. Dihydroxy derivatives o EETs, ormed by hydrolysis, may regulate vascular tone by inhibiting the Na / K ATPase in vascular smooth muscle cells and may a ect renal unction by regulating ion absorption and secretion. Future research may reveal more def nitive unctions or EETs in human physiology.

Isoprostanes Phospholipid-esterif ed arachidonic acid is susceptible to ree-radical-mediated peroxidation, and release o these modif ed lipids rom the phospholipid by phospholipase A2 gives rise to the isoprostanes (Fig. 43-1). During oxidative stress, isoprostanes are ound in the blood at levels much higher than those o cyclooxygenase products. Two isoprostanes in particular, 8-epi-PGF2 and 8-epi-PGE2, are potent vasoconstrictors. Isoprostanes can activate NF B, phospholipase C , protein kinase C, and calcium ux. Because the rate o ormation o isoprostanes depends on cellular oxidation conditions, isoprostane levels may be indicative o oxidative stress in a wide range o pathologic conditions. Urinary isoprostane might serve as a biomarker o oxidative stress in ischemic syndromes, reper usion injury, atherosclerosis, and hepatic diseases.

Metabolic Inactivation o Local Eicosanoids Prostaglandins, leukotrienes, thromboxanes, and lipoxins are inactivated by hydroxylation, -oxidation (resulting in a loss o two carbons), or -oxidation (to dicarboxylic acid derivatives). These degradation processes render the molecules more hydrophilic and excretable in the urine.

Integrated Inf ammation Schema As described above, eicosanoids are generated locally in numerous complex reactions. It is not necessary to remember each mediator but rather to understand the general scheme o these biosynthetic pathways. This section, along with Table 43-5, provides a concise overview o the physiologic unctions o eicosanoids relevant to in ammation and host de ense.

C h a p t e r 4 3 Pharmacology o Eicosanoids

803

A H

H

COOH

H(O)O

Aspirin-acetylated COX-2 COOH

Eicos a pe nta e noic a cid (EPA)

18-hydrope roxy-EPA 5-Lipoxygenase HOOC O(O)H

5(6)-Epoxyresolvin Esynthase reaction

COOH

O

OH

HO

5(6)-Epoxy-18-hydroxy-EPA

5-Hydrope roxy, 18-hydroxy-EPA

Enzym atic epoxide hydrolysis

Peroxidase

OH OH COOH

HO OH COOH

Re s olvin E1 (RvE1) Re gula te s ne utrophil infiltra tion Re gula te s de ndritic ce ll function a nd IL-12 production P romote s re s olution of infla mma tion Re duce s colitis P rote cts from os te ocla s t-me dia te d bone de s truction

HO

Re s olvin E2 (RvE2) Re gula te s ne utrophil infiltra tion

FIGURE 43-6. Resolvins, protectins, and maresins: biosynthesis and actions of novel families of omega-3-derived mediators. A. EPA is the precursor to E-series resolvins. B and C. DHA is the precursor to D-series resolvins, protectins, and maresins. Some o the major endogenous anti-inf ammatory and pro-resolving unctions are listed below some o the mediators. In addition, resolvin D1 regulates neutrophil in ltration and resolvin D2 enhances microbial phagocytosis and clearance. (continued)

804 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY B HOOC

HOOC

HO OH HO

HO

OH

OH

Re s olvin D1 (RvD1)

Re s olvin D2 (RvD2)

7(8)-Epoxide inte rme dia te

5-Lipoxygenase H

H HOOC

Lipoxygenase COOH

OOH

5-Lipoxygenase

Docos a he xa e noic a cid (DHA)

17-Hydrope roxy-DHA

4(5)-Epoxide inte rme dia te

OH HOOC

COOH OH OH OH

Re s olvin D3 (RvD3)

FIGURE 43-6. (continued)

HO OH

Re s olvin D4 (RvD4)

C h a p t e r 4 3 Pharmacology of Eicosanoids C H

H

H

H

COOH

COOH

Docos a he xa e noic a cid (DHA)

Docos a he xa e noic a cid (DHA) 12/15-Lipoxygenases

Lipoxygenase

O(O)H HOOC

COOH

17-Hydrope roxy-DHA

OOH

14-hydrope roxy-DHA

Enzym atic epoxidation and hydrolysis

Enzym atic epoxidation and conversion

COOH

COOH

OH

OH OH

OH

P rote ctin D1 (P D1)

Re gula te s ne utrophil a nd T-ce ll infiltra tion Re gula te s TNF a nd inte rfe ron production P romote s re s olution of infla mma tion Re duce s pe ritonitis a nd a irwa y infla mma tion P rote cts bra in from is che mia /re pe rfus ion injury Mitiga te s kidne y is che mia injury

FIGURE 43-6. (continued)

Ma re s in 1 (Ma R1)

Re gula te s ne utrophil infiltra tion P romote s re s olution of infla mma tion

805

806 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY P la te le t

TABLE 43-5 Roles o Eicosanoids in the Stages o Inf ammation ACTION

EICOSANOIDS INVOLVED

Vasoconstriction

PGF2 , TxA2, LTC4, LTD4, LTE4

Vasodilation (erythema)

PGI2, PGE1, PGE2, PGD2, LXA4, LXB4, LTB4

Edema (swelling)

PGE2, LTB4, LTC4, LTD4, LTE4

Chemotaxis, leukocyte adhesion

LTB4, HETE, LXA4, LXB4

LXA4 LXB4

AA A

LTC4, LTD4, LTE4

Pain and hyperalgesia

PGE2, PGI2, LTB4

Local heat and systemic ever

PGE2, PGI2, LXA4

Le ukocyte

Lipoxins, resolvins, protectins, maresins

Acute in ammation is the result o an intricate network o molecular and cellular interactions induced by responses to a variety o stimuli, such as trauma, ischemia, in ectious agents, or antibody reactions. Acute superf cial in ammation generates local pain, edema, erythema, and warmth; in ammation in visceral organs can have similar signs and symptoms (which, in certain cases, can result in swelling against an organ capsule) and result in severe impairment o organ unction. Leukotrienes and lipoxins, as well as thromboxanes, prostaglandins, and prostacyclins, are critical or generating, maintaining, mediating, and resolving inf ammatory responses. The in ammatory cascade is initiated when cells in a particular region are exposed to a oreign substance or are damaged. That insult stimulates a local cytokine cascade (including interleukins or TNF), which increases COX-2 mRNA and enzyme levels. COX-2 then acilitates production o the proin ammatory and vasoactive eicosanoids. Locally high concentrations o PGE2, LTB4, and cysteinyl leukotrienes promote the accumulation and inf ltration o in ammatory cells by increasing blood ow and vascular permeability. LTB4 and 5-HETE are also important in attracting and activating neutrophils. LTB4, biosynthesized and released by activated neutrophils at the site o in ammation, recruits and activates additional neutrophils and lymphocytes so that these cells adhere to endothelial suraces and transmigrate into the interstitial spaces. Increased vascular permeability also results in uid leak and cellular inf ltration, causing edema. With the aggregation o a multitude o in ammatory cells, transcellular biosynthetic routes are exploited to generate eicosanoids (Fig. 43-7). In transcellular biosynthesis, eicosanoid intermediates are donated rom one cell type to another to generate a greater diversity o local chemical mediators. This demonstrates the importance o cellular adhesion and cell–cell interaction in in ammatory and immune responses. Feedback mechanisms are designed to ensure that the inammatory response cannot proceed unchecked. Lipoxins help resolve in ammation and promote the return o the

LTA4 LTA4 hydrolase

LTB4

P ros ta cyclin Prostacyclin synthase

COX

Resolution o inf ammation

LTA4

5-Lipoxygenase/FLAP

Increased vascular permeability

LTC 4

AA

LTA4

LTC 4

LTC4 synthase

Endothe lia l ce ll

FIGURE 43-7. Examples o transcellular biosynthesis. Transcellular biosynthesis is used to generate lipoxins and cysteinyl leukotrienes locally. In the example shown here, the leukocyte (neutrophil) obtains arachidonic acid (AA) rom platelets and uses AA to synthesize leukotriene A4 (LTA4) and leukotriene B4 (LTB4). Leukotriene A4 is trans erred rom the leukocyte to platelets and endothelial cells, which synthesize and secrete leukotriene C4 (LTC4). Platelets also synthesize lipoxins (LXA4, LXB4) rom leukotriene A4, and endothelial cells synthesize prostacyclin using AA rom endogenous stores. Note that the eicosanoids synthesized within each cell type are determined by the enzymatic repertoire o that cell type: or example, neutrophils synthesize primarily LTA4 and LTB4 because they express 5-lipoxygenase and LTA4 hydrolase, whereas endothelial cells biosynthesize prostacyclin and LTC4 because they express COX-1, COX-2, prostacyclin synthase, and LTC4 synthase.

tissues, organs, and organism to homeostasis. COX-2derived eicosanoids may also unction in wound healing and resolution. Hence, the temporal sequence o events is critical to an organized in ammatory response. PGE2 inhibits the unctions o B and T lymphocytes and NK cells, while LTB4 and the cysteinyl leukotrienes regulate T-cell proli eration. PGE2 and PGI2 are potent pain sensitizers, and lipoxins reduce nociception. These actors mediate and regulate the transition rom acute to chronic in ammation (Figs. 43-2, 43-4, and 43-6).

PATHOPHYSIOLOGY OF EICOSANOIDS In ammation and the immune response are the body’s mechanisms or combating oreign invaders and trauma. The overall scheme is designed to remove the inciting stimulus and resolve tissue damage. In some cases, the response mechanism itsel causes local tissue damage, such as when activated neutrophils inadvertently release proteases and reactive oxygen species into the local milieu. In other settings, i the in ammatory reactions persist or too long or i the immune system misidentif es a component o sel as oreign, misdirected responses can cause signif cant and chronic tissue injury. Prof led brie y in the ollowing discussion are selected in ammatory diseases in which eicosanoids are implicated, including asthma, in ammatory bowel disease, rheumatoid

C h a p t e r 4 3 Pharmacology of Eicosanoids

arthritis, glomerulonephritis, cancer, and cardiovascular disease. Other diseases not discussed here, but having a possible eicosanoid-related in ammatory basis, include certain skin disorders, reper usion injuries, Alzheimer’s disease, and adult respiratory distress syndrome.

Asthma Asthma is an airway in ammatory disorder typif ed by intermittent attacks o dyspnea, coughing, and wheezing. Symptoms result rom chronic airway in ammation, hyperreactivity, constriction, and obstruction. In asthma, antigens in the lungs stimulate cytokine cascades leading to the generation o both prostaglandins (e.g., PGD2) and leukotrienes. LTB4 attracts in ammatory cells and promotes cellular aggregation. LTB4 acts particularly on B lymphocytes to cause cell activation, proli eration, and di erentiation. LTB4 also promotes expression o Fc RII receptors (i.e., receptors or the Fc portion o IgE antibodies) on mast cells and basophils; these receptors bind IgE that is released by antigen-stimulated B lymphocytes. LTC4 and LTD4 are extremely potent bronchoconstrictive compounds, more than 1,000 times as potent as histamine. These cysteinyl leukotrienes also cause the airway epithelium to secrete mucus, while impairing the clearance o mucus by inhibiting the beating o cilia on airway epithelium. Mucus secretion is exacerbated by neutrophils and eosinophils, which become part o the in ammatory exudate clogging the airways. LTD4 and LTE4 also recruit eosinophils to asthmatic airways; eosinophils integrate signals rom T lymphocytes and, when activated, release actors that damage the airway epithelium and enhance local airway in ammation. In mouse models o asthma in which either the 5-LOX or CysLT1 gene is knocked out, reduced airway hyperresponsiveness and leukocyte inf ltration are observed. These results underscore the important role o leukotrienes in the pathogenesis o asthma. The role o leukotriene inhibitors in asthma treatment is discussed below; or additional in ormation, re er to Chapter 48, Integrative In ammation Pharmacology: Asthma.

Inf ammatory Bowel Disease Crohn’s disease and ulcerative colitis are idiopathic, chronic, relapsing, ulcerative, and in ammatory diseases o the gastrointestinal tract. Although the diseases are pathologically distinct, elevated LTB4 production in the a ected mucosa results in abnormal leukocyte inf ltration into the parenchyma in both conditions. Chronic in ammation and leukocyte inf ltration lead to progressive mucosal damage, with overt histologic changes. Crohn’s disease is characterized by ocal damage, f ssuring ulcers, and granulomas, while mucosal in ammation and colonic dilatation are ound in ulcerative colitis. Both diseases increase the risk o adenocarcinoma o the colon in the a ected areas. Stable analogues o lipoxin A4 are e ective treatments in mouse models o Crohn’s disease and bowel in ammation and may represent a promising new pharmacologic approach to the treatment o in ammatory bowel disease.

Rheumatoid Arthritis Rheumatoid arthritis is a chronic, systemic, autoimmune, and in ammatory disease that primarily attacks the joints but also a ects the skin, cardiovascular system, lungs, and

807

muscles. Rheumatoid arthritis a ects up to 1.5% o North Americans and is three times more prevalent in emales than in males. Autoimmune targeting o normal joint proteins results in in ammation, with resultant local release o cytokines, TNF, growth actors, and interleukins, all o which induce COX-2 expression. Levels o COX-2 enzyme and PGE2 are markedly elevated in the synovial f uid o a ected joints. PGE2 stimulates pain pathways, and other COX2-derived eicosanoids and 5-LOX-derived leukotrienes activate the surrounding endothelium to recruit in ammatory cells. Macrophages release collagenase and proteases, while lymphocyte activity leads to immune complex ormation; both processes urther damage joint tissue and provide substrates that accelerate chronic in ammation. Common f ndings include synovitis, leukocytosis, rheumatoid nodules, and the presence o rheumatoid actor (a circulating antibody directed against IgG). As a Native American woman in her f th decade, Ms. G is in a higher risk group or rheumatoid arthritis. Autoimmune destruction o her joints resulted in the f ndings o a high erythrocyte sedimentation rate (consistent with a state o chronic in ammation), synovial leukocytosis, radiographic f ndings o bone loss, and the progressive loss o joint mobility and unction. For additional in ormation on rheumatoid arthritis, re er to Chapter 46, Pharmacology o Immunosuppression.

Glomerulonephritis Glomerulonephritis delineates a large group o in ammatory renal conditions that may ultimately lead to renal ailure through deterioration o renal hemodynamics and glomerular f ltration. Local complement activation promotes neutrophil and macrophage inf ltration. Inf ltration o the glomerulus is a characteristic early pathologic f nding that correlates with abnormal levels o LTB4, which is biosynthesized by LTA4 hydrolase in the kidney mesangium, and which acilitates neutrophil adhesion to the glomerular mesangium and epithelium. LTA4 is also a substrate or the biosynthesis o LTC4 and LTD4. All o the cysteinyl leukotrienes (LTC4, LTD4, LTE4, and LTF4) promote endothelial and mesangial proli eration. Cysteinyl leukotrienes also directly a ect glomerular unction; specif cally, LTC4 and LTD4 decrease renal blood ow and glomerular f ltration rate (GFR) by vasoconstricting arterioles and contracting mesangial spaces. Studies with inhibitors have conf rmed the roles o leukotrienes in glomerulonephritis: LOX inhibitors administered at early stages o glomerulonephritis prevent glomerular in ammation and evidence o structural damage, and both LOX inhibitors and LTD4 receptor antagonists increase GFR and decrease proteinuria. Interestingly, the kidney mesangium expresses both LTA4 hydrolase and 12-LOX, con erring the ability to synthesize either LTB4 or LXA4 rom leukocyte-derived LTA4. At low concentrations, LTA4 is used primarily or LTB4 ormation; this condition corresponds to the initiation o in ammation. Conversely, when LTA4 concentrations are relatively high, as in long-standing in ammation, LTA4 is converted mostly into LXA4, which provides an autoinhibitory, counterregulatory impact on the in ammatory response. In the glomerulus, LXA4 counteracts the deleterious proin ammatory consequences o leukotrienes as well as the e ect o leukotrienes on GFR, in part by raising a erent arteriolar ow via vasodilation.

808 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Cancer Long-term epidemiologic studies have suggested a correlation between chronic NSAID therapy and decreased incidence o colorectal cancer. Human colorectal adenomas and carcinomas express abundant COX-2; similar results have been ound with gastric adenocarcinomas and breast tumors. In these tissues, COX-2 is believed to generate PGE2 and other eicosanoids that promote tumor growth. The perinuclear localization o the COX-2 enzyme (Table 43-1) suggests the potential or an intracellular unction o eicosanoid products in oncogenesis. Some eicosanoid derivatives can bind to homologues o the retinoic acid receptor (RXR) amily o transcription actors, which are involved in many unctions including the regulation o cell growth and di erentiation. Overexpression o COX-2 would generate eicosanoids that could ood RXR signaling pathways and provide excessive growth stimuli. A COX-2 inhibitor is being sought as a prophylactic therapy or patients with amilial adenomatous polyposis, who are at increased risk or colorectal cancer (see “COX-2 Inhibitors” section). There is also evidence that aspirin may be a chemoprotective agent in colorectal cancer.

Cardiovascular Disease Platelet-derived thromboxane A2 is an important mediator o thrombosis in acute coronary syndromes and other cardiovascular diseases; the COX inhibitor aspirin is an e ective antiplatelet agent in the prophylaxis and treatment o these diseases (see below and Chapter 23, Pharmacology o Hemostasis and Thrombosis). Intravascular leukotriene production during the rupture o atheromatous plaques is also thought to contribute to the pathophysiology o acute coronary syndromes. Genetic polymorphisms in 5-lipoxygenase, FLAP, and LTA4 hydrolase may be linked to myocardial in arction.

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic intervention in eicosanoid biosynthesis and action is particularly use ul or controlling in ammation and restoring homeostasis. Pharmacologic interventions can be directed at any o the number o steps outlined above to achieve the desired e ects with tissue, spatial, and temporal selectivity. Strategies include altering the expression o key enzymes, competitively and noncompetitively inhibiting the activity o specif c enzymes (e.g., PGE2 synthase), activating receptors with exogenous receptor agonists, and preventing receptor activation with exogenous receptor antagonists. As with all aspects o medicine, therapeutic benef ts must be weighed against the possible adverse e ects.

Phospholipase Inhibitors Inhibition o phospholipase A2 prevents the release o arachidonic acid rom cellular phospholipids, the rate-limiting step in eicosanoid biosynthesis. In the absence o proin ammatory mediators derived rom arachidonic acid, in ammation is limited. Glucocorticoids (also known as corticosteroids, including prednisone , prednisolone , and dexamethasone ) are a mainstay o therapy in a multitude o autoimmune and in ammatory

diseases. Glucocorticoids induce a amily o secreted calcium- and phospholipid-dependent proteins called lipocortins . Lipocortins inter ere with the action o phospholipase A2 and thereby limit the availability o arachidonic acid. Annexins, such as annexin 1 and annexin 1-derived peptides, are also induced by glucocorticoids. In turn, annexins act at G protein-coupled receptors on leukocytes to block proin ammatory responses and enhance endogenous anti-inammatory mechanisms; one anti-in ammatory mechanism involves activation o the lipoxin A4 receptor. Glucocorticoids also inhibit the action o COX-2 and the ormation o prostaglandins by several mechanisms: (1) repressing COX-2 gene and enzyme expression; (2) repressing the expression o cytokines that activate COX-2; and (3) as noted above, limiting the available pool o COX-2 substrate (arachidonic acid) by indirectly blocking phospholipase A2. Because o this proound and global suppression o immune and in ammatory responses, glucocorticoids are used to treat a number o autoimmune conditions (see Chapter 46). Small-molecule inhibitors o specif c phospholipases are under investigation; these compounds may o er the potential or decreased adverse e ects compared to those associated with glucocorticoid use. See Chapter 29, Pharmacology o the Adrenal Cortex, or a more extensive discussion o the e ects o glucocorticoids.

Cyclooxygenase Inhibitors Cyclooxygenase pathway inhibitors are some o the most requently prescribed drugs in medicine. The nonsteroidal anti-in ammatory drugs (NSAIDs) and acetaminophen are the most commonly used agents in this class.

Traditional Nonselective Inhibitors: NSAIDs

NSAIDs are clinically important because o their combined anti-in ammatory, antipyretic, and analgesic properties. The ultimate goal o most NSAID therapies is to inhibit the COX-mediated generation o proin ammatory eicosanoids and to limit the extent o in ammation, ever, and pain. The antipyretic activity o NSAIDs is likely related to their ability to decrease the levels o PGE2, particularly in the region o the brain surrounding the hypothalamus. Despite the benef ts o current NSAIDs, these drugs only suppress the signs o the underlying in ammatory response but may not necessarily reverse or resolve the in ammatory process. A multitude o NSAIDs have been developed over the last century; most are polycyclic carboxylic acid derivatives. Except or aspirin, all NSAIDs act as reversible, competitive inhibitors o cyclooxygenase (Fig. 43-2). These drugs block the hydrophobic channel o the cyclooxygenase protein where the substrate arachidonic acid binds, thereby preventing conversion o arachidonic acid to PGG2. Traditional NSAIDs inhibit both COX-1 and COX-2 to di erent degrees. Because o inhibition o COX-1, long-term NSAID therapy has many deleterious e ects. The cytoprotective roles o the COX-1 eicosanoid products are eliminated, leading to a spectrum o NSAID-induced gastropathy including dyspepsia, gastrotoxicity, subepithelial damage and hemorrhage, gastric mucosal erosion, rank ulceration, and gastric mucosal necrosis. As in Ms. G’s case, patients with gastric ulceration can have bleeding into the stomach, where reaction o hemoglobin with stomach acid results in hematemesis, or vomiting o material that has the color and

C h a p t e r 4 3 Pharmacology o Eicosanoids

consistency o “co ee grounds.” Regulation o blood ow to the kidney is similarly perturbed, decreasing GFR and potentially causing renal ischemia, papillary necrosis, interstitial nephritis, and renal ailure. The results o studies on the e ects o COX-2 inhibitors (see below) have prompted reinvestigation o the e ects o COX-1 inhibitors and traditional NSAIDs, with the f nding that these classes o drugs may also be associated with cardiac risks. The US Food and Drug Administration (FDA) asked nonprescription NSAID manu acturers to update labeling with specif c in ormation about cardiovascular risks and to “remind patients o the limited dose and duration o treatment o these products.” Epidemiologic studies suggest that up to 20–30% o hospitalizations o patients over the age o 60 may be due to complications o NSAID use. The organic acid unctionality o NSAIDs con ers important pharmacokinetic properties on these drugs, including near-complete absorption rom the gut, binding to plasma albumin, accumulation in cells at the site o in ammation, and e f cient renal excretion. NSAIDs can be divided into short ( 6 hours) and long ( 10 hours) hal -li e classes. NSAIDs with long elimination hal -lives include naproxen, salicylate , piroxicam, and phenylbutazone . Chemical classif cation o the NSAIDs is based on the structure o a key moiety in each subclass (Fig. 43-8). The ollowing discussion groups the NSAIDs by chemical class; a discussion o the choice o a particular NSAID or a given clinical situation ollows the descriptions o the individual agents.

Salicylate class

Propionic acid class

O OH

OH O

O

Ibuprofen

O

Aspirin Acetic acid class (Phenyl acetic acids)

Oxicam class

O

OH

HO

O

Cl

N H

H N O

Cl

N

N

S O

Piroxicam Diclofenac

Acetic acid class (Indole acetic acids)

Aminophenol class O

O

OH

NH O

N O

OH

Cl

Salicylates

Salicylates include aspirin (acetylsalicylic acid) and its derivatives. Aspirin is the oldest o the NSAIDs and is widely used to treat mild to moderate pain, headache, myalgia, and arthralgia. In contrast to other NSAIDs, aspirin acts in an irreversible manner by acetylating the active-site serine residue in both COX-1 and COX-2. Acetylation o COX-1 destroys the enzyme’s cyclooxygenase activity, preventing the ormation o COX-1-derived prostaglandins, thromboxanes, and prostacyclin. Salicylates (along with indomethacin, piroxicam, and ibupro en) may also inhibit the neutrophil oxidative burst by reducing NADPH oxidase activity. Daily low-dose aspirin is used as an antithrombogenic agent for management of acute coronary syndromes, chronic atherosclerotic disease, and ischemic stroke. Recall that aspirin is antithrombogenic because o its irreversible inhibition o COX, which prevents platelets rom biosynthesizing TxA2. Within an hour o oral aspirin administration, the COX-1 activity in existing platelets is irreversibly destroyed. Platelets, lacking nuclei, cannot synthesize new protein. There ore, the irreversibly acetylated COX-1 enzymes cannot be replaced by reshly synthesized proteins, and the platelets’ cyclooxygenase activity is irreversibly inhibited or their circulating li etime (about 10 days). Although aspirin also irreversibly inhibits vascular endothelial cell COX-1 and COX-2, the endothelial cells can synthesize new COX protein and thus can rapidly resume synthesis o PGI2. A single administration of aspirin thus decreases for several days the amount of thromboxane that can be generated, shifting the vascular TxA2-PGI2 balance toward PGI2-mediated vasodilation, platelet inhibition, and antithrombogenesis.

809

O

Indomethacin

Acetaminophen

Fenamate class

Ketone class

OH

O H N

O

Nabumetone Mefenamate

FIGURE 43-8. Structural classes of NSAIDs. NSAIDs are generally hydrophobic molecules, most o which have a carboxylic acid group. NSAIDs are categorized by class depending on one or more o the key moieties in the structure. The moiety that is common to members o each class is highlighted by a box. The structure helps to determine the pharmacokinetic properties o each particular NSAID. Note that acetaminophen is not actually an NSAID, because it has only weak anti-inf ammatory properties; this drug is included here because, like NSAIDs, acetaminophen is commonly used or its analgesic and antipyretic e ects.

Aspirin-mediated inhibition o COX-2 prevents the generation o prostaglandins. Unlike COX-1, which is totally inactivated by aspirin, the aspirin-modif ed COX-2 enzyme retains a distinct part o its catalytic activity and can orm a new product, 15-(R)-HETE, rom arachidonic acid. By analogy to lipoxin biosynthesis (Fig. 43-5), 5-LOX then converts 15-(R)-HETE to 15-epi-lipoxins, which are relatively stable stereoisomers (carbon 15-position epimers) o lipoxins that are collectively called aspirin-triggered lipoxins (ATLs).

810 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

15-Epi-lipoxins mimic the unctions o lipoxins as antiin ammatory agents. 15-Epi-lipoxins may represent another endogenous mechanism o anti-in ammation, and their production mediates at least part o the anti-in ammatory e ects o aspirin. Development o 15-epi-lipoxin analogues could lead to anti-in ammatory drugs that do not have the adverse e ects associated with COX-1 inhibition. Aspirin is generally well tolerated. Its major toxicities are the gastropathy and nephropathy common to all NSAIDs. Long-term aspirin therapy can lead to gastrointestinal ulceration and hemorrhage, nephrotoxicity, and hepatic injury. NSAIDs should be used cautiously, i at all, in patients with renal insu f ciency and heart ailure. Two unique toxicities are aspirin-induced airway hyperreactivity in asthmatics (so-called aspirin-sensitive asthma) and Reye’s syndrome . The prevalence o aspirin sensitivity among patients with asthma is approximately 10%. Exposure to aspirin in these patients leads to ocular and nasal congestion along with severe airway obstruction. Aspirin-sensitive patients are also reactive to some other NSAIDs, including indomethacin, naproxen, ibupro en, me enamate, and phenylbutazone. One possible etiology o aspirin/NSAID sensitivity in asthmatics is that exposure to these drugs leads to increased levels o leukotrienes, which are implicated in the pathogenesis o asthma (see Fig. 43-1). Reye’s syndrome is a condition characterized by hepatic encephalopathy and liver steatosis in young children. Aspirin therapy during the course o a ebrile viral in ection has been implicated as a potential etiology o the liver damage. Aspirin is generally not administered to children because o the ear o Reye’s syndrome, and acetominophen is widely used in children instead o aspirin.

postsurgical patients; however, in part due to its potency and adverse e ects, ketorolac is used or no more than 3–5 days. The acetic acid NSAIDs are mostly used to relieve symptoms in the long-term treatment o rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and other musculoskeletal disorders. Indomethacin also has specif c uses in the treatment o gout and pericarditis and in promoting the closure o a patent ductus arteriosus in newborns by inhibiting the vasodilatory eicosanoids PGE2 and PGI2. Use o acetic acid NSAIDs causes gastrointestinal ulceration and, rarely, hepatitis and jaundice.

Propionic Acid Derivatives

Ketone NSAIDs Nabumetone is a ketone prodrug that is oxidized in vivo to the

Propionic acid NSAIDs include ibupro en, naproxen, ketopro en, and f urbipro en. Ibupro en is a relatively potent analgesic used in rheumatoid arthritis (as in the case o Ms. G, to relieve intermittent pain), osteoarthritis, ankylosing spondylitis, gout, and primary dysmenorrhea. Naproxen has a long plasma hal -li e, is 20 times more potent than aspirin, directly inhibits leukocyte unction, and causes less severe gastrointestinal adverse e ects than aspirin. Epidemiologic studies have suggested that naproxen may be the least cardiotoxic NSAID, but the current evidence or this f nding is insu f cient to support a separate clinical indication. Acetic Acid Derivatives

Acetic acid NSAIDs include the indoleacetic acids— indomethacin, sulindac , and etodolac —and the phenylacetic acids diclo enac and ketorolac (a substituted phenylacetic acid derivative). Besides inhibiting cyclooxygenase, many o the acetic acid NSAIDs promote the incorporation o unesterif ed arachidonic acid into triglyceride, thus reducing the availability o the substrate or cyclooxygenase and lipoxygenase. Indomethacin is a direct inhibitor o neutrophil motility, but it is not tolerated by patients as well as ibupro en. Diclo enac also reduces intracellular arachidonic acid concentrations by altering cellular atty acid transport. Diclo enac is a more potent anti-in ammatory than indomethacin and naproxen and is used widely in the treatment o pain associated with renal stones. Ketorolac is a strong analgesic that is available in both intravenous and intramuscular preparations. It is used particularly in

Oxicam Derivatives Piroxicam is as e f cacious as aspirin, naproxen, and ibupro-

en in the treatment o rheumatoid arthritis and osteoarthritis and may be better tolerated. Piroxicam has additional e ects in the modulation o neutrophil unction by inhibiting collagenase, proteoglycanase, and the oxidative burst. Because o its extremely long hal -li e, piroxicam can be administered once daily. As with other NSAIDs, piroxicam displays gastrointestinal adverse e ects such as gastric ulceration, and it prolongs the bleeding time because o its antiplatelet e ect.

Fenamate Derivatives

The two enamate NSAIDs are me enamate and mecloenamate . Both inhibit cyclooxygenases but also antagonize prostanoid receptors to various degrees. Because enamates have less anti-in ammatory activity and are more toxic than aspirin, there is little advantage to their use. Me enamate is used only or primary dysmenorrhea, and mecloenamate is used in the treatment o rheumatoid arthritis and osteoarthritis.

active acid orm. Compared to other nonselective NSAIDs, nabumetone has pre erential activity against COX-2. The incidence o gastrointestinal adverse e ects is relatively low, although headache and dizziness are requently reported.

Acetaminophen

Acetaminophen is sometimes classif ed with the NSAIDs,

but it is technically not an NSAID: although acetaminophen has analgesic and antipyretic e ects similar to aspirin, the anti-in ammatory e ect o acetaminophen is insignif cant because o its weak inhibition o cyclooxygenases. Nonetheless, acetaminophen therapy is widely used, especially in patients (such as children) who are at risk or the adverse e ects o aspirin. The most important adverse e ect o acetaminophen is hepatotoxicity, and clinicians are advised both to monitor and reduce the dose o acetaminophen in combination with other analgesics and to reduce the total daily dose o acetaminophen. Modif cation o acetaminophen by hepatic cytochrome P450 enzymes produces a reactive metabolite, which is normally detoxif ed by conjugation with glutathione. An overdose o acetaminophen can overwhelm glutathione stores, leading to cellular and oxidative damage and, in severe cases, to acute hepatic necrosis (see Chapter 6, Drug Toxicity).

Selection of the Appropriate NSAID

The anti-in ammatory, analgesic, and antipyretic e ects o the NSAIDs vary among the many agents. However, despite

C h a p t e r 4 3 Pharmacology of Eicosanoids

di erences in chemistry, tissue selectivity, enzyme selectivity, pharmacokinetics, and pharmacodynamics, the di erences in e f cacy may not be clinically signif cant. Overall, the rationale and choice o NSAID do not generally make a substantial di erence in treating rheumatoid arthritis or osteoarthritis. However, success ul NSAID therapy is still considered more o an art than a science, and therapy or each patient should be directed at achieving the desired anti-inammatory, analgesic, and antipyretic e ects while minimizing adverse e ects. The adverse gastric e ects o long-term NSAID therapy can be reduced by co-administration o histamine H2 receptor antagonists or proton pump inhibitors (re er to Chapter 47, Integrative In ammation Pharmacology: Peptic Ulcer Disease).

O H2 N

O S

S

N

N O

CF 3

O

Celecoxib

Rofecoxib

O

OH

COX-2 Inhibitors

As noted earlier, long-term NSAID therapy can be associated with severe gastrointestinal adverse e ects that are thought to be caused by inhibition o gastrointestinal COX-1. It was hypothesized that selective inhibition o COX-2 could have the theoretical advantage o inhibiting the chemical mediators responsible or in ammation while maintaining the cytoprotective e ects o the products o COX-1 activity.

O

O

811

O N H

S

O

N N

N

S O

H2 N

S

O

O

Meloxicam

Valdecoxib

FIGURE 43-9. COX-2 selective inhibitors. COX-2 selective inhibitors are

COX-2 Selective Inhibitors

Although COX-2 was identif ed only in the 1990s, intense research swi tly led to the development o COX-2 selective inhibitors or clinical use. Compared with COX-1, COX-2 has a larger hydrophobic channel through which substrate (arachidonic acid) enters the active site. Subtle structural di erences between COX-2 and COX-1 allowed the development o drugs that act pre erentially on COX-2. The COX-2 selective inhibitors—celecoxib, rofecoxib, valdecoxib, and meloxicam (Fig. 43-9)—are sul onic acid derivatives that exhibit 100- old greater selectivity or COX-2 than or COX-l. The relative inhibition o the two cyclooxygenase isozymes in any given tissue is also a unction o drug metabolism, pharmacokinetics, and possibly enzyme polymorphisms. The COX-2 selective inhibitors have antiin ammatory, antipyretic, and analgesic properties similar to the traditional NSAIDs, but they do not share the antiplatelet actions o the COX-1 inhibitors. Various coxibs had been approved or treatment o osteoarthritis, rheumatoid arthritis, acute pain in adults, and primary dysmenorrhea. However, relative to other NSAIDs, the sa ety prof le o the COX-2 selective inhibitors is uncertain. At the present time, only celecoxib is an approved drug in the United States. Ro ecoxib was withdrawn by the manu acturer rom the worldwide market in 2004 because o an increase in myocardial in arction and stroke with prolonged use; valdecoxib was then withdrawn in 2005. The increased thrombogenicity o COX-2 inhibitors, uncovered in clinical use, may be due to prolonged inhibition o vascular COX-2 within endothelial cells, leading to reduced PGI2 ormation. In addition, inhibition o COX-2 may generate problems in wound healing, angiogenesis, and the resolution o in ammation. COX-2 selective inhibitors are much more expensive than equivalent doses o many NSAIDs, especially aspirin and indomethacin. Note that Ms. G’s physician attempted to take advantage o the relative gastrointestinal sa ety o COX-2 selective inhibitors when she was switched rom ibupro en to a COX-2 inhibitor, in part because o symptomatic and endoscopic evidence

hydrophobic sulfonic acid derivatives. Like traditional NSAIDs, these molecules block the hydrophobic channel leading to the active site of cyclooxygenase and thus inhibit the enzyme. Note that COX-2 selective inhibitors are generally larger molecules than NSAIDs. These drugs preferentially inhibit COX-2 compared to COX-1 because the hydrophobic channel of COX-2 is larger than that of COX-1. (That is, COX-2 selective inhibitors are too bulky to access the smaller hydrophobic channel of the COX-1 enzyme.) The COX-2 selective inhibitors display approximately 100-fold greater selectivity for COX-2 compared to COX-1.

o NSAID-induced gastropathy. It is being increasingly recognized, however, that the COX-2 inhibitors may not have as signif cant an advantage over traditional NSAIDs as previously thought in reducing gastropathy and gastrointestinal bleeding. For example, one study with ro ecoxib demonstrated a f ve old increase in signif cant upper gastrointestinal bleed compared to placebo. One possible mechanism or this toxicity could be the adverse e ect o COX-2 inhibitors on the healing o gastric ulcerations. Celecoxib remains the only FDA-approved COX-2 selective inhibitor. Currently approved indications include osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis ( 2 years o age), ankylosing spondylitis, acute pain in adults, and primary dysmenorrhea. Celecoxib is also considered as an adjunct to usual care (e.g., surgery, endoscopic surveillance) to reduce the number o adenomatous colorectal polyps in individuals with familial adenomatous polyposis . Celecoxib decreases the activity o peroxisome proli erator-activated receptor (PPAR ), a transcription actor that heterodimerizes with the RXR transcription actors involved in growth regulation. It is not yet clear whether COX-2 inhibitors bind directly to PPAR or whether their action leads to the production o other molecules that inhibit PPAR . In either case, inhibition o PPAR prevents signaling through the PPAR pathway and thus removes a potent mitogenic stimulus that could promote the development o colon cancer. Like other coxibs, celecoxib carries a label warning o increased, possibly atal, and possibly dose- and durationdependent cardiovascular thrombotic events (myocardial

812 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

in arction and stroke). Celecoxib also increases the risks o hypertension, edema, and heart ailure, particularly at higher doses. Celecoxib is contraindicated in the treatment o pain associated with coronary artery bypass surgery. A primary consideration in prescribing analgesic therapy with a coxib is whether the patient has a concurrent need or an anti-in ammatory agent. I the patient requires primarily analgesia, then acetaminophen may su f ce, perhaps in combination with adjunct analgesics or adjunct therapies (e.g., or arthropathies, consider physical therapy or surgical intervention). I , however, there is an established indication or chronic anti-in ammatory therapy and there is also a risk actor or gastropathy (e.g., history o ulcer disease, elderly, concurrent antiplatelet or anticoagulant or glucocorticoid therapy), then a coxib or a combination regimen with an NSAID and a proton pump inhibitor may be considered. In all cases, the risks o coxibs in patients at risk or ischemic heart disease and cerebrovascular disease must be considered as part o the overall risk/benef t analysis. It was hoped that the second-generation COX-2 inhibitors in development—such as parecoxib (a water-soluble prodrug orm o valdecoxib), etoricoxib, and lumiracoxib—would demonstrate increased selectivity or COX-2 over COX-l and would not have the adverse cardiovascular e ects o the existing COX-2 inhibitors. However, none o these agents has achieved FDA approval, and urther clinical development in this class o drugs remains in question. Cytokine Inhibitors

The proin ammatory cytokines TNF- and IL-1 enhance prostaglandin production and up-regulate COX-2. Novel molecular technologies have provided the ability to inhibit the action o these cytokines and thus to inhibit the process whereby an injurious stimulus activates COX-2 and initiates the in ammatory response. Five antibody-based TNF- antagonists are currently available: etanercept, inf iximab, adalimumab, golimumab, and certolizumab pegol. Etanercept consists o the extracellular domain o the TNF- receptor coupled to human IgG1; in iximab is a humanized mouse monoclonal antibody directed against TNF- ; and adalimumab, golimumab, and certolizumab pegol are humanized monoclonal IgG1 antibodies or Fab antibody ragments directed against TNF- . TNF- antagonists were f rst approved or treatment o rheumatoid arthritis. With ew adverse e ects, these drugs halt joint destruction and bone erosion, decrease pain, calm swollen and tender joints, and limit overall disease progression in rheumatoid arthritis. Anti-TNF antibody drugs have also been approved or use in a variety o other autoimmune diseases (see Chapter 46), such as ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, juvenile idiopathic arthritis ( 4 years o age or adalimumab and 2 years o age or etanercept), Crohn’s disease (adalimumab, certolizumab pegol, and in iximab), and ulcerative colitis (in iximab). Years o experience with this class o drugs has shown an increased risk o serious in ections, including disseminated or extrapulmonary tuberculosis, invasive ungal in ections (Aspergillus and endemic ungi such as Histoplasma), hepatitis B virus reactivation, and opportunistic in ections. Patients are routinely tested or latent tuberculosis prior to initiation o therapy and must be monitored or active tuberculosis during treatment. Other adverse e ects include a small but possibly increased risk o lymphoma, demyelinating disease, heart ailure, and pancytopenia.

Lipoxins, ATLs, and lipoxin-stable analogues also block the actions o TNF- , providing a potential new treatment approach (see below). Anakinra is a recombinant orm o the human IL-1 receptor produced in Escherichia coli; this drug is approved or use in patients with rheumatoid arthritis who have ailed one or more disease-modi ying antirheumatic agents. Canakinumab is a recombinant monoclonal IgG1 antibody directed against human IL-1 beta. Additional IL-1 antagonists are being developed or use in in ammatory and autoimmune diseases. For more in ormation on these agents, re er to Chapter 46.

Prostanoid Receptor Mimetics Several applications or prostanoid receptor agonists are listed in the Drug Summary Table at the end o this chapter.

Thromboxane Antagonists Both TxA2 receptor antagonists and thromboxane synthase inhibitors could theoretically represent power ul and selective agents capable o inhibiting platelet activity and protecting against thrombosis and vascular disease. Thromboxane antagonists could serve as “super” platelet inhibitors in the management o patients with cardiovascular disease. TxA2 receptor antagonists, unlike aspirin, would also be expected to block the vasoconstrictive action o the isoprostanes. Compounds such as dazoxiben and pirmagrel inhibit thromboxane synthase, and ridogrel is a TxA2 receptor antagonist. These thromboxane antagonists have not yet ound clinical utility, however, because the clinical benef t o these drugs is not signif cantly greater than that o aspirin, which is ar less expensive; in addition, other inhibitors o platelet aggregation, which antagonize the P2Y12 subtype o platelet ADP receptor, are in widespread clinical use already (see Chapter 23).

Leukotriene Inhibition Lipoxygenase Inhibition

Inhibition o 5-lipoxygenase has the potential to represent a major therapeutic modality in diseases involving leukotrienemediated pathophysiology, including asthma, in ammatory bowel disease, and rheumatoid arthritis. Lipoxygenase inhibition is an attractive therapeutic approach in these diseases because leukotrienes are potent, locally acting mediators. Several strategies are possible or the design o lipoxygenase inhibitors, based on the structure, unction, and mechanism o the lipoxygenase enzymes. Suicide inhibitors o lipoxygenase (e.g., derivatives o arachidonic acid with triple bonds instead o double bonds), which become covalently bound to the active site and render it inactive, have been developed but are not available or clinical use. Radical scavengers such as catechols, butylated hydroxytoluene (BHT), and -tocopherol trap the radical intermediates in the lipoxygenase reaction and thereby prevent the unctioning o the enzyme, but these nonspecif c compounds cannot be used clinically or lipoxygenase inhibition. Drugs that impair or alter the ability o lipoxygenase to utilize its nonheme iron would be expected to inhibit the activity o the enzyme. The only lipoxygenase inhibitor available or clinical use is zileuton (Fig. 43-10A), a benzothiophene derivative o N-hydroxyurea that inhibits 5-LOX by chelating its nonheme iron. In asthma, zileuton induces bronchodilation, improves symptoms, and generates longlasting improvement in pulmonary unction tests. Zileuton is

C h a p t e r 4 3 Pharmacology o Eicosanoids

biosynthesis, are currently in development. Adenosine , acting via its receptors on neutrophils, inhibits LTB4 biosynthesis by regulating arachidonic acid release and, possibly, by inter ering with the in ux o calcium. Furthermore, adenosine is thought to have a role in limiting cell and tissue injury during in ammation. High cell turnover at in ammatory sites generates high local concentrations o adenosine, which may decrease LTB4 biosynthesis and reduce leukocyte recruitment and activation. Selective adenosine receptor agonists could be considered or development as pharmacologic agents in the control o in ammation.

O

A HO

N

NH2

S

Zileuton

B

Leukotriene Receptor Antagonists O

O

O S

N H

O N

HN

O O

Zafirlukast

COO H

S Cl

813

N

HO

Montelukast

FIGURE 43-10. Leukotriene pathway inhibitors. A. Zileuton is a 5-lipoxygenase inhibitor that blocks the biosynthesis o leukotrienes rom arachidonic acid. B. Zaf rlukast and montelukast are CysLT1 receptor antagonists. All three drugs are approved or the prophylaxis and chronic treatment o asthma. None o these drugs is e ective in the treatment o acute asthma attacks.

e ective in the treatment o asthma induced by cold, drugs, and allergens. However, because o its low bioavailability, low potency, and signif cant adverse e ects such as liver toxicity, zileuton is not as widely used as the other antileukotriene asthma drugs (see below).

5-Lipoxygenase Activating Protein (FLAP) Inhibition

Inter ering with the role o FLAP could represent an alternative approach to the selective inhibition o 5-LOX activity and leukotriene unction. Recall that 5-LOX is activated a ter the enzyme translocates to the nuclear membrane and docks with FLAP, and that FLAP binds arachidonic acid released by phospholipase A2 and shuttles it to the 5-LOX active site. FLAP inhibitors have been developed that both prevent and reverse LOX binding to FLAP and block the arachidonic acid binding site, but no FLAP inhibitors are currently available or clinical use.

Leukotriene Synthesis Inhibitors

Other than zileuton, no specif c inhibitors o the enzymes involved in leukotriene synthesis are available or clinical use. Specif c LTA4 hydrolase inhibitors, which block LTB4

Leukotriene receptor antagonism represents a receptor-based mechanism or inhibiting leukotriene-mediated bronchoconstriction and other e ects (Fig. 43-4). Cysteinyl leukotriene receptor (CysLT1) antagonists are e ective against asthma induced by antigen, exercise, cold, or aspirin. These agents signif cantly improve bronchial tone, pulmonary unction tests, and asthma symptoms. Montelukast and zaf rlukast (Fig. 43-10B) are the currently available cysteinyl leukotriene receptor antagonists; the main clinical application or these antagonists is in the treatment o asthma. More potent CysLT1 antagonists are in development, including pobilukast, tomelukast, and verlukast. Further research will likely elucidate cysteinyl leukotriene receptor subtypes and their respective tissue distributions, which could o er the possibility o tissue-targeted antagonism and the application o these tissue-selective antagonists to other conditions such as rheumatoid arthritis, in ammatory bowel disease, and various allergic disorders.

Lipoxins, Aspirin-Triggered Lipoxins, Resolvins/Protectins/Maresins, and LipoxinStable Analogues Lipoxins, ATLs, and the omega-3-derived resolvins, protectins, and maresins all o er the potential to antagonize the inammatory actions o leukotrienes and other in ammatory mediators and to promote resolution o in ammation. Stable oral and parenteral analogues o these compounds could represent a novel approach to treatment o in ammation, since they are agonists o endogenous anti-in ammation and pro-resolution pathways rather than direct enzyme inhibitors or receptor antagonists. Because lipoxins are endogenous regulators, they would be expected to have selective actions with ew adverse e ects. Stable analogues o lipoxins and ATLs are currently being developed, and second-generation lipoxin-stable analogues have shown e f cacy in enhancing the resolution o recurring bouts o acute in ammation in skin in ammation and gastrointestinal in ammation models. Peptido-conjugates o the proresolving mediators have recently been identif ed that stimulate tissue regeneration and enhance clearance and killing o microbes. This approach to the treatment o in ammation remains to be established in human trials.

CONCLUSION AND FUTURE DIRECTIONS Eicosanoids are critical mediators o homeostasis and o many pathophysiologic processes, especially those involving host de ense and in ammation. Arachidonic acid is the

814 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

important substrate and is converted into prostaglandins, thromboxanes, prostacyclin, leukotrienes, lipoxins, isoprostanes, and epoxyeicosatetraenoic acids. Prostaglandins have diverse roles in vascular tone regulation, gastrointestinal regulation, uterine physiology, analgesia, and in ammation. Prostacyclin and thromboxane coordinately control vascular tone, platelet activation, and thrombogenesis. Leukotrienes (LTC4, LTD4) are the chie mediators o bronchoconstriction and airway hyperactivity; LTB4 is a major activator o leukocyte chemotaxis and inf ltration. Lipoxins antagonize the e ects o leukotrienes, reduce the extent o in ammation, and activate resolution pathways. Pharmacologic interventions at many critical points in these pathways are use ul in limiting in ammatory sequelae. Glucocorticoids inhibit several steps in eicosanoid generation, including the rate-determining step involving phospholipase A2. However, chronic glucocorticoid use is associated with many serious adverse e ects, including osteoporosis, muscle wasting, and abnormal carbohydrate metabolism. Cyclooxygenase inhibitors block the f rst step o prostanoid synthesis and prevent the generation o prostanoid mediators o in ammation. Lipoxygenase inhibitors, FLAP inhibitors, leukotriene synthesis inhibitors, and leukotriene receptor antagonists prevent leukotriene signaling, thereby limiting in ammation and its deleterious e ects. Future drug development e orts will allow selective targeting o eicosanoid pathways involved in many clinical conditions. Systems biology has revealed mechanisms underlying in ammatory disease and has created a new discipline o resolution pharmacology. Essential omega-3 atty acids, in particular EPA and DHA, are precursors to pro-resolving and anti-in ammatory SPM that serve a physiologic role leading to programmed resolution o in ammation (Fig. 43-6). These new bioactive mediators are many times more potent than

their respective omega-3 precursors and, hence, may mediate the essential and benef cial e ects o omega-3 atty acids. In the near uture, resolvins and protectins may be developed as new therapeutic agents to promote resolution o in ammation.

Suggested Reading Brink C, Dahlen SE, Drazen J, et al. International Union o Pharmacology XXXVII. Nomenclature or leukotriene and lipoxin receptors. Pharmacol Rev 2003;55:195–227. (International consensus report on eicosanoid receptors and their antagonists.) Buckley CD, Gilroy DW, Serhan CN. Proresolving lipid mediators and mechanisms in the resolution o acute in ammation. Immunity 2014;40:315–327. (Reviews advances in the role o eicosanoid pathways and novel lipid mediators in resolution programs o in ammation.) Dalli J, Ramon S, Norris PC, et al. Novel proresolving and tissue regenerative resolvin and protectin sulf do-conjugated pathways. FASEB J 2015;29: 2120–2136. (Reports two new amilies o pro-resolving molecules involving sulf do-conjugates o protectins and maresins.) Psaty BM, Furberg CD. COX-2 inhibitors—lessons in drug sa ety. N Engl J Med 2005;352:1133–1135. (Reviews issues surrounding withdrawal o COX-2 selective inhibitors.) Serhan CN. Pro-resolving lipid mediators are leads or resolution physiology. Nature 2014;510:92–101. (Reviews the chemical entities and pathways involved in resolution o in ammation and homeostasis.) Serhan CN, Chiang N, Dalli J. The resolution code o acute in ammation: novel pro-resolving lipid mediators in resolution. Semin Immunol 2015;27:200–215. (Reviews the chemical entities and pathways involved in resolution o in ammation and homeostasis.) Sostres C, Gargallo CJ, Lanas A. Aspirin, cyclooxygenase inhibition and colorectal cancer. World J Gastrointest Pharmacol Ther 2014;5:40–49. (Reviews antitumor pharmacology o aspirin and clinical trials evidence o the e ect o aspirin on colorectal cancer; discusses the concept o aspirin as a chemopreventive medication.) Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Ann Rev Pharmacol Toxicol 1998;38:97–120. (Historic overview o prostaglandin research, including discussion o the pharmacologic manipulation o these pathways.)

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818 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

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His ta mine INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819 INTRODUCTIO 819– 81 9–82 820 820 PHY LOGY OF HIST PHYSIOLOGY STAMINE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819 8119 Histamine Hist Hi istamine staam minee Synthe Syynt Sy nnthe theesis e ssisss,, S esi Storage Storagge, g , and annd R Release . . . . . . . . . . . . . . 819 819 19 Actions A Ac c tions ct ioons ooff H Histami Histam iiss ttam a min ne ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 819 819 19 Histamine H Hi sstami stttaa miin ine ne ne Re R Recept Recep Rece e c e ppttor oors rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821 821 21 PATH P PA ATH AT THOP OPHYSIOLOGY OPHY PHYSI HYS YSIOL SIO OLO LOGY OGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 822 822 82 Clinical C Cl linic linical innical nic ical cal M Manifestations Manifesta a nif an i esta ife e s taat es ati atio ttio io ions ioons onns of of H Histamine His is ist s tam st am mine ine ne Pat atho hophysiology hoph phyysi ys ioolo loogy ogy . . . . . 82 823 823 23 α q/1 Hist Hi sttamine a mine in ne and a nd An Anaaaphyla xisβ . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 823 q lax 1a1x γ PHARMACOLOGIC MACOLO M LOGI LO OGIC O GI CLASSES CLAS CLA LA ASS AS SSES SA AND ND AGE ENTS . . . . . . . . . . . . . . . . 82 8233 GDP GD H1-Antihistami ta mine tamines ne s .G . . . .P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8233 Mechanism o Actionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823

Ina ctive s ta te

Class ssiif ca cati tion o Fi ti Firs rsts tt a ndd Second-Generation H1-Antihistaminess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823 Pharmacologic P Ph ha har ha rrma macoooloogic E ects and Clinical Use sess . . . . . . . . . . . . . . 82 8255 Pharmacokine Ph aarrm rm maac a cco ok oki k nneeeticss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 85 Advers A Ad dve dv veerrrss e E ec e c ttss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825 Other O Ot tth thee r An Antihistam A ntihhist histtam ta mi miines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 CONCLUSION C CO ON O NCL CLU LUS L U US SION A SI SIO AND AN ND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 827 ND Suggested S ugg gggαested Readin ding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82 8277 qq/1 q/111d R

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INTRODUCTION Histamine is a biogenic amine ound in many tissues, including mast cells, basophils, lymphocytes, neurons, and gastric enterochroma f n-like cells. It is an autacoid—that is, a molecule secreted locally to increase or decrease the activity o nearby cells. Histamine is a major mediator o allergic and inf ammatory processes: it also has signi cant roles in the regulation o gastric acid secretion, neurotransmission, and immune modulation. Knowledge o the diverse actions o histamine has led to the development o a number o widely used pharmacologic agents that regulate the e ects o histamine in pathologic states. This chapter ocuses on the pharmacologic actions o H1-antihistamines; H2-antihistamines are discussed in Chapter 47, Integrative In ammation Pharmacology: Peptic Ulcer Disease.

PHYSIOLOGY OF HISTAMINE Histamine Synthesis, Storage, and Release Histamine is synthesized rom the amino acid L-histidine. The enzyme histidine decarboxylase catalyzes the decarboxylation o histidine to 2-(4-imidazolyl)ethylamine, commonly known as histamine (Fig. 44-1). The synthesis o histamine occurs in mast cells and basophils o the immune system, enterochroma f n-like (ECL) cells in the gastric mucosa, and certain neurons in the central nervous system (CNS) that use histamine as a neurotransmitter. Oxidative pathways in the liver rapidly degrade circulating histamine to inert metabolites. One major metabolite o histamine, imidazole acetic acid, can be measured in the urine to determine the amount o histamine that has been released systemically.

Histamine synthesis synthesis and storage can be divided into two “pools”: a slowly turning over pool and a rapidly turning over pool. The slowly turning over pool is located in mast cells and basophils. Histamine is stored in large granules in these in ammatory cells, and the release o histamine involves complete degranulation o the cells. Degranulation can be triggered by allergic processes, anaphylaxis, or cellular destruction rom trauma, cold, or other insults. This pool is termed slowly turning over because several weeks are required to replenish the stores o histamine a ter degranulation has occurred. The rapidly turning over pool is located in gastric ECL cells and in histaminergic CNS neurons. These cells synthesize and release histamine as required or gastric acid secretion and neurotransmission, respectively. Unlike mast cells and basophils, ECL cells and histaminergic neurons do not store histamine. Instead, the production and release o histamine in these cells depend on physiologic stimuli. In the gut, or example, histidine decarboxylase is activated a ter the ingestion o ood.

Actions of Histamine Histamine has a broad spectrum o actions involving many organs and organ systems. To understand the roles o histamine, it is use ul to consider the physiologic e ects o histamine in each tissue (Table 44-1). These e ects include actions on bronchial smooth muscle, vascular smooth muscle, vascular endothelium, a erent nerve terminals, heart, gastrointestinal tract, and CNS. On smooth muscle, histamine causes some muscle bers to contract and others to relax. In the human respiratory system, histamine causes bronchoconstriction (the e ect varies in other species). However, the sensitivity o bronchial 819

820 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Elle n , a g e n e ra lly h e a lth y 76-ye a r-o ld g ra n d m o th e r, s u ffe r s fro m a lle rg ic rh in itis . Eve ry s p rin g , s h e d e ve lo p s a ru n n y n o s e , itch y e ye s , a n d s n e e zin g . To re lie ve h e r s ym p to m s , s h e ta ke s a n o ve rr-th th e -co co u n te r a n tih is ta m in e , d ip h e n h yd ra m in e . S h e is a n n o ye d b y th e u n p le a s a n t e ffe cts th a t a cco m p a n y h e r a lle rg y m e d ica tio n . Eve ry tim e s h e ta ke s h e r a n tih is ta m in e , Elle n fe e ls d ro w s y a n d h e r m o u th fe e ls d ry. S h e m a ke s a n a p p o in tm e n t w ith h e r d o cto r, w h o s u b s e q u e n tly a d vis e s Elle n to ta ke lo ra ta d in e . Up o n ta kin g h e r n e w

smooth muscle to histamine varies among individuals; those with asthma may be up to 1,000 times more sensitive to histamine-mediated bronchoconstriction than nonasthmatic individuals. Other smooth muscles—such as those in the bowel, bladder, iris, and uterus—also contract on exposure to histamine, but these e ects are not thought to play a large role physiologically or clinically. In vascular smooth muscle, histamine dilates postcapillary venules and terminal arterioles. Veins, however, constrict on exposure to histamine. The vasodilatory e ect on the NH2

N HN

HO

O

His tidine Decarboxylation (L-histidine decarboxylase)

NH2

N HN

His ta mine

Ring methylation (Imidazole-Nmethyltransferase)

NH2

N

Oxidative deamination (mainly Diamine oxidase)

OH

N O

HN

N

Me thyl his ta mine

ImAA

Oxidation (Monoamine oxidase)

OH

N N

Conjugation with ribose

O

Me thyl ImAA

ImAA ribos ide

FIGURE 44-1. Histamine synthesis and degradation. Histamine is synthesized from histidine in a decarboxylation reaction catalyzed by L-histidine decarboxylase. The liver metabolizes histamine into inert by-products. Histamine can be methylated on the imidazole ring or oxidatively deaminated. These degradation products can then undergo further oxidation or conjugation with ribose. Diamine oxidase is also known as histaminase. ImAA, imidazole acetic acid.

a lle rg y m e d ica tio n , Elle n ’s s ym p to m s a re re lie ve d a n d s h e e xp e rie n ce s n o d ro w s in e s s o r o th e r a d ve rs e e ffe cts .

Questions 1 . Why does Ellen develop seasonal allergic rhinitis? 2 . What is the mechanism of action of diphenhydramine and loratadine? 3 . Why does diphenhydramine cause drowsiness and dry mouth, and why doesn’t loratadine cause drowsiness or dry mouth?

postcapillary venule bed is the most prominent e ect o histamine on the vasculature. During in ection or injury, histamineinduced venule dilation engorges the local microvasculature with blood, enhancing the access o immune cells that initiate repair processes to the damaged area. This engorgement explains the erythema observed in inf amed tissues. Histamine also causes contraction o vascular endothelial cells. Histamine-induced endothelial cell contraction causes these cells to separate rom one another, allowing release o plasma proteins and f uid rom postcapillary venules and thereby causing edema . Thus, histamine is a key mediator o local responses at sites o injury. Peripheral sensory nerve terminals also respond to histamine. The sensations o itch and pain result rom a direct depolarizing action o histamine on a erent nerve terminals. This e ect is responsible or the pain and itch experienced a ter an insect bite, or example. The combined actions o histamine on vascular smooth muscle, vascular endothelial cells, and peripheral nerve terminals are responsible or the wheal-and-f are response noted a ter histamine release in the skin. Endothelial cell contraction causes the edematous wheal response, while the red, pain ul f are results rom vasodilation and sensory nerve stimulation. Histamine also causes a similar process to occur in the nasal mucosa. Endothelial cell contraction, increased vascular permeability, glandular hypersecretion, and stimulation o irritant receptors contribute to mucosal edema and rhinorrhea, as well as the itching and sneezing typical o allergic rhinitis. The cardiac e ects o histamine consist o minor increases in the orce and rate o cardiac contraction. Histamine enhances Ca2 inf ux into cardiac myocytes, leading to increased inotropy. The increase in heart rate is caused by an increase in the rate o phase 4 depolarization in sinoatrial nodal cells. The primary role o histamine in the gastric mucosa is to potentiate gastrin-induced acid secretion. Histamine is one o three molecules that stimulate acid secretion in the stomach, the others being gastrin and acetylcholine. Activation o histamine receptors in the stomach leads to an increase in intracellular Ca2 in parietal cells and results in increased secretion o hydrochloric acid by the gastric mucosa. Finally, histamine unctions as a neurotransmitter in the CNS. Histaminergic neurons originate in the tuberomammillary nucleus o the hypothalamus and project di usely throughout the brain and spinal cord. Although the unctions o histamine in the CNS are not well understood, histamine

C h a p t e r 4 4 Histamine Pharmacology 821

TABLE 44-1 Major Physiologic Actions o Histamine TISSUE

EFFECT OF HISTAMINE

CLINICAL MANIFESTATIONS

RECEPTOR SUBTYPE

Lungs

Bronchoconstriction

Asthma-like symptoms

H1

Vascular smooth muscle

Postcapillary venule dilation Terminal arteriole dilation Venoconstriction

Erythema

H1

Vascular endothelium

Contraction and separation of endothelial cells

Edema, wheal response

H1

Peripheral nerves

Sensitization of afferent nerve terminals

Itch, pain

H1

Heart

Minor increase in contractility and heart rate

Minor

H2

Stomach

Increased gastric acid secretion

Peptic ulcer disease, heartburn

H2

CNS

Neurotransmitter

Circadian rhythms, wakefulness

H3

CNS, central nervous system.

is believed to be important in the maintenance o sleep–wake cycles (circadian rhythms), cognitive processes (attention, memory, and learning), and eeding behaviors (appetite suppression).

Histamine Receptors Histamine actions are mediated by the binding of histamine to one of four receptor subtypes: H1, H2, H3, and H4. All our subtypes are seven-transmembrane, G protein-coupled receptors, and all demonstrate constitutive activity independent o agonist binding. The receptor iso orms di er in their expression levels, second messenger pathways, and tissue distributions (Table 44-2). The H1 receptor activates G protein-mediated hydrolysis o phosphatidylinositol-4,5-bisphosphate (PIP2), leading to increased intracellular concentrations o inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers the release o Ca2 rom intracellular stores, increasing cytosolic Ca2 concentration and activating downstream pathways. DAG activates protein kinase C, leading to phosphorylation o numerous cytosolic target proteins. In some tissues, such as bronchial smooth muscle, the increase in cytosolic Ca2 causes smooth muscle contraction by Ca2 /calmodulin-mediated activation o myosin light chain kinase, which phosphorylates myosin light chain. In other tissues, especially precapillary arteriolar sphincters and postcapillary venules, the increase in cytosolic Ca2 causes smooth muscle relaxation by inducing the synthesis o nitric oxide (see Chapter 22, Pharmacology o Vascular Tone). H1 receptor stimulation also leads to the activation o NF B, an important and ubiquitous transcription actor that promotes the expression o adhesion molecules and proin ammatory cytokines. H1 receptors are expressed primarily on vascular endothelial cells and smooth muscle cells. These receptors mediate inf ammatory and allergic reactions . Tissue-specif c responses to H1 receptor stimulation include (1) edema, (2) erythema, (3) bronchoconstriction, and (4) sensitization o primary a erent nerve terminals. H1 receptors are also expressed on postsynaptic neurons in the tuberomammillary nucleus o the hypothalamus, cerebral cortex, and limbic system. These neurons appear to be involved in the control o circadian rhythms, wake ulness, and energy metabolism.

The major function of the H2 receptor is to mediate gastric acid secretion in the stomach. This receptor subtype is expressed on parietal cells in the gastric mucosa, where histamine acts synergistically with gastrin and acetylcholine to regulate acid secretion (see Chapter 47). H2 receptors are also expressed on cardiac muscle cells, on some immune cells, and on certain postsynaptic neurons in the CNS. H2 receptors on parietal cells activate a G protein-dependent cyclic AMP cascade, leading to enhanced proton pumpmediated delivery o protons into the gastric uid. Whereas H1 and H2 receptor subtypes have been well characterized, H3 and H4 receptor subtypes and their downstream actions are areas o active investigation. H3 receptors are predominantly located on presynaptic neurons in distinct regions o the CNS, including the cerebral cortex, basal ganglia, and tuberomammillary nucleus o the hypothalamus. H3 receptors appear to unction as both autoreceptors and heteroreceptors, thereby limiting the synthesis and release o histamine as well as other neurotransmitters, including dopamine, acetylcholine, norepinephrine, GABA, and serotonin. This complex interaction between histamine and various neurotransmitter systems contributes to histamine’s wide-

TABLE 44-2 Histamine Receptor Subtypes RECEPTOR SUBTYPE

POSTRECEPTOR SIGNALING MECHANISM

TISSUE DISTRIBUTION

H1

Gq/11 → Increased IP3, DAG, and intracellular Ca 2 , activated NF B

Smooth muscle, vascular endothelium, brain

H2

Gs → Increased cAMP

Gastric parietal cells, cardiac muscle, mast cells, brain

H3

Gi/o → Decreased cAMP

CNS, gastric mucosa

H4

Gi/o → Decreased cAMP, increased intracellular Ca 2

Hematopoietic cells

G, G protein; cAMP, cyclic adenosine monophosphate; IP3, inositol trisphosphate; DAG, diacylglycerol; NF B, nuclear factor kappa B; CNS, central nervous system.

822 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

spread e ects on CNS unctions, including wake ulness, appetite, and memory. The downstream e ects o H3 receptor activation are mediated via a decrease in cAMP. H4 receptors are primarily localized to cells o hematopoietic origin, primarily mast cells, eosinophils, dendritic cells, and basophils. H4 receptors share 40% homology with H3 receptors and bind many H3 receptor agonists, although with lower a f nity. Coupling o the H4 receptor to Gi/o leads to decreased cAMP and activation o phospholipase C , and downstream events result in increased intracellular Ca2 . H4 receptors are o particular interest because they are thought to play an important role in in ammation; activation o H4 receptors mediates histamine-induced leukotriene B4 production, adhesion molecule up-regulation, and chemotaxis o mast cells, eosinophils, and dendritic cells. H4 receptors also appear to play a role in modulating itch and pain.

PATHOPHYSIOLOGY Histamine is an essential mediator o immune and inf ammatory responses. Histamine plays a prominent role in the IgE-mediated type I hypersensitivity reaction, also known as the allergic reaction. In a localized allergic reaction, an allergen (antigen) f rst penetrates an epithelial sur ace (e.g., skin, nasal mucosa). The allergen can also be delivered systemically, as in the case o an allergic response to penicillin.

With the aid o T-helper (TH) cells, the allergen stimulates B lymphocytes to produce IgE antibodies that are specif c or that allergen. The IgE then binds to Fc receptors on mast cells and basophils, in a process known as sensitization. Once these immune cells are “sensitized” with IgE antibodies, they are able to detect and respond rapidly to a subsequent exposure to the allergen. Upon such an exposure, the allergen binds to and cross-links the IgE/Fc receptor complexes, triggering cell degranulation (Fig. 44-2). Histamine released by mast cells and basophils binds to H1 receptors on vascular smooth muscle cells and vascular endothelial cells. Activation o these receptors increases local blood ow and vascular permeability. This completes the initial stage o the in ammatory response. Prolonged inammation requires the activity o other immune cells. The histamine-induced local vasodilation allows such immune cells greater access to the injured area, while the increased vascular permeability acilitates movement o the immune cells into the tissue. Mast cell degranulation can also occur as a response to local tissue damage in the absence o a humoral immune response. For example, trauma or chemical damage can physically disrupt the mast cell membrane and thereby initiate the degranulation process. Histamine release allows or enhanced access o macrophages and other immune cells, which can begin to repair the damaged area.

A Initia l e xpos ure Alle rge n

B ce ll

IgE Ma s t ce ll

Ma s t ce ll

Ca pilla ry

Gra nule s IgE

B S ubs e que nt e xpos ure Alle rge n

Ede ma

Cros s -linke d IgE

His ta mine

De gra nula te d ma s t ce ll

Ma s t ce ll de gra nula tion

FIGURE 44-2. Pathophysiology of the IgE-mediated hypersensitivity reaction. Allergen-induced mast cell degranulation requires two separate exposures to the allergen. A. On initial exposure, the allergen must penetrate epithelial sur aces so that it can encounter cells o the immune system. Activation o the immune response causes B lymphocytes to secrete allergen-specif c IgE antibodies. These IgE molecules bind to Fc receptors on mast cells, leading to sensitization o the mast cells. B. On subsequent exposure, the multivalent allergen cross-links two IgE/Fc receptor complexes on the mast cell sur ace. Receptor cross-linking causes the mast cell to degranulate. Local histamine release results in an in ammatory response, shown here as edema.

C h a p t e r 4 4 Histamine Pharmacology 823

Clinical Manifestations of Histamine Pathophysiology The IgE-mediated hypersensitivity reaction is responsible or initiation o certain in ammatory disorders, including allergic rhinitis and acute urticaria (hives). In the introductory case, Ellen su ered rom allergic rhinitis, with a runny nose, itchy eyes, and sneezing. In allergic rhinitis, an environmental allergen, such as pollen, crosses the nasal epithelium and enters the underlying tissue. There, the allergen encounters previously sensitized mast cells and cross-links IgE/Fc receptor complexes on the mast cell sur ace. Consequently, the mast cell degranulates and releases histamine, which binds to H1 receptors in the nasal mucosa and local tissues. Stimulation o the H1 receptors causes blood vessel dilation and increases vascular permeability, leading to edema. This swelling in the nasal mucosa is responsible or the nasal congestion that is experienced in allergic rhinitis. The accompanying itching, sneezing, runny nose, and tearing result rom the combined action o histamine and other in ammatory mediators, including kinins, prostaglandins, and leukotrienes. These molecules initiate the hypersecretion and irritation characteristic o allergic rhinitis. Mast cell activation also occurs in acute urticaria. Here, an allergen, such as penicillin, enters the body, either through ingestion or parenterally, and reaches the skin through the circulation. Histamine release results in a disseminated wheal-and- are response, creating pruritic, erythematous, and edematous plaques on the skin.

Histamine and Anaphylaxis Systemic mast cell degranulation can cause the li ethreatening condition known as anaphylaxis . Typically, anaphylactic shock is initiated in a previously sensitized individual by a hypersensitivity reaction to an insect bite, an antibiotic such as penicillin, or ingestion o certain highly allergenic oods such as peanuts. An allergen that is distributed systemically, either by intravenous injection or by absorption into the circulation, can stimulate mast cells and basophils to release massive amounts o histamine throughout the body. The resulting systemic vasodilation and extravasation o plasma into the interstitium cause severe hypotension. Systemic histamine release also causes bronchoconstriction and epiglottal swelling, which can be lethal within minutes i not treated rapidly by the administration o epinephrine, as described below.

PHARMACOLOGIC CLASSES AND AGENTS Histamine pharmacology employs three approaches, each o which leads to blockade o histamine action (Table 44-3). The f rst, and the most requently used, approach is to administer antihistamines , which typically are inverse agonists or competitive antagonists selective or the H1, H2, H3, or H4 receptor. H1-antihistamines are discussed below in detail: their mechanism o action involves stabilization o the inactive con ormation o the H1 receptor to decrease signaling events that would lead to the in ammatory response. A second strategy is to prevent mast cell degranulation induced by binding o an antigen to the IgE/Fc receptor complex on mast cells. Cromolyn and nedocromil use this strategy

TABLE 44-3 Strategies of Histamine Pharmacology

STRATEGY

EXAMPLE OF PHARMACOLOGIC AGENT

EXAMPLE OF DISEASE TREATED

Administer inverse agonist of histamine receptor

Diphenhydramine, loratadine

Allergies

Prevent mast cell degranulation

Cromolyn, nedocromil

Asthma

Administer physiologic antagonist to counter the pathological effects of histamine

Epinephrine

Anaphylaxis

to prevent asthma attacks (see Chapter 48, Integrative Inammation Pharmacology: Asthma); these compounds disrupt the chloride current through mast cell membranes, which is a key step in the degranulation process. The third strategy is to administer a drug that unctionally counteracts the e ects o histamine. An example o this approach is the use o epinephrine to treat anaphylaxis. Epinephrine is an adrenergic agonist that induces bronchodilation and vasoconstriction (see Chapter 11, Adrenergic Pharmacology); these actions counter the bronchoconstriction, vasodilation, and hypotension caused by histamine in anaphylactic shock.

H1-Antihistamines Mechanism o Action

Historically, H1-antihistamines were re erred to as H1 receptor antagonists, based on experiments in tracheal smooth muscle that showed a drug-induced parallel shi t in the histamine concentration–response relationship (see Chapter 2, Pharmacodynamics). Recently, however, advances in histamine pharmacology have shown that H1-antihistamines are inverse agonists rather than receptor antagonists. H1 receptors appear to coexist in two con ormational states—the inactive and active con ormations—that are in equilibrium with one another in the absence o histamine or antihistamine (Fig. 44-3). In the basal state, the receptor tends toward constitutive activation. Histamine acts as an agonist or the active con ormation o the H1 receptor, and histamine binding shi ts the equilibrium urther toward the active receptor state. In comparison, antihistamines are inverse agonists . Inverse agonists bind pre erentially to the inactive con ormation o the H1 receptor and shi t the equilibrium toward the inactive state. Thus, even in the absence o endogenous histamine, inverse agonists reduce constitutive receptor activity (see Chapter 2).

Classif cation o First- and Second-Generation H1Antihistamines The f nding that histamine is a major mediator o the allergic hypersensitivity reaction led to the discovery o the f rst H1-antihistamines by Bovet and Staub in 1937. Clinically use ul drugs that inhibit the actions o histamine began to appear in the 1940s. Currently, H1-antihistamines parse into two categories: f rst-generation and second-generation H1-antihistamines (see the Drug Summary Table or details o H1-antihistamine classif cation).

824 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY A

αq/11 β

αq/11

γ

GDP

H1-antihistamines are less selective or the H1 receptor and may additionally bind cholinergic, -adrenergic, and serotonergic receptors at therapeutic doses. The second-generation H1-antihistamines can be structurally categorized into our main subclasses—alkylamines, piperazines, phthalazinones, and piperidines. Widely used secondgeneration H1-antihistamines include loratadine , cetirizine ,

GTP

Ina ctive s ta te

Active s ta te N

X

N

Agonis t (his ta mine ) B

His ta mine Ge ne ra l s tructure (X = C, O, or omitte d)

Alkylamines

αq/11 β

γ

GDP

αq/11

Ethers or ethanolamines

Cl

GTP

O

N

Ina ctive s ta te

N

Active s ta te N

Inve rs e a gonis t (H1-antihistamines)

H1-antihistamine

C

Diphenhydramine

Chlorpheniramine

Ethylenediamines N

N

αq/11 β GDP

Ina ctive s ta te

γ

αq/11

N

N

GTP

Active s ta te

Phenothiazines

N

S Tripelennamine

Promethazine Piperazines

FIGURE 44-3. Simplif ed two-state model o H1 receptor. A. H1 receptors coexist in two con ormational states—the inactive and active states—which are in con ormational equilibrium with one another. B. Histamine acts as an agonist or the active con ormation o the H1 receptor and histamine binding shi ts the equilibrium toward the active con ormation. C. Antihistamines act as inverse agonists that bind and stabilize the inactive con ormation o the H1 receptor, thereby shi ting the equilibrium toward the inactive receptor state.

N N

N Cyclizine

The basic structure o the f rst-generation H1-antihistamines consists o two aromatic rings linked to a substituted ethylamine backbone. These drugs are divided into six main subgroups based on their substituted side chains—ethanolamines, ethylenediamines, alkylamines, piperazines, phenothiazines, and piperidines (Fig. 44-4). Diphenhydramine , hydroxyzine , chlorpheniramine , and promethazine are among the most requently used f rst-generation H1-antihistamines. Firstgeneration H1-antihistamines are neutral at physiologic pH and readily cross the blood–brain barrier, where they block the actions o histaminergic neurons in the CNS. Compared to the second-generation H1-antihistamines, f rst-generation

Piperidines

Cyproheptadine

FIGURE 44-4. Structure o f rst-generation H1-antihistamines. The general structure o the f rst-generation H1-antihistamines consists o a substituted ethylamine backbone with two terminal aromatic rings. (Note the similarity between the ethylamine moiety in these drugs and the ethylamine side chain o histamine shown in Fig. 44-1.) Each o the six subclasses (denoted by blue boxes) is a variation on this general structure. First-generation H1-antihistamines are neutral compounds at physiologic pH and readily cross the blood–brain barrier. In contrast, second-generation H1-antihistamines (e.g., loratadine, cetirizine, exo enadine) are ionized at physiologic pH and do not appreciably cross the blood–brain barrier (not shown). This di erence in blood–brain barrier penetration underlies the di erential extent o sedation associated with use o the f rst- and second-generation H1-antihistamines.

C h a p t e r 4 4 Histamine Pharmacology 825

and fexofenadine . Newer second-generation H1-antihistamines include levocetirizine , the active enantiomer o cetirizine, and desloratadine , an active metabolite o loratadine. Secondgeneration H1-antihistamines are ionized at physiologic pH and do not appreciably cross the blood–brain barrier. The di erences in lipophilicity and selectivity between the f rst- and second-generation H1-antihistamines account or their di erential adverse e ect prof les, notably, the tendency to cause CNS depression (drowsiness) and dry mouth (anticholinergic e ect).

Pharmacologic Effects and Clinical Uses

Antihistamines are used in a broad spectrum o clinical conditions, including allergy, itching, nausea, vomiting, motion sickness, and insomnia. Despite the known contributions o histamine to bronchoconstriction and anaphylaxis, the currently available antihistamines have a limited role in the treatment o asthma or anaphylactic reactions. Allergy Disorders

H1-antihistamines are most use ul in the treatment o allergic disorders to relieve symptoms o rhinitis, conjunctivitis, urticaria, and pruritus. H1-antihistamines strongly block the increased capillary permeability necessary or the ormation o edema and are there ore more e ective when used prophylactically than a ter an allergic reaction has begun. The anti-in ammatory properties o H1-antihistamines are attributable to suppression o the NF B pathway and subsequent reduction in proin ammatory cytokine transcription, chemotaxis, and adhesion molecule expression. The f rst- and second-generation H1-antihistamines are equally e f cacious in the treatment o chronic urticaria and allergic rhinitis. However, due to their avorable adverse e ect prof les, second-generation antihistamines are generally pre erred or long-term clinical use. Although most oral H1-antihistamines do not appreciably relieve symptoms o nasal congestion, topical nasal antihistamines, such as olopatadine and azelastine , have been shown to be benef cial, particularly when they are combined with intranasal corticosteroids. The most recently approved topical H1 antihistamine or allergy disorders is bepotastine besilate ophthalmic solution, which alleviates both ocular and nonocular symptoms o allergic conjunctivitis. Generalized Itching Hydroxyzine and doxepin are potent antipruritic agents, and

their clinical e ectiveness is likely related to their pronounced CNS e ects. Doxepin, a tricyclic antidepressant, is best used in patients with depression, since even small doses can cause con usion and disorientation in nondepressed patients. Compared to oral H1-antihistamines, topical H1-antihistamines (including nasal and ophthalmic preparations) have a more rapid onset o action, but they require multiple administrations each day. Cutaneous preparations o antihistamines, administered or pruritic dermatoses, may paradoxically cause allergic dermatitis.

Nausea and Motion Sickness

First-generation H1-antihistamines can be used to counter motion sickness as well as chemotherapy- and migraine-related nausea and vomiting. By inhibiting histaminergic signals rom the vestibular nucleus to the vomiting center in the medulla, H1-antihistamines such as dimenhydrinate , diphenhydramine , meclizine , and promethazine are use ul as antiemetic agents.

Insomnia

Due to their prominent CNS depressive e ects, f rstgeneration H1-antihistamines such as diphenhydramine , doxylamine , and pyrilamine are also used to treat insomnia. While e ective in promoting sleep, the increased incidence o adverse e ects, including the tendency to cause next-day sedation, limits their use ulness in clinical practice. Standard doses o f rst-generation antihistamines have been shown to cause a reduction in alertness and psychomotor per ormance similar in magnitude to social alcohol consumption. For this reason, f rst-generation antihistamines are contraindicated in individuals who are required to maintain alertness or precision. Several commonly prescribed psychiatric medications, including trazodone (antidepressant) and quetiapine (antipsychotic), are also requently utilized in the treatment o insomnia due to their antihistaminergic properties in the CNS. Limited Use: Asthma and Anaphylaxis

H1-antihistamines have limited e f cacy in bronchial asthma and should not be used as monotherapy or asthma. While H1-antihistamines appear to inhibit constriction o bronchial smooth muscle in guinea pigs, this e ect is much less pronounced in humans because o contributions rom other mediators such as leukotrienes and serotonin. H1-antihistamines alone are also ine ective or systemic anaphylaxis or severe angioedema with laryngeal swelling. In these conditions, the contributions rom other local mediators are una ected by H1-antihistamine treatment, and epinephrine remains the treatment o choice.

Pharmacokinetics

Oral H1-antihistamines are well absorbed rom the gastrointestinal tract, and they reach peak plasma concentrations in 2–3 hours. The duration o e ect varies depending on the particular H1-antihistamine agent. Most H1-antihistamines are metabolized by the liver, and dose adjustments should be considered in patients with severe liver disease. As inhibitors o hepatic cytochrome P450 enzymes, H1-antihistamines may a ect the metabolism o other drugs utilizing the cytochrome P450 system. Co-administration o agents competing or the same enzymes can reduce the metabolism o an H1-antihistamine and increase its serum level.

Adverse Effects

The major adverse e ects o H1-antihistamines are CNS toxicity, cardiac toxicity, and anticholinergic e ects. Adverse e ect prof les o second-generation H1-antihistamines have been thoroughly investigated, but long-term sa ety studies o f rst-generation H1-antihistamines are lacking despite their use or over six decades. Because o their high lipophilicity, f rst-generation H1-antihistamines readily penetrate the blood–brain barrier. These drugs antagonize the neurotransmitter e ects o histamine on H1 receptors in the CNS (especially the hypothalamus) and the periphery. As noted above, the high CNS penetration accounts or the sedating action o these drugs. In the introductory case, Ellen experienced sedation when she took diphenhydramine or her allergic rhinitis. Factors that increase the risk o CNS toxicity include low body mass, severe hepatic or renal dys unction, and concomitant use o drugs, such as alcohol, that impair CNS unction.

826 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

The low CNS penetration o second-generation H1antihistamines is attributable to several eatures o the molecules. First, as noted earlier, these compounds are ionized at physiologic pH and so do not di use readily across membranes. Second, they exhibit high binding a f nity to albumin as well as to the P-glycoprotein e ux pump on the luminal sur ace o the vascular endothelium, thereby limiting their distribution into the CNS. Second-generation H1-antihistamines are o ten pre erred or extended use because o their limited sedative e ects. For example, the second-generation H1-antihistamines loratadine, desloratadine, and exo enadine are the only oral H1-antihistamines permitted or use by airline pilots. H1-antihistamines that prolong the QT interval can cause cardiac toxicity, especially in patients with preexisting cardiac dys unction. Some earlier second-generation H1-antihistamines had serious cardiotoxic e ects at high plasma concentrations. Two o these drugs, ter enadine and astemizole, were withdrawn by the US Food and Drug Administration (FDA) because they caused prolonged QT intervals that sometimes led to ventricular arrhythmias. The mechanism by which H1-antihistamines prolong the QT interval is thought to involve inhibition o the IKr current and not blockade o the H1 receptor. The human ether-a-go-go-related gene (hERG) encodes the subunit o the potassium channel mediating the IKr current, and in vitro testing using variants o hERG is now available or assessing whether a medication has the potential to inhibit the IKr current. Anticholinergic adverse e ects, which are more prominent with f rst-generation than with second-generation H1-antihistamines, include pupillary dilation, dry eyes, dry mouth, and urinary retention and hesitancy. Elderly individuals may demonstrate increased sensitivity to the anticholinergic and sedative e ects o f rst-generation H1antihistamines and may also experience more drug–drug interactions due to a greater number o concomitant medications. The -adrenergic blockade and subsequent hypotension associated with some f rst-generation antihistamines urther predispose older individuals to alls. Young children also appear to be more susceptible to adverse e ects related to antihistamine use. Because o the un avorable adverse e ect prof le and limited evidence or the e f cacy o antihistamines in young children, the FDA advises against the use o cough and cold preparations containing antihistamines in children less than 2 years o age. Overdose o f rst-generation H1-antihistamines can cause severe CNS depression presenting as somnolence, ataxia, and coma. In young children and the elderly, where paradoxical stimulation is more common, acute poisoning may cause hallucinations, irritability, and convulsions be ore progressing to respiratory ailure and cardiovascular collapse. The CNS e ects are also generally accompanied by marked anticholinergic symptoms, such as dehydration, pupillary dilation, and ever.

Other Antihistamines Competitive antagonists and inverse agonists have also been developed against the H2, H3, and H4 receptors. Considerable interest was generated by the development o selective H2 receptor antagonists that inhibit histamine-induced gastric acid secretion. H2 receptor antagonists, which are discussed in detail in Chapter 47, di er in structure rom H1-antihistamines in that they contain an intact f ve-membered ring (instead o two or more bulky aromatic rings) and an uncharged, bulky side chain (Fig. 44-5; see also Fig. 47-5). These agents act

N

H N

S

H N N

HN

C

N

Cimetidine

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FIGURE 44-5. Structure of H2 receptor antagonists. H2 receptor antagonists have a thioethanolamine backbone (highlighted in blue box) that is N-substituted with a bulky side chain and that terminates in a single f ve-membered ring. (Compare the bulky N-substituted side chain o the H2 antagonists with the simple tertiary amine o the H1-antihistamines in Fig. 44-4, and compare the small f ve-membered imidazole or uran ring o the H2 antagonists with the pair o bulky aromatic rings o the H1-antihistamines.) These structural di erences enable cimetidine, ranitidine, and other H2 antagonists to bind selectively to H2 receptors in the gastric mucosa, thereby decreasing the production o gastric acid.

as reversible, competitive antagonists o histamine binding to H2 receptors on gastric parietal cells and thereby reduce gastric acid secretion. Clinical indications include acid reux disease (heartburn) and peptic ulcer disease. Many o these agents are also available over the counter or the symptomatic treatment o heartburn. Cimetidine and ranitidine are two o the most commonly used H2 receptor antagonists. A signif cant adverse e ect o cimetidine involves inhibition o cytochrome P450-mediated drug metabolism, which can result in undesirable elevations in the serum levels o certain concomitantly administered drugs. H2 receptors are also expressed in the CNS and in cardiac muscle, but the therapeutic doses o H2 receptor antagonists are su f ciently low that CNS and cardiovascular adverse e ects are negligible. The pharmacology o H3 and H4 receptors is an active area o investigation. To date, no drugs selectively directed against H3 and H4 receptors have been approved or clinical use. H3 receptors are thought to provide feedback inhibition o certain e ects o histamine in the CNS and in ECL cells. In animal studies, H3 receptor antagonists induce wake ulness and improve attention, e ects that are thought to be mediated by overstimulation o cortical H1 receptors. H3 receptor antagonists that have been developed or experimental use include thioperamide , clobenpropit, ciproxifan, and proxyfan. Pitolisant, ormerly known as tripolisant, is an H3 receptor-selective inverse agonist in late-phase clinical development or the treatment o narcolepsy. Similar to H3 receptors, H4 receptors couple with Gi/o to decrease intracellular cAMP concentrations. Because H4 receptors are selectively expressed on cells o hematopoietic origin, especially mast cells, basophils, and eosinophils, there is considerable interest in elucidating the role o the H4 receptor in the in ammatory process. H4 receptor antagonists represent a promising area o drug development to treat in ammatory conditions that involve mast cells and eosinophils.

C h a p t e r 4 4 Histamine Pharmacology 827

CONCLUSION AND FUTURE DIRECTIONS Histamine plays a key role in diverse physiologic processes including allergy, in ammation, neurotransmission, and gastric acid secretion. Drugs targeting H1 and H2 receptors have substantially increased the pharmacologic options or treatment o allergy and peptic ulcer disease. While most H1-antihistamines demonstrate similar e f cacy in the treatment o allergic rhinitis and urticaria, signif cant di erences exist in the adverse e ect prof les o f rst- and second-generation H1-antihistamines. The more recent elucidation o the H3 and H4 receptor subtypes has renewed interest in the role o histamine in CNS-related disorders. H3-specif c receptor targeting may provide new therapies or a number o cognitive, neuroendocrine, and neuropsychiatric conditions. Clinical and preclinical research is currently underway evaluating prototypic H3 antagonists in pathological processes such as sleep–wake disorders (narcolepsy and insomnia), neuropsychiatric diseases (Alzheimer’s disease, ADHD, dementia, depression, and schizophrenia), neurologic disorders (epilepsy), nociceptive processes (neuropathic pain), and eeding and energy homeostasis (obesity and diabetes). The H4 receptor is also an emerging molecular target or drug development, as it is thought to play an important role in in ammatory conditions involving mast cells and eosinophils. Agents directed against H4 receptors might one day be employed to treat a variety o in ammatory conditions, such as asthma, allergic rhinitis, in ammatory bowel disease, and rheumatoid arthritis.

Acknowledgment We thank Joseph C. Kvedar, Cindy Chambers, Ashish Sahasrabudhe, and Robert R. Rando or their valuable contributions to the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Bhowmik M, Khanam R, Vohora D. Histamine H3 receptor antagonists in relation to epilepsy and neurodegeneration: a systemic consideration o recent progress and perspectives. Br J Pharmacol 2012;167:1398–1414. (Comprehensively reviews the current state o H3 receptor antagonist research with a ocus on epilepsy and neurodegenerative disorders.) Leurs R, Church MK, Taglialatela M. H1-antihistamines: inverse agonism, anti-in ammatory actions and cardiac e ects. Clin Exp Allergy 2002;32:489–498. (Mechanism-based discussion o H1-antihistamines as inverse agonists.) Nicolas JM. The metabolic prof le o second-generation antihistamine. Allergy 2000;55:46–52. (Discussion o di erences among second-generation drugs.) Simons FE.Advances in H1-antihistamines. N Engl J Med 2004;351:2203–2217. (Comprehensively summarizes the mechanism o action and clinical uses o H1-antihistamines.) Thurmond RL, Gel and EW, Dun ord PJ. The role o histamine H1 and H4 receptors in allergic in ammation: the search or new antihistamines. Nat Rev Drug Discov 2008;7:41–53. (Reviews the role o histamine in inf ammation and immune modulation, with emphasis on the role o the H4 receptor.) Zampeli E, Tiligada E. The role o histamine H4 receptor in immune and in ammatory disorders. Br J Pharmacol 2009;157:24–33. (Reviews H4 receptor biology and pharmacology.)

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INTRODU INT UCTION & CA CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830–831 8 Hydroxyureaa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838 B tyratee s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Buty 8388 PHYSIOL LOGY OF HEMATOPOIESIS H ATOPOIES TOPOI TO TOPOIE PO OIE S . . . . . . . . . . . . . . . . . . . . . . . . 830 8 A ennntss That Age att Stimul Stimulate Stimula Stim tim Lee uk ukoocyte Production o . . . . . . . . . . . . . 83 8388 Centraa l Role of Hema Ce H attopoieti poie oie icc Growth h Facto Factor Factors Fac ac r . . . . . . . . . . . 830 8 Re combinant Rec binant nant Hu Hum H m man G-CS CSFs SFs (Fi Filg lgra rast stim im a nd d Muult ltiiil ililine ineeage a ge Gro rowth wth h Fact F Factors act . . . . . . . . . . . . . . . . . . . . . . . . . 832 8 E os inophilobla Eos inophi ino inophi philob hillobla lo lob ob bbllla s t Monob Monobla la s t My M e lobla lob la s t Mee ga ka ry r yo yob yob b laP a sEG t P r roe ro oe o e r ryth ythro ythrob thro rob o b bla la a s t ) . . . . . . . . 838 -Filgrastim) F and GM-CSF M (Sargramostim) m Linee a ge-Specif cif c Growth w Fact ctor orss . . . . . . . . . . . . . . . . . . . . . 83 8333 Agents A ggents That Stimulate i Platelet e Production . . . . . . . . . . . . . . . 838 Eryt Er ythr hrroc yt ytee Pr Prod oduc ucti ction tion on ((Erythropoiesis) (Eryt Erythropoiesis) y hrop opoi oies esis is)) . . . . . . . . . . . . . . . . . 833 883 333 Blo oThrombopoi o d and ti tiss s ue uieeti s in Rec ecep epto torr A Agon Ag onis ists tss . . . . . . . . . . . . . . . . . . . 839 Eryythhropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833 Interleukin-11 1 (rhIL-11 [Oprelvekin]) p pr . . . . . . . . . . . . . . . . . . . 839 Leuk Le ukooc yyt ytee Pr Prod oduc ucttion i (Myelopo ion (M Myyelopoiesis M esis and L Lymphopoiesi Lymph y poiesis) iss) s . . 835 Immunomodulatory mm modula latory A Agenntss with Graa nulocyte-Stim S mulating ulating la tingg Fa Fact F torrss . . . . . . . . . . . . . . . . . . . . . 835 85 Antineoplastic A oplastic Ap Applicaa tio ons . . . . . . . . . . . . . . . . . . . . . . . . . . 8833399 Lym mpphocyte-Stimu S mulating ulating l ting lating lati ng F Fa Fact a c torrs . . . . . . . . . . . . . . . . . . . . . . 835 85 Inter In n errons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839 839 83 Platelett Production t (Thrombopo Thromb Thrombo rom mb oieesis) is) s) . . . . . . . . . . . . . . . . . . . 836 86 Levamisolee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839 Thrombopoietinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836 Interleukin-22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 836 Tretinoin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839 Agents That Stimulate Erythrocyte Production. . . . . . . . . . . . 836 Erythropoiesis-Stimulating Agents (ESAs)) . . . . . . . . . . . . . 837 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 840 Agents That Induce Fetal Hemoglobin (HbF). . . . . . . . . . . . . . 837 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .840 5-Azacytidine and Decitabinee . . . . . . . . . . . . . . . . . . . . . . . . 837

INTRODUCTION Many clinical situations are characterized by def ciencies o red blood cells, white blood cells, and/or platelets—cells o the hematopoietic system. This chapter describes the pharmacologic agents that can be used to stimulate production o hematopoietic cells; it is also important to note the nonpharmacologic alternatives, which could include trans usion and bone marrow transplantation. Blood cell production is controlled physiologically by hematopoietic growth actors, a diverse but unctionally overlapping group o glycoproteins produced by the body in response to certain signals. For example, hypoxia stimulates the synthesis and release o the erythroid lineage growth actor erythropoietin, which in turn stimulates the production o erythrocytes in an attempt to relieve the hypoxia. The main pharmacologic strategy used to stimulate the production o blood cells is to administer exogenous growth actors or synthetic growth actor analogues. This chapter provides an introduction to the cells o the hematopoietic system, the growth actors that stimulate their production, and the pharmacologic agents used to increase blood cell production. An outline o the immunomodulatory agents used in anticancer therapy is also presented. 830

PHYSIOLOGY OF HEMATOPOIESIS The cells o the hematopoietic system are unctionally diverse (Table 45-1). Red blood cells, or erythrocytes , carry oxygen; many types o white blood cells, rom granulocytes and macrophages to lymphocytes , f ght in ection and help protect against cancer; and platelets help control bleeding. Nonetheless, these cells have one eature in common: they all develop rom a common cell in the bone marrow called the pluripotent hematopoietic stem cell (Fig. 45-1). Hematopoietic stem cells are induced to di erentiate along committed lineages into red blood cells, white blood cells, or platelets through interactions with glycoproteins called hematopoietic growth factors .

Central Role of Hematopoietic Growth Factors Hematopoietic growth actors and cytokines constitute a heterogeneous group o molecules that regulate blood cell production, maturation, and unction. Nearly 36 such actors have been identif ed, ranging in size rom 9 to 90 kDa. The membrane-associated receptors or these actors

C h a p t e r 4 5 Pharmacology o Hematopoiesis and Immunomodulation

Fi ty-tw o -ye a r-o ld Mr s . M p re s e n ts w ith a lu m p in h e r le t b re a s t. S u b s e q u e n t m a m m o g ra m , co re b io p s y, a n d lu m p e cto m y le a d to th e d ia g n o s is o in f ltra tin g d u cta l ca rcin o m a th a t is lo ca lize d b u t lym p h n o d e –p o s itive . S h e b e g in s a d ju va n t ch e m o th e ra p y w ith d o xo ru b icin a n d cyclo p h o s p h a m id e . Te n d a ys a te r th e f rs t cycle o ch e m o th e ra p y, h e r w h ite b lo o d ce ll co u n t (WBC) d ro p s , a s e xp e cte d ; ove r th e n e xt 9 d a ys , h e r WBC re cove rs to its n o rm a l va lu e . By th e th ird cycle o ch e m o th e ra p y, Mrs . M is m o d e ra te ly a n e m ic, w ith a h e m a to crit o 28% (n o rm a l, 37–48%), a n d s h e e e ls q u ite a tig u e d . Se ve n d a ys a te r th e o u rth cycle o ch e m o th e ra p y, h e r WBC p lu m m e ts to 800 ce lls p e r m icro lite r ( L) o b lo o d (n o rm a l, 4,300–10,800 ce lls / L), a n d h e r a b s o lu te n e u tro p h il co u n t (ANC) is 300 ce lls / L. In th is s e ttin g , s h e d e ve lo p s s h a kin g ch ills a n d a e ve r to 102°F. S h e is a d m itte d to th e h o s p ita l, w h e re s h e re ce ive s p a re n te ra l a n tib io tics , a n d s h e re m a in s th e re o r 5 d a ys u n til h e r ANC ris e s to a n a cce p ta b le le ve l. Mrs . M co m p le te s h e r cycle s o d oxo ru b icin a n d cyclo p h o s p h a m id e ch e m o th e ra p y, co n tin u e s ch e m o th e ra p y w ith p a clita xe l, a n d re ce ive s lo ca l ra d ia tio n th e ra p y. Mrs . M is w e ll o r 2 ye a rs b u t th e n p re s e n ts w ith p a in in th e le t le g . Wo rku p re ve a ls th a t th e

831

ca n ce r h a s m e ta s ta s ize d to h e r le t e m u r a n d live r. S h e b e g in s ch e m o th e ra p y w ith d o xo ru b icin a n d d o ce ta xe l b u t a g a in d e ve lo p s s e ve re n e u tro p e n ia a n d e ve r. S h e b e co m e s s h o rt o b re a th w h ile clim b in g s ta irs , a n d h e r h e m a to crit is 27%. He r iro n s to re s a re n o rm a l. Th e re a te r, h e r ch e m o th e ra p y is s u p p le m e n te d w ith p e g yla te d re co m b in a n t h u m a n G-CS F (PEG-f lg ra s tim ) a n d a n a n a lo g u e o h u m a n e ryth ro p o ie tin (d a rb e p o e tin ). Ne u tro p e n ia a n d e ve r d o n o t re cu r, a n d b y 4 w e e ks a te r th e in itia tio n o e ryth ro p o ie tin th e ra p y, h e r h e m a to crit ris e s to 34.5% a n d s h e h a s n o rm a l e xe rcis e to le ra n ce . Th e ch e m o th e ra p y yie ld s e xce lle n t p a llia tive re s u lts . On e ye a r la te r, s h e is s till in re m is s io n a n d le a d in g a n a ctive li e .

Questions 1 . Are G-CSF and erythropoietin multilineage growth actors or lineage-specif c growth actors? 2 . How does erythropoietin increase the number o erythrocytes in the blood? 3 . How do analogues o hematopoietic growth actors such as darbepoetin and PEG-f lgrastim di er rom endogenous, “natural” hematopoietic growth actors? 4 . What are the important adverse e ects o erythropoietin?

TABLE 45-1 Hematopoietic Cells, Growth Factors, and Growth Factor Analogues CELL TYPE

MAJ OR FUNCTION(S)

LINEAGE-SPECIFIC GROWTH FACTOR

Red blood cell (erythrocyte)

Oxygen transport

Erythropoietin (EPO)

Anemia

Epoetin al a, PEG-epoetin beta, darbepoetin al a

Platelet (thrombocyte)

Hemostasis

Thrombopoietin (TPO)

Thrombocytopenia

Eltrombopag, romiplostim, IL-11

Monocyte/macrophage

Phagocytosis o bacteria and cellular and chemical debris, stimulation o T lymphocytes

M-CSF

—

—

Neutrophil

Phagocytosis o bacteria, immune stimulation

G-CSF

Neutropenia

Filgrastim, PEG-f lgrastim, sargramostim

Eosinophil

Control o parasites

IL-5

—

—

B lymphocyte

Production o antibody, stimulation o T lymphocytes

Specif c interleukins

Various immunodef ciency syndromes

—

T lymphocyte

Killing o virus- and bacteriain ected cells, control o immune responses

Specif c interleukins

Various immunodef ciency syndromes

rhIL-2

NK cell

Killing o cancer cells

—

—

—

DEFICIENCY STATE

THERAPEUTIC AGENTS

NK, natural killer; M-CSF, monocyte colony-stimulating actor; G-CSF, granulocyte colony-stimulating actor; IL-5, interleukin-5; PEG, polyethylene glycol; IL-11, interleukin-11; rhIL-2, recombinant human interleukin-2.

832 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Bone marrow

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P luripote nt he ma topoie tic s te m ce ll

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M

u

Triline a ge mye loid s te m ce ll (CFU-S )

Lymphoid s te m ce ll

IL-3, GM-CS F, IL-6 GM-CS F

TP O, IL-11

l

l

s

IL-5

S CF, Flt3L, IL-7

CFU-G/M

CFU-Mix

P ro-B

P ro-NK

P ro-T

IL-5

M-CS F

G-CS F

Flt3L

IL-15

IL-7

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CFU-G

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TP O

EP O Thymus CFU-E

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Eos inophilobla s t

Monobla s t

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Me ga ka ryobla s t P roe rythrobla s t

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Blood and tissues

Eos inophil

Monocyte / ma cropha ge

Ne utrophil

P la te le ts

Erythrocyte

B ce ll

NK ce ll

T ce ll

FIGURE 45-1. Development o cells o the hematopoietic system. Mature cells o the hematopoietic system all develop rom pluripotent stem cells that reside in the bone marrow. The type o mature cell that develops is dependent on the extracellular milieu and the exposure o stem cells and progenitor cells to specif c growth actors. The pluripotent hematopoietic stem cell di erentiates into a trilineage myeloid stem cell (CFU-S) or a lymphoid stem cell. Depending on the growth actors that are present, CFU-S cells di erentiate into granulocytes (eosinophils, neutrophils), monocyte/macrophages, platelets, or erythrocytes. Lymphoid stem cells di erentiate into B cells, natural killer (NK) cells, or T cells. Except or the terminal di erentiation o pro-T cells to mature T cells, which takes place in the thymus, the di erentiation o all hematopoietic stem cells, progenitor cells, and precursor cells occurs in the bone marrow. O the growth actors illustrated here, G-CSF, GM-CSF, erythropoietin (EPO), and IL-11 are currently used as therapeutic agents. BFU, burst- orming unit; CFU, colony- orming unit; CSF, colony-stimulating actor; IL, interleukin; SCF, stem cell actor; TPO, thrombopoietin.

belong to at least six receptor superfamilies, and genes encoding the factors are found on 11 different chromosomes. Conceptually, growth factors can be divided into two groups: multilineage (also called general, early-acting, or pleiotropic ) growth factors, which stimulate multiple lineages, and lineage-specif c (also called lineage-dominant or late-acting) growth factors, which stimulate differentiation and survival of a single lineage. Many growth factors and

cytokines act synergistically with one another, sometimes with overlapping effects.

Multilineage Growth Factors

Multilineage growth factors include stem cell actor (also called steel actor or KIT ligand), interleukin-3 (IL-3), granulocyte-monocyte colony-stimulating actor (GM-CSF), insulin-like growth factor 1, IL-9, IL-11, and others. Many of

C h a p t e r 4 5 Pharmacology of Hematopoiesis and Immunomodulation

these growth actors are discussed below with respect to the development o individual hematopoietic cell types. The relevant pharmacologic principle is that multilineage growth actors might be appropriate or treating conditions such as pancytopenia in which multiple hematopoietic lineages are a ected. The ability o multilineage growth actors to stimulate multiple lineages results rom two eatures o their molecular and cellular physiology. First, the receptors or these growth actors are both structurally related and modular; this commonality makes them somewhat interchangeable. Second, the signal transduction cascades activated by binding o these growth actors to their receptors involve a common amily o signaling proteins, the JAK-STAT proteins. In the myeloproli erative diseases polycythemia vera, essential thrombocytosis, and myeloid metaplasia with myelof brosis, the JAK2 kinase is constitutively activated by a point mutation that leads to a single amino acid substitution (V617F) in the protein product o the gene. These diseases are characterized by clonal proli eration o all lineages, highlighting the general role o the JAK-STAT pathway in hematopoiesis. Pharmacologists have exploited the commonalities o multilineage growth actor signaling to design synthetic growth actors with novel properties (see below).

Lineage-Specif c Growth Factors

For a growth actor to be lineage-specif c, at least one o two conditions must be met: (1) the expression o the growth actor’s receptor(s) must be limited to progenitor and/or precursor cells within a single lineage and/or (2) the growth actor must induce inhibitory or apoptotic signals in cells o other lineages. Erythropoietin is one example o a lineage-specif c growth actor; thrombopoietin, whose actions are essentially limited to the platelet lineage, is another. Other so-called lineage-specif c growth actors are more properly considered lineage-selective, because they have secondary e ects on lineage(s) other than the lineage o their primary action. Such actors include G-CSF, which primarily promotes the di erentiation o neutrophils, and a number o interleukins , which have selective actions on certain myeloid and lymphoid lineages (see below). From a pharmacologic perspective, lineage-specif c growth actors represent selective therapeutics that can be used to treat a def ciency o a single hematopoietic cell type. Some growth actors may also have unique e ects against certain cancers, perhaps due to their prodi erentiation and promaturation properties.

Erythrocyte Production (Erythropoiesis) Erythrocytes are uniquely suited to their role o transporting oxygen rom the lungs to the tissues o the body. These cells contain high concentrations o hemoglobin, a protein that binds and releases oxygen molecules in response to the partial pressure o oxygen in the blood and tissues. Each hemoglobin molecule consists o our similar polypeptide chains, and each chain contains a binding site or molecular oxygen. The major orm o adult hemoglobin, which has two alpha and two beta chains ( 2 2), is called hemoglobin A (HbA). Fetal hemoglobin, or hemoglobin F (HbF), contains gamma ( ) chains instead o chains ( 2 2). HbF predominates during the latter 6 months o etal li e and has a higher a f nity or oxygen than HbA does, which helps acilitate the trans er o oxygen rom mother to etus. A ter

833

birth, DNA methylation inactivates the globin gene, and expression o the globin gene rises. It is important to note that expression o the , , and globin chains is regulated independently, making possible a multitude o hemoglobinopathies in which the or chains are abnormal or underexpressed because o an inherited mutation. In sickle cell anemia , a point mutation in the globin gene results in the production o an abnormal hemoglobin— hemoglobin S (HbS)—that polymerizes upon deoxygenation, causing morphologic “sickling” o erythrocytes and leading to hemolytic anemia, pain ul vaso-occlusive crises, and pro ound end-organ damage. This autosomal recessive disease is the most common inherited blood disorder in the United States, a ecting more than 70,000 individuals. Another common hemoglobinopathy is thalassemia , in which the chain is structurally and unctionally normal but underexpressed. Upon their release rom the bone marrow, normal erythrocytes circulate in the blood with a li espan o approximately 120 days. The number o erythrocytes in the blood is determined by the balance between new erythrocyte production in the bone marrow and erythrocyte loss due to cell destruction (hemolysis) and bleeding. This number is measured clinically as either the hemoglobin level (the concentration o hemoglobin per unit volume o blood) or the hematocrit (the percentage o blood volume that is composed o erythrocytes). The normal hemoglobin level ranges rom 14 g/dL to 17 g/dL in men and 12 g/dL to 15 g/dL in women, and the normal hematocrit ranges rom 42% to 50% in men and 37% to 46% in women. These gender di erences are o ten attributed to increased blood loss through physiologic—that is, menstrual—bleeding in women and enhanced erythropoiesis induced by androgens (through unclear mechanisms) in men. A hemoglobin level or hematocrit below the normal range is def ned as anemia .

Erythropoietin

Erythrocyte production, or erythropoiesis , proceeds under the control o several growth actors. The major growth actor controlling erythropoiesis is erythropoietin, a heavily glycosylated protein that is produced mainly by the liver in the etus and by the kidney a ter birth. A lineage-specif c growth actor, erythropoietin has received great clinical attention because it stimulates all but the earliest intermediates in the erythroid lineage but does not signif cantly a ect other lineages. Its physiologic importance is attested to by experiments in mice and pathologic conditions in humans, both o which show that the absence o erythropoietin results in severe anemia. Furthermore, rare activating mutations o the erythropoietin receptor have been described in patients with primary amilial and congenital polycythemia, a disorder mani ested by isolated erythrocytosis and increased responsiveness to erythropoietin. Such was the case or Eero Mantyranta, a Finnish cross-country skier who won several gold medals in the 1964 Olympics but was accused o blood “doping” (receiving erythrocyte trans usions to artif cially increase oxygen-carrying capacity) because o an abnormally high hematocrit. He was exonerated 30 years later when researchers identif ed an activating mutation o the erythropoietin receptor in samples rom him and his amily. Given the role o erythrocytes in transporting oxygen, it is not surprising that erythropoietin production is triggered

834 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY Low O2

Normal or high O2 CoCl2 Iron che la tion Antioxida nts

HIF-1α P HD

HIF-1α P HD

O 2 or CO HIF-1α

VHL comple x

OH

Ub Ub Ub Ub Ub

HIF-1α

OH

Nucle us

26S prote a s ome HIF-1α HIF-1β

Tra ns cription of VEGF, P DGF-β, TGF-α, EP O ge ne s

Ub Ub

HIF-1α fra gme nts

FIGURE 45-2. Regulation of erythropoietin synthesis. Synthesis o erythropoietin by the kidney is increased when the oxygen content o the blood is low and decreased when the oxygen content o the blood is normal or high. The physiologic O2 sensor is an iron-containing dioxygenase, prolyl hydroxylase (PHD). (In vitro experiments using CoCl2, iron chelation, antioxidants, and CO demonstrated the identity o the O2 sensor as an iron-containing protein.) Under conditions o normal or high O2 (left panel), activated PHD hydroxylates proline residues on hypoxia-inducible actor 1 (HIF-1 ). This post-translational modif cation enhances HIF-1 binding to the ubiquitin ligase pVHL(VHLcomplex), leading to ubiquitination (Ub) and proteolytic degradation o HIF-1 by the 26S proteasome. Under low oxygen conditions (right panel ) the prolyl hydroxylase is inactivated, allowing HIF-1 to accumulate, translocate to the nucleus, and induce the expression o a number o genes, including the gene encoding erythropoietin (EPO). In pathologic conditions, such as chronic kidney disease, the cells o the kidney that normally synthesize EPO are injured. These injured cells cannot synthesize adequate amounts o EPO, even under conditions o hypoxia, and anemia ensues. Recombinant human EPO can be administered exogenously to supply the missing growth actor and thereby treat the anemia. See text or discussion o the risks and benef ts o EPO treatment in patients with anemia and chronic kidney disease.

by hypoxia. Erythropoietin expression is strongly induced by hypoxia-inducible factor 1 alpha (HIF-1 ), which binds to an enhancer element in the erythropoietin gene and activates gene transcription (Fig. 45-2). The level o HIF-1 within a cell is heavily in uenced by the local oxygen tension. Under normal or high oxygen conditions, HIF-1 is hydroxylated by prolyl hydroxylase (PHD) via its Fe (II)-dependent dioxygenase activity. Prolyl hydroxylation o HIF-1 acilitates its binding to the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex, thus targeting HIF-1 or proteasomal degradation. Under hypoxic conditions, prolyl hydroxylation o HIF-1 does not occur, HIF-1 does not associate with pVHL, and HIF-1 instead translocates to the nucleus, where it enhances transcription o the hypoxia-inducible genes, including erythropoietin. In the rare autosomal recessive disease amilial erythrocytosis 2 (also called Chuvash polycythemia, a ter the ethnic population o the mid-Volga River region in which it was f rst described), both germline copies o pVHL are mutated so as to prevent association with HIF-1 , reducing the degradation o HIF-1 and leading to elevated levels o erythropoietin and other target genes.

A ter transcription and translation, the 166-amino acid, 18-kDa erythropoietin protein is glycosylated to 34–39 kDa, its terminal arginine is cleaved, and the protein is secreted and transported in the circulation to the bone marrow. There, it binds to erythropoietin receptors expressed on the sur ace o BFU-E and all subsequent progenitor and precursor cells in the erythroid lineage, including the erythrocyte’s immediate precursor cell, the reticulocyte . Then, through a complex intracellular signaling cascade mediated by JAK-STAT, erythropoietin receptor activation enhances the proli eration and di erentiation o erythroid-lineage cells, including the terminal di erentiation o reticulocytes to erythrocytes. Erythropoiesis completes a negative eedback loop on erythropoietin production, because the more erythrocytes in the blood (i.e., the higher the hemoglobin level and hematocrit) the higher the oxygen-carrying capacity o the blood. In the absence o cardiopulmonary disease, the higher oxygencarrying capacity resolves the hypoxia and thereby removes the stimulus or increased erythropoietin production. Table 45-2 lists the mechanisms o several prominent pathologic conditions that stimulate or inhibit erythropoiesis.

C h a p t e r 4 5 Pharmacology o Hematopoiesis and Immunomodulation

TABLE 45-2 Pathologic Conditions That Stimulate or Inhibit Erythropoiesis CONDITION

MECHANISM

Stimulate Erythropoiesis Bleeding Hemolysis High altitude Pulmonary disease

Induce tissue hypoxia

J AK2-activating mutations in myeloproli erative disorders

Increase intracellular J AKSTAT signaling

Inhibit Erythropoiesis Chronic kidney disease

Decreases erythropoietin synthesis in kidney

Iron, olate, or vitamin B12 def ciency Chronic in ammatory conditions Sideroblastic anemia Thalassemia Malignant inf ltration o bone marrow Aplastic anemia, pure red cell aplasia Drug-induced bone marrow toxicity

Decrease erythroblast di erentiation and erythrocyte production

Leukocyte Production (Myelopoiesis and Lymphopoiesis) White blood cells, or leukocytes , are essential cells o the immune system. There are two main categories o leukocytes, corresponding to the two main branches o the immune system. Cells o the innate branch o the immune system include granulocytes (neutrophils , eosinophils , and basophils ), monocyte/macrophages , and variants o the macrophage lineage. Neutrophils target bacteria, eosinophils target parasites, and basophils participate in hypersensitivity responses. Macrophages also target bacteria, but these cells and their variants—dendritic cells , Langerhans cells , and osteoclasts , among others—have important additional unctions. Macrophages play key roles in stimulating and regulating both the innate and adaptive branches o the immune system during in ection and in clearing biological debris. Dendritic cells and Langerhans cells are important or initiating and targeting the immune response. These cells transport antigen rom the site o inoculation to lymph nodes, where lymphocyte responses are coordinated. Osteoclasts are essential or bone resorption. Cells o the adaptive branch o the immune system are called lymphocytes . The two types o lymphocytes are B cells, which make antibodies, and T cells, and which target virus-in ected and neoplastic cells (among other unctions). “Adaptive” re ers to the ability o these cells to recognize and respond to specif c in ectious agents and other targets (see Chapter 42, Principles o In ammation and the Immune System). All white blood cells develop rom pluripotent hematopoietic stem cells (Fig. 45-1). Under the in uence o growth actors, these stem cells di erentiate into either myeloid stem cells or lymphoid stem cells . Myeloid stem cells urther di erentiate into the various cells o the innate branch o the immune system (as well as erythrocytes and platelets), while lymphoid stem cells di erentiate into cells o the adaptive branch o the immune system. The growth actors that regulate these di erentiation pathways are discussed in the ollowing sections.

835

Granulocyte-Stimulating Factors

The di erentiation o pluripotent stem cells into myeloid stem cells is ostered by certain multilineage growth actors such as stem cell actor and IL-3. Further di erentiation o myeloid stem cells into neutrophils and monocyte/ macrophages is controlled by the multilineage growth actor granulocyte-monocyte colony-stimulating factor (GM-CSF) and the lineage-specif c growth actors granulocyte colonystimulating factor (G-CSF) and monocyte colony-stimulating factor (M-CSF). The di erentiation o myeloid stem cells into eosinophils is controlled by interleukin-5 (IL-5). GM-CSF has relatively broad e ects on cells o the myeloid lineage. Produced mainly by macrophages and T cells, this 18- to 28-kDa glycoprotein stimulates the di erentiation o myeloid stem cells and progenitor cells into morphologically recognizable precursors o eosinophils, monocyte/ macrophages, and neutrophils. GM-CSF also enhances the activity o these mature leukocytes and promotes the di erentiation o macrophages into Langerhans cells. Some o the e ects o GM-CSF are indirect. For example, the e ects o GM-CSF on neutrophil production and unction may result not only rom direct GM-CSF stimulation o neutrophil precursors but also rom GM-CSF-stimulated secretion o other cytokines (such as TNF and IL-1) by other cells. Like other hematopoietic growth actors, GM-CSF signals through the JAK-STAT signaling pathway. G-CSF has e ects that are more lineage-selective than those o GM-CSF. G-CSF is an 18-kDa glycoprotein that, like GM-CSF, signals through the JAK-STAT signaling cascade. G-CSF is released into the circulation by monocytes, macrophages, epithelial cells, and f broblasts at sites o inection. In the bone marrow, G-CSF stimulates the production o neutrophils, which in turn enhance the ability o the immune system to f ght in ection. Locally released G-CSF stimulates neutrophil-mediated phagocytosis. The e ects o M-CSF (also known as CSF1) are restricted to the di erentiation and activation o monocyte/ macrophages and their various related cells (including a subset o osteoclasts). In a positive eedback loop, these are also the cells that produce M-CSF. M-CSF exists in alternatively spliced 70–80 kDa and 40–50 kDa iso orms. A rare benign tumor called pigmented villonodular synovitis has recently been shown to contain a genetic translocation involving CSF1. This translocation leads to dysregulated expression o CSF1 and the ormation o an in ammatory mass composed predominantly o proli erating macrophages and histiocytes. IL-5 is produced by a subset o helper T cells. This growth actor selectively promotes the di erentiation, adhesion, degranulation, and survival o eosinophils. As such, IL-5 is believed to play an important role in the pathophysiology o allergic reactions and asthma.

Lymphocyte-Stimulating Factors

Regulatory proteins called interleukins control lymphocyte development and activation. To date, more than 30 members o this amily have been def ned. Family members are numbered IL-1, IL-2, and so orth. Interleukins regulate not only lymphocyte di erentiation but also multiple and overlapping aspects o innate and adaptive immune responses, including stimulation o T cells and macrophages. Several interleukins are described above as granulocyte-stimulating actors; others are discussed below in the context o platelet production. IL-2 and IL-7 are two interleukins critical to white blood cell di erentiation. IL-2 is a 45-kDa protein produced by

836 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

T cells. Because it drives proli eration o T cells and B cells, IL-2 once received much attention as a potential immunostimulant. Investigations o this hypothesis showed, however, that mice lacking IL-2 exhibit lymphoproliferative rather than lymphopenic diseases. This unexpected f nding underscores the principle that growth actors and immune cells have diverse unctions in vivo, including, as in this case, regulatory or suppressive (tolerogenic) e ects as well as stimulatory e ects. This f nding also points out that uncontrolled proli eration can ensue i di erentiation is not regulated normally, a process that may underlie some types o cancer. IL-7, produced by cells in the spleen, thymus, and bone marrow stroma, is a multilineage lymphostimulatory growth actor that enhances the growth and di erentiation o B cells and T cells. The interferons constitute a second amily o regulatory proteins that modulate lymphocyte growth and activity. Like the interleukins, these proteins can stimulate the activity o T cells and macrophages. Inter erons have prominent antiviral actions and are sometimes used in the treatment o in ections such as hepatitis B and C (see Chapter 38, Pharmacology o Viral In ections). Other e ects o inter erons include promoting the terminal di erentiation o lymphocytes, suppressing cell division (in some situations), and exerting direct cytotoxic e ects on cells under stress. The three types o inter erons—called IFN- , IFN- , and IFN—have di erent biological actions. The cellular e ects o inter erons, like those o hematopoietic growth actors, are mediated by specif c cell sur ace receptors and JAK-STAT signal transduction cascades.

Platelet Production (Thrombopoiesis) Platelets—sometimes called thrombocytes —are essential or clot ormation. These small cells, which lack a nucleus and do not synthesize new proteins, have a hal -li e o about 10 days in the circulation. The production o platelets, like that o all ormed elements o the hematopoietic system, is controlled by both multilineage and lineage-specif c growth actors (Fig. 45-3). The most important multilineage growth actors that stimulate platelet production are IL-11, IL-3, GM-CSF, stem cell actor, and IL-6. Not surprisingly, these Me ga ka ryobla s t

Mye loid s te m ce ll

IL-11 TP O

IL-11

P la te le ts

TP O

IL-3 GM-CS F S CF Early

IL-6

actors also stimulate the production o erythrocytes because platelets and erythrocytes share a common progenitor, the CFU-Mix cell. Whether CFU-Mix cells become erythrocytes or platelets depends on their subsequent exposure to lineage-specif c growth actors. Di erentiation into BFU-E and other cells o the erythroid lineage is promoted by erythropoietin, while di erentiation into CFU-Mega cells and then into megakaryocytes (which then orm platelets) is promoted by the lineage-specif c growth actor thrombopoietin (Fig. 45-1).

Thrombopoietin

Thrombopoietin (TPO) is produced in the liver and, to a lesser extent, in the proximal convoluted tubule o the kidney. Like erythropoietin, thrombopoietin is a heavily glycosylated protein (35 kDa) that has its major e ect on a single cell lineage; also like erythropoietin, thrombopoietin signals through a JAK-STAT transduction cascade. However, unlike erythropoietin, thrombopoietin is not regulated in its activity at the level o gene expression, because thrombopoietin is expressed constitutively. Instead, by an interesting unctional mechanism, circulating levels o thrombopoietin are regulated by the thrombopoietin receptor (also known as Mpl), which is the protein product o the gene c-mpl. Structurally and unctionally, the thrombopoietin receptor resembles the receptors or IL-3, erythropoietin, and GMCSF. It is ound both on platelet progenitors—CFU-S, CFUMix, CFU-Mega, and megakaryocytes—and on platelets themselves. Thrombopoietin has di erent e ects on these cell types, however. On platelet progenitors, the binding o thrombopoietin to its receptor promotes cell growth and di erentiation. In contrast, thrombopoietin receptors on platelets act as molecular sponges to bind excess thrombopoietin and thereby prevent platelet overproduction i platelets are in adequate supply. Thrombopoietin also enhances platelet unction by sensitizing these cells to the proaggregatory e ects o thrombin and collagen (see Chapter 23, Pharmacology o Hemostasis and Thrombosis).

PHARMACOLOGIC CLASSES AND AGENTS The hematopoietic growth actors used clinically can be divided into two groups. First, recombinant or synthetic growth actor analogues are used to treat def ciencies o the various hematopoietic cell populations. This group includes the G-CSF and erythropoietin analogues administered to Mrs. M. Second, some growth actors have therapeutic use in the treatment o various malignancies.

Agents That Stimulate Erythrocyte Production Late

S ta ge of me ga ka ryocytopoie s is

FIGURE 45-3. Growth factors involved in platelet production. Several growth factors are involved in platelet production (megakaryocytopoiesis). IL-11 acts primarily in the early stages; this growth factor stimulates production of GM-CSF and acts synergistically with IL-3 and stem cell factor (SCF) to increase the proliferation and differentiation of megakaryocyte progenitors. IL-6 and thrombopoietin (TPO) act primarily in the late stages of megakaryocytopoiesis. Both recombinant human IL-11 (oprelvekin) and TPO receptor agonists (eltrombopag and romiplostim) can be used therapeutically to increase platelet production.

The erythroid lineage-specif c actions o erythropoietin make this growth actor an obvious candidate or use in the treatment o some orms o anemia. Anemia can result rom any o a large number o underlying conditions that either interrupt the normal process o erythropoiesis or result in the premature loss or destruction o mature erythrocytes (Table 45-2). One common indication or erythropoietin therapy is chronic kidney disease, in which loss o unctional kidney tissue results in elimination o the cells that, in normal physiology, are responsible or erythropoietin production. Another potential indication or erythropoietin is anemia induced by anticancer therapies, which can

C h a p t e r 4 5 Pharmacology of Hematopoiesis and Immunomodulation

be directly toxic to bone marrow or kidney or can induce a state o relative resistance to endogenous erythropoietin by mechanisms that may involve proin ammatory cytokines, oxidative stress, and antierythropoietin antibodies. (Cancer can also cause anemia through bleeding, poor nutrition, and inf ltration o the bone marrow by tumor cells; these causes can o ten be diagnosed and treated directly.) Chemotherapyinduced anemia and its associated symptoms, such as those experienced by Mrs. M, can there ore be treated with erythropoietin under some circumstances.

Erythropoiesis-Stimulating Agents (ESAs)

There are currently three erythropoiesis-stimulating agents (ESAs) in clinical use in North America: recombinant human erythropoietin (rhEPO) (also known as epoetin alfa ), methoxy polyethylene glycol (PEG)-epoetin beta , and darbepoetin alfa ( ormerly known as novel erythropoiesis stimulating protein or NESP). Like endogenous erythropoietin, epoetin al a, PEG-epoetin beta, and darbepoetin al a act by stimulating the erythropoietin receptor and inducing erythropoiesis. rhEPO increases the hematocrit level by at least 6% in hal to three-quarters o patients receiving the drug, depending on the etiology o the anemia and the dose o rhEPO administered. rhEPO and darbepoetin are very similar in structure; in act, the two agents di er only in the number o sialic acid (carbohydrate) groups that are attached to the protein. The development o darbepoetin began with the observation that more sialic acid groups con er higher potency on erythropoietin. Darbepoetin’s two extra sialic acid groups also give this drug a three old longer hal -li e than erythropoietin, enabling less requent administration. PEG-epoetin beta’s polyethylene glycol coating gives this drug a longer hal -li e than either erythropoietin or darbepoetin. All three agents are proteins and must there ore be administered parenterally. In addition to its well-characterized role in stimulating erythropoiesis, erythropoietin may also play a role in glial and neuronal cell survival ollowing noxious stimuli or ischemic injury. Clinical studies o the neuroprotective e ects o erythropoietin are ongoing. Administration o erythropoietin to nonanemic or mildly anemic patients can lead to polycythemia, blood hyperviscosity, and stroke or myocardial in arction. Eighteen young cyclists died unexpectedly a ter the illegal introduction o erythropoietin into the world o pro essional cycling in the 1980s, possibly as a consequence o these adverse e ects. Another serious adverse e ect o certain preparations o recombinant erythropoietin became evident between 1998 and 2003. More than 200 patients who received one ormulation o recombinant erythropoietin developed pure red cell aplasia and were ound to have developed neutralizing antibodies against erythropoietin. The exact cause o the immune response is not well understood; one hypothesis involves the exposure o erythropoietin neoantigens as a result o partial denaturation o the therapeutic protein preparation. Erythropoietin and darbepoetin may also induce hypertension, and the use o these drugs is contraindicated in patients with uncontrolled hypertension. The mechanism responsible or erythropoietin-induced hypertension remains to be elucidated. Clinical studies have ound that patients with anemia and chronic kidney disease are at higher risk or death, serious cardiovascular events, and stroke when they are treated with

837

erythropoiesis-stimulating agents to target a hemoglobin level o greater than 11 g/dL. The mechanisms responsible or these e ects are an area o active investigation. Current US Food and Drug Administration (FDA) guidelines recommend that the use o erythropoiesis-stimulating agents should be considered when the hemoglobin level o patients with chronic kidney disease is less than 10 g/dL, and that the dosing should be individualized to use the lowest dose o ESA su f cient to reduce the need or erythrocyte trans usions. Studies have suggested that erythropoietin may also decrease survival and increase the risk o tumor progression or recurrence in patients with breast, non-small cell lung, head and neck, lymphoid, and cervical cancers, despite an improvement in the patients’ chemotherapy-induced anemia. The mechanisms and implications o these f ndings remain controversial. Potential explanations could include expression o the erythropoietin receptor on some cancer cells, synergistic toxicity due to combining erythropoietin therapy with chemotherapy and radiation therapy, and increased thrombogenicity associated with the elevated hemoglobin levels induced by erythropoietin therapy. These observations have led the FDA to alter the label o erythropoiesis-stimulating agents such that they are no longer indicated or patients receiving myelosuppressive chemotherapy administered with curative intent. In the palliative setting, it is important or physicians to discuss with patients the potential benef ts and risks o hematopoietic support with these agents.

Agents That Induce Fetal Hemoglobin (HbF) Sickle cell disease is marked by acute pain crises, increased susceptibility to in ection, and pro ound hemolytic anemia. Sickle hemoglobin (HbS)-containing erythrocytes are the root cause o these clinical mani estations o disease, which begins in childhood when HbS is f rst produced. Newborns and in ants with sickle cell disease are asymptomatic because etal globin gene expression persists or many months a ter birth, keeping etal hemoglobin (HbF) levels high. (In patients with sickle cell disease, typical HbF levels are 15% o total hemoglobin as late as age 2 and 1–5% o total hemoglobin in adults.) Consistent with this observation, adults in whom HbF expression persists at high levels have less requent pain crises and milder anemia than those with low HbF expression. These observations have made increasing HbF levels a tantalizing therapeutic goal. In principle, there are two approaches or increasing HbF: stimulating HbF expression in adults and preventing the switch rom etal (HbF) to adult (HbS) hemoglobin expression in children. Two drugs in current clinical practice, 5-azacytidine and hydroxyurea , use the f rst approach; the butyrates , a class o drugs that are still in clinical trials, may utilize both approaches. Early studies suggest that 5-azacytidine and hydroxyurea could be synergistic with butyrates and with erythropoietin, although erythropoietin should be used with caution in patients with sickle cell disease because it stimulates erythropoiesis in HbS- as well as HbF-containing cells.

5-Azacytidine and Decitabine

5-Azacytidine and its congener 5-aza-2′-deoxycytidine (decitabine ) are DNA demethylating agents that can increase

HbF production to greater than 20% o total globin expres-

838 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

sion in patients with sickle cell disease or thalassemia. (Theoretical studies suggest that an HbF level o 30–40% would render a patient asymptomatic.) 5-Azacytidine and decitabine are thought to act by reversing methylation o the globin gene, but this mechanism remains unproven. Concern over the unknown mechanism o action and ear o long-term cancer risk (both agents also inter ere with normal DNA synthesis; see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance) have hindered the acceptance o these drugs as a prophylactic therapy in sickle cell disease.

Hydroxyurea

The 1990s saw the f rst use o hydroxyurea to treat sickle cell disease. A cytostatic agent that blocks cell division by inhibiting ribonucleotide reductase, hydroxyurea had previously been used to treat clonal hematological disorders such as chronic myelogenous leukemia and polycythemia vera (see Chapter 39). From this experience, hydroxyurea was known to be relatively sa e or long-term administration, even in children; suppression o white blood cell and platelet production was known to be its main adverse e ect. The induction o HbF by hydroxyurea is slower than that by 5-azacytidine; nevertheless, hydroxyurea has proved to be e ective in about 60% o patients with sickle cell disease. In these patients, hydroxyurea increases HbF levels to 20% or more, decreases the requency o pain ul crises by 50% ( rom 4.5 to 2.5 per year, on average), and decreases the number o trans usions required by patients who have three or more crises per year. However, hydroxyurea does not prevent end-organ damage or stroke. In 1998, hydroxyurea was approved by the FDA or use in the treatment o sickle cell disease. Despite its long history o use, hydroxyurea’s mechanism o action in sickle cell disease remains uncertain. The current hypothesis is that hydroxyurea blocks the division o HbS-expressing erythroid precursors and that this somehow triggers reversion to a etal pattern o hemoglobin expression in an attempt to maintain erythrocyte production. It has been shown that the mechanism by which hydroxyurea increases HbF expression is independent o ribonucleotide reductase inhibition.

Butyrates

Butyrates (e.g., arginine butyrate, phenylbutyrate) are short-chain atty acids that inhibit histone deacetylases, the enzymes that modi y DNA to make it inaccessible to transcription actors. Butyrates have increased HbF levels rom 2% to more than 20% in early clinical trials, although these agents are apparently not e ective in patients whose baseline HbF level is less than 1%. Butyrates prevent the switch rom HbF to HbS in experimental animals, and children born to diabetic mothers (whose blood contains elevated levels o butyrates) have higher than normal levels o HbF. Butyrates are thought to act by allowing certain transcription actors to maintain or resume activity. Although this mechanism could explain the increased HbF production in response to butyrates, it does not explain the selectivity o butyrates or HbF over HbS production in patients with sickle cell disease.

Agents That Stimulate Leukocyte Production A low neutrophil count, or neutropenia , is most o ten the result o inter erence with progenitor cell proli eration and di erentiation

into mature white blood cells (myelosuppression). Neutropenia requently accompanies leukemia and other malignancies that invade the bone marrow, and it is a common adverse e ect o cancer chemotherapy. Less common causes o neutropenia include bone marrow transplantation, congenital neutropenia, and HIVor zidovudine-associated neutropenia. Three agents have been approved or use in the treatment o cancer- and chemotherapyinduced neutropenia: recombinant human G-CSF (f lgrastim); its pegylated, long-acting orm, PEG-G-CSF (PEG-f lgrastim); and recombinant human GM-CSF (sargramostim).

Recombinant Human G-CSFs (Filgrastim and PEGFilgrastim) and GM-CSF (Sargramostim)

Filgrastim and sargramostim are almost identical to the natural growth actors G-CSF and GM-CSF, respectively, and they act by the same mechanisms as the endogenous proteins. Although GM-CSF is a multilineage growth actor, the major clinical e ect o GM-CSF or G-CSF administration is a dose-independent increase in the absolute neutrophil count. (GM-CSF also causes a mild and dose-dependent increase in eosinophils.) As noted above, G-CSF and GM-CSF enhance the microbicidal activity o neutrophils in addition to stimulating their production. For Mrs. M (see introductory case), PEG-f lgrastim hastened the recovery o her neutrophils a ter chemotherapy and enhanced the ability o her neutrophils to combat in ection. G-CSF and GM-CSF also mobilize hematopoietic stem cells rom the bone marrow into the peripheral circulation; or this reason, they are o ten used be ore harvesting peripheral blood stem cells or transplantation. The immunostimulatory e ects o GM-CSF have ostered research into its ability to increase antitumor immune activity. A f lgrastim analogue has been conjugated to polyethylene glycol (PEG). This analogue, PEG-f lgrastim, is metabolized more slowly than the native molecule. PEG-f lgrastim can there ore be administered as a single injection that is unctionally equivalent to multiple daily doses o f lgrastim. The main adverse e ect o recombinant human G-CSF is bone pain, which resolves upon discontinuation o the drug. The theoretical risk that G-CSF could induce acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) remains controversial. In general, observational studies do not support an increased risk, but a study o breast cancer patients treated with chemotherapy did demonstrate a f veold increased incidence o AML/MDS in patients who received G-CSF. O note, however, these patients also received a higher dose o cyclophosphamide than patients who did not develop AML/MDS. GM-CSF is associated with ever, arthralgia, edema, and pleural and pericardial e usion. G-CSF and GM-CSF are proteins and must be administered parenterally, typically by daily injection over the course o several weeks.

Agents That Stimulate Platelet Production A low platelet count, or thrombocytopenia , is an important adverse e ect o many cancer chemotherapeutic agents, occasionally limiting the doses that can be delivered with acceptable sa ety and tolerability. The complications o thrombocytopenia include increased bleeding risk and platelet trans usion requirement; in turn, platelet trans usion is associated with an increased risk o in ection, ebrile reaction, and, rarely, gra t-versus-host disease.

C h a p t e r 4 5 Pharmacology of Hematopoiesis and Immunomodulation

Research into the pharmacologic management o chemotherapy-induced thrombocytopenia initially ocused on the thrombopoietin (TPO) analogues recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) (see below). To date, however, only recombinant human IL-11 (rhIL-11 or oprelvekin) has been approved by the FDA or this

indication. These agents all have the potential to increase megakaryocytopoiesis (platelet production) in a dose-dependent manner; although these drugs stimulate some multipotent as well as committed precursor cells, they do not signif cantly increase the hematocrit or white blood cell count. Importantly, these agents must all be administered prophylactically because there is a 1–2 week delay rom drug administration to a clinically signif cant increase in platelet count.

Thrombopoietin Receptor Agonists

Cloning o the thrombopoietin gene in 1994 led to the development o two thrombopoietin analogues. The f rst, rhTPO, was a ull-length, glycosylated analogue; the second, PEG-rHuMGDF, consisted o the N-terminal 163 amino acids o thrombopoietin conjugated to polyethylene glycol (PEG). Like natural thrombopoietin, both rhTPO and PEGrHuMGDF bind to Mpl (the endogenous receptor or thrombopoietin, named or its role in murine myeloproli erative leukemia), and activation o Mpl is the basis or the e ect o these drugs. Both rhTPO and PEG-rHuMGDF were tested as prophylactic agents to minimize chemotherapy-induced thrombocytopenia, and both caused a two old to ten old increase in platelet count. One potential caution was that stimulation o platelet production could lead to thrombosis i the platelets that are produced are also activated. A small trial o PEG-rHuMGDF suggested that this drug was sa e to use in treating the thrombocytopenia associated with AML, even though AML cells may also express the TPO receptor. However, the heavily bioengineered variants o natural TPO (e.g., PEG-rHuMGDF) were dropped rom clinical development because o an excess risk o developing anti-TPO autoantibodies, which could suppress natural platelet production. The testing o ull-length rhTPO was subsequently dropped as well, even though there were no reports o neutralizing antibodies in patients who received this lightly bioengineered agent. Two newer TPO receptor agonists are approved by the FDA or treatment o thrombocytopenia due to re ractory immune thrombocytopenic purpura (ITP), an autoimmune disease caused by autoantibodies directed against the patient’s own platelets. These drugs are eltrombopag, a small-molecule TPO receptor agonist, and romiplostim, a recombinant IgG1 Fc-peptide usion protein that also binds and activates the TPO receptor. By activating the TPO receptor, both molecules induce a transient increase in the platelet count. However, worsening thrombocytopenia may develop a ter cessation o treatment with these agents, and bone marrow toxicity (mani esting as bone marrow f brosis and other conditions) has also been reported.

Interleukin-11 (rhIL-11 [Oprelvekin])

Recombinant human IL-11 (rhIL-11), also called oprelvekin, is the only drug currently approved or the prevention o severe thrombocytopenia in patients receiving myelosuppressive chemotherapy. Oprelvekin is produced in Escherichia coli and di ers rom natural IL-11 only in its lack o the

839

N-terminal proline residue. rhIL-11 causes a dose-dependent increase in the platelet count and in the number o megakaryocytes in the bone marrow. The practical goal o treatment with oprelvekin is to maintain the platelet count above 20,000/ L (normal range, 150,000–450,000/ L) in order to minimize the risk o li e-threatening bleeding. However, the use o rhIL-11 is associated with signif cant adverse e ects, especially atigue and uid retention. Atrial f brillation has also been observed, and rhIL-11 should be used with caution in any patient with underlying heart disease. The undesirable actions o rhIL-11 likely result rom pleiotropic e ects o this actor on receptors distributed outside the hematopoietic system. It is unclear whether the therapeutic benef t o this agent outweighs the risk o systemic adverse e ects.

Immunomodulatory Agents with Antineoplastic Applications Interferons

Clinical investigation has led to the use o inter erons as therapeutic agents against a number o di erent malignancies, with moderate success. However, the multiple and overlapping e ects o these proteins have made it di f cult to determine the drugs’ mechanism o action in any given clinical situation. Induction o antitumor-directed immunity, terminal di erentiation o cycling tumor cells, and direct cytotoxic e ects have all been hypothesized to play important roles in the treatment o di erent malignancies. Inter erons are also used to treat certain viral in ections and are discussed in greater detail in Chapter 38.

Levamisole

Levamisole was known as an antihelminthic agent or decades

be ore its anticancer e ects were discovered. In combination with the antimetabolite 5- uorouracil (see Chapter 39), this drug is now approved or use in the treatment o colon cancer. Although its mechanism o action remains uncertain, levamisole is thought to cause macrophages and T cells to secrete cytokines (such as IL-1) and other actors that suppress tumor growth.

Interleukin-2

Interleukin-2 (IL-2) is approved by the FDA or the treatment o melanoma. At therapeutic doses, however, this cytokine has relatively low e f cacy and relatively high toxicity. See Chapter 46, Pharmacology o Immunosuppression, or more in ormation about IL-2.

Tretinoin

Tretinoin, or all-trans retinoic acid (ATRA), is a ligand o the

retinoic acid receptor (RAR). ATRA is used in the treatment o acute promyelocytic leukemia. This disease is characterized by a translocation t(15;17) in which part o the RAR gene is used to the PML gene, creating a usion protein that induces a block to di erentiation and thereby allows development o the leukemia. Treatment with ATRA stimulates di erentiation o these cells into more normal granulocytes. In some patients, the induction o di erentiation can lead to li e-threatening overproduction o white blood cells. ATRA can also induce a rapidly progressive syndrome o ever, acute respiratory distress with pulmonary inf ltrates, edema and weight gain, and multisystem organ ailure. Therapy with high doses o glucocorticoids is o ten an e ective treatment or this ATRA syndrome.

840 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

CONCLUSION AND FUTURE DIRECTIONS The production o cells o the hematopoietic system—red blood cells (erythrocytes), white blood cells (neutrophils, monocytes, lymphocytes, and other cell types), and platelets—is controlled by a variety o proteins called growth factors and cytokines. Cancer chemotherapy, malignant inf ltration o the bone marrow, and other conditions can cause def ciencies in these cell populations (anemia, neutropenia, and/or thrombocytopenia). The agents currently used to treat these def ciencies are mainly recombinant analogues o the natural growth actors or agonists o the growth actor receptors. Thus, the erythropoietin analogues rhEPO, PEG-epoetin beta, and darbepoetin treat anemia; the G-CSF and GM-CSF analogues f lgrastim, PEG-f lgrastim, and sargramostim treat neutropenia; and rhIL-11 and the thrombopoietin receptor agonists eltrombopag and romiplostim treat thrombocytopenia. Several agents a ecting the hematopoietic system are also used to treat sickle cell disease, a common autosomal recessive disease caused by a point mutation in the globin gene. These agents (hydroxyurea, 5-azacytidine, and decitabine) increase expression o etal hemoglobin (HbF) and thereby restore normal erythrocyte structure and unction. Several other drugs, including recombinant orms o the immunostimulatory inter eron proteins, levamisole, and retinoic acid, are used to treat certain cancers, although their precise mechanisms o action remain unknown. Other agents that activate hematopoiesis continue to be identif ed. Animal studies suggest that pharmacologic inhibitors o the repressor BCL11A could potentially induce HbF and thereby ameliorate the hematologic and pathologic mani estations o sickle cell disease. Preclinical evidence suggests that daily injections o a parathyroid hormone analogue (PTH 1-34) promote blood cell development, perhaps

by activating stimulatory receptors on osteoblasts that neighbor hematopoietic stem cells. These observations have led to clinical trials o PTH in enhancing stem cell production or transplantation and in protecting hematopoietic stem cells rom the cytotoxic e ects o chemotherapy. Studies designed to tease apart the complex overlapping unctionalities o hematopoiesis-regulating proteins are likely to provide a source o more selective pharmacologic interventions in the uture.

Suggested Reading Bennett CL, Djulbegovic B, Norris LB, Armitage JO. Colony-stimulating actors or ebrile neutropenia during cancer therapy. N Engl J Med 2013;368:1131–1139. (Reviews the clinical uses of G-CSF and GM-CSF in cancer therapy.) Hankins J, Aygun B. Pharmacotherapy in sickle cell disease—state o the art and uture prospects. Br J Haematol 2009;145:296–308. (Reviews use of hydroxyurea and decitabine.) Kaushansky K. Lineage-specif c hematopoietic growth actors. N Engl J Med 2006;354:2034–2045. (Reviews hematopoietic growth factors.) Kuter DJ. The biology o thrombopoietin and thrombopoietin receptor agonists. Int J Hematol 2013;98:10–23. (Reviews treatment of thrombocytopenia, including use of romiplostim and eltrombopag.) P e er MA, Burdmann EA, Chen CY, et al. A trial o darbepoetin al a in type 2 diabetes and chronic kidney disease. N Engl J Med 2009;361:2019– 2032. (Clinical trials of erythropoiesis-stimulating agents in patients with anemia and chronic kidney disease.) Singh AK, Szczech L, Tang KL, et al. Correction o anemia with epoetin al a in chronic kidney disease. N Engl J Med 2006;355:2085–2098. (Investigates quality of life in patients with chronic kidney disease treated with epoetin alfa to target hemoglobin levels of 13.5 g/dL vs. 11.3 g/dL.) Smith TJ, Khatcheressian J, Lyman GH, et al. Update o recommendations or the use o white blood cell growth actors: an evidence-based clinical practice guideline. J Clin Oncol 2006;24:3187–3205. (American Society of Clinical Oncology guidelines for the use of myeloid growth factors.) Xu J, Peng C, Sankaran VG, et al. Correction o sickle cell disease in adult mice by inter erence with etal hemoglobin switching. Science 2011;334:993–996. (Evidence that inactivation of BCL11A could represent a viable therapeutic strategy for sickle cell disease.)

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C h a p t e r 4 5 Pharmacology of Hematopoiesis and Immunomodulation 841

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Cytokine s Cyto Cy toki kin n e re cce p tor

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T ce ll

MHC cl cla s s II TCR TC 3

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Pharmacology of Immunosuppression CD CD4 D4

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An tige i n-pr pree s e n nti tin n g ce ll

Eliza b e t h A. Bre zin s ki, Lloyd B. Klicks t e in , a n d Ap ril W. Arm s t ro n g 2 l C

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INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844–845 PATHOPHYSIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8444 Transplantatioon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8444 Soli So lidd Organ Rejectionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844 Graft-Versus-Host Disease (GVHD)) . . . . . . . . . . . . . . . . . . . 846 Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846 PHARMA MACO COLO LOGI GIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 846 Inhibitors o Gene Express ssio ionn . . . . . . . . . . . . . . . . . . . . . . . . . . 847 Glucocortiico coid idss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 8477 Cytotoxic Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848 848 84 Antimee ta tabo boli lite tess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848 Alkylating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850 500 Specif c Lymphocyte-Signaling Inhibitors . . . . . . . . . . . . . . . . 850 Cyclosporine and Tacrolimus. . . . . . . . . . . . . . . . . . . . . . . . . 850 85 85 mTOR TOR Inhibitors I hibi s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851 Cytokine and Cytokine Receptor Inhibition . . . . . . . . . . . . . . . 852 TNF- Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 852 IL-12/IL-23p40 Cytokine Inhibitors . . . . . . . . . . . . . . . . . . . . . 854 IL-1 Cytokine and IL-1 Receptor Inhibitorss . . . . . . . . . . . . . 854

INTRODUCTION Patients with autoimmune disease and patients who have received transplanted tissues or organs typically require therapy with immunosuppressive drugs. Immunosuppressive agents have been in use or more than 50 years, beginning with corticosteroids, antimetabolites, and alkylating agents. These early agents assisted in the treatment o previously incurable conditions, but their lack o specif city led to many serious adverse e ects. Over the past 20 years, the f eld o immunosuppression has shi ted to specif c inhibitors o immunity that a ect distinct immune pathways. This shi t is important both because o the greater e f cacy and reduced toxicity o these agents and because, as the mechanisms o these agents are discovered, insights are gained into the operation o the immune system.

PATHOPHYSIOLOGY Transplantation The f rst transplant per ormed success ully in humans was a kidney transplant between identical twins. No immunosuppression was used, and the individuals did well. Currently, most organ transplantation occurs between unrelated individuals, termed an allograft. Donor and recipient tissues 844

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Ce ll s urfa cee ree ccee pto p tor

n

s io n IL-17 -177 Cy -1 Cytoki Cyt tokkine nee aan and nndd IL-17 IL 17 Receptor s . . . . . . . . . . . 854 8 o f cInhibitors enhibitors l ls IL-6 Receptor Receptor ec tor to or Inhibitor Inh nhhib hibitorr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854 8 De plet Deple ple let eetio ti tio ion on o S Sp pecif cif c Immune mmu muun Cel ells el . . . . . . . . . . . . . . . . . . . . . 854 ell 8 Polyclonal P olycl olyc l l Anttiibod ib die iess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855 8 Monoclonal Antibo bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855 8 85 LFA-3 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8855 5 B-Lymphocyte yte Stim Stimulato ti torr . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855 8 Antibodyy–D Drug rug Co Conj njug njugates jugates juugat gaatttes ess . . . . . . . . . . . . . . . . . . . . . . . . . . 856 8 Inhi In hibi bition i o Costimulation u o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856 Aba Ab Abat baatac acep eptt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856 Belatace B tace acept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 acep 8566 7 Bloc Blockade o Ce Celll Adhesion dhe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856 Natali Nata Nataliz Natal taliz alizumab b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856 Inhib hibi hibition hibit ibition o C Compl Co o ple lement Activation . . . . . . . . . . . . . . . . . . . . . 856 Inhibition Inhibitio tio tion ion io on o Imm on Immune mm munee Checkpoints . . . . . . . . . . . . . . . . . . . . . . . 857 m CTLA-4 Blockadee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857 PD-1 Blockadee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 857 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .857

express di erent major histocompatibility complex (MHC) class I molecules, one class o alloantigens, and recipient immune cells there ore recognize the transplanted tissues as oreign. This is termed alloimmunity, and it occurs when the recipient’s immune system attacks a transplanted organ. In the case o a bone marrow or stem cell transplant, graftversus-host disease (GVHD) can result when donor lymphocytes mount an assault on recipient tissues.

Solid Organ Rejection

Transplant rejection o solid organs can be divided into three major phases according to the time to onset. These phases, hyperacute , acute , and chronic rejection, are caused by di erent mechanisms and are there ore treated di erently. The ollowing three sections examine each o these processes, and Table 46-1 summarizes their di erences. Hyperacute Rejection Hyperacute rejection is mediated by pre ormed recipient an-

tibodies against donor antigen. Because these antibodies are present at the time o organ implantation, hyperacute rejection occurs almost immediately a ter reper usion o the transplanted organ. In act, the surgeon can observe the changes in the organ minutes a ter restoration o blood ow. The normal, healthy, pink appearance o the transplanted organ rapidly

C h a p t e r 4 6 Pharmacology o Immunosuppression

Mr. W is 59 ye a rs o ld w h e n h e u n d e rg o e s rig h t kid n e y tra n s p la n ta tio n in th e s p rin g o 20 0 0 o r ch ro n ic kid n e y a ilu re re s u ltin g ro m lo n g -s ta n d in g u n co n tro lle d hyp e rte n s io n . His in d u ctio n im m u n o s u p p re s s a n t re g im e n co n s is ts o a n tith ym o cyte g lo b u lin . Th e a n tith ym o cyte g lo b u lin is co -a d m in is te re d w ith a g lu co co rtico id , a n a n tih is ta m in e , a n d a n a n tip yre tic. On p o s to p e ra tive d a y 1, h e is tra n s itio n e d to a m a in te n a n ce im m u n o s u p p re s s a n t re g im e n o ta cro lim u s , m yco p h e n o la te m o e til, a n d p re d n is o n e . Pro g re s s d u rin g th e f rs t 2 m o n th s p o s t-tra n s p la n t is e xce lle n t, b u t th e n Mr. W d e ve lo p s h e a d a ch e s a n d la b o ra to ry te s tin g s h o w s a n in cre a s e in h is cre a tin in e . Th e g lu co co rtico id d o s e is in cre a s e d a n d h is cre a tin in e im p rove s . Th re e m o n th s a te r s u rg e ry, Mr. W is a d m itte d to th e h o s p ita l w ith e ve r, d e cre a s e d u rin e o u tp u t, a n d g ra t te n d e rn e s s . La b o ra to ry te s tin g a ga in s h o w s in cre a s e d s e ru m cre a tin in e a n d th e ta cro lim u s s e ru m le ve l is w ith in th e ra p e u tic lim its . In e ctio u s w o rku p is n e ga tive a n d re n a l u ltra s o u n d s h o w s n o e vid e n ce o o b s tru ctio n . A re n a l a llo g ra t b io p s y d e m o n s tra te s tu b u litis a n d in tim a l a rte ritis , e vid e n ce o a cu te ce llu la r re je ctio n . He is tre a te d w ith h ig h -d o s e p u ls e d g lu co co rtico id th e ra p y, w h ich p ro d u ce s a d ve rs e e e cts o a m e ta llic ta s te a n d a b d o m in a l d is co m o rt. Mr. W’s re n a l u nction im p rove s a nd h e is dis cha rge d . Ove r th e n e xt e w w e e ks , h is s te ro id d o s e is s lo w ly ta p e re d d o w n to h is m a in te n a n ce d o s e .

becomes cyanotic, mottled, and f accid. This rapid change is the result o complement activation by antibody binding to endothelial cells o the transplanted organ, resulting in thrombosis and ischemia. Most commonly, hyperacute rejection is mediated by recipient antibodies that react with blood group antigens in donor organs (e.g., type AB donor in a type O recipient). Matching o blood types between donor and recipient prevents hyperacute rejection; there ore, drug therapy or hyperacute rejection is typically not necessary. Hyperacute rejection also occurs in xenotransplantation (i.e., organ transplantation between species, such as a pig heart transplanted into a human recipient), due to the presence o pre ormed human antibodies that react against antigenic proteins and carbohydrates expressed by the donor species.

845

In De ce m b e r 2010, Mr. W a rrive s a t clin ic o r h is re g u la r a n n u a l e xa m in a tio n . He is in g o o d h e a lth o n a m a in te n a n ce im m u n o s u p p re s s a n t re g im e n o ta cro lim u s , m yco p h e n o la te m o e til, a n d p re d n is o n e , a n d h is m o n th ly cre a tin in e va lu e s h a ve re m a in e d n o rm a l. Th e re h a s b e e n n o e vid e n ce o re je ctio n s in ce 20 0 0. Re n a l u ltra s o u n d s h o w s re m a rka b ly n o rm a l g ra t s tru ctu re , p e rh a p s a s a re s u lt o th e a g g re s s ive hyp e rte n s io n m a n a g e m e n t b y h is p h ys icia n s . Ho w e ve r, h is g lu co s e le ve ls a re co n s is te n tly e le va te d o n ro u tin e la b o ra to ry te s tin g , a n in d ica tio n th a t h e m a y h a ve d e ve lo p e d n e w-o n s e t d ia b e te s a te r tra n s p la n ta tio n . Be ca u s e o h is n e wo n s e t d ia b e te s , Mr. W’s ta cro lim u s a n d p re d n is o n e d o s e s a re d e cre a s e d a n d h e is s ta rte d o n o ra l th e ra p y to m a n a g e h is d ia b e te s . Ove r th e n e xt 2 ye a rs , h is g lu co s e a n d h e m o g lo b in A1c le ve ls re m a in a t g o a l a n d h e is a b le to e n jo y tim e w ith h is a m ily.

Questions 1 . How does each o the drugs prescribed or Mr. W reduce the likelihood o rejection? 2 . Why is antithymocyte globulin administered with glucocorticoids, an antihistamine, and an antipyretic? 3 . Why does Mr. W’s pulsed steroid dose need to be slowly tapered down to his maintenance dose level? 4 . What is the likely cause o Mr. W’s new-onset diabetes? Why were his tacrolimus and prednisone doses decreased?

Acute Rejection

Acute rejection has cellular and humoral components. Acute cellular rejection is mediated by cytotoxic T cells and causes interstitial as well as vascular damage. This cellular response is most commonly seen in the initial months a ter transplantation. Immunosuppression o T cells is highly e ective at preventing or limiting activation o the recipient immune system by the transplanted organ, thereby preventing acute cellular rejection. In acute humoral rejection, recipient B cells become sensitized to donor antigens in the transplanted organ and produce antibodies against these alloantigens a ter a period o 7–10 days. The antibody response is typically directed against endothelial cells and is thus also known as acute vascular rejection. Like acute cellular rejection, acute humoral

TABLE 46-1 Modes of Immune Rejection HYPERACUTE REJ ECTION

ACUTE REJ ECTION

CHRONIC REJ ECTION

Mechanism

Pre ormed recipient antibodies react with donor antigen and activate complement

Cellular—Donor antigen activates recipient T cells Humoral—Donor antigen stimulates recipient antibody response

Unknown, but thought to be caused by chronic inf ammation resulting rom activated T-cell responses to donor antigen

Time course

Minutes to hours

Weeks to months

Months to years

How suppressed

Matching o donor and recipient blood types

Immunosuppression

Currently cannot be suppressed

846 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

rejection can usually be prevented by immunosuppression o the recipient a ter transplantation. Even with immunosuppression, however, episodes o acute rejection can occur months or even years a ter transplantation. Transplant recipients experiencing acute rejection are usually asymptomatic, and symptoms o ever or malaise are usually nonspecif c. Chronic Rejection Chronic rejection is believed to be both cellular and humoral

in nature and does not occur until months or years a ter transplantation. Because hyperacute and acute rejection are generally well controlled by donor/recipient matching and immunosuppressive therapy, chronic rejection is now the most common li e-threatening pathology associated with organ transplantation. Chronic rejection is thought to result rom chronic inammation caused by the response o activated recipient T cells to donor antigen. Activated T cells release cytokines that recruit macrophages into the gra t. The macrophages induce chronic in ammation that leads to intimal proli eration o the vasculature and scarring o the gra t tissue. The chronic changes eventually lead to irreversible organ ailure. Other contributing nonimmune actors can include ischemia-reper usion injury and in ection. No e ective treatment regimens are currently available to eliminate chronic rejection. It is believed, however, that several experimental therapies have a reasonable chance o reducing chronic rejection. Especially promising is the possibility o developing tolerance through elimination o costimulation (see below).

Graft-Versus-Host Disease (GVHD)

Leukemia, primary immunodef ciency, and other conditions can be treated with bone marrow or peripheral stem cell transplantation. In this procedure, hematopoietic and immune unction is restored a ter the patient’s bone marrow has been eradicated by aggressive chemotherapy and/or radiation therapy. GVHD is a major complication o allogeneic bone marrow or stem cell transplantation. GVHD is an alloimmune in ammatory reaction that occurs when transplanted immune cells attack the cells o the recipient. The severity o GVHD ranges rom mild to li e-threatening and typically involves the skin (rash), gastrointestinal tract (diarrhea), lungs (pneumonitis), and liver (veno-occlusive disease). GVHD can o ten be ameliorated by removing T cells rom the donor bone marrow be ore transplantation. Mild to moderate GVHD can also be benef cial when donor immune cells attack recipient tumor cells that have survived the aggressive chemotherapy and radiation therapy. (In the case o leukemia, this is called the graft-versus-leukemia effect, or GVL.) There ore, although removing donor T cells rom the “gra t” reduces the risk o GVHD, this may not be the best approach or marrow transplants used in antineoplastic therapy.

Autoimmunity Autoimmune diseases occur when the host immune system attacks its own tissues, mistaking sel -antigen or oreign. The typical result is chronic in ammation in the tissue(s) expressing the antigen. Autoimmune diseases are most commonly due to a breakdown o sel -tolerance, both central and peripheral. Central tolerance re ers to the specif c clonal deletion o autoreactive T and B cells during their development rom precursor cells in the thymus (T cells) and bone marrow (B cells). Central

tolerance ensures that the majority o immature autoreactive T and B cells do not develop into sel -reactive clones. The thymus and bone marrow do not express every antigen in the body, however; a number o proteins are expressed only in specif c tissues. For this reason, peripheral tolerance is also important. Peripheral tolerance results rom deletion o autoreactive T cells by Fas-Fas ligand-mediated apoptosis, activation o T suppressor cells, or induction o T-cell anergy due to antigen presentation in the absence o costimulation. Although breakdown in tolerance lies at the center o virtually all autoimmune diseases, the inciting stimulus leading to loss o tolerance is o ten unknown. Genetic actors may play a role, in that the presence o certain MHC subtypes may predispose T cells to the loss o sel -tolerance. For example, human leukocyte antigen (HLA)-B27 is causally related to many orms o autoimmune spondylitis. Several other autoimmune diseases are linked to specif c HLA loci, supporting an association, i not a causal role, or genetic predisposition to autoimmunity. Molecular mimicry, in which epitopes rom in ectious agents are similar to sel -antigens, can also lead to a breakdown o tolerance and may be the mechanism underlying poststreptococcal glomerulonephritis. Several other processes, including ailure o T-cell apoptosis, polyclonal lymphocyte activation, and exposure o cryptic sel -antigens, have also been hypothesized to lead to autoimmunity. The details o these mechanisms are beyond the scope o this book; however, the result o each is a loss of tolerance. Once sel -tolerance has been compromised, the specif c expression o autoimmunity can take one o three general orms (Table 46-2). In some diseases, production o autoantibodies against a specif c antigen causes antibody-dependent opsonization o cells in the target organ, with subsequent cytotoxicity. One example is Goodpasture’s syndrome, which results rom autoantibodies against collagen type IV in the renal glomerular basement membrane. In some autoimmune vasculitis syndromes, circulating antibody–antigen complexes deposit in blood vessels, causing in ammation and injury to the vessels. Two examples o immune complex disease are mixed essential cryoglobulinemia and systemic lupus erythematosus. Finally, T-cell-mediated diseases are caused by cytotoxic T cells that react with a specif c sel -antigen, resulting in destruction o the tissue(s) expressing that antigen. One example is type 1 diabetes mellitus, in which the cytotoxic T cells react against sel -antigens in pancreatic -cells. The pharmacologic therapy or autoimmune diseases does not yet match the exquisite specif city o the o ending biological process. Most currently available pharmacologic agents cause generalized immunosuppression and do not target the specif c pathophysiology. Better understanding o the molecular pathways leading to autoimmune diseases should reveal new pharmacologic targets that can be used to suppress the specif c autoimmune response be ore disease arises.

PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic suppression o the immune system utilizes eight mechanistic approaches (Fig. 46-1): 1. Inhibition o gene expression to modulate in ammatory responses 2. Depletion o expanding lymphocyte populations with cytotoxic agents

C h a p t e r 4 6 Pharmacology o Immunosuppression

847

TABLE 46-2 Representative Examples of Autoimmune Diseases, Categorized by Type of Tissue Damage Antibody to Self-Antigen SYNDROME

AUTOANTIGEN

CONSEQUENCE

Acute rheumatic ever

Streptococcal cell wall antigens that crossreact with cardiac muscle

Arthritis, myocarditis

Autoimmune hemolytic anemia

Rh blood group antigens

Destruction o erythrocytes

Goodpasture’s syndrome

Renal glomerular basement membrane collagen type IV

Glomerulonephritis, pulmonary hemorrhage

Immune thrombocytopenic purpura

Platelet GPIIb-IIIa

Excessive bleeding

Pemphigus vulgaris

Epidermal cadherin

Blistering o skin

SYNDROME

AUTOANTIGEN

CONSEQUENCE

Mixed essential cryoglobulinemia

Rheumatoid actor IgG complexes

Systemic vasculitis

Systemic lupus erythematosus

DNA, histones, ribosomes, snRNP, scRNP

Glomerulonephritis, vasculitis, arthritis

SYNDROME

AUTOANTIGEN

CONSEQUENCE

Experimental autoimmune encephalitis, multiple sclerosis

Myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein

Brain invasion by CD4 T cells, several CNS def cits

Rheumatoid arthritis

Unknown—possible synovial joint antigens

J oint in ammation and destruction

Type 1 diabetes mellitus

Pancreatic -cell antigens

Immune-Complex Disease

T-Cell-Mediated Disease

-Cell destruction, insulin-dependent diabetes mellitus

Rh, Rhesus actor; DNA, deoxyribonucleic acid; IgG, immunoglobulin G; CNS, central nervous system; snRNP, small nuclear ribonucleoprotein; scRNP, small cytoplasmic ribonucleoprotein.

3. Inhibition o lymphocyte signaling to block activation o lymphocytes and expansion o lymphocyte populations 4. Neutralization o cytokines and cytokine receptors essential or mediating the immune response 5. Depletion o specif c immune cells, usually via cellspecif c antibodies 6. Blockade o costimulation to induce anergy 7. Blockade o cell adhesion to prevent migration and homing o in ammatory cells 8. Inhibition o innate immunity, including complement activation

Inhibitors of Gene Expression Glucocorticoids

Glucocorticoids have broad anti-in ammatory e ects.

The intimate relationship between cortisol and the immune system is discussed in Chapter 29, Pharmacology o the Adrenal Cortex. Brie y, glucocorticoids are steroid hormones that exert their physiologic actions by binding to the cytosolic glucocorticoid receptor. The glucocorticoid– glucocorticoid receptor complex translocates to the nucleus and binds to glucocorticoid response elements (GREs) in the promoter region o specif c genes, either up-regulating or down-regulating gene expression.

Glucocorticoids have important metabolic e ects on essentially all cells o the body and, in pharmacologic doses, suppress the activation and unction o innate and adaptive immune cells. Glucocorticoids down-regulate the expression o many in ammatory mediators, including key cytokines such as tumor necrosis actor (TNF)- , interleukin-1 (IL-1), and IL-4. The role o glucocorticoids in suppressing eicosanoid biosynthesis and signaling is discussed in Chapter 43, Pharmacology o Eicosanoids. The overall e ect o glucocorticoid administration is pro oundly anti-in ammatory and immunosuppressive, explaining the use o glucocorticoids or the treatment o numerous in ammatory diseases such as rheumatoid arthritis and transplant rejection. Long-term glucocorticoid administration has important adverse e ects. Diabetes, reduced resistance to in ection, osteoporosis, cataracts, increased appetite leading to weight gain, hypertension and its sequelae, and the masking o in ammation must all be closely monitored in patients receiving glucocorticoids. Abrupt cessation o glucocorticoid therapy can result in acute adrenal insu f ciency because the hypothalamus and pituitary gland require weeks to months to reestablish adequate ACTH production. During this time, the underlying disease can worsen because o disinhibition o the immune system. To prevent the latter complications, glucocorticoid dosage should be tapered slowly as therapy is terminated. This is why Mr. W’s dose o glucocorticoids was

848 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Antimetabolites

4

Cytokine s Cytokine re ce ptor T ce ll

MHC cla s s II TCR 3

1

CD4

B7

CD28 5

6

Antige n-pre s e nting ce ll 2 C

l

o n

Ce ll s urfa ce re ce ptor

al e

xp

an sio

n of ce

lls

Antimetabolites have been a mainstay o immunosuppressive treatment or many years. Their power ul suppressive e ect on immune cells is accompanied by many adverse e ects related to their lack o selectivity. The older antimetabolites, such as azathioprine and methotrexate, a ect all rapidly dividing cells and can have toxic e ects on the gastrointestinal mucosa and bone marrow. Newer antimetabolites, such as mycophenolate mo etil and lef unomide , cause ewer adverse e ects. Mycophenolate mo etil may also be relatively selective or immune cells, urther reducing its toxicity. Antimetabolites typically a ect both cell-mediated and humoral immunity, rendering patients more susceptible to in ection than would occur i only one o these immune systems were a ected. Antimetabolites are widely used in the treatment o cancer, and their mechanisms o action are described in detail in Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance. Here, we ocus on the antimetabolites used or immunosuppression and brie y discuss the anti-in ammatory aspects o their mechanisms o action. Azathioprine Azathioprine (AZA) was the f rst drug to be used or sup-

7

Tis s ue s

pression o the immune system a ter organ transplantation, and it remains a mainstay or this indication. AZA is a prodrug o the purine analogue 6-mercaptopurine (6-MP), which is slowly released as AZA reacts nonenzymatically with sul hydryl compounds such as glutathione (Fig. 46-2). The slow release of 6-MP from AZA favors immunosuppression. Although AZA prolongs organ gra t survival, this drug is less e f cacious than mycophenolate mo etil in improving the long-term survival o kidney allogra ts. AZA and 6-MP are also used as immunosuppressants in the treatment o in ammatory bowel disease, acute lymphoblastic leukemia, and autoimmune skin disorders.

FIGURE 46-1. Overview o mechanisms o pharmacologic immunosuppression. The molecular mechanisms by which immune cells are activated and function provide eight major points for pharmacologic intervention by immunosuppressive agents. Blockade of T-cell activation can be accomplished by (1) inhibition of gene expression, (2) selective attack on clonally expanding lymphocyte populations, (3) inhibition of intracellular signaling, (4) neutralization of cytokines and cytokine receptors required for T-cell stimulation, (5) selective depletion of T cells (or other immune cells), (6) inhibition of costimulation by antigen-presenting cells, and (7) inhibition of lymphocyte–target cell interactions. Suppression of innate immune cells and complement activation may also block the initiation of immune responses (not shown).

NO 2

N N

S N

N N

N H

Azathioprine

tapered slowly a ter his pulsed steroid therapy or treatment o acute cellular rejection.

Cytotoxic Agents Cytotoxic agents are used both or immunosuppression and or antineoplastic chemotherapy. The therapeutic goal in both cases is the elimination o pathogenic cells. Two classes o cytotoxic agents, antimetabolites and alkylating agents , are commonly used as immunosuppressants. Antimetabolites are structural analogues o natural metabolites that inhibit essential pathways involving these metabolites. Alkylating agents inter ere with DNA replication and gene expression by alkylation o DNA.

+

Gluta thione

S N

HN N

N H

Mercaptopurine

FIGURE 46-2. Formation o mercaptopurine rom azathioprine. Azathioprine is a prodrug form of the antimetabolite 6-mercaptopurine. Mercaptopurine is formed by the cleavage of azathioprine in a nonenzymatic reaction with glutathione. Although mercaptopurine can also be used directly as a cytotoxic agent, azathioprine has higher oral bioavailability and a longer duration of action and is more immunosuppressive than mercaptopurine.

C h a p t e r 4 6 Pharmacology of Immunosuppression

Methotrexate Methotrexate is a olate analogue used since the 1950s to

treat malignancies. Since that time, methotrexate has also become an extremely versatile drug in treating a wide variety o immune-mediated diseases, including rheumatoid arthritis and psoriasis. In addition, methotrexate is used or the prevention o GVHD. The precise mechanism by which methotrexate exerts its anti-in ammatory e ect is uncertain. Although methotrexate inhibits dihydro olate reductase, the combination o methotrexate and low-dose olate is as e ective as methotrexate alone in the treatment o rheumatoid arthritis. (High-dose olinic acid does inter ere with the e f cacy o methotrexate, however.) Methotrexate may also act as an anti-in ammatory agent by increasing adenosine levels. Adenosine is a potent endogenous anti-in ammatory mediator that inhibits neutrophil adhesion, phagocytosis, and superoxide generation. Methotrexate also causes apoptosis o activated CD4 and CD8 T cells but not o resting T cells. Other immunosuppressive agents, including 5- uorouracil, 6-mercaptopurine, and mycophenolic acid, also promote apoptosis o activated T cells. Methotrexate may be such a versatile drug because o its combined antineutrophil, antiT-cell, and antihumoral e ects.

Mycophenolic Acid and Mycophenolate Mo etil Mycophenolic acid (MPA) is an inhibitor o inosine mono-

phosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the ormation o guanosine (see Fig. 39-3). Because MPA has low oral bioavailability, it is usually administered as a sodium salt or in its prodrug orm, mycophenolate mo etil (MMF), both o which have greater oral bioavailability (Fig. 46-3). MMF is increasingly used in the treatment o immune-mediated disease because o its high selectivity and pro ound e ect on lymphocytes.

O

OH O

O O

O

N O

Mycophenolate mofetil Plasma esterases

O

OH OH

O O

O

Mycophe nolic a cid

FIGURE 46-3. Mycophenolic acid and mycophenolate mo etil. Mycophenolate mofetil (MMF) has higher oral bioavailability than mycophenolic acid (MPA). Orally administered MMF is absorbed into the circulation, where plasma esterases rapidly cleave the ester bond to yield MPA. Both agents inhibit inosine monophosphate dehydrogenase type II (IMPDH II), an enzyme crucial for de novo synthesis of guanosine. Because of its higher oral bioavailability, MMF (or the sodium salt of MPA) is typically used.

849

MPA and MMF both act primarily on lymphocytes. Two main actors contribute to this selectivity. First, as discussed in Chapter 39, lymphocytes are dependent on the de novo pathway o purine synthesis, whereas most other tissues rely heavily on the salvage pathway. Because IMPDH is required or de novo synthesis o guanosine nucleotides but not or the salvage pathway, MPA selectively a ects cells such as lymphocytes that rely on de novo purine synthesis. Second, IMPDH is expressed in two iso orms: type I and type II. MPA pre erentially inhibits type II IMPDH, the iso orm expressed mainly in lymphocytes. Together, these actors con er on MPA and MMF selectivity against T and B cells, with relatively low toxicity to other cells. Inhibition o IMPDH by MPA reduces intracellular guanosine levels and increases intracellular adenosine levels, with many downstream e ects on lymphocyte activation and activity. MPA has a cytostatic e ect on lymphocytes and it can also induce apoptosis o activated T cells, leading to the elimination o reactive clones o proli erating cells. Because guanosine is required or some glycosylation reactions, the reduction in guanosine nucleotides leads to decreased expression o adhesion molecules that are required or recruitment o several immune cell types to sites o in ammation. Furthermore, because guanosine is a precursor o tetrahydrobiopterin (BH4), which regulates inducible nitric oxide synthase (iNOS), the reduction in guanosine levels leads to decreased NO production by neutrophils. Endothelial NOS (eNOS), which controls vascular tone and is regulated by Ca2 and calmodulin, is not a ected by changes in guanosine levels, again demonstrating the considerable selectivity o MPA. As noted above, clinical studies comparing MMF and AZA have shown MMF to be more e f cacious in preventing acute rejection o kidney transplants. Animal models show that chronic rejection is also reduced more e ectively in recipients treated with MMF than in those treated with AZA or cyclosporine. The e f cacy o MMF in treating chronic rejection may be related to its inhibition o both the lymphocyte and the smooth muscle cell proli eration characteristic o chronic rejection. MMF is also e f cacious in the treatment o autoimmune disease. In rheumatoid arthritis, levels o rheumatoid actor, immunoglobulin, and T cells are all reduced by treatment with MMF. MMF is requently used in the initial therapy o lupus nephritis. There have also been isolated reports o success ul treatment o myasthenia gravis, psoriasis, autoimmune hemolytic anemia, and in ammatory bowel disease with MMF. The most common adverse e ect o MMF is gastrointestinal discom ort, which is dose-dependent and can include nausea, diarrhea, so t stools, anorexia, and vomiting. Lef unomide

Activated lymphocytes both proli erate and synthesize large quantities o cytokines and other e ector molecules, and these processes require increased DNA and RNA synthesis. There ore, agents that reduce intracellular nucleotide pools have suppressive e ects on these activated cells. Lef unomide is an inhibitor o pyrimidine synthesis, specif cally blocking the synthesis o uridylate (UMP) by inhibiting dihydroorotate dehydrogenase (DHOD). DHOD is a key enzyme in the synthesis o UMP (Fig. 46-4), which is essential or the synthesis o all pyrimidines. (See Chapter 39 or a review o

850 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY O-

O

O H2 N

FIGURE 46-4. Inhibition o pyrimidine synthesis by lef unomide. De novo

O

pyrimidine synthesis depends on the oxidation o dihydroorotate to orotate, a reaction that is catalyzed by dihydroorotate dehydrogenase. Lef unomide inhibits dihydroorotate dehydrogenase and thereby inhibits pyrimidine synthesis. Because lymphocytes are dependent on de novo pyrimidine synthesis or cell proli eration and clonal expansion a ter immune cell activation, depletion o the pyrimidine pool inhibits lymphocyte expansion. Experimentally, lef unomide appears to inhibit pre erentially the proli eration o B cells; the mechanism o this pre erential action is unknown.

O-

N H

N-Ca rba moyla s pa rta te H+ Dihydroorotase H2 O O

pyrimidine synthesis.) Experimentally, le unomide has been shown to be most e ective in reducing B-cell populations, and a signif cant e ect on T cells has also been observed. Le unomide is currently approved or use in rheumatoid arthritis. The drug has also shown signif cant e f cacy in the treatment o other immune diseases, including systemic lupus erythematosus and myasthenia gravis. Le unomide has antiviral activity against cytomegalovirus (CMV) and has been used to treat this in ection in cases o drug-resistant CMV and in transplant patients. Le unomide prolongs transplant gra t survival and limits GVHD in animal models. The most signif cant adverse e ects o le unomide are diarrhea and reversible alopecia. Le unomide undergoes signif cant enterohepatic circulation, resulting in a prolonged pharmacologic e ect. I le unomide must be removed quickly rom a patient’s system, cholestyramine may be administered. By binding to bile acids, cholestyramine interrupts the enterohepatic circulation and causes a rapid “washout” o le unomide.

HN O-

N H

O

O

Dihydroorota te NAD+ Dihydroorotate dehydrogenase

Leflunomide

NADH O HN O

O-

N H O

Orota te P RP P Orotate phosphoribosyl transferase

Alkylating Agents

PPi

Cyclophosphamide Cyclophosphamide (Cy) is a highly toxic drug that alkylates

O HN

-

O

OP

O

O-

N

O

O O H

O

H

H

H OH OH Orotidyla te

H+ Orotidylate decarboxylase CO 2 O HN

-

O

OP

O

N

O O

O H

DNA. The mechanism o action and uses o Cy are discussed extensively in Chapter 39; there ore, the discussion here is limited to Cy’s utility in treating diseases o the immune system. Because Cy has a major suppressive e ect on B-cell proli eration but can enhance T-cell responses, the use o Cy in immune diseases is limited to disorders o humoral immunity, particularly systemic lupus erythematosus. Another use under consideration or Cy is the suppression o antibody ormation against xenotransplant gra ts. Adverse e ects o Cy are severe and widespread, including leukopenia, cardiotoxicity, pulmonary toxicity, and increased risk o cancer because o mutagenicity. The risk o bladder cancer is especially notable because Cy produces a carcinogenic metabolite, acrolein, which is concentrated in the urine. When high-dose Cy is administered by intravenous in usion, acrolein can be detoxif ed by co-administration o mesna (a sul hydryl-containing compound that neutralizes the reactive moiety o acrolein).

H

H

Speci c Lymphocyte-Signaling Inhibitors

OH

H OH

Cyclosporine and Tacrolimus

Uridyla te (UMP )

The discovery in 1976 that cyclosporine (CsA; also re erred to as cyclosporin A) is a specif c inhibitor o T-cell-mediated immunity enabled widespread whole-organ transplantation. In act, CsA made heart transplantation a legitimate alternative in the treatment o end-stage heart ailure. CsA is a cyclic decapeptide isolated rom a soil ungus, Tolypocladium inf atum. CsA inhibits the production o IL-2 by activated T cells. IL-2 is an important cytokine that acts in an autocrine and

C h a p t e r 4 6 Pharmacology of Immunosuppression

Cyclosporine

Tacrolimus (FK506)

Cyclophilin

FKBP

Ca lcine urin (ina ctive )

Ca 2 +

Ca lmodulin Ca lcine urin (a ctive ) P

Ina ctive NFAT

Active NFAT

IL-2 mRNA

851

increase their biosynthesis o extracellular matrix components, resulting in interstitial f brosis. Tacrolimus (also known as FK506) is a more potent immunosuppressant than CsA; although its structure di ers rom that o CsA, it acts by a similar mechanism (Fig. 46-5). Tacrolimus is a macrocyclic triene isolated rom the soil bacterium Streptomyces tsukubaensis. Tacrolimus acts by binding to FK-binding proteins (FKBP), and the tacrolimus– FKBP complex inhibits calcineurin. Tacrolimus inhibits IL-3, IL-4, IFN- , and TNF- production in vitro, and it appears to inhibit cell-mediated immunity without suppressing B-cell or natural killer (NK) cell unction. Tacrolimus is generally 50–100 times more potent than CsA, but, like CsA, it is nephrotoxic. Tacrolimus can also cause new-onset diabetes mellitus in post-transplant patients. In the introductory case, tacrolimus was a probable contributor to Mr. W’s new-onset diabetes. This was the rationale or lowering both the tacrolimus and prednisone doses in Mr. W’s immunosuppressive regimen a ter he developed diabetes mellitus. Tacrolimus is approved as an immunosuppressant or transplantation. A topical ormulation is used or the treatment o atopic dermatitis and other eczematous diseases.

mTOR Inhibitors

Sirolimus , also known as rapamycin, is a macrocyclic triene IL-2 ge ne Nucle us

FIGURE 46-5. Mechanisms of action of cyclosporine and tacrolimus. The actions of cyclosporine and tacrolimus (also known as FK506) are mediated by blockade of intracellular T-cell signaling. In normal T-cell signaling (bottom), stimulation of T cells increases the level of intracellular calcium, and Ca 2 / calmodulin activates the calcineurin-mediated dephosphorylation of the cytoplasmic transcription factor NFAT. Activated NFAT translocates to the nucleus, where it induces IL-2 gene transcription. Cyclosporine and tacrolimus cross the plasma membrane and bind to the cytoplasmic immunophilins cyclophilin and FK-binding protein (FKBP), respectively (top). Both the cyclosporine– cyclophilin and tacrolimus–FKBP complexes bind to calcineurin, preventing the activation of calcineurin phosphatase activity by Ca 2 /calmodulin.

paracrine manner to cause activation and proli eration o T cells (Fig. 46-5). Activated T cells increase their production o IL-2 via a pathway that begins with dephosphorylation o a cytoplasmic transcription actor, NFAT (nuclear factor of activated T cells ). NFAT is dephosphorylated by the cytoplasmic phosphatase calcineurin. Upon dephosphorylation, NFAT translocates to the nucleus and enhances transcription o the IL-2 gene. CsA acts by binding to cyclophilin, and the CsA–cyclophilin complex binds to calcineurin and inhibits its phosphatase activity. By inhibiting calcineurin-mediated NFAT dephosphorylation, CsA prevents translocation o NFAT to the nucleus and thereby suppresses IL-2 production. CsA is approved or use in organ transplantation, psoriasis, and rheumatoid arthritis. CsA is also used occasionally in the treatment o rare autoimmune diseases that are not responsive to other immunosuppressants. An ophthalmic preparation o CsA is approved or the treatment o chronic dry eyes. The use ulness o CsA is limited by severe adverse e ects, including nephrotoxicity, hypertension, in ection, neurotoxicity, and hepatotoxicity. The mechanism o CsA toxicity is complex but may include stimulation o trans orming growth actor- (TGF- ) production. TGF- causes cells to

isolated rom the soil bacterium Streptomyces hygroscopicus. Although they are structurally similar and are both used to prevent and treat organ rejection, tacrolimus and sirolimus have di erent mechanisms o action. Both bind to FKBP, but the sirolimus–FKBP complex does not inhibit calcineurin; instead, it blocks the IL-2 receptor signaling required or T-cell proli eration (Fig. 46-6). Sirolimus–FKBP binds to and inhibits molecular target o rapamycin (mTOR), a serine-threonine kinase that phosphorylates p70 S6 kinase and PHAS-1 (among other substrates). p70 S6 kinase and PHAS-1 regulate translation, the ormer by phosphorylating proteins (including the ribosomal S6 protein) involved in protein synthesis and the latter by inhibiting the activity o a actor (eIF4E) required or translation. By inhibiting mTOR, sirolimus–FKBP inhibits protein synthesis and arrests cell division in the G1 phase (Fig. 46-6). Major adverse e ects o sirolimus include hypertension, interstitial lung disease, and leukopenia. Notably, however, the nephrotoxicity associated with CsA and tacrolimus is not observed with sirolimus. Everolimus and zotarolimus are mTOR inhibitors that are structurally related to sirolimus. Everolimus is approved or prevention o kidney transplant rejection and treatment o renal cell carcinoma, hormone-receptor positive, HER2negative breast cancer, progressive neuroendocrine tumors o pancreatic origin, and renal angiomyolipoma and tuberous sclerosis complex, while zotarolimus is used only in drugeluting stents. Recent studies have shown that sirolimus, everolimus, and zotarolimus inhibit mTOR complex 1 but are relatively weak inhibitors o mTOR complex 2; newer drugs are being developed to inhibit both complexes. Sirolimus-, everolimus-, and zotarolimus-eluting stents have been approved or use in the treatment o coronary artery disease. In this unique drug delivery system, the mTOR inhibitor elutes rom stents during the f rst ew weeks a ter stent placement, locally inhibiting proli eration o coronary artery smooth muscle cells and thereby reducing the rate o in-stent restenosis that results rom neointimal proli eration o vascular smooth muscle cells.

852 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Sirolimus

IL-2

IL-2 re ce ptor

mTOR FKBP p70 S 6 kina s e

P HAS -1

Tra ns la tion of s e le cte d mRNAs ne e de d for ce ll cycle progre s s ion

FIGURE 46-6. Mechanism of action of sirolimus. IL-2 receptor signal transduction involves a complex set o protein–protein interactions that lead to increased translation o selected mRNAs encoding proteins required or T-cell proli eration. Specif cally, IL-2 receptor activation initiates an intracellular signaling cascade that leads to phosphorylation o the molecular target o rapamycin (mTOR). mTOR is a kinase that phosphorylates and thereby regulates the activity o PHAS-1 and p70 S6 kinase. PHAS-1 inhibits the activity o a actor (eIF4E) required or translation, and p70 S6 kinase phosphorylates proteins involved in protein synthesis (not shown). The net e ect o mTOR activation is to increase protein synthesis, thereby promoting the transition rom G1 to S phase o the cell cycle. Sirolimus (also known as rapamycin) crosses the plasma membrane and binds to intracellular FK-binding protein (FKBP). The sirolimus–FKBP complex inhibits mTOR, thereby inhibiting translation and causing T cells to arrest in G1. Everolimus and zotarolimus are sirolimus analogues that act by the same mechanism.

metabolites, thereby increasing phagocytic killing activity. TNF- also stimulates production o acute-phase proteins, has pyrogenic e ects, and osters local containment o the in ammatory response. Some o these e ects are indirect and are mediated by other cytokines induced by TNF- . TNF- has been implicated in numerous autoimmune diseases. Rheumatoid arthritis, psoriasis, and Crohn’s disease are three disorders in which inhibition o TNF- has demonstrated therapeutic e f cacy. Rheumatoid arthritis illustrates the central role o TNF- in the pathophysiology o autoimmune diseases (Fig. 46-7). Although the initial stimulus or joint in ammation is still debated, it is thought that macrophages in a diseased joint secrete TNF- , which activates endothelial cells, other monocytes, and synovial f broblasts. Activated endothelial cells up-regulate adhesion molecule expression, resulting in recruitment o in ammatory cells to the joint. Monocyte activation has a positive eedback e ect on T-cell and synovial f broblast activation. Activated synovial f broblasts secrete interleukins, which recruit additional in ammatory cells. With time, the synovium hypertrophies and orms a pannus that leads to destruction o bone and cartilage in the joint, causing the characteristic de ormity and pain o rheumatoid arthritis. Five therapies inter ering with TNF activity have been approved. Etanercept is a soluble TNF receptor dimer that links the extracellular, ligand-binding domain o human

Ma cropha ge

TNF Endothe lia l ce lls Monocyte /ma cropha ge

Cytokine and Cytokine Receptor Inhibition Cytokines are critical signaling mediators in immune unction. Cytokines are also pleiotropic; that is, they exert di erent e ects depending on the target cell and overall cytokine milieu. For this reason, pharmacologic uses o cytokines or cytokine inhibitors may have unpredictable e ects. Anticytokine therapy has been in clinical use or immunologically mediated diseases or more than a decade. The f rst anticytokine agent approved or use was etanercept, an anti-TNF drug developed or rheumatoid arthritis. During the initial clinical studies, some patients with severe, drugre ractory rheumatoid arthritis literally got up rom their wheelchairs and walked a ter receiving etanercept. This dramatic e f cacy ushered in a new era o biological therapies or autoimmune disease, and the number o new drugs that inhibit proin ammatory cytokines continues to grow rapidly. An alternative approach to block the action o in ammatory cytokines is to target the cytokine receptor.

TNF- Inhibitors

Tumor necrosis factor (TNF)- is a cytokine central to many

aspects o the in ammatory response. Macrophages, mast cells, and activated TH cells (especially TH1 cells) secrete TNF- . TNF- stimulates macrophages to produce cytotoxic

IL-1 Activa te d e ndothe lia l ce lls

IL-8

S ynovia l fibrobla s t

T ce ll

Ma trix me ta lloprote a s e s P GE 2 IL-6 Ca rtila ge de gra da tion

Le ukocyte a dhe s ion a nd dia pe de s is

FIGURE 46-7. Proposed roles for tumor necrosis factor in rheumatoid arthritis. Tumor necrosis actor (TNF) is secreted by activated macrophages in an a ected joint, where this cytokine has multiple proin ammatory e ects. First, TNF activates endothelial cells to up-regulate their expression o cell sur ace adhesion molecules (shown as projections on endothelial cells) and undergo other phenotypic changes that promote leukocyte adhesion and diapedesis. Second, TNF has a positive eedback e ect on nearby monocytes and macrophages, promoting their secretion o cytokines such as IL-1. In turn, IL-1 activates T cells (among other unctions), and the combination o IL-1 and TNF stimulates synovial f broblasts to increase their expression o matrix metalloproteases, prostaglandins (especially PGE2), and cytokines (such as IL-6) that degrade the joint cartilage. Synovial f broblasts also secrete IL-8, which promotes neutrophil diapedesis.

C h a p t e r 4 6 Pharmacology o Immunosuppression

TNF receptor type II to the Fc domain o human immunoglobulin G1 (IgG1); inf iximab is a partially humanized mouse monoclonal antibody directed against human TNF- ; and adalimumab is a ully human IgG1 monoclonal antibody directed against TNF- (Fig. 46-8). Certolizumab pegol is a pegylated anti-TNF- monoclonal antibody ragment that lacks the Fc portion o the antibody; as a result, unlike in iximab and adalimumab, certolizumab does not cause antibody-dependent cell-mediated cytotoxicity or f x complement in vitro. Golimumab is a ully human IgG1 monoclonal antibody directed against TNF- that has a longer hal -li e than the other anti-TNF agents.

Extra ce llula r doma in of huma n p75 TNF re ce ptor

s s s s

Fc doma in of huma n IgG1

C H2

C H2

C H3

C H3

Etanercept

VH

VH C H1

VL

C H1

VL

CL

CL s s s s C H2

C H2

C H3

C H3

Infliximab

Huma n

Mous e

FIGURE 46-8. Anti-tumor necrosis actor agents. Shown is the molecular domain organization o etanercept and in iximab. Etanercept consists o the extracellular domain o the human tumor necrosis actor (TNF) receptor used to the Fc domain o human IgG1. This “decoy” receptor binds TNFand TNF- in the circulation, preventing the access o these cytokines to target tissues. In iximab is a partially humanized monoclonal antibody directed against TNF- . The variable heavy chain (VH) and variable light chain (VL) regions are derived rom mouse antihuman sequences, while the remainder o the antibody (the constant regions, denoted by CH and CL) is composed o human antibody sequences. This modif cation o the original mouse anti-TNF monoclonal antibody reduces the likelihood o developing neutralizing antibodies against in iximab. Additional TNF inhibitors include adalimumab, a ully human monoclonal antibody; certolizumab pegol, a pegylated monoclonal antibody ragment; and golimumab, a ully human monoclonal antibody (not shown).

853

The monoclonal antibody-based TNF- inhibitors illustrate the desirability o treatment with “humanized” or “ ully human” antibodies as opposed to mouse or other nonhuman antibodies. Because mouse antibodies are oreign, treatment with them can induce the production o antibodies against the mouse-specif c regions o the therapeutic antibody. The presence o these antiantibodies can reduce drug e f cacy by sequestering the therapeutic antibody be ore it can exert its desired e ect. To address this problem, one approach is to humanize therapeutic antibodies. In this approach, the portions o the antibody not involved in binding to the antigen are changed to the corresponding human sequences. Antibodies can be partially or fully humanized, depending on the extent o these changes. Humanization limits the likelihood o production o human antibodies against the therapeutic antibody, increasing the clinical e ectiveness o the antibody and allowing its long-term use (see Chapter 54, Protein Therapeutics). A more recent approach to the preparation o therapeutic antibodies is to prepare the antibody in an experimental animal bearing a human immune system or to use an in vitro human antibody system. This strategy generates ully human antibodies that do not require urther manipulation to render them nonimmunogenic. Although all f ve o the anti-TNF agents target TNF- , etanercept is somewhat less selective than the others because it binds to both TNF- and TNF- . In iximab, adalimumab, certolizumab, and golimumab are selective or TNF- and do not bind TNF- . The Fc portions o in iximab, adalimumab, and golimumab may also have specif c activity with respect to complement f xation and binding to Fc receptors on e ector cells. The immune e ector actions o these agents may be relevant to their mechanisms o action because TNF- is expressed on the sur ace o cells, especially macrophages, and the cell sur ace orm is cleaved to yield the soluble cytokine. Anti-TNF agents with immune e ector unctions may have biological e ects di erent rom those o agents that do not bind Fc receptors or f x complement. The indications or TNF inhibitors have recently expanded to include conditions spanning the f elds o dermatology, rheumatology, and gastroenterology. Etanercept is approved or use in rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis. In iximab is approved or use in rheumatoid arthritis, Crohn’s disease, ulcerative colitis, plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis. Adalimumab is approved or use in rheumatoid arthritis, juvenile idiopathic arthritis, Crohn’s disease, ulcerative colitis, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, and hidradenitis suppurtiva. Certolizumab is approved or the treatment o rheumatoid arthritis, Crohn’s disease, psoriatic arthritis, and ankylosing spondylitis. Golimumab is approved or use in adults with rheumatoid arthritis (in combination with methotrexate), ulcerative colitis, psoriatic arthritis, and ankylosing spondylitis. Although high levels o TNF- are likely mediators o underlying pathophysiologic processes, treatment with an anti-TNF agent o ten improves disease symptoms without reversing the underlying pathophysiology. There ore, upon drug discontinuation, maintenance o clinical response is uncertain. Etanercept, in iximab, adalimumab, certolizumab, and golimumab are proteins and must be administered parenterally. Orally active inhibitors o TNF- and inhibitors o TNF- converting enzyme (TACE) are under investigation. Several important adverse e ects must be considered when administering TNF inhibitors. All patients should

854 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

undergo screening or tuberculosis be ore initiating therapy because o increased risk o reactivating latent tuberculosis. Any patient who develops an in ection while taking a TNF inhibitor should undergo evaluation and aggressive antibiotic treatment. Additionally, patients diagnosed with a severe in ection are recommended to temporarily interrupt treatment with TNF inhibitors. Epidemiologic surveillance has suggested that there may be an increased risk o demyelinating disease with anti-TNF therapy, although it has not yet been determined whether the relationship is causal.

antagonist, once-weekly injections o rilonacept are su f cient or e f cacy in the treatment o CAPS. Canakinumab is a human IgG1 monoclonal antibody directed against IL-1 that is approved or use in CAPS and systemic juvenile idiopathic arthritis. Perhaps because o its specif city or IL-1 , canakinumab may be administered just once monthly and still exhibit ull e f cacy. No studies have been conducted to compare the e f cacy o the three anti-IL1 therapies.

IL-12/IL-23p40 Cytokine Inhibitors

Interleukin-17 (IL-17, also known as IL-17A) is a cytokine that

New biological therapies or the treatment o T-cell-mediated diseases include antibodies to IL-12 and IL-23. IL-12 and IL-23 are cytokines involved in natural killer cell activation and CD4 T-cell di erentiation and activation. IL-12, a heterodimer composed o p40 and p35 subunits, directs the di erentiation o naïve T cells into TH1 cells, which secrete IL-2, IFN- , and TNF- . IL-23 is also a heterodimer that has the same p40 subunit covalently linked to a p19 subunit. IL-23 directs the di erentiation o naïve T cells into TH17 cells, which secrete IL-17 and IL-22. Ustekinumab is a high-a f nity human IgG1 monoclonal antibody that binds to the p40 subunit shared by IL-12 and IL-23. Ustekinumab is approved or use in psoriasis and psoriatic arthritis and is in clinical trials or the treatment o Crohn’s disease, sarcoidosis, and multiple sclerosis. Adverse e ects include an increased risk of infection.

IL-1 Cytokine and IL-1 Receptor Inhibitors

Interleukin-1 (IL-1) is an ancient cytokine, expressed in both

vertebrates and invertebrates, that serves as a bridge between innate and adaptive immunity. Two orms o IL-1, IL-1 and IL-1 , are encoded on di erent genes. In humans, IL-1 has primarily an immune role, while IL-1 may be involved in maintenance o epithelial cell unction. Human genetic data and studies with IL-1 antagonists point to a nonredundant role or IL-1 as an in ammatory mediator. Herea ter, thereore, we use the term IL-1 to re er to IL-1 . Most IL-1 is generated by activated mononuclear cells. IL-1 stimulates IL-6 production, enhances adhesion molecule expression, and stimulates cell proli eration. Modulation o IL-1 activity in vivo is accomplished in part by an endogenous IL-1 receptor antagonist (IL-1ra). Anakinra , a recombinant orm o IL-1ra, is approved or use in rheumatoid arthritis. Anakinra has modest e ects on pain and swelling but signif cantly reduces bony erosions, possibly because it decreases osteoclast production and blocks IL-1-induced metalloproteinase release rom synovial cells. Several rare syndromes are mediated in part by increased levels o IL-1. Collectively, these syndromes are termed cryopyrin-associated periodic syndromes (CAPS). Anakinra is approved or use in the CAPS syndrome neonatal-onset multisystem in ammatory disease, and the CAPS syndromes Muckle-Wells syndrome and Hibernian ever have also been treated e ectively with anakinra. Anakinra may cause neutropenia and increase susceptibility to infection. The rapid clearance o this small recombinant peptide and its competitive binding mechanism may explain the need or daily injections o the drug to achieve e f cacy. Rilonacept is a recombinant, soluble IL-1 receptor Fc usion protein that is approved or use in CAPS. Rilonacept binds both IL-1 and IL-1 and also endogenous IL-1ra. Possibly because o its binding to the endogenous receptor

IL-17 Cytokine and IL-17 Receptor Inhibitors

is mainly produced by IL-23-induced TH17 cells. IL-17 plays a central role in in ammation by stimulating the production o key in ammatory mediators, including IL-6, TNF- , and IL-1 . Keratinocytes, f broblasts, macrophages, and neutrophils are among the cell types that respond to IL-17-induced proin ammatory cytokine production. IL-17 also stimulates the proli eration and survival o neutrophils, T cells, and B cells. Ixekizumab and secukinumab are two ully human monoclonal antibodies directed against IL-17A that are in late-phase clinical trials or the management o psoriasis and rheumatoid arthritis. IL-17 exerts its proin ammatory e ect by signaling through two receptor subtypes, IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC). Brodalumab is an antiIL-17 receptor monoclonal antibody that acts as a competitive inhibitor o the IL-17RA subunit. This drug is under active investigation or the treatment o psoriasis, rheumatoid arthritis, and Crohn’s disease.

IL-6 Receptor Inhibitor

The cytokine interleukin-6 (IL-6) was initially identif ed as a actor that di erentiates activated B cells into immunoglobulin-producing cells. IL-6 is a key mediator in many physiologic and pathologic processes, including the acutephase in ammatory response, angiogenesis, neutrophil migration, di erentiation o helper T cells, bone and cartilage metabolism, lipid metabolism, and cancer. Lymphocytes, monocytes, f broblasts, keratinocytes, endothelial cells, mesangial cells, and adipocytes all produce IL-6, while cells o hematopoietic origin express the IL-6 receptor. Elevated serum levels o IL-6 have been implicated in the pathogenesis o many autoimmune and in ammatory conditions. Tocilizumab is a monoclonal antibody directed against the IL-6 receptor that is approved or use in polyarticular juvenile idiopathic arthritis and systemic juvenile idiopathic arthritis and in patients with rheumatoid arthritis who have had an inadequate response to anti-TNF drugs. The drug is administered every 4 weeks as an intravenous in usion.

Depletion o Specif c Immune Cells Appropriately targeted antibodies deplete the immune system o reactive cells and thereby provide e ective therapy or autoimmune diseases and transplant rejection. When the adaptive immune system reacts to an antigen, the resulting immunologic response includes the clonal expansion o cells specif cally reactive against that antigen. Treatment with exogenous antibodies directed against cell sur ace molecules that are expressed selectively on reactive immune cells can pre erentially deplete the immune system o these reactive cells. Antibodies that target cell sur ace receptors expressed selectively on malignant cells o immune origin are discussed in Chapter 40, Pharmacology o Cancer: Signal Transduction.

C h a p t e r 4 6 Pharmacology of Immunosuppression

Polyclonal Antibodies Antithymocyte Globulin Antithymocyte globulin (ATG) is a preparation o antibodies in-

duced by injecting rabbits or horses with human thymocytes. The rabbit or horse antibodies are polyclonal and target many antigens on human T cells. Because ATG targets essentially all T cells and leads to pro ound lymphocyte depletion, ATG treatment results in broad immunosuppression that can predispose to in ection. ATG is approved or use in prevention or treatment o renal transplant rejection, and the equine-derived material is also approved or treatment o aplastic anemia. ATG is administered intravenously once daily or up to 28 days. ATG therapy is o ten complicated by ever and headache as prominent mani estations o the cytokine release syndrome . This syndrome, common to many antibody drugs that target lymphocytes, results rom activation o T cells and release o T-cell cytokines be ore the antibody-coated T cells can be cleared by macrophages. The cytokine release syndrome typically occurs a ter the f rst ew doses o ATG therapy, and the syndrome dissipates as T cells are eliminated. However, administration o successive ATG doses can also be complicated by the development o antibodies against rabbit- or horsespecif c epitopes on the administered immunoglobulins. ATG is generally co-administered with glucocorticoids, an antihistamine, and an antipyretic to mitigate in usion reactions. Note that Mr. W received medications in these three classes when starting induction ATG therapy a ter kidney transplant.

Monoclonal Antibodies OKT3 OKT3 (muromonab-CD3, anti-CD3) is a mouse monoclonal

antibody directed against human CD3, one o the cell sur ace signaling molecules important or activation o the T-cell receptor. CD3 is specif cally expressed on T cells (both CD4 and CD8 cells). Treatment with OKT3 depletes the available pool o T cells via antibody-mediated activation o complement and clearance o immune cells. Because OKT3 targets all T cells, OKT3 treatment can result in pro ound immunosuppression. Also, because OKT3 binds to CD3, and CD3 is important or T-cell activation, OKT3 therapy can broadly activate T cells, resulting in the cytokine release syndrome. Another limitation is that OKT3 is a mouse antihuman antibody (see above). OKT3 was approved or use in acute renal transplant rejection but was subsequently (in 2010) withdrawn voluntarily rom the market by the manu acturer.

Anti-CD20 mAb Rituximab is a chimeric, partially humanized anti-CD20

monoclonal antibody. CD20 is expressed on the sur ace o all mature B cells, and administration o rituximab causes proound depletion o circulating B cells. Originally approved or the treatment o CD20 non-Hodgkin’s lymphoma (see Chapter 40), rituximab has also been approved or use in rheumatoid arthritis re ractory to TNF inhibitors, granulomatosis with polyangiitis, and microscopic polyangiitis. Several additional anti-CD20 antibodies are in clinical development; ofatumumab and obinutuzumab are ully human anti-CD20 monoclonal antibodies that recognize an epitope distinct rom that bound by rituximab. O atumumab and obinutuzumab are approved or use in chronic lymphocytic leukemia. Anti-CD25 mAb Basiliximab is a monoclonal antibody directed against CD25,

the high-a f nity IL-2 receptor. IL-2 mediates early steps in

855

T-cell activation. Because CD25 is expressed only on activated T cells, anti-CD25 antibody therapy selectively targets T cells that have been activated by an MHC-antigen stimulus. Basiliximab is administered prophylactically in renal transplantation to inhibit acute organ rejection. It is also used as a component o general immunosuppressive regimens a ter organ transplantation. Basiliximab is typically administered in a two-dose regimen, with the f rst administration 2 hours be ore transplantation surgery and the second dose 4 days a ter transplantation. This type o dosing regimen, in which drug is administered or a limited period immediately a ter transplantation, is re erred to as induction therapy. Daclizumab is another antibody with the same mechanism o action that was voluntarily withdrawn rom the market by the manu acturer in 2009. Anti-CD52 mAb Campath-1 (CD52) is an antigen expressed on most mature

lymphocytes and on some lymphocyte precursors. An antibody against this antigen was originally tested in rheumatoid arthritis and ound to cause prolonged and sustained depletion o all T cells, o ten lasting or years. The reason or the sustained lymphocyte depletion is unknown. Anti-CD52 mAb therapy did lead to some improvement in the symptoms o arthritis; however, the sustained depletion o lymphocytes and concern about in ections precluded urther study o this antibody in autoimmune conditions. Under the generic name alemtuzumab, anti-CD52 monoclonal antibody had been approved as an adjunctive therapy in the treatment o B-cell chronic lymphocytic leukemia—a condition in which sustained suppression o the leukemic cells is desirable. However, alemtuzumab has been voluntarily withdrawn rom the market in the United States and Europe.

LFA-3

LFA-3 (also called CD58) is the counter-receptor or CD2, an

antigen expressed at high levels on the sur ace o memory e ector T cells. Interaction o CD2 on T cells with LFA-3 on antigen-presenting cells promotes increased T-cell proli eration and enhanced T-cell-dependent cytotoxicity. Because the memory e ector T-cell population is increased in patients with psoriasis, a pharmacologic agent that disrupts the CD2– LFA-3 interaction was tested or use in psoriasis. Alefacept is an LFA-3/Fc usion protein that interrupts CD2–LFA-3 signaling by binding to T-cell CD2 and thereby inhibits T-cell activation. Additionally, the Fc portion o ale acept may activate NK cells to deplete the immune system o memory e ector T cells. Clinically, ale acept signif cantly decreases the severity o chronic plaque psoriasis. Because CD2 is expressed on other adaptive immune cells, however, administration o ale acept also causes a dosedependent reduction in CD4 and CD8 T-cell populations. Patients taking ale acept may have an increased risk o serious in ection and an increased risk o malignancy, primarily skin cancer. Ale acept was voluntarily withdrawn rom the market by the manu acturer in 2011.

B-Lymphocyte Stimulator

Belimumab is a human monoclonal antibody directed against

the B-lymphocyte stimulator (BLyS) cytokine. BLyS binding to normal B cells activates signaling cascades that stimulate cell survival and cell di erentiation into antibodyand autoantibody-producing cells. Belimumab blocks the

856 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

normal unction o BLyS, resulting in B-cell apoptosis. As mentioned previously, autoantibodies against sel -antigens are one mechanism o tissue injury and in ammation in systemic lupus erythematosus. Reducing the number o circulating B cells leads to a subsequent decrease in autoantibody production and reduced disease activity.

The major adverse e ects o abatacept are exacerbations of preexisting chronic obstructive lung disease and increased susceptibility to infection. Abatacept should not be administered concurrently with TNF inhibitors because the combination carries an unacceptably high risk o in ection.

Antibody–Drug Conjugates

Belatacept is a close structural congener o abatacept that

Brentuximab vedotin is a chimeric human monoclonal

antibody–drug conjugate directed against CD30. CD30 is a membrane glycoprotein in the TNF receptor amily that signals through multiple mechanisms, including the NF B pathway, to promote cell proli eration and survival. CD30 is expressed on activated CD4 and CD8 T cells and on B cells. It is also highly expressed on Reed-Sternberg cells in Hodgkin’s lymphoma and on anaplastic large cell lymphoma cells. The specif c role o CD30 in the pathogenesis o lymphoma is under investigation. In brentuximab vedotin, the anti-CD30 antibody is linked to the antimitotic drug monomethyl auristatin E (MMAE) through a valine-citrulline dipeptide. This linker dipeptide is enzymatically cleaved a ter endocytosis at the target site to release MMAE into the cytoplasm. MMAE prevents microtubule polymerization, causing cell cycle arrest in the G2 to M phase and subsequent apoptosis in CD30-expressing cells. Brentuximab vedotin is approved or the treatment o relapsing Hodgkin’s lymphoma a ter ailure o multi-agent chemotherapy or ailure o autologous stem cell transplant. It is also approved or systemic anaplastic large cell lymphoma a ter ailure o at least one multi-agent chemotherapy regimen. Additional anti-CD30 monoclonal antibodies, bispecif c antibodies, and antibody–drug conjugates are under active investigation or the treatment o Hodgkin’s lymphoma.

Inhibition of Costimulation Costimulation re ers to the paradigm that cells o the im-

mune system typically require two signals or activation (see Chapter 42, Principles o In ammation and the Immune System). I a f rst signal is provided in the absence o a second signal, the target immune cell may become anergic rather than activated. Because induction o anergy could lead to long-term acceptance o an organ gra t or limit the extent o an autoimmune disease, inhibition o costimulation represents a viable strategy or immunosuppression. Several therapeutic agents inhibit costimulation by blocking the second signal required or cell activation, and more such agents are under development.

Abatacept

Abatacept consists o CTLA-4 used to an IgG1 constant

region. Abatacept complexes with costimulatory B7 molecules on the sur ace o antigen-presenting cells. When the antigen-presenting cell interacts with a T cell, MHC:antigen– TCR interaction (“signal 1”) occurs, but the complex o B7 with abatacept prevents delivery o a costimulatory signal (“signal 2”), and the T cell develops anergy or undergoes apoptosis. By this mechanism, abatacept therapy appears to be e ective in down-regulating specif c T-cell populations. Abatacept is approved or the treatment o juvenile idiopathic arthritis and rheumatoid arthritis that is re ractory to methotrexate or TNF inhibitors. Clinically, abatacept signif cantly improves symptoms o rheumatoid arthritis in patients who ail to respond to methotrexate or TNF inhibitors.

Belatacept

has increased a f nity or B7-1 and B7-2. In a large clinical trial, belatacept was as e ective as cyclosporine at inhibiting acute rejection in renal transplant recipients. Belatacept is approved as an immunosuppressant or renal transplantation in patients who are seropositive or Epstein-Barr virus (EBV).

Blockade of Cell Adhesion The recruitment and accumulation o in ammatory cells at sites o in ammation is an essential element o most autoimmune diseases; the only exceptions to this rule are autoimmune diseases that are purely humoral, such as myasthenia gravis. Drugs that inhibit cell migration to sites o in ammation may also inhibit antigen presentation and cytotoxicity, thus providing multiple potential mechanisms o therapeutic action.

Natalizumab

Alpha-4 integrins are critical to immune-cell adhesion and homing. The 4 1 integrin mediates immune-cell interactions with cells expressing vascular cell adhesion molecule 1 (VCAM-1), while the 4 7 integrin mediates immune-cell binding to cells expressing mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Natalizumab is a monoclonal antibody directed against 4 integrin that inhibits immune-cell interactions with cells expressing VCAM-1 or MAdCAM-1. Natalizumab was approved or the treatment o relapsing multiple sclerosis. During postmarketing surveillance o the drug, however, several patients treated with natalizumab developed progressive multi ocal leukoencephalopathy (PML), a rare demyelinating disorder caused by in ection with JC virus. This f nding resulted in voluntary withdrawal o the drug. A ter urther FDA investigation, it was decided to resume testing o natalizumab and to add a warning to the product label regarding the possible association. Natalizumab was subsequently reapproved or use in the treatment o multiple sclerosis and Crohn’s disease.

Inhibition of Complement Activation The complement system mediates multiple innate immune responses (see Chapter 42). Recognition o oreign proteins or carbohydrates leads to sequential activation o complement proteins and eventual assembly o the membrane attack complex, a multiprotein structure that can cause cell lysis. Patients with paroxysmal nocturnal hemoglobinuria (PNH) have acquired de ects in complement regulatory proteins, leading to inappropriate activation o complement and complementmediated lysis o erythrocytes. Eculizumab is a humanized monoclonal antibody directed against C5, a complement protein that mediates late steps in complement activation and triggers assembly o the membrane attack complex. Eculizumab is approved or treatment o PNH; it signif cantly decreases hemoglobinuria and the need or erythrocyte transusions in patients with this disorder. Eculizumab is also approved or treatment o atypical hemolytic uremic syndrome. Genetic evidence indicates that complement activation may play an etiologic role in age-dependent macular degeneration,

C h a p t e r 4 6 Pharmacology of Immunosuppression

suggesting that inhibitors o the complement cascade could be use ul local therapies or this disease.

Inhibition of Immune Checkpoints As discussed above in the “Inhibition o Costimulation” section, the immune system uses costimulatory signals to activate antigen-specif c immune responses (see also Chapter 42). The immune system also uses immune checkpoint signals to inhibit such responses. Inhibitory checkpoint molecules include cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and several others. In general, ligation o these molecules on T cells inhibits the immune response; the signaling pathways that mediate these inhibitory responses are subjects o active investigation. Some tumors have been ound to up-regulate PD-1 ligands and thereby inhibit T-cell immune surveillance o tumors. Studies have shown that inhibition o CTLA-4 and/or PD-1 augments T-cell activation, proli eration, and cytokine production, and can be used to enhance the anti-tumor immune response.

CTLA-4 Blockade

lpilimumab is a f rst-generation immune checkpoint inhibi-

tor. This recombinant, humanized IgG1 monoclonal antibody binds to CTLA-4 and blocks the interaction o CTLA-4 with its ligands B7-1 and B7-2 (see Chapter 42). In clinical studies, ipilimumab showed an overall survival benef t in patients with unresectable or metastatic melanoma who had been previously treated with one or more anticancer therapies. Ipilimumab was approved or use in 2011 in advanced melanoma that is unresectable or metastatic. Patients taking ipilimumab should be monitored or possible immunerelated hepatotoxicity and endocrinopathies.

PD-1 Blockade

Nivolumab and pembrolizumab are recombinant, humanized

IgG4 monoclonal antibodies that bind to PD-1 and block the interaction o PD-1 with its ligands PD-L1 and PD-L2. By inhibiting the inhibition o T-cell proli eration and cytokine production that results rom binding o PD-1 to PD-L1 and PD-L2, blockade o PD-1 by nivolumab and pembrolizumab releases PD-1 pathway-mediated inhibition o the immune response, including the anti-tumor immune response. In appropriate mouse tumor models, blocking PD-1 activity results in decreased tumor growth. Nivolumab and pembrolizumab are indicated or the treatment o unresectable or metastatic melanoma with disease progression ollowing treatment with ipilimumab and, i the tumor is positive or a B-RAF V600 mutation, a B-RAF inhibitor. These therapies are also approved or metastatic non-small cell lung cancers that express PD-L1 and show disease progression on or a ter platinum-containing chemotherapy. For non-small cell lung cancer with sensitizing EGFR mutations or ALK rearrangements, disease progression on therapies targeted or these genomic tumor aberrations should be demonstrated prior to treatment with nivolumab or pembrolizumab.

CONCLUSION AND FUTURE DIRECTIONS Several approaches are available or the pharmacologic suppression o adaptive immunity, ranging rom the relatively low-selectivity approaches represented by glucocorticoids and cytotoxic agents to the more selective approaches

857

represented by cell-signaling inhibitors and antibody therapies. Glucocorticoids induce pro ound suppression o the in ammatory response and immune system but cause many adverse e ects, most o which are due to drug e ects on cells outside the immune system. Glucocorticoid receptor modulators are being sought that retain the anti-in ammatory e ects o glucocorticoids but have less severe adverse e ects on metabolism and bone mineral homeostasis. Cytotoxic agents target DNA replication; although immune cells are highly susceptible to these drugs, so, too, are other normal cells such as those in the gastrointestinal epithelium. The cytotoxic agent mycophenolate mo etil is highly selective, both because lymphocytes depend on de novo purine synthesis and because mycophenolic acid pre erentially targets the inosine monophosphate dehydrogenase isoenzyme expressed in lymphocytes. Lymphocyte-signaling inhibitors—such as cyclosporine, tacrolimus, sirolimus, and everolimus, which target intracellular signal transduction pathways necessary or T-cell activation—are also reasonably selective. Many new inhibitors o intracellular signaling in lymphocytes are under investigation; inhibition o the Janus kinase amily appears particularly promising. Cytokine inhibitors interrupt soluble signals mediating immune-cell activation. TNF inhibitors— such as etanercept, in iximab, and adalimumab—represent an expanding class o drugs. Promising new targets include cytokine pathways associated with TH17 cells, which are targeted by the IL-17A inhibitors ixekizumab and secukinumab and the IL-17 receptor antagonist brodalumab, among others. The concept o preventing immune-cell activation has been extended to the blockade o costimulation represented by abatacept and belatacept. Speci c depletion o B cells is a well-established therapy or lymphomas and rheumatoid arthritis: belimumab, a f rst-in-its-class antibody against a crucial B-cell survival actor, is used in the treatment o systemic lupus erythematosus. Speci c depletion o T cells may be benef cial in organ transplantation: antithymocyte globulin is directed against T-cell-specif c epitopes. Several antibody therapeutics and small molecules are available that block immune-cell adhesion and homing, and more such agents are under development. Immune checkpoint inhibitors are an exciting new class o anticancer therapies. Disclosure

Lloyd B. Klickstein is an employee and stockholder o Novartis, Inc., which manu actures or distributes drugs discussed in this chapter, including cyclosporine, mycophenolate sodium, everolimus, canakinumab, and basiliximab. April W. Armstrong serves as investigator and/or consultant to AbbVie, Amgen, Celgene, Janssen, Lilly, Merck, Novartis, and Pf zer.

Suggested Reading Benedetti G, Miossec P. Interleukin 17 contributes to the chronicity o in ammatory diseases such as rheumatoid arthritis. Eur J Immunol 2014;44: 339–347. (Discusses role that IL-17 may have in inf ammatory diseases.) Intleko er AM, Thompson CB. At the bench: preclinical rationale or CTLA-4 and PD-1 blockade as cancer immunotherapy. J Leukoc Biol 2013;94:25–39. (Discusses the molecular mechanisms by which CTLA-4 and PD-1 unction to turn o established immune responses.) Mahoney KM, Rennert PD, Freeman GJ. Combination cancer immunotherapy and new immunomodulatory targets. Nat Rev Drug Discov 2015;14:561–584. (Discusses CTLA-4, PD-1, and other potential drug targets that mediate inhibition o the anti-tumor immune response.) Murphy K, Travers P, Walport M. Janeway’s immunobiology: the immune system in health and disease. 8th ed. New York: Garland Publishing; 2012. (Discussion o autoimmunity and transplantation immunity.)

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C h a p t e r 4 6 Pharmacology of Immunosuppression 863

H. pylor lorri

Anti tibi bio ticc s

Co ating g a g e nts nt

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Integrative Inf ammation Pharmacology: Peptic Ulcer Disease K+

H+/K / + ATP ATP a s e

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INTRODUCTION & CASES 1 & 2. . . . . . . AC INT . .C. . . . . . . . . . . . . . 886 8644–865 T tN Agents A g That Neutralize Neutral Neutr uttrraa lliz utr uutra lize zee Acid A id . . . . . . . . . . . . . . . . . . . . . . . . . . . 873 H DAG A Ga Agents Agent A tssDA Th That T Tha ha att Promote P Prromot Promot Promo mote o M Mucosal ossaaall D Defense De 8733 CCKB . . . . . . . . . . . . . . . . . 87 PHYSIOLOGY OF GASTRIC ACID SEC ECRE RETI TION . . . . . . . 2. . . . . . . . 864 64M 3 Coating Aggent ents t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 8733 Neurohormonal Controol of Ne Secretio . . . . . . . . 864 H2Gblot cke cic keAcid rrss d Secretion P LC C Prostaglandins nss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873 Phas ases es of Ga st stri tricc Ac A id Secretion . His . . . ta . .mine . . . . . . . . . . . . . . . . . 866 Hi Agen Ag ennts Thatt Moddify Risk Facttorss . . . . . . . . . . . . . . . . . . . . . . . . 874 Protective Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 8666 Die Di ie ieet, Tobacco, and Alc lcohol c l . . . . . . . . . . . . . . . . . . . . . . . . . . 874 PATHOPHYSIOLOGY OF PEPTIC ULCER DISEASE SE . . . . . . . . . . . 867 Treatment T Tre Tr re off H. py ylori rii IInfection on . . . . . . . . . . . . . . . . . . . . . . . 874 Helicobacter pylori. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 867 AC Ch ACh Ga tr G a s trin trin CONCLUSION ON AND FUTURE TURE DIRECTION DIRECTIONS . . . . . . . . . . . . . . . . . . 874 NSAI NS AIDs Ds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868 CONC Ne N vgees ve Acid Hypersecretion . . . . . . . . . . . . . . . . . . . . ECL . . . . .ce . .ll. . . . . . . 868 Su ue rgg este tedd Re Read adin ingg B . . ood . . . . vve . e. s. s. e. l. . . . . . . . . . . . . . . . . . . . . . . . 874 Blood Other Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 869 Agents That Decrease Acid Secretion . . . . . . . . . . . . . . . . . . . 869 H2 Receptor Antagonistss . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 Proton Pump Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870

INTRODUCTION A peptic ulcer is a break in the mucosa o the stomach (gastric ulcer) or duodenum (duodenal ulcer). Four-and-a-hal million people in the United States su er rom active peptic ulcer disease, and 500,000 new cases o peptic ulcer disease are diagnosed each year. The li etime prevalence o peptic ulcer disease is approximately 10%, and the estimated annual cost or treatment exceeds $1 billion. There are several pathophysiologic mechanisms or peptic ulcer disease, and clinical management o ten requires multiple pharmacologic strategies. This chapter describes the physiology o gastric acid secretion and the pathophysiology underlying the ormation o peptic ulcers. The pharmacologic agents used in the treatment o peptic ulcer disease are then discussed in relation to the pathophysiology that is interrupted by these drugs.

PHYSIOLOGY OF GASTRIC ACID SECRETION Neurohormonal Control o Gastric Acid Secretion Hydrochloric acid is secreted into the stomach by parietal cells , which are located in oxyntic glands in the undus and 864

γ

GTP

body o the stomach. The parietal cell actively transports H across its apical canalicular membranes via H /K ATPases (proton pumps) that exchange intracellular H or extracellular K . Three neurohormonal secretagogues regulate this process: histamine , gastrin, and acetylcholine (ACh). Each o these secretagogues binds to and activates specif c receptors on the basolateral membrane o the parietal cell, thereby initiating the biochemical changes necessary or active transport o H out o the cell. Histamine , released by enterochroma f n-like (ECL) cells located in and adjacent to the oxyntic glands and by mast cells in the lamina propria, binds to histamine H2 receptors on the parietal cell. H2 receptor activation stimulates adenylyl cyclase and increases intracellular cyclic adenosine monophosphate (cAMP). In turn, cAMP activates cAMP-dependent protein kinase (protein kinase A [PKA]). PKA phosphorylates and activates proteins responsible or tra f cking o cytoplasmic tubulovesicles containing H /K ATPase to the apical membrane o the cell. The H /K ATPase does not pump H into the tubulovesicles because the permeability o the vesicular membrane to K is low. A ter usion o the tubulovesicles with the apical membrane, the availability o extracellular K allows the H /K ATPase to pump H rom the parietal cell into the gastric lumen. Concurrent with the tra f cking o cytoplasmic tubulovesicles to the apical membrane, cellular

C h a p t e r 4 7 Integrative Inf ammation Pharmacology: Peptic Ulcer Disease

865

To m is a 24-ye a r-o ld g ra d u a te s tu d e n t. He is in good health, although he sm okes a p p roxim a te ly tw o p a cks o ciga re tte s an d d rin ks f ve cu p s o co e e a d ay. He is cu rren tly u n d er s tre ss b ecau s e o the im ppee nd in g d ea dlin e o r h is co m p u ter s cie n ce th es is. Fo r th e p a s t 2 w e e ks , To m h a s n o te d a b u rn in g p a in in h is u p p e r a b d o m e n th a t o ccu rs 1–2 h o u rs a te r e a tin g . In a d d itio n , th e p a in re q u e n tly a wa ke n s h im a t a p p roxim a te ly 3:0 0 AM. His p a in is u s u a lly re lie ve d b y e a tin g a n d b y ta kin g o ve r-th e -co u n te r a n ta cid s . Wh e n th e p a in in cre a s e s in in te n s ity, To m d e cid e s to vis it h is in te rn is t, Dr. S m ith , a t Un ive rs ity He a lth Se rvice s . Dr. S m ith n o te s th a t th e a b d o m in a l e xa m in a tio n is n o rm a l e xce p t o r e p iga s tric te n d e rn e s s . Dr. S m ith d is cu s s e s d ia g n o s tic o p tio n s w ith To m , in clu d in g a n u p p e r g a s tro in te s tin a l x-ra y s e rie s a n d a n e n d o s co p ic e xa m in a tio n . To m ch o o s e s to u n d e rg o th e e n d o s co p ic e xa m in a tio n . Du rin g th e e xa m in a tio n , a n u lce r is id e n tif e d in th e p ro xim a l

p o rtio n o th e d u o d e n u m o n th e p o s te rio r wa ll. Th e u lce r is 0.5 cm in d ia m e te r. A m u co s a l b io p s y o th e ga s tric a n tru m is p e r o rm e d o r d e te ctio n o He lico b a cte r p ylo ri. To m is d ia g n o s e d w ith a d u o d e n a l u lce r. Dr. S m ith p re s crib e s o m e p ra zo le , a p ro to n p u m p in h ib ito r. Th e n e xt d a y, w h e n th e p a th o lo g y re p o rt in d ica te s th e p re s e n ce o a n H. p ylo ri in e ctio n , Dr. S m ith p re s crib e s b is m u th , cla rith ro m ycin , a n d a m oxicillin in a d d itio n to th e p ro to n p u m p in h ib ito r. Dr. S m ith a ls o a d vis e s To m to s to p s m o kin g a n d d rin kin g co e e .

Ma ria n n e is a 54-ye a r-o ld a d m in is tra to r in a p rin tin g s h o p w h o typ e s 4–5 h o u rs a d a y. S h e d e ve lo p s ca rp a l tu n n e l s yn d ro m e a n d b e g in s to ta ke s e ve ra l a s p irin d a ily o r th e p a in . On e m o n th la te r, Ma ria n n e d e ve lo p s a b u rn in g p a in in h e r u p p e r a b d o m e n . A te r vo m itin g “ co e e g ro u n d s ” m a te ria l a n d n o ticin g th a t h e r b o w e l m ove m e n ts a re b la ck, s h e d e cid e s to vis it th e e m e rg e n cy ro o m o h e r lo ca l h o s p ita l. Th e o n -ca ll ga s tro e n te ro lo g is t p e r o rm s a n e n d o s co p y a n d co n f rm s th a t Ma ria n n e h a s a ga s tric u lce r th a t h a s re ce n tly b le d . Th e g a s tro e n te ro lo g is t e xp la in s to Ma ria n n e th a t s h e h a s a p e p tic u lce r. Ma ria n n e’s b re a th te s t is n e ga tive o r H. p ylo ri, a n d s h e is to ld th a t a s p irin is th e m o s t like ly ca u s e . Ma ria n n e is tre a te d w ith a n ta cid s a n d ra n itid in e (a n H2 re ce p to r a n ta g o n is t) a n d is to ld to s to p ta kin g n o n s te ro id a l a n ti-in a m m a to ry d ru g s (NS AIDs ),

in clu d in g a s p irin . Th e g a s tro e n te ro lo g is t re vie w s w ith Ma ria n n e th e lis t o p a in -re lie vin g m e d ica tio n s th a t a re co n s id e re d to b e NSAIDs . Tw o w e e ks p a s s . Ma ria n n e in o rm s h e r g a s tro e n te ro lo g is t th a t th e p a in in h e r w ris t h a s b e co m e u n b e a ra b le a n d th a t s h e m u s t co n tin u e ta kin g a s p irin to b e a b le to typ e a t w o rk a n d ke e p h e r jo b . Th e ga s tro e n te ro lo g is t te lls Ma ria n n e th a t s h e ca n ta ke a s p irin a s lo n g a s s h e s w itch e s h e r a n tiu lce r m e d ica tio n ro m a n H2 a n ta g o n is t to a p ro to n p u m p in h ib ito r.

activation mobilizes an apical membrane K channel to provide the extracellular K or this process (Fig. 47-1). Gastrin is secreted into the bloodstream by G cells in the gastric antrum, and acetylcholine is released rom postganglionic nerves with cell bodies located in the submucosa (Meissner’s plexus). Both o these secretagogues bind to their respective G protein-coupled receptors on the parietal cell and thereby activate phospholipase C and increase intracellular calcium levels (Ca2 ) (Fig. 47-1). Beyond the involvement o phospholipase C and intracellular Ca2 , the signaling pathways by which parietal cell stimulation by gastrin and ACh leads to H /K ATPase activation remain to be ully elucidated; protein kinase C is likely to be involved. In addition to its relatively minor role in stimulating the

Questions 1 . What risk actors did Tom have or the development o peptic ulcer disease? What is the role o H. pylori in this disease? 2 . Why was Tom given clarithromycin rather than metronidazole or treatment o his H. pylori in ection? 3 . Why was Tom also treated with a proton pump inhibitor?

Questions 4 . Why was an H2 antagonist prescribed or Marianne’s ulcer, and why was her medication switched to a proton pump inhibitor when she insisted on using aspirin?

parietal cell directly, gastrin has a major role in stimulating the release o histamine by ECL cells (see below). While histamine, gastrin, and ACh increase acid secretion by parietal cells, somatostatin-secreting D cells and prostaglandins limit the extent o gastric acid secretion. Somatostatin decreases acid secretion via three mechanisms: (1) inhibition o gastrin release rom G cells by a paracrine mechanism, (2) inhibition o histamine release rom ECL cells and mast cells, and (3) direct inhibition o parietal cell acid secretion. Prostaglandin E2 (PGE2) enhances mucosal resistance to tissue injury by (1) reducing basal and stimulated gastric acid secretion and (2) enhancing epithelial cell bicarbonate secretion, mucus production, cell turnover, and local blood f ow.

866 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY Lume n

Cl−

K+

Apica l me mbra ne

Ca na liculus K+

H+/K+ ATP a s e P a rie ta l ce ll Tubulove s icle conta ining ina ctive H+/K+ ATP a s e

Tra ns loca tion/ fus ion

Endopla s mic re ticulum

ATP H+

ADP

Ca 2+

P rote in kina s e s ATP cAMP

IP 3 Ba s ola te ra l me mbra ne

αs GTP

AC

β

γ

H2

β

αq

γ

M3

β

γ

GTP

DAG

CCKB P LC

His ta mine Ga s trin

ACh

ECL ce ll

Ne rve

Blood ve s s e l

FIGURE 47-1. Control of parietal cell acid secretion. Stimulation o parietal cell acid secretion is modulated by paracrine (histamine), neuroendocrine (acetylcholine [ACh]), and endocrine (gastrin) pathways, which activate their respective receptors (H2, M3, and CCKB). H2 receptor activation increases cAMP, which activates protein kinase A. M3 and CCKB receptor activation stimulates release o Ca 2 by the Gq-mediated IP3/DAG pathway; these signals may also stimulate protein kinase C activity. Protein kinase activation results in translocation o cytoplasmic tubulovesicles containing inactive H /K ATPase to the apical membrane. Fusion o tubulovesicles with the apical membrane activates H /K ATPase, which pumps H ions into the stomach lumen. An apical membrane Cl channel couples Cl e f ux to H e f ux, and an apical membrane K channel recycles K out o the cell. The net result o this process is the rapid extrusion o HCl into the stomach lumen. In addition to its direct e ect on CCKB receptors on parietal cells, gastrin also stimulates CCKB receptors on ECL cells to promote histamine release (not shown).

Phases of Gastric Acid Secretion

Protective Factors

Gastric secretions increase considerably during a meal. There are three phases o gastric acid secretion. The cephalic phase includes responses to sight, taste, smell, and thought o ood. “Sham eedings,” experiments in which ood is chewed but not swallowed, trigger an increase in acid secretion mediated by vagal stimulation and increased gastrin secretion. Mechanical distension o the stomach and ingestion o amino acids and peptides stimulate the gastric phase . Distension activates stretch receptors in the wall o the stomach that are linked to short intramural nerves and vagal f bers. Luminal nutrients, such as amino acids, are strong stimulants or gastrin release. Gastrin travels via the blood to the oxyntic mucosa and stimulates ECL cells to release histamine. An important negative eedback on acid secretion in this phase is acid (pH 3)-mediated inhibition o gastrin release rom antral G cells. Acid secretion is also inhibited by release o somatostatin rom antral D cells. The intestinal phase involves stimulation o gastric acid secretion by digested protein in the intestine. Gastrin plays a major role in mediating this phase as well.

Factors that protect the gastric mucosa include gastric mucus, prostaglandins (discussed above and in Chapter 43, Pharmacology o Eicosanoids), gastric and duodenal bicarbonate, restitution (repair), and blood ow. The epithelial cells o the stomach secrete mucus , which acts as a lubricant that protects the mucosal cells rom abrasions. Composed o hydrophilic glycoproteins that are viscous and have gelorming properties, the mucus layer enables ormation o an uninterrupted layer o water at the luminal sur ace o the epithelium. Together, the mucus and water layers attenuate potential damage due to the acidic environment o the gastric lumen. Prostaglandins stimulate mucus secretion, whereas NSAIDs and anticholinergic medications inhibit mucus production. In addition, H. pylori disrupts the mucus layer (see below). Bicarbonate protects the gastric epithelium by neutralizing gastric acid. Bicarbonate is secreted by epithelial cells at the luminal sur ace o the gastric mucosa, in gastric pits, and at the luminal sur ace o the duodenal mucosa. Bicarbonate secretion in the duodenum serves to neutralize acid entering the intestine rom the stomach.

C h a p t e r 4 7 Integrative Inf ammation Pharmacology: Peptic Ulcer Disease

Restitution re ers to the ability o the gastric mucosa to

undergo repair. Damage is repaired through migration o undamaged epithelial cells along the basement membrane to f ll de ects created by the sloughing o injured cells. The f nal protective actor is blood f ow . Blood ow to the gastric mucosa removes acid that has di used across a damaged mucus layer.

PATHOPHYSIOLOGY OF PEPTIC ULCER DISEASE A peptic ulcer is a break in the lining o the stomach or duodenum. The break can involve the mucosa, muscularis mucosa, submucosa, and in some cases, the deeper layers o the muscle wall. This compromise o mucosal integrity can cause pain, bleeding, obstruction, per oration, and even death. Peptic ulcers are caused by an imbalance between protective actors and damaging actors in the gastrointestinal mucosa. This section describes the main pathophysiologic mechanisms involved in ulcer ormation, the two most common o which are H. pylori in ection and NSAID use.

Helicobacter pylori H. pylori, a Gram-negative, spiral-shaped bacterium, is the

most common cause o non-NSAID-associated peptic ulcer disease. H. pylori has been ound in the gastric antrum o a signif cant number o patients with duodenal ulcers and gastric ulcers, including Tom in the introductory case. Eradication o H. pylori leads to lower recurrence and relapse rates in patients with ulcers. The latter f nding, together with the act that many ulcer patients are in ected with H. pylori, constitute the major evidence or H. pylori’s causal role in peptic ulcer disease. H. pylori lives in the acidic environment o the stomach. The initial in ection is transmitted by the oral route. Upon ingestion, the microaerophilic bacterium uses its our to six agellae to move in corkscrew ashion through the gastric mucus layer. H. pylori attaches to adhesion molecules on the sur ace o gastric epithelial cells. In the duodenum, H. pylori attaches only to areas containing gastric epithelial cells that have arisen as a result o excess acid damage to

Ure a s e a ctivity

867

the duodenal mucosa (gastric metaplasia). H. pylori is able to live in such a hostile environment partly because o its production o the enzyme urease , which converts urea to ammonia. The ammonia bu ers the H and orms ammonium hydroxide, creating an alkaline cloud around the bacterium and protecting it rom the acidic environment o the stomach. H. pylori’s virulence actors cause damage to the host. Urease is one o these damaging actors because it is an antigen that causes a strong immune response. In addition, ammonium hydroxide produced by urease causes gastric epithelial cell injury. Other virulence actors include lipopolysaccharides (endotoxins), which are components o the bacteria’s outer membrane, as well as a lipase and a protease that are secreted by the bacteria and degrade the gastric mucosa. Cytotoxicity caused by H. pylori has also been linked to two major proteins: cytotoxin-associated gene A (Cag A) and vacuolating cytotoxin (VacA). The cag pathogenicity island is linked to expression o Cag A. This pathogenicity island, which is present in the majority o H. pylori isolates, contains approximately 32 genes that encode a bacterial type IV secretion system. The secretion system inserts into gastric epithelial cells o the host and transports Cag A (and other virulence actors) into the epithelial cells. Once inside the host cell, Cag A undergoes tyrosine phosphorylation by host kinases. Both unphosphorylated and phosphorylated Cag A in uence host signaling pathways and host cellular unctions, including acid secretion, cytokine release, cellular proli eration and apoptosis, cell polarity, and cell motility. Compared to strains o H. pylori that do not express Cag A, H. pylori strains expressing Cag A have been linked to a higher incidence o duodenal ulcers, gastric ulcers, and gastric cancer. The persistence o H. pylori can be traced, in part, to the inappropriate immune response that it elicits. Instead o the normal TH2 mucosal immunity response, which controls luminal in ections by means o secretory (IgA) antibody, the H. pylori organism elicits a TH1 response. Cytokines associated with the TH1 response induce in ammation and epithelial cell damage. Several additional mechanisms characterize H. pyloriinduced peptic ulcer disease (Fig. 47-2). Acid secretion is

G ce ll

pH

Ga s trin Ce ll prolife ra tion S oma tos ta tin Acid s e cre tion

Helicobacter pylori D ce ll Infla mma tory me dia tors

P a rie ta l ce ll

Duode na l ulce r dis e a s e

FIGURE 47-2. The role o H. pylori in duodenal peptic ulcer disease. Two o the mechanisms by which H. pylori in ection predisposes to peptic ulcer disease are illustrated. First, the inf ammatory mediators elicited by H. pylori inhibit somatostatin secretion by D cells in the antrum o the stomach. Decreased D cell somatostatin secretion causes disinhibition o gastrin release rom G cells. Second, the ammonium hydroxide produced by H. pylori-derived urease increases gastric pH, which in turn stimulates gastrin secretion. Activation o gastrin release by both o these mechanisms leads to parietal cell proli eration, which increases the unctional capacity o the gastric mucosa to secrete H ions and thereby predisposes to the development o duodenal ulcer disease.

868 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

increased in patients with H. pylori-associated duodenal ulcers. This is thought to result rom increased levels o circulating gastrin, causing parietal cell proli eration and increased acid production. Gastrin secretion is elevated by two mechanisms: (1) the ammonia generated by H. pylori produces an alkaline environment near the G cells and thereby stimulates gastrin release and (2) the number o antral D cells is lower than normal in H. pylori-in ected patients, resulting in decreased somatostatin production and increased gastrin release. H. pylori also decreases duodenal bicarbonate secretion and thereby weakens the protective mechanisms o the duodenal mucosa. The presence o H. pylori in ection can be detected using the 13C-urea breath test, which is based on the organism’s production o urease. In this test, urease converts ingested 13 C-urea to 13CO2 i H. pylori is present in the stomach, and the 13CO2 is detected in the breath. The 13C-urea breath test is currently the best diagnostic test or H. pylori; other methods o detection include a stool antigen test, histologic examination o a gastric mucosal biopsy (as was per ormed in Tom’s case), and serologic testing or H. pylori antibodies. Serologic testing is o limited utility because the blood test or antibodies remains positive indef nitely. Thus, a positive serologic test does not di erentiate an active in ection rom exposure to the organism in the distant past.

NSAIDs More than 100,000 patients are hospitalized each year or NSAID-associated gastrointestinal complications, and gastrointestinal bleeding has a 5–10% mortality rate in these patients. The gastrointestinal tract is the most common target or the adverse e ects o NSAID use. NSAID-associated gastrointestinal damage is attributable to both topical injury and systemic effects o the NSAID (Fig. 47-3). Most NSAIDs are weak organic acids. In the acidic environment o the stomach, these drugs are neutral compounds that can cross the plasma membrane and enter gastric epithelial cells. In the neutral intracellular environment, the drugs are re-ionized and trapped. The resulting intracellular damage is responsible or the local gastrointestinal injury associated with NSAID use. NSAIDs also cause systemic injury to the gastrointestinal lining, largely because o decreased mucosal prostaglandin synthesis. As described in detail in Chapter 43, two cyclooxygenase enzymes catalyze the ormation o prostaglandins rom arachidonic acid. In general, cyclooxygenase-1 (COX-1) is constitutively expressed and produces the gastric prostaglandins responsible or mucosal integrity, whereas cyclooxygenase-2 (COX-2) is induced by in ammatory stimuli. Inhibition o COX-1 by NSAIDs can lead to mucosal ulceration because inhibition o PGE2 synthesis removes one o the protective mechanisms maintaining the integrity o the gastric mucosa. Although COX-2 selective NSAIDs (coxibs) may carry a lower risk o ulcer ormation than nonselective NSAIDs , the coxibs appear to be associated with an increase in myocardial in arction and stroke. Several o the COX-2 selective NSAIDs have been voluntarily withdrawn (ro ecoxib and valdecoxib), and use o the third has been voluntarily limited (celecoxib). The adverse cardiovascular e ects o the COX-2 selective inhibitors may result rom their suppression o prostacyclin production by vascular endothelial cells (catalyzed by COX-1 and COX-2), allowing thromboxane produced by platelets

A S ys te mic e ffe cts Inhibition of cyclooxyge na s e

Ga s tric a cid s e cre tion Bica rbona te /mucus production Blood flow

P ros ta gla ndins

NSAID Expre s s ion of inte rce llula r a dhe s ion mole cule s in ga s tric va s cula r e ndothe lium

Ne utrophil a dhe re nce to va s cula r e ndothe lia l ce lls

Mucos a l da ma ge due to ne utrophilde rive d fre e ra dica ls a nd prote a s e s

B Topica l injury

S toma ch lume n

Ga s tric e pithe lia l ce ll

(pH ~ 2)

(pH ~ 7) O

O OH

O-

H+ + O

O O

O

Ce ll da ma ge

NSAID (aspirin) we a k a cid

FIGURE 47-3. Role of NSAIDs in peptic ulcer disease. NSAID-associated peptic ulcer disease is a result o both systemic e ects and topical injury. A. Systemic e ects: NSAIDs inhibit cyclooxygenase and thereby decrease the production o prostaglandins. Because prostaglandins activate Gi, and thereby decrease the generation o cAMP in gastric parietal cells, decreased prostaglandin production causes increased gastric acid secretion. Decreased prostaglandins also decrease bicarbonate production, mucus production, and blood f ow in the stomach. An additional systemic e ect involves the increased expression o intercellular adhesion molecules (ICAMs) in the vascular endothelium o the stomach, which increases neutrophil adherence to the vascular endothelial cells. Neutrophils release ree radicals and proteases that cause mucosal damage. B. Topical e ects: NSAIDs induce local injury via ion trapping. From the lumen o the stomach, the drug enters the gastric epithelial cell in its protonated (uncharged) orm. In the neutral environment o the cytoplasm, the NSAID is ionized and trapped inside the cell, causing cell damage.

(catalyzed by COX-1) to exert an unopposed prothrombotic e ect (see Chapter 43). NSAIDs may induce ulcers not only by inhibition o prostaglandin synthesis but also by other systemic mechanisms. For example, NSAIDs increase expression o intercellular adhesion molecules in the vascular endothelium o the gastric mucosa, and increased adherence o neutrophils to the vascular endothelium causes release o ree radicals and proteases that damage the mucosa.

Acid Hypersecretion Acid hypersecretion is an important causative actor in some patients with peptic ulcer disease. Zollinger-Ellison syndrome and Cushing’s ulcers are two clinical examples in which hyperacidity leads to peptic ulcer disease. In Zollinger-Ellison syndrome, a gastrin-secreting tumor o the non-beta cells o the endocrine pancreas leads to increased acid secretion. In Cushing’s ulcers, seen in patients with severe head injuries, heightened vagal (cholinergic) tone causes gastric hyperacidity (see Fig. 47-1).

C h a p t e r 4 7 Integrative Inf ammation Pharmacology: Peptic Ulcer Disease

Other Factors

Agents That Decrease Acid Secretion

Gastric chie cells secrete pepsin, a digestive enzyme, as the inactive precursor pepsinogen. Studies have suggested a role or pepsin in ulcer ormation. Cigarette smoking is associated with peptic ulcer disease; the mechanism is thought to involve impairment o mucosal blood ow and healing and inhibition o pancreatic bicarbonate production. Ca eine ingestion (increased acid secretion), alcoholic cirrhosis, glucocorticoid use, and genetic in uences are also associated with peptic ulcer disease. Finally, chronic psychological stress may occasionally be an important cause o peptic ulcer disease. In Case 1, Tom smoked cigarettes, drank a lot o co ee, and was under stress to f nish his computer science thesis. These actors may have contributed to his development o an ulcer.

H2 Receptor Antagonists

The discovery o H2 receptor antagonists by Black and colleagues in the 1970s signif cantly changed the treatment o peptic ulcer disease. These investigators identif ed a second histamine receptor (H1 was the f rst; see Chapter 44, Histamine Pharmacology) and elucidated its role in gastric acid secretion. H2 receptor antagonists (also called H2 blockers ) reversibly and competitively inhibit the binding o histamine to H2 receptors, resulting in suppression o gastric acid secretion. H2 receptor antagonists also indirectly decrease gastrin- and acetylcholine-induced gastric acid secretion. Four H2 receptor antagonists are available: cimetidine , ranitidine , famotidine , and nizatidine (Fig. 47-5). H2 receptor antagonists are absorbed rapidly rom the small intestine. Peak plasma concentrations are achieved within 1–3 hours. Elimination o H2 receptor antagonists involves both renal excretion and hepatic metabolism. It is there ore important to decrease the dose o these drugs in patients with liver or kidney ailure. An exception is nizatidine, which is eliminated primarily by the kidney. All our drugs are well tolerated in general. Occasional minor adverse e ects include diarrhea, headache, muscle pain, constipation, and atigue. H2 receptor antagonists may

PHARMACOLOGIC CLASSES AND AGENTS Several pathophysiologic mechanisms can lead to peptic ulcer disease, and clinical management requires consideration o multiple pharmacologic options. The available agents can be divided into drugs that (1) decrease acid secretion, (2) neutralize acid, (3) promote mucosal de ense, and (4) modi y risk actors (Fig. 47-4).

Bismuth

Antacids H. pylori

Antibiotics

869

Lume n

Coating agents

Cl−

Proton pump inhibitors

K+

Ca na liculus

Mucus ce ll

Mucus ce ll

+

K

H+/K+ ATP a s e P a rie ta l Tra ns loca tion/ fus ion ce ll Tubulove s icle conta ining ina ctive H+/K+ ATP a s e

Endopla s mic re ticulum

ATP H+

ADP

Ca 2+

P rote in kina s e s ATP cAMP

IP 3

αs GTP

AC

β

γ

H2

H2 blockers His ta mine

β

αq

γ

γ

GTP

DAG

M3

CCKB P LC

ACh ECL ce ll

β

Ga s trin Ne rve

Blood ve s s e l

FIGURE 47-4. Sites of action of drugs used to treat peptic ulcer disease. H2 receptor antagonists (H2 blockers) inhibit activation o the histamine H2 receptor by endogenous histamine. Proton pump inhibitors decrease the activity o the H /K ATPase on the canalicular membrane o the parietal cell. Antacids neutralize acid in the stomach lumen. Coating agents provide a protective layer on the epithelial sur ace o the gastric mucosa. Bismuth and antibiotics act to eradicate H. pylori rom the mucus layer coating the gastric mucosa. H. pylori in ection is an important contributing actor in the pathogenesis o peptic ulcer disease.

870 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

an H2 receptor antagonist other than cimetidine may be preerred when the patient is prescribed multiple medications. Cimetidine crosses the placenta and is secreted into breast milk and is there ore not recommended or use during pregnancy or when nursing. Cimetidine can have antiandrogenic e ects because o its action as an antagonist at the androgen receptor, resulting in gynecomastia (enlarged breasts) and impotence in men and, rarely, galactorrhea (discharge o milk) in women.

NH2

N HN

His ta mine (imida zole ring)

N

H N

S

H N N

HN

Proton Pump Inhibitors

C

Proton pump inhibitors block the parietal cell H /K ATPase

N

Cimetidine (imida zole ring)

O S

N

H N

H N

NO 2

Ranitidine (fura n ring) NH2 O N N H2 N

S

N

O S

NH2

S NH2

Famotidine (thia zole ring)

N N

S

NO 2

H N HN

S

Nizatidine (thia zole ring)

FIGURE 47-5. Histamine H2 receptor antagonists. H2 receptor antagonists

(proton pump). Compared to H2 receptor antagonists, proton pump inhibitors are superior at suppressing acid secretion and promoting peptic ulcer healing. Omeprazole is the prototype proton pump inhibitor. Several other proton pump inhibitors have also been developed, including esomeprazole (the [S]-enantiomer o omeprazole), rabeprazole , lansoprazole , dexlansoprazole (the [R]-enantiomer o lansoprazole), and pantoprazole (Fig. 47-6). All o the proton pump inhibitors are prodrugs that require activation in the acidic environment o the parietal cell canaliculus. Oral ormulations o these drugs are entericcoated to prevent premature activation. The prodrug is converted to its active sulfenamide orm in the acidic canalicular environment, and the sul enamide reacts with a cysteine residue on the H /K ATPase to orm a covalent disulf de bond (Fig. 47-7). Covalent binding o the drug inhibits the activity o the proton pump irreversibly, leading to prolonged and nearly complete suppression o acid secretion. In order or acid secretion to resume, the parietal cell must synthesize new H /K ATPase molecules, a process that requires approximately 18 hours. The six available proton pump inhibitors have similar rates o absorption and oral bioavailability. Rabeprazole and lansoprazole appear to have a signif cantly aster onset o action than omeprazole and pantoprazole. Comparisons o e ectiveness suggest that esomeprazole inhibits acid secretion more e ectively than other proton pump inhibitors at therapeutic doses.

share moieties related to histamine, providing a structural rationale for inhibition of the H2 receptor. For a more detailed description of the structure of these agents, see the legend to Figure 44-5.

Clinical Indications

induce con usion and hallucinations in some patients. These adverse e ects in the central nervous system are uncommon, however, and are typically associated with intravenous administration o the H2 receptor antagonist. Additional adverse e ects specif c to cimetidine, the f rst H2 receptor antagonist to be developed, are discussed below. Several clinically signif cant drug–drug interactions can occur with H2 receptor antagonists. For example, ketoconazole, a drug that requires an acidic medium or gastric absorption, has reduced uptake in the alkaline environment created by H2 receptor antagonists. As a second example, H2 receptor antagonists compete or renal tubular secretion o procainamide and certain other drugs. Cimetidine inhibits many cytochrome P450 enzymes and thus can inter ere with the hepatic metabolism o numerous drugs. For example, cimetidine can decrease the metabolism o lidocaine, phenytoin, quinidine, theophylline, and war arin, acilitating the accumulation o these drugs to toxic levels. Cimetidine appears to inhibit P450 enzymes to a greater extent than the other H2 receptor antagonists, and

Proton pump inhibitors are used to treat H. pylori-associated ulcers and hemorrhagic ulcers and to allow continued use o NSAIDs in a patient with a known peptic ulcer. Proton pump inhibitors are pre erred or the treatment o peptic ulcer disease when there is an accompanying H. pylori in ection because they contribute to eradication o the in ection by inhibiting the growth o H. pylori. Proton pump inhibitors are also e ective in preventing recurrent hemorrhagic ulcers. Clot ormation involves processes that are impaired in acidic environments, and the proound suppression o gastric acid secretion by proton pump inhibitors helps maintain clot integrity in the ulcer bed. For example, an intravenous in usion o omeprazole is able to maintain the intragastric pH above 6.0, thereby supporting platelet aggregation and clot stability (see below). Proton pump inhibitors are superior to H2 receptor antagonists (ranitidine) or healing o NSAID-associated gastric and duodenal ulcers when the patient continues NSAID use, most likely because proton pump inhibitors are better able to sustain a constant increase in gastric pH. Several considerations may avor the use o H2 receptor antagonists over proton pump inhibitors. H2 receptor antagonists

C h a p t e r 4 7 Integrative Inf ammation Pharmacology: Peptic Ulcer Disease

871

O

NH

N

S N

O

O

Omeprazole

Ca na liculus

O

NH

N

S

K+

N

O

P a rie ta l ce ll

pH < 2.0 Omeprazole

O

ATP H+

ADP

Esomeprazole Omeprazole

O N

HN

pH 7.1 (cytopla s m)

Fre e ly cros s e s ce ll me mbra ne

S N

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FIGURE 47-6. Proton pump inhibitors. The proton pump inhibitors are a amily o structurally related prodrugs that are all activated by the mechanism shown in Figure 47-7. Note that esomeprazole is the (S)-enantiomer o omeprazole, which is ormulated as a racemic mixture o (R)- and (S)-enantiomers. Dexlansoprazole (not shown) is the (R)-enantiomer o lansoprazole.

have been in use longer than proton pump inhibitors, and their adverse effects are better studied. This may be an especially important consideration for pregnant women because H2 receptor antagonists (with the exception of cimetidine) have proven to be safe in pregnancy, whereas the safety of proton pump inhibitors in pregnancy is less certain. In addition, H2 receptor antagonists are generally less expensive than proton pump inhibitors. The possibility that proton pump inhibitors may cause gastric carcinoid tumors is sometimes raised as a concern for long-term proton pump inhibitor therapy, although this association has not been observed in humans.

O

Active s ulfe na mide

O

S ulfe na mide -H+/K+ ATP a s e comple x (ina ctive e nzyme )

FIGURE 47-7. Mechanism of action of omeprazole, a proton pump inhibitor. Omeprazole reely enters the cytoplasm o the parietal cell (pH 7.1) in uncharged orm. In the acidic environment o the parietal cell canalicular system (pH 2.0), omeprazole is converted to its active sul enamide orm. The sul enamide reacts with a cysteine residue on the H /K ATPase to orm a covalent disul de bond. Covalent modi cation o the H /K ATPase inhibits the activity o the proton pump and thereby prevents acid secretion.

In Case 1, Tom was given a proton pump inhibitor because he was found to have an associated H. pylori infection. In Case 2, the gastroenterologist recommended a proton pump inhibitor to allow for the concomitant use of an NSAID. Formulations

Four of the six proton pump inhibitors (omeprazole, esomeprazole, lansoprazole, and pantoprazole) are available in intravenous dosage forms. Intravenous formulations of proton pump inhibitors are useful clinically because this delivery route bypasses the harsh acidic environment of the stomach and upper duodenum. Intravenous delivery allows more of the drug to reach its site of action in the parietal cell canaliculus without degradation. For example, esomeprazole has a twofold higher peak concentration and a 66–83% greater area under the plasma concentration curve (AUC) when the dose is delivered intravenously instead of orally. The US Food and Drug Administration (FDA) has approved intravenous formulations of lansoprazole (7-day limit), esomeprazole (10-day limit), and pantoprazole (10-day limit) for treatment

872 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

o erosive esophagitis in patients unable to take oral medications. Intravenous pantoprazole is also approved or treatment o the gastrin-induced hypersecretory state associated with Zollinger-Ellison syndrome. The intravenous ormulation should be reserved or patients who require pro ound acid suppression or who are unable to take oral medications. Patients with erosive esophagitis and patients with compromised gastrointestinal absorption are also candidates or therapy with intravenous proton pump inhibitors. One appropriate indication or an intravenous proton pump inhibitor would be upper gastrointestinal hemorrhage with endoscopic evidence o a visible blood vessel, because gastric acid impairs clot ormation (see above). An oral ormulation should be substituted or the intravenous in usion once the bleeding has stopped. The enteric coating on oral proton pump inhibitors helps prevent drug metabolism in the stomach but is also responsible or slower onset o drug action. One newer oral ormulation o a proton pump inhibitor (omeprazole/sodium bicarbonate) in a powder or capsule orm may be use ul in obtaining aster suppression o stomach acid while providing equivalent duration o action. However, the sodium load or the patient is considerable (300 mg sodium per packet o medication and 460 mg sodium per capsule). The potential adverse e ects o the high sodium load may make this an unacceptable drug or patients with cardiac, renal, or hepatic disease. The powder ormulation, however, may be use ul or patients who are unable to swallow a tablet or capsule. Metabolism and Excretion

The six available proton pump inhibitors have similar rates o metabolism. Five o these drugs are metabolized

by cytochrome P450 enzymes in the liver (specif cally, by CYP2C19 and CYP3A4). Rabeprazole is largely metabolized through a nonenzymatic reduction pathway. Box 47-1 describes the e ect o pharmacogenetic di erences on the P450-mediated metabolism o omeprazole, lansoprazole, esomeprazole, and pantoprazole. A ter metabolism o proton pump inhibitors by the liver, the metabolites are excreted via the kidney. Patients with chronic kidney disease generally do not require adjustment o the standard dose. However, patients with liver ailure should be treated with lower doses o these drugs. Elderly patients do not generally require dose reduction even though plasma clearance is reduced, because the plasma hal -li e is short and accumulation does not typically occur. Elderly patients with concomitant renal and liver dys unction should receive lower doses to avoid an increased risk o adverse e ects. Proton pump inhibitors cross the human placental barrier. A recent meta-analysis o human studies did not indicate an increased rate o mal ormations in children born to women who took proton pump inhibitors during the f rst trimester o pregnancy. Adverse Effects

Proton pump inhibitors are generally well tolerated. Adverse e ects may include headache, nausea, disturbed bowel unction, and abdominal pain. A potential concern is the large increase in plasma gastrin associated with proton pump inhibitor use. Because gastric acid is a physiologic regulator o gastrin secretion by G cells in the gastric antrum, the decreased acid secretion caused by proton pump inhibitor therapy leads to increased gastrin release. The trophic e ects

B O X 4 7 - 1 Me ta b o lis m o f Pro to n Pu m p In h ib ito rs An individual’s response to treatment with a proton pump inhibitor (PPI) may vary rom a marked decrease in acid secretion to little change in acid secretion. The pharmacogenetics o drug metabolism is the major actor responsible or this variation. Omeprazole, lansoprazole, esomeprazole, dexlansoprazole, and pantoprazole are extensively metabolized in the liver to less active or inactive metabolites; o these f ve PPIs, omeprazole is the most extensively metabolized and pantoprazole is the least extensively metabolized. Metabolism o the PPIs involves two cytochrome P450 isoenzymes: CYP2C19 and CYP3A4 (also called P450 2C19 and P450 3A4, 4 respectively). CYP2C19 is responsible or the major metabolism o PPIs, while CYP3A4 unctions as an ancillary metabolic pathway when the main pathway through CYP2C19 is saturated. Studies have shown that individuals have di erent rates o metabolism and clearance o these drugs because o genetic polymorphisms in their CYP2C19 isoenzymes. Two polymorphisms o CYP2C19 (CYP2C19m1 and CYP2C19m2) are associated with decreased enzyme activity. Carriers o two copies o the polymorphisms are “poor metabolizers” o PPIs. Carriers o one copy o the polymorphisms are “intermediate to extensive metabolizers”; their rate o CYP2C19-mediated drug metabolism is reduced but not to the extent o individuals with two copies o the polymorphisms. These polymorphisms exist

most commonly in Asian populations: 20% o some Asian populations are poor metabolizers, whereas only 2–6% o Caucasian populations are poor metabolizers. Compared to the majority o individuals (“extensive metabolizers”) taking the same dose o omeprazole, lansoprazole, esomeprazole, dexlansoprazole, or pantoprazole, “poor metabolizers” exhibit decreased clearance o the PPI, leading to higher plasma concentrations o the drug and greater degrees o acid suppression. Fortunately, the standard recommended doses o PPIs take these di erences into account, and most patients reach a su f cient degree o acid suppression regardless o the variability in metabolism o these drugs. Pharmacogenetic di erences in PPI metabolism can lead to potentially signif cant drug–drug interactions, however. To date, only omeprazole has been ound to interact with other drugs metabolized by CYP2C19. Although clinically signif cant interactions do not generally occur, awareness should be high i patients are taking omeprazole concomitantly with war arin, phenytoin, diazepam, or carbamazepine. In the uture, screening or the presence o CYP2C19 polymorphisms could allow physicians to determine which PPI is most appropriate or each patient and what dosage should most e ectively avor acid suppression while avoiding drug–drug interactions. ■

C h a p t e r 4 7 Integrative Inf ammation Pharmacology: Peptic Ulcer Disease

o gastrin can induce hyperplasia o ECL cells and parietal cells in the gastric mucosa. Although rats treated or long periods with omeprazole developed gastric carcinoid tumors, these tumors have not been observed in humans. Patients with Zollinger-Ellison syndrome usually develop ECL and parietal cell hyperplasia, and some develop carcinoid tumors, but no increase in carcinoid tumors has been ound in Zollinger-Ellison patients taking proton pump inhibitors. Hypergastrinemia can also result in rebound hypersecretion o acid upon discontinuation o the proton pump inhibitor. Several recent studies suggest that proton pump inhibitors may decrease the clinical e f cacy o the antiplatelet agent clopidogrel. One rationale or this potential drug– drug interaction could be that proton pump inhibitors and clopidogrel share a common metabolic pathway mediated by the cytochrome P450 isoenzyme CYP2C19 in the liver: most proton pump inhibitors are metabolized by CYP2C19 (see above), and clopidogrel is converted rom a prodrug to the active drug orm by the same enzyme. The clinical importance o this interaction remains uncertain, however, as observational studies have revealed con icting results, and at least one large clinical trial has ound no signif cant di erence in adverse clinical outcomes (cardiovascular death, myocardial in arction, or stroke) between individuals treated with clopidogrel alone and individuals treated concomitantly with clopidogrel and a proton pump inhibitor. Some studies suggest an increased risk o hip racture in patients who take proton pump inhibitors or an extended period o time. Research on this topic has yielded con icting evidence to date: some studies suggest that proton pump inhibitor therapy may decrease gastric absorption o insoluble calcium by raising gastric pH, but other studies suggest that omeprazole may decrease bone resorption by inhibiting osteoclastic vacuolar H /K ATPase. Use o proton pump inhibitors during hospital admission has been shown to increase the risk or hospital-acquired pneumonia, Clostridium di f cile in ection, and enteric in ections with Salmonella and Escherichia coli. This increased risk may be related to compromise o a normal de ense mechanism (i.e., gastric acid) by the proton pump inhibitor, allowing ingested organisms to escape acid-mediated destruction. Theoretically, anticholinergic agents could be used to antagonize M3 muscarinic ACh receptors on parietal cells and thereby decrease gastric acid secretion. However, anticholinergic agents are not used in the treatment o peptic ulcer disease because they are not as e ective as H2 receptor antagonists or proton pump inhibitors and they have many adverse anticholinergic e ects.

Agents That Neutralize Acid Antacids are used on an as-needed basis or symptomatic

relie o dyspepsia. These agents neutralize hydrochloric acid by reacting with the acid to orm water and salts. The most widely used antacids are mixtures o aluminum hydroxide and magnesium hydroxide . The hydroxide ion reacts with hydrogen ions in the stomach to orm water, while the magnesium and aluminum react with bicarbonate in pancreatic secretions and with phosphates in the diet to orm salts. Common adverse e ects associated with these antacids include diarrhea (magnesium) and constipation (aluminum). When antacids containing aluminum and magnesium are taken together, constipation and diarrhea may be

873

avoided. Antacids containing aluminum can bind phosphate; the resulting hypophosphatemia can cause weakness, malaise, and anorexia. In patients with chronic kidney disease, aluminum-containing antacids have been reported to cause neurotoxicity. Patients with chronic kidney disease should avoid magnesium-containing antacids because they can lead to hypermagnesemia. Sodium bicarbonate reacts rapidly with HCl to orm water, carbon dioxide, and salt. Antacids containing sodium bicarbonate have high amounts o sodium; in patients with hypertension or uid overload, sodium-containing antacids can result in signif cant sodium retention. Calcium carbonate is less soluble than sodium bicarbonate; it reacts with gastric acid to produce calcium chloride and carbon dioxide. Calcium carbonate is use ul not only as an antacid but also as a calcium supplement or prevention o osteoporosis. The high calcium content o this antacid ormulation may cause constipation.

Agents That Promote Mucosal Defense Agents that promote mucosal de ense are used in the symptomatic relie o peptic ulcer disease. These drugs include coating agents and prostaglandins.

Coating Agents

Sucralfate , a complex salt o sucrose sul ate and aluminum

hydroxide, is a coating agent used to alleviate the symptoms o peptic ulcer disease. Sucral ate has little ability to alter gastric pH. Instead, in the acidic environment o the stomach, this complex orms a viscous gel that binds to positively charged proteins and thereby adheres to gastric epithelial cells (including areas o ulceration). The gel protects the luminal sur ace o the stomach rom degradation by acid and pepsin. Because sucral ate is poorly soluble, there is little systemic absorption and no systemic toxicity. Constipation is one o the ew adverse e ects. In addition, sucral ate may bind to drugs such as quinolone antibiotics, phenytoin, and war arin and thereby limit their absorption. Colloidal bismuth is a second coating agent used in peptic ulcer disease. Bismuth salts combine with mucus glycoproteins to orm a barrier that protects an ulcer rom urther damage by acid and pepsin. Bismuth agents may stimulate mucosal bicarbonate and prostaglandin E2 secretion and thereby also protect the mucosa rom acid and pepsin degradation. Colloidal bismuth has been ound to impede the growth o H. pylori and is requently used as part o a multidrug regimen or the eradication o H. pylori-associated peptic ulcers (see below).

Prostaglandins

Prostaglandins can be used in the treatment o peptic ulcer disease (see Chapter 43), specif cally in the treatment o NSAID-induced ulcers. NSAIDs are ulcerogenic because they inhibit prostaglandin synthesis and thereby interrupt the “gastroprotective” unctions o PGE2, which include reduced gastric acid secretion and enhanced bicarbonate secretion, mucus production, and blood ow. Misoprostol is a prostaglandin analogue used to prevent NSAID-induced peptic ulcers. Its most requent adverse e ects are abdominal discom ort and diarrhea. In clinical practice, these adverse e ects o ten inter ere with patient adherence. Misoprostol is contraindicated in women who are (or may be) pregnant because o the possibility o

874 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

generating uterine contractions that could result in abortion (see Chapter 30, Pharmacology o Reproduction).

Agents That Modify Risk Factors Diet, Tobacco, and Alcohol

As in Case 1, diet therapy typically involves recommendations to avoid ca eine-containing products because o their ability to increase acid secretion. Avoidance o alcohol and cigarette smoking is also advised. Excessive alcohol intake is directly toxic to the mucosa and is associated with erosive gastritis and an increased incidence o peptic ulcers. Cigarette smoking is thought to decrease the production o duodenal bicarbonate and diminish mucosal blood ow, leading to a delay in ulcer healing.

Treatment of H. pylori Infection

Elimination o H. pylori can lead to cure o H. pyloriassociated peptic ulcers. Treatment or H. pylori in ection uses broad-spectrum antibiotics, such as amoxicillin or tetracycline combined with metronidazole or clarithromycin, together with bismuth citrate and a proton pump inhibitor or ranitidine. Common regimens involve triple therapy with amoxicillin, clarithromycin, and a proton pump inhibitor, or quadruple therapy with tetracycline, metronidazole, a proton pump inhibitor, and bismuth. H. pylori may develop resistance to antibiotic therapy. Metronidazole resistance has been reported in the United States in patients with H. pylori in ections. Resistance to clarithromycin is less common. Three point mutations in the clarithromycin-binding site on H. pylori 23S rRNA (A2143G, A2142G, and A2142C) appear to be responsible or clarithromycin resistance, and the A2143G mutation has been associated with a very low bacterial eradication rate. Levo oxacin has recently been suggested as a use ul alternative drug (together with amoxicillin) in second-line therapeutic regimens in patients with resistance to clarithromycin. In Case 1, Tom was given clarithromycin rather than metronidazole because the ormer drug is less commonly associated with drug resistance. The adverse e ects o therapy or H. pylori in ection include hypersensitivity reactions to penicillin analogues, nausea, headache, and antibiotic-induced diarrhea caused by superin ection with C. di f cile. These e ects, together with the complicated dosing schedules associated with triple therapy and quadruple therapy, can lead to nonadherence. Resistance to H. pylori is a growing concern, and antibiotic regimens will need to evolve in order to meet the challenge.

CONCLUSION AND FUTURE DIRECTIONS Peptic ulcer disease is responsible or signif cant morbidity and mortality in the United States. Because more than one pathophysiologic mechanism is o ten involved in the disease, multiple pharmacologic agents may be required or its prophylaxis and treatment (Fig. 47-4). Pharmacologic agents active against peptic ulcer disease decrease acid secretion, promote mucosal de ense, and modi y risk actors. Use o intravenous proton

pump inhibitors and screening or cytochrome P450 polymorphisms may allow enhancement and customization o pharmacologic therapy or patients at risk. Improved treatment o H. pylori in ection has the potential to decrease the overall incidence o peptic ulcer disease. COX-2 inhibitors have allen short o expectations because o adverse cardiovascular e ects. It remains to be seen whether new NSAIDs can be developed that do not promote peptic ulcer ormation and have an acceptable cardiovascular e ect prof le. Future directions will ocus on unraveling and understanding the potential adverse e ects that have recently been attributed to proton pump inhibitors. Given the prominence o this class o drugs in current medical practice, the interactions with thienopyridine antiplatelet agents (clopidogrel and prasugrel), the e ects on bone ormation and resorption, the risk o hospital acquired in ections, and the risk o enteric in ections will need to be care ully explored.

Suggested Reading Barletta JF, Sclar DA. Proton pump inhibitors increase the risk or hospitalacquired Clostridium di f cile in ection in critically ill patients. Crit Care 2014;18:714–717. (Case-control study showing that proton pump inhibitors are independent risk actors or the development o Clostridium di f cile in ection in ICU patients.) Cardoso RN, Benjo AM, DiNicolantonio JJ, et al. Incidence o cardiovascular events and gastrointestinal bleeding in patients receiving clopidogrel with and without proton pump inhibitors: an update meta-analysis. Open Heart 2015;2:e000248. (Updated review o potential interaction o proton pump inhibitors and clopidogrel.) Chan FKL, Lau JYW. Treatment o peptic ulcer disease. In: Feldman M, Friedman LS, Brandt LJ, eds. Sleisenger and Fordtran’s gastrointestinal and liver disease. 9th ed. Philadelphia: WB Saunders; 2010:869–886. (Clinical overview o the management o peptic ulcer disease.) De Francesco V, Margiotta M, Zullo A, et al. Clarithromycin-resistant genotypes and eradication o Helicobacter pylori. Ann Intern Med 2006;144: 94–100. (Discusses clarithromycin-resistant genotypes in H. pylori.) Forte JG, Zhu L. Apical recycling o the gastric parietal cell H,K-ATPase. Annu Rev Physiol 2010;72:273–296. (Detailed review o the membrane recycling pathway responsible or translocation o cytoplasmic tubulovesicles and their usion with the apical membrane o gastric parietal cells.) Herzig SJ, Howell MD, Ngo LH, Marcantonio ER. Acid-suppressive medication use and the risk or hospital-acquired pneumonia. JAMA 2009;301:2120–2128. (Epidemiologic data suggesting an association between proton pump inhibitors and development o pneumonia.) Johnson DA, Oldf eld EC. Reported side e ects and complications o longterm proton pump inhibitor use: dissecting the evidence. Clin Gastroenterol Hepatol 2013;11:458–464. (Recent evidence regarding possible adverse e ects o proton pump inhibitors.) Kopic S, Murek M, Geibel JP. Revisiting the parietal cell. Am J Physiol Cell Physiol 2010;298:C1–C10. (Detailed review o parietal cell physiology and ion transport, ocusing on ion transporters in the apical and basolateral membranes.) McColl K. E ect o proton pump inhibitors on vitamins and iron. Am J Gastroenterol 2009;104:S5–S9. (Physiology o proton pump inhibitors and absorption o nutrients.) Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R. Translocation o Helicobacter pylori Cag A into gastric epithelial cells by type IV secretion. Science 2000;25:1487–1500. (Study describing the mechanisms responsible or cagA virulence.) Targownik LE, Leslie WD, Davison KS, et al. The relationship between proton pump inhibitor use and longitudinal change in bone mineral density: a population-based study rom the Canadian Multicentre Osteoporosis Study (CaMos). Am J Gastroenterol 2012;107:1361–1369. (Clinical data on the possible association between proton pump inhibitors and osteoporosis.)

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876 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Nu u c le us

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48

Integrative Inf ammation Pharmacology: Asthma Zile uto n

Gluta th io n e Le uk ko trie n e A4

J o s h u a M . Ga la n t e r a n d S t e p h e n La za ru s Le u ko trie ne C 4

Le u ko trie n e C 4 s yntha ynth ha s e E Epo Epoxide p oxide o xide id INTRODUCTI CT ON N & CASE . . . . . ((ma . .ma . . . .s. t. .ce . . . llls . .s. ,. . . . . . . . . 877–878 lls, yd drol ro la la s eOF A PhHYSIOLOGY AIRW AI R AY SMOOTH MUSCLE TONE e os inophils ) AND IMMUNE FUNCTION N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8777 77 (ne utrophils, utrophils sNC , oth h Mu Muscle Contraction . . . . . . . . 877 777 monocyte mPhysiology onocy yteosAirway ) ay Smoot

Methy M Me eth thyl th hylxa x nthines and Phosphodiesterase r Inhibitors . . . . . 88 8866 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887 Maa Magnesium M Tra ns port Tra porte por.te t e r AntiAnti-In i-IInff ammatory Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8887 Co Corticosteroids Cor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887 Cromolynss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888 C Immune Functio ionn in thhe Air irwa wayy . . . . . . . . . . . . . . . . . . . . . . . . . 87 8799 Le uk kottriene i Pathhway-M Modi diffyin ingg Ag Agen ents nts . . . . . . . . . . . . . . . 888 PATHOPHYSIOLOGY OF ASTHMA A . . . . . . . . . . . . . . . . . . . . . . . . . 879 8 Ext Extra E x t tra r a c ce e l llu l u la l a r Anti-IgE gE Ant ntib ibod odie iess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889 Lroncho e uk u kootri ot o tri rie ie ne n e BDisease Asth As thma ma a s a B constrictive . . . . . . . . . . . . . . . 881 4 p. .a. ce c. .e. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889 Drug Delivery. . . s. .pa Asthma as an Inf ammatory o Disease . . . . . . . . . . . . . . . . . . . . 881 TH2 Cells and the Originn of Asthma . . . . . . . . . . . . . .Le ukotr uk ukotrie otr neeClinical C 4 Management o Asthma . . . . . . . . . . . . . . . . . . . . . . . . 889 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 889 Plasma Cells, IgE, Ma Mast Mas astt C Ce Cells, ells, aandd Leuk Leukotrieness . . . . . . . . 882 CO Leu Eosinophilss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882 S Suggested uggest g tedd R Reading e adi ding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .891 891 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . . . . 882 Bronchodilators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884 -Adrenergic Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884 Anticholinergics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 885

INTRODUCTION Asthma is a chronic disease characterized by inf ammation o the airways and exaggerated airway smooth muscle constriction. The symptoms o asthma include dyspnea and wheezing as well as mucus production and cough, particularly at night. Asthma is both an obstructive lung disease and an inf ammatory disease; the obstructive component is characterized by bronchoconstriction, whereas the inf ammatory component is marked by airway edema, goblet cell hyperplasia, mucus secretion, and in ltration and cytokine release by immune and inf ammatory cells. Although the airway obstruction is generally reversible during acute asthma attacks, over time, the disease may cause airway remodeling and permanent deterioration in pulmonary unction. Medications used to treat asthma act in one o two ways: by relaxing bronchial smooth muscle or by preventing and reducing inf ammation. This chapter approaches asthma as both a bronchoconstrictive and an inf ammatory disease. A ter discussing the physiologic control o bronchial tone and the unction o immune pathways in the airways, the chapter turns to the pathophysiology o asthma. Current therapies are then discussed, including the pharmacology o both bronchodilators and airway anti-inf ammatory agents.

PHYSIOLOGY OF AIRWAY SMOOTH MUSCLE TONE AND IMMUNE FUNCTION Asthma involves dys unction in the pathways that regulate both smooth muscle tone and immune unction in the airways. It is there ore important to review the normal physiology o these systems be ore discussing the pathophysiology o asthma.

Physiology of Airway Smooth Muscle Contraction As discussed in Chapter 9, Principles o Nervous System Physiology and Pharmacology, involuntary responses o smooth muscle are regulated by the autonomic nervous system. In the airways, sympathetic (adrenergic) tone causes bronchodilation and parasympathetic (cholinergic) tone causes bronchoconstriction. Bronchial smooth muscle tone is also regulated by nonadrenergic, noncholinergic (NANC) bers that innervate the respiratory tree. Sympathetic innervation o the lung is concentrated primarily on pulmonary blood vessels and the submucosal glands. There is little direct sympathetic innervation o the bronchial smooth muscle. However, airway smooth muscle 877

878 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

WY is a 51-ye a r-o ld m a n w ith a lo n g s ta n d in g h is to ry o a s th m a a n d a lle rg ie s , f rs t d ia g n o s e d a t th e a g e o 6. His a s th m a h a d b e e n m a n a g e d s u cce s s u lly o r m a n y ye a rs o n a re g im e n o tw ice -dd a ily in h a le d u tica s o n e (a n in h a le d co rtico s te ro id ) a n d a lb u te ro l (a -a d re n e rg ic a g o n is t) a s n e e d e d w h e n e ve r h e d e ve lo p e d s h o rtn e s s o b re a th o r w h e e zin g . Ove r th e p a s t ye a r, Mr. Y h a s n o tice d w o rs e n in g s ym p to m s a n d m o re re q u e n t a s th m a a tta cks . He h a s h a d s h o rtn e s s o b re a th , w h e e zin g , a n d ch e s t tig h tn e s s w h e n ru n n in g to ca tch th e b u s . He h a s a ls o d e ve lo p e d a s ig n if ca n t a m o u n t o co u g h in g , e s p e cia lly a t n ig h t, a n d h a s o u n d h im s e l u s in g a lb u te ro l s e ve ra l tim e s a d a y. On e h a zy, h o t s u m m e r d a y, Mr. Y d e ve lo p s s u b s ta n tia l co u g h in g , w h e e zin g , a n d s h o rtn e s s o b re a th a t re s t. He ta ke s tw o p u s o h is a lb u te ro l in h a le r b u t f n d s th a t it p rovid e s o n ly m in im a l re lie . He ca lls h is d o cto r b u t h a s tro u b le e ve n s p e a kin g in u ll s e n te n ce s . His d o cto r a d vis e s h im to g o to th e e m e rg e n cy d e p a rtm e n t (ED) im m e d ia te ly. On p re s e n ta tio n to th e ED, Mr. Y is im m e d ia te ly g ive n a lb u te ro l via n e b u lize r a n d a la rg e d o s e o in tra ve n o u s m e thylp re d n is o lo n e (a co rtico s te ro id ). Alth o u g h h e is n o w m o re co m o rta b le , h e co n tin u e s to “ e e l tig h t” a n d h e h a s q u ie t b re a th s o u n d s o n e xa m . Fo rtu n a te ly, w ith co n tin u e d a d m in is tra tio n s o n e b u lize d a lb u te ro l a n d a tre a tm e n t w ith in h a le d ip ra tro p iu m (a n a n tich o lin e rg ic a g e n t), Mr. Y b e g in s to e e l b e tte r ove r th e n e xt e w h o u rs . He is h o s p ita lize d o r 2 d a ys a n d s e n t h o m e o n a ta p e rin g d o s e o p re d n is o n e , a n o ra l co rtico s te ro id . At a o llo w -u p a p p o in tm e n t w ith h is p u lm o n o lo g is t, Mr. Y is co n ce rn e d th a t h is a s th m a h a s s ig n if ca n tly w o rs e n e d . Eve n th o u g h th e a cu te e ve n t h a s p a s s e d , h e co n tin u e s to h a ve re q u e n t a s th m a

cells express 2-adrenergic receptors (and, to a lesser extent, 1-adrenergic receptors) that are responsive to circulating catecholamines. 2-Adrenergic receptors are activated by epinephrine , which is secreted by the adrenal medulla and causes bronchodilation. Exogenous epinephrine was one o the rst pharmacotherapies or asthma and was available until recently in some over-the-counter ormulations. Newer, 2-selective adrenergic agonists, such as the albuterol used by Mr. Y, are now considered the rst-line bronchodilators or treatment o acute asthmatic symptoms. The vagus nerve provides parasympathetic innervation to the lungs. Airway smooth muscle cells express muscarinic receptors , especially the excitatory M3 subtype o muscarinic receptor. Parasympathetic postganglionic neurons release acetylcholine, which stimulates these muscarinic receptors and induces bronchoconstriction. Parasympathetic neurons are dominant in maintaining airway smooth muscle tone, and anticholinergic agents can cause bronchorelaxation. These agents are used primarily

s ym p to m s , a n d h is e xa m in a tio n a n d p u lm o n a ry u n ctio n te s ts s u g g e s t th a t h is p u lm o n a ry u n ctio n is s ig n if ca n tly re d u ce d . His p u lm o n o lo g is t d is cu s s e s m e d ica tio n a d h e re n ce a n d th e p ro p e r wa y to u s e in h a le rs , in clu d in g u s e o a s p a ce r w ith th e u tica s o n e in h a le r a n d th e n e e d o r Mr. Y to rin s e h is m o u th a te r its u s e . Mr. Y’s p u lm o n o lo g is t a ls o in cre a s e s th e in te n s ity o h is d ru g re g im e n b y a d d in g s a lm e te ro l, a lo n g -a ctin g -a g o n is t, a s w e ll a s m o n te lu ka s t, a cys te inyl le u ko trie n e re ce p to r a n ta g o n is t. Th re e m o n th s la te r, Mr. Y re p o rts th a t h is b a s e lin e s ym p to m s h a ve im p rove d b u t th a t h e h a s h a d a n in te rva l a s th m a e xa ce rb a tio n re q u irin g tre a tm e n t w ith p re d n is o n e . Be ca u s e h is a s th m a is s till n o t u n d e r a d e q u a te co n tro l a n d h is la b o ra to ry te s ts s h o w a n e le va te d Ig E le ve l, Mr. Y’s p h ys icia n re co m m e n d s th a t h e s ta rt o m a lizu m a b , a n a n ti-Ig E m o n o clo n a l a n tib o d y. Mr. Y n o w re ce ive s tw ice -m o n th ly in je ctio n s o o m a lizu m a b a n d h e h a s h a d o n ly o n e m ild a s th m a e xa ce rb a tio n in th e 6 m o n th s h e h a s b e e n o n th is th e ra p y.

Questions 1 . Why did Mr. Ydevelop asthma? 2 . Why was Mr. Y initially managed with both an inhaled corticosteroid (f uticasone), taken twice daily, and a -adrenergic agonist (albuterol), taken only as needed? 3 . Why was it pre erable to keep Mr. Yon a maintenance regimen o an inhaled corticosteroid (f uticasone) instead o a systemic corticosteroid? Why was it necessary to administer systemic corticosteroids (methylprednisolone intravenously and prednisone orally) to treat his asthma exacerbations? 4 . How does omalizumab, an anti-IgE monoclonal antibody, prevent exacerbations o asthma?

in the treatment o chronic obstructive pulmonary disease (see Box 48-1) but can also be used in acute asthma exacerbations (as was the case with Mr. Y) or when -adrenergic agonists are contraindicated. Airway NANC bers are primarily under parasympathetic control. These bers can be either stimulatory (causing bronchoconstriction) or inhibitory (causing bronchodilation). NANC bers do not release either norepinephrine or acetylcholine but instead release neuropeptides. Bronchoconstricting peptides released by NANC bers include neurokinin A, calcitonin gene-related peptide , substance P, bradykinin, tachykinin, and neuropeptide Y; the bronchodilating peptide vasoactive intestinal polypeptide (VIP) is also released by NANC bers, as is the bronchodilating gasotransmitter nitric oxide (NO). Although no pharmacologic agents have yet been developed to take advantage o the NANC system, nitric oxide is a marker o the intensity o airway inf ammation, and NO measurements have been used to assess the severity o asthma and titrate therapy accordingly.

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

879

B O X 4 8 - 1 Ph a rm a co lo g y o f Ch ro n ic Ob s tru ctive Pu lm o n a ry Dis e a s e Chronic obstructive pulmonary disease (COPD) describes a spectrum o disorders that result in obstructive lung disease. Unlike asthma, COPD is generally not reversible. COPD is caused by an abnormal inf ammatory response to an inhaled environmental insult. In 90% o cases, this insult to the lungs is tobacco smoke. Clinically, COPD is divided into two requently overlapping diseases: emphysema and chronic bronchitis. Pulmonary emphysema re ers to alveolar enlargement caused by destruction o alveolar walls and loss o pulmonary elastic recoil, whereas chronic bronchitis is a clinical diagnosis made on the basis o a chronic cough or 3 or more months during 2 consecutive years that cannot be attributed to another cause. As noted above, COPD is caused by an abnormal response to inhalation o tobacco smoke or other toxic agents. In contrast to asthma, where CD4 T lymphocytes, B lymphocytes, mast cells, and eosinophils are the primary inf ammatory cells, the inf ammatory response to tobacco smoke is primarily neutrophilic and monocytic. Tobacco smoke stimulates resident alveolar macrophages to produce chemokines that attract neutrophils. These neutrophils and resident macrophages release proteinases, particularly matrix metalloproteinases. The proteinases degrade elastin, which provides elastic recoil to the alveoli, as well as other proteins that compose the matrix supporting the lung parenchyma. Cell death ollows, due to impaired attachment o alveolar cells to the degraded matrix and to the toxic actions o inf ammatory cells and the environmental insult. The result is that alveoli degrade and coalesce, orming the characteristic enlargement o air spaces typical o emphysema. There is also enhanced mucus production and brosis, although the mechanisms underlying these pathologic phenomena have not been well characterized. Although it is tempting to think that inf ammation in COPD could be held in check by inhaled corticosteroids, steroids are un ortunately o limited bene t in this disease. The lack o steroid

Immune Function in the Airway As described in Chapter 42, Principles o Inf ammation and the Immune System, T lymphocytes play a key role in controlling the immune response. T lymphocytes are classi ed as CD8 TC (cytotoxic) cells , which are mediators o cellular adaptive immunity, and CD4 TH (helper) cells , which regulate adaptive immune responses. TH cells are subclassi ed as TH1 and TH2 cells based on the cytokines they produce. TH1 cells, which produce predominantly interferon- , IL-2, and TNF- , avor a cellular immune response involving T lymphocytes. TH2 cells, on the other hand, produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 and avor a humoral immune response involving antibody production by B cells. Because cytokines produced by activated TH1 and TH2 cells are mutually inhibitory, any given immune stimulus elicits predominantly one or the other response (Fig. 48-1). All individuals continually inhale environmental aeroallergens such as pollens, cat dander, dust mites, and a host o

e cacy likely results rom the act that the inf ammatory cells responsible or COPD are macrophages and neutrophils, which are less responsive than lymphocytes and eosinophils to the actions o corticosteroids. Moreover, the activity o histone deacetylase is impaired in COPD, so the inhibition o proinf ammatory transcription actors is limited. A number o studies have examined the e ects o inhaled corticosteroids on lung unction in COPD, but none have ound a statistically signi cant bene t. However, inhaled corticosteroids have been ound to reduce the requency and severity o acute exacerbations o COPD. There ore, while corticosteroids are not routinely recommended or the treatment o COPD, they may be indicated in patients who develop requent, severe exacerbations. Because cysteinyl leukotrienes, mast cells, and IgE have no role in the pathophysiology o COPD, speci c treatments or asthma that target these pathways are not use ul in COPD. Interestingly, although leukotriene B4 (LTB4) is a potent chemotactic actor or neutrophils, clinical studies o LTB4 antagonism have not shown a bene t to date. Bronchodilators produce only a modest improvement in airf ow in patients with COPD. However, even a small improvement in airf ow can signi cantly improve symptoms in patients with COPD, especially in those whose lungs have become hyperinf ated. Asthma is punctuated by acute attacks, while most patients with COPD have chronic breathlessness that is worsened with exertion. There ore, short-acting “reliever” medications are less bene cial than long-acting drugs in COPD. Both -adrenergic agonists and inhaled anticholinergic agents cause bronchodilation in COPD. However, many patients with COPD have concomitant coronary artery disease, so anticholinergic agents may be pre erred in this subset o patients. There is evidence that the bronchodilatory e ects o -agonists and anticholinergic agents (and theophylline) are additive; there ore, patients with severe COPD may bene t rom combination therapies such as ormoterol and tiotropium. ■

other antigens. These allergens are phagocytosed by antigenpresenting cells lining the airways. The antigens are recognized as oreign by TH cells and generate a low-level TH1 response mediated primarily by inter eron- , as well as a low-level IgG antibody response. However, in asthma, an exaggerated TH2 response o ten predominates, generating airway inf ammation and bronchial hyperresponsiveness (Fig. 48-1).

PATHOPHYSIOLOGY OF ASTHMA Asthma is a complex disease characterized by airway inlammation, airway smooth muscle hyperresponsiveness, and symptomatic bronchoconstriction. Because the most prominent clinical eature o asthma is bronchoconstriction, a simplistic approach to understanding the disease ocuses on airway smooth muscle contraction. At its most undamental level, however, asthma is an in lammatory

880 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY Alle rge n

Airway Airwa y e pithe lium

Goble t ce ll

Na ïve T-ce ll Antige n-pre s e nting ce ll

CD4 IL-12

IL-4 Activa te d TH2 lymphocyte

CD4 IL-10

TH2 cytokine s (IL-4, IL-5, IL-6, IL-9, IL-10, IL-13)

E i hil Eos inophil (MBP, ECP, le ukotrie ne s , cytokine s )

Activa te d TH1 lymphocyte

CD4

IFN-γ

Ma s t ce ll (his ta mine , le ukotrie ne s , cytokine s )

P la l s ma ce ll (IgE)

TH1 cytokine s (IFN-γ, IL-2, TNF-α)

Infla mma tory ce ll inde pe nde nt me cha nis ms (IL-13)

Low-le ve l IgG re s pons e (phys iologic re s pons e )

M h ge Ma cropha Low le ve l TH1 re s pons e (phys iologic re s pons e )

Blood ve s s e ls

Goble t ce ll hype rpla s ia Airwa y e de ma

S ubmucos a l gla nd Epithe lium

S ube pithe lia l fibros is Ca rtila ge

S mooth mus cle hype rpla s ia a nd/or hype rtrophy

S mooth mus cle Asthma

Normal airway (no asthma)

FIGURE 48-1. Origins of the asthmatic immune response. In nonatopic individuals, antigens derived rom allergens are presented by antigen-presenting dendritic cells to engender a low-level, physiologic TH1 response. This response does not cause airway inf ammation or bronchoconstriction (right side). Inter eron- , produced by activated TH1 lymphocytes, inhibits a TH2 response. In individuals susceptible to asthma, allergen-derived antigens that are presented to immature CD4 T cells cause these cells to di erentiate into activated TH2 lymphocytes. The TH2 lymphocytes release cytokines that recruit other inf ammatory cells, including eosinophils, mast cells, and IgE-producing B cells. Together, these cells produce an inf ammatory response in the airway. Activated TH2 cells also induce an asthmatic response directly, in part through release o IL-13. The net result—airway hyperresponsiveness, mucus production by goblet cells, airway edema, subepithelial brosis, and bronchoconstriction—constitutes the asthmatic response (left side).

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

disease o the airways, and treatment o the underlying in lammation is crucial to maintain normal airway unction. There ore, as detailed below, the treatment o asthma employs both bronchodilators and anti-in lammatory agents.

Asthma as a Bronchoconstrictive Disease

As thma tic re s pons e (hype rre s pons ive ne s s )

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Hype rs e ns itivity Norma l re s pons e

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The propensity or the airways o asthmatic patients to constrict in response to a wide variety o stimuli, including allergens, environmental irritants, exercise, cold air, and in ections, is termed hyperresponsiveness . Two eatures o airway hyperresponsiveness separate the response to stimuli in asthmatic patients rom the nonasthmatic response: hypersensitivity and hyperreactivity. Hypersensitivity describes a normal response at abnormally low levels o stimuli (i.e., the airways o asthmatic patients constrict to stimuli that do not elicit a response in healthy individuals). Hyperreactivity describes an exaggerated response at normal levels o stimuli (i.e., the airways o asthmatic patients respond too vigorously). In Figure 48-2, hypersensitivity describes a shi t o the stimulus–response curve to the le t, while hyperreactivity describes an upward shi t. The overall response to stimuli in asthmatic patients represents the combination o hypersensitivity and hyperreactivity. The causes o airway hyperresponsiveness in asthma have not been completely elucidated. The hyperreactive response may be explained by alterations in airway smooth muscle mass due to the increase in size (hypertrophy) and number (hyperplasia) o myocytes that occurs in response to inf ammation (Fig. 48-1). The hypersensitivity response is due to alterations in smooth muscle excitation–contraction coupling. Possible mechanisms o altered coupling in asthma include greater responsiveness o intracellular calcium release channels, increased calcium sensitization, and changes in the expression o ion channels, receptors, and second messengers.

S timulus (e .g., me tha choline ) FIGURE 48-2. Airway hyperresponsiveness in asthma. Nonasthmatics have a low-level response to a stimulus that produces mild smooth muscle contraction at high exposures (normal response). An asthmatic patient has airways that mani est exaggerated smooth muscle contraction (bronchoconstriction) at low doses o stimulus (hyperresponsiveness). The two components o hyperresponsiveness are hypersensitivity (a normal response at abnormally low doses o stimulus) and hyperreactivity (an exaggerated response at normal doses o stimulus).

881

Asthma as an Inf ammatory Disease Although the primary symptoms (wheezing and shortness o breath) o most asthmatic patients are due to bronchoconstriction, the underlying cause o asthma is an allergic inf ammation o the airways. The inf ammatory process is visible histologically as airway edema, goblet cell hyperplasia, subepithelial brosis, mucus hypersecretion, and in ltration by a variety o inf ammatory cells, including TH2 lymphocytes, antigen-presenting cells, plasma cells, mast cells, neutrophils, and eosinophils (Fig. 48-1). Airway inf ammation can lead to a chronic cough in asthmatic individuals, even in those who do not develop symptomatic bronchoconstriction (this diagnosis is known as cough variant asthma ). Many inf ammatory mediators and cytokines govern the interplay among the various immune cells. Antiinf ammatory medications, particularly corticosteroids, are mainstays in the pharmacologic treatment o asthma. As the complex pathophysiology o asthma has been urther elucidated, more targeted therapies that block speci c inf ammatory pathways are being developed.

TH2 Cells and the Origin of Asthma

Although the exact causes o asthma are not ully understood, one theory suggests that asthma, like other allergic diseases, is the result o an immune imbalance avoring TH2 lymphocytes over TH1 lymphocytes. TH2 lymphocytes contribute to asthma through three mechanisms. First, in patients with a hereditary predisposition to atopy ( rom the Greek, meaning “out o place”), an allergen can trigger a type I hypersensitivity response. In normal (nonatopic) individuals, an allergen is phagocytosed by antigenpresenting cells, stimulating a low-level TH1 response and the production o appropriate amounts o IgG antibodies directed against the allergen. In atopic individuals, however, the same allergen induces a strong TH2 response mediated through the release o IL-4, which induces B cells to produce exaggerated amounts o IgE antibodies directed against the allergen (Fig. 48-1). The IgE antibodies bind to high-a nity IgE receptors on mast cells, and subsequent cross-linking o the IgE receptors upon reexposure to the allergen causes mast cell degranulation and triggers an allergic reaction (Fig. 48-2, and see below). Second, TH2 cells can directly induce a type IV hypersensitivity reaction through the production o IL-13 (and, to a lesser degree, IL-4). In the airway, IL-13 causes goblet cell hyperplasia, increased mucus production, and smooth muscle hyperplasia and/or hypertrophy. IL-13 also stimulates B cells to produce IgE (Fig. 48-1). Third, TH2 lymphocytes recruit eosinophils by producing IL-5 as well as GM-CSF and IL-4. These cytokines (especially IL-5) induce eosinophil proli eration and release rom the bone marrow and promote eosinophil survival in the circulation and tissues. As in many patients with asthma, Mr. Y had a high level o circulating eosinophils and elevated levels o serum IgE. What causes the imbalance between TH1 and TH2 lymphocytes in patients with asthma? Although the exact reasons remain to be ully elucidated, they likely involve environmental e ects on genetically susceptible individuals. Epidemiologic studies have ound that exposures to tuberculosis and viruses such as measles and hepatitis A are protective against the development o asthma. Having older siblings and/or encountering other children through attendance at a day care acility (both o which are associated with

882 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

increased exposure to in ectious agents) are also associated with a decreased incidence o asthma. Living in a rural environment (where there is substantial contact with bacterial endotoxins) is also protective. One leading theory suggests that a “Western li estyle,” including decreased exposure early in li e to microbes that engender TH1-lymphocyte responses, contributes to the development o asthma and other allergic diseases in susceptible individuals. Although this “hygiene hypothesis” is probably too simplistic to explain the origins o a complex disease such as asthma, it orms a use ul model or thinking about the disease and is a possible explanation or the dramatic rise in the incidence o asthma in the Western hemisphere. It is impossible to know exactly what caused Mr. Y’s asthma; however, the act that he had allergic rhinitis and elevated levels o IgE suggests that he had an atopic predisposition triggered by environmental allergens.

Plasma Cells, IgE, Mast Cells, and Leukotrienes

As noted above, an IgE-mediated type I hypersensitivity response is one mechanism by which allergens cause the pathologic and clinical mani estations o asthma (Fig. 48-3). The allergic response is initiated when a dendritic cell phagocytoses an inhaled allergen. The dendritic cell presents the processed allergen to TH2 cells and activates them. The activated TH2 cells bind to and activate B lymphocytes via CD40 on the B-cell sur ace. Activated TH2 cells also generate IL-4 and IL-13, which induce B-cell trans ormation into IgE-producing plasma cells. IgE circulates brief y in the bloodstream be ore binding to high-a f nity IgE receptors (Fc RI) on mast cells. Upon reexposure, the allergen binds to and cross-links the IgE–Fc RI complexes, thereby activating the mast cell. The activated mast cell degranulates, releasing its pre ormed inf ammatory mediators. These molecules include histamine , proteolytic enzymes, and certain cytokines (such as platelet-activating actor). The activated mast cell also releases arachidonic acid rom its plasma membrane and produces leukotrienes and prostaglandin D2 (Fig. 48-4). Acutely, mast cell degranulation produces bronchoconstriction and airway inf ammation. Histamine released by the mast cells promotes capillary leakage, leading to airway edema. Mast cells also release leukotriene C4 (LTC4), which is subsequently converted into LTD4 and LTE4 (see Chapter 43, Pharmacology o Eicosanoids). These three leukotrienes, called cysteinyl leukotrienes , are central to the pathophysiology o asthma because they induce marked bronchoconstriction. Leukotriene D4 is 1,000 times more potent than histamine in producing bronchoconstriction. Leukotrienes also cause mucus hypersecretion, capillary leakage, and vasogenic edema and recruit additional inf ammatory cells. The e ect o the leukotrienes, though slower in onset, is more power ul and sustained than that o the pre ormed mediators. Because o their delayed yet potent inf ammatory e ect, leukotrienes were once called slow-reacting substance o anaphylaxis (SRS-A) be ore their actual structures were identi ed. Mast cells recruit other inf ammatory cells via the release o cytokines. This produces a delayed reaction that develops 4 to 6 hours a ter exposure to allergen (Fig. 48-3). Mast cells also release tryptase , a protease that activates receptors on epithelial and endothelial cells, inducing the expression o adhesion molecules that attract eosinophils

and basophils. Tryptase is also a smooth muscle mitogen, causing hyperplasia o airway smooth muscle cells and contributing to airway hyperresponsiveness. The production o IL-1, IL-2, IL-3, IL-4, IL-5, GM-CSF, inter eron- , and TNF- by mast cells contributes to chronic inf ammation and the chronic asthmatic reaction. Finally, mast cells release proteases and proteoglycans that act on supporting airway structures to produce chronic changes in the airway (also called airway remodeling). Unlike the reversible component o bronchoconstriction that characterizes the acute asthmatic reaction, airway remodeling induced by chronic inf ammation may cause irreversible impairment in pulmonary unction.

Eosinophils

The major physiologic role o eosinophils is to de end against parasitic in ections. Eosinophils originate in the bone marrow and are stimulated by IL-4, IL-5, and GM-CSF produced by TH2 lymphocytes and mast cells. Eosinophils migrate rom the bloodstream to the airway by binding to speci c adhesion molecules, particularly VCAM-1, and by traveling along chemokine gradients to sites o inf ammation. Once recruited to the airway, eosinophils have a complex, multi unctional role in asthma. Activated eosinophils secrete cytotoxic granules that cause local tissue damage and induce airway remodeling, lipid mediators and neuromodulators that a ect airway tone, and cytokines and chemokines that recruit other inf ammatory cells. The toxic granules o eosinophils contain a number o cationic proteins—including major basic protein (MBP), eosinophilic cationic protein (ECP), eosinophil peroxidase , and eosinophil-derived neurotoxin—that are directly damaging to the bronchial epithelium. For example, ECP can breach the integrity o target cell membranes by orming ion-selective, voltage-insensitive pores, and eosinophil peroxidase catalyzes the production o highly reactive oxygen species that oxidize target cell proteins and induce apoptosis. Eosinophils also produce matrix metalloproteinases that contribute to airway remodeling. Eosinophils contribute both directly and indirectly to airway hyperresponsiveness. MBP and ECP a ect smooth muscle tone and induce hyperresponsiveness by increasing intracellular calcium concentrations and inhibiting the inhibitory M2 subtype o muscarinic receptors, thereby increasing vagal tone. Eosinophil-derived cysteinyl leukotrienes and neuropeptides (such as substance P) increase vasodilation, vascular permeability, mucus hypersecretion, and airway smooth muscle contraction. Finally, eosinophils are immunomodulatory cells that can ampli y the immune response in asthma. Eosinophils up-regulate endothelial adhesion molecules and thereby recruit other inf ammatory cells. Eosinophils are also antigenpresenting cells capable o urther activating T lymphocytes.

PHARMACOLOGIC CLASSES AND AGENTS The pharmacologic agents used to treat asthma are divided into two broad categories: relievers and controllers (also called preventers ). This distinction emphasizes the clinical uses o these agents and helps patients understand and adhere to the prescribed regimen. This classi ication scheme also relates to the mechanisms o action o drugs

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

883

Airway Airwa y e pithe lium

Alle rge n MHC cla s s II mole cule

TH2

T-ce ll re ce ptor

Ne urotrophins

IgE cros s -linke d by a lle rge n

Antige npre s e nting ce ll

FcεRI-bound IgE Ma s t ce ll

Omalizumab IgE

His ta mine -re le a s ing fa ctor, ne urope ptide s , IL-9

IL-4, IL-13

IL-4 IL-5

IL-5

IL-4

Ne uron

P la s ma ce ll

ECP

Ma s t ce ll

His ta mine , le ukotrie ne s , pla te le t-a ctiva ting fa ctor

Eos iinophil hil IL-5

Ne urope ptide s

His ta mine , le ukotrie ne s , cytokine s

MBP, ECP, le ukotrie ne s , cytokine s

Chronic a s thma tic re a ction

Acute a s thma tic re a ction

Bronchocons triction

Bronchocons triction

Va s oge nic e de ma

Airwa y e de ma

Mucus hype rs e cre tion

Mucus production

Chronic infla mma tion Airwa y re mode ling

FIGURE 48-3. The allergic response in asthma. Asthma produces acute and chronic inf ammatory responses in the airways. Antigen-presenting cells phagocytose and process allergens, presenting the antigens to CD4 T cells. These cells di erentiate into cytokine-producing TH2 lymphocytes. The activated TH2 cells release IL-4, IL-13, and IL-5, which recruit B cells and eosinophils. The B cells di erentiate into IgE-producing plasma cells. The IgE binds to Fc RI receptors on mast cells and antigen-presenting cells. Upon reexposure to the allergen, the IgE-bound Fc RI is cross-linked, inducing the mast cell to degranulate and release pre ormed and newly generated inf ammatory mediators including histamine, cysteinyl leukotrienes, platelet-activating actor, and other cytokines. These cytokines cause acute airway inf ammation and produce acute asthmatic symptoms (an asthma “attack” or exacerbation). Chronically, activated TH2 cells and mast cells produce circulating IL-5 that recruits eosinophils, and TH2 cells release products that stimulate local mast cells and neurons. Together, the inf ammatory mediators and catabolic enzymes produced by eosinophils, mast cells, and neurons cause chronic airway inf ammation and lead to airway remodeling. Omalizumab is a humanized monoclonal antibody directed against the Fc RI-binding domain o IgE. By preventing IgE rom binding to the IgE receptor (Fc RI) on mast cells, omalizumab inhibits mast cell degranulation upon reexposure to allergen and thereby modulates the acute asthmatic reaction. Omalizumab also down-regulates Fc RI on antigen-presenting cells, thereby diminishing antigen processing and presentation to CD4 lymphocytes. Because ewer immature T cells are induced by allergen to di erentiate into TH2 lymphocytes, the chronic asthmatic reaction is also blunted.

for asthma. In general, bronchodilators, which alleviate smooth muscle bronchoconstriction, are used as relievers, and anti-inflammatory medications, which decrease airway inflammation, are used as controllers. There is also evidence that some medications—methylxanthines ,

for example—have both bronchodilatory and antiinflammatory effects. At the start of the introductory case, Mr. Y used fluticasone (an inhaled corticosteroid) as a controller, with albuterol (a short-acting 2-agonist) as a reliever.

884 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY Nucle us

results in bronchoconstriction. Because sympathomimetics cause rapid relaxation o airway smooth muscle, 2-adrenergic agonists are particularly e ective in relieving acute asthma symptoms.

Cytos ol P ros ta gla ndins

Aspirin

Cyclooxygenase 5-Lipoxygenase activating protein (FLAP)

PLA2

Ara chidonic a cid

2-Adrenergic

5-Lipoxygenase Zileuton

Gluta thione Le ukotrie ne A4

Le ukotrie ne C 4 Epoxide hydrolase (ne utrophils , monocyte s )

Leukotriene C4 synthase (ma s t ce lls , e os inophils )

Tra ns porte r

Extra ce llula r s pa ce

Le ukotrie ne B4 Le ukotrie ne C 4 Le ukotrie ne D4

Le ukotrie ne E 4 BLT1

Cys LT1 Montelukast Zafirlukast

Leukocyte Che mota xis

-Adrenergic Agonists

Airway S mooth mus cle contra ction Eos inophil migra tion Airwa y e de ma

FIGURE 48-4. The leukotriene pathway in asthma. Leukotrienes are some o the most potent bronchoconstrictors known and are important mediators o inf ammation in the airway. Drugs that inhibit leukotriene production or leukotriene receptor binding have a role in asthma therapy. Leukotrienes are ormed when arachidonic acid is released rom the inner leaf et o the plasma membrane by the action o phospholipase A2 (PLA2). Arachidonic acid is converted to leukotriene A4 by the action o 5-lipoxygenase. 5-Lipoxygenase is activated by the membrane-bound enzyme 5-lipoxygenase activating protein (FLAP). Leukotriene A4 is converted to leukotriene C4 by the action o leukotriene C4 synthase in mast cells and eosinophils, and leukotriene C4 is transported out o the cell. Leukotriene C4 is converted to leukotriene D4 and then to leukotriene E4; all three o these cysteinyl leukotrienes bind to CysLT1 receptors expressed on airway smooth muscle cells, leading to bronchoconstriction and airway edema. Leukotriene A4 is converted to leukotriene B4 by epoxide hydrolase in neutrophils and monocytes. Leukotriene B4 is transported out o the cell and binds to BLT1 receptors expressed on leukocytes, leading to leukocyte chemotaxis and recruitment. The leukotriene pathway can be inhibited by the 5-lipoxygenase inhibitor zileuton or by the CysLT1 receptor antagonists montelukast and za rlukast.

Bronchodilators Bronchodilators a ect airway smooth muscle tone by acting on autonomic nervous system receptors and signaling pathways. Sympathetic activation (mediated primarily by 2-adrenergic receptors) results in bronchodilation, while parasympathetic stimulation (mediated by muscarinic acetylcholine receptors)

stimulation relaxes airway smooth muscle and leads to bronchodilation. Both systemic and aerosolized agents that stimulate 2-adrenergic receptors are e ective therapies or asthma. One early treatment or asthma involved the subcutaneous administration o adrenaline (epinephrine ). By the middle o the twentieth century, epinephrine was made into an inhaled ormulation that is still available today. The nonselective adrenergic agonist ephedrine (Ma-Huang) has been used as a remedy or asthma or centuries by practitioners o traditional Chinese medicine. Epinephrine is a nonselective adrenergic agonist that binds to -, 1-, and 2-adrenergic receptors (see Chapter 11, Adrenergic Pharmacology). Although it is an e ective bronchodilator, epinephrine also causes cardiac stimulation via 1-receptors, leading to tachycardia, palpitations, and potentially arrhythmias, and peripheral vasoconstriction via -receptors, leading to hypertension. Isoproterenol is structurally related to epinephrine. While epinephrine stimulates both - and -adrenergic receptors, isoproterenol stimulates only -adrenergic receptors. Isoproterenol stimulates both 1- and 2-receptors and there ore causes both bronchodilation and cardiac stimulation, but because it does not stimulate -receptors, it does not cause peripheral vasoconstriction. Isoproterenol is not used requently in current practice because o the availability o agents that are more selective or 2-receptors and the potential adverse cardiac e ects at higher doses o isoproterenol. Indeed, an epidemic o asthma-associated deaths in Great Britain in the mid-1960s was attributable to the use o highdose isoproterenol inhalers, possibly due to the combined e ect o hypoxemia rom asthma and cardiac stimulation rom isoproterenol. The f rst agents to o er relative 2 selectivity were isoetharine and metaproterenol, although both drugs had moderate 1 e ects. The newer drugs terbutaline , albuterol (also re erred to as salbutamol), pirbuterol, and bitolterol bind to 2-adrenergic receptors 200–400 times more strongly than to 1-receptors and cause signif cantly milder cardiac e ects than the less selective adrenergic agonists. Albuterol was the f rst o the strongly 2-selective agents to be available in inhaled orm, urther reducing systemic e ects. Modern inhaled 2-selective agonists were the f rst drugs to allow regular treatment o asthma with an acceptable adverse e ect prof le. Nonetheless, at high doses, especially i taken orally, even these drugs can cause cardiac stimulation and tachycardia. In addition, since 2-adrenergic receptors are expressed in peripheral skeletal muscle, activation o these receptors by 2-selective agents can result in a tremor. Albuterol is a racemic mixture o two stereoisomers: R-albuterol (or levalbuterol) and S-albuterol. Levalbuterol, which is available as a pure enantiomer, has tighter binding to 2-receptors and is more 2-selective. In contrast, the S isomer induces airway hyperresponsiveness in animal models, although in clinical practice, this e ect has not been signif cant. Although racemic albuterol and levalbuterol produce similar response and adverse e ect prof les or most

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

885

β2 Agonist β2 -Adre ne rgic re ce ptor

FIGURE 48-5. Mechanism of

and theophylline in asthma. In airway smooth muscle cells, activation o protein kinase A by cAMP leads to phosphorylation o a number o intracellular proteins and thus to smooth muscle relaxation and bronchodilation. Any therapy that increases the level o intracellular cAMP is expected to lead to bronchodilation. In practice, this is accomplished in one o two ways: by increasing the production o cAMP or by inhibiting the breakdown o cAMP. cAMP production is stimulated by 2-agonist-mediated activation o 2-adrenergic receptors, which are G protein-coupled receptors. cAMP breakdown is inhibited by theophylline-mediated inhibition o phosphodiesterase.

Ade nylyl cycla s e

2-agonists

patients, a subset o patients may be more sensitive to the 1 e ects o S-albuterol and may experience decreased tachycardia and palpitations when taking levalbuterol. -Adrenergic receptors are coupled to the stimulatory G protein Gs (see Chapter 11). The subunit o Gs activates adenylyl cyclase, which catalyzes the production o cyclic adenosine monophosphate (cAMP). In the lung, cAMP causes a decrease in the intracellular calcium concentration and, via activation o protein kinase A, inactivates myosin light chain kinase and activates myosin light chain phosphorylase (Fig. 48-5). In addition, the 2-agonists open large-conductance calcium-activated potassium channels (KCa) and thereby hyperpolarize airway smooth muscle cells. The combination o decreased intracellular calcium, increased membrane potassium conductance, and decreased myosin light chain phosphorylation leads to smooth muscle relaxation and bronchodilation. There is signi cant variability in clinical response among patients using 2-agonists. Some o this variability may be mediated through variants in the gene or the 2adrenergic receptor. Researchers studying the e ect o single nucleotide polymorphisms (SNPs) in the gene have ound a common genetic variant that is associated with increased susceptibility to nocturnal asthma. Subjects homozygous or this genetic variant who receive regularly scheduled albuterol doses develop a decline in their peak expiratory f ow rate (a measure o bronchoconstriction), while subjects without the polymorphism develop increased peak f ow rates with scheduled albuterol use. Although the pharmacogenetics o the 2-adrenergic receptor are complicated and have yielded inconsistent associations, it is likely that some o the variability in drug response results rom genetic inf uences. Most 2-adrenergic agonists have a rapid onset o action (15 to 30 minutes), a peak e ect at 30 to 60 minutes, and a duration o action o approximately 4 to 6 hours. This time course o drug action makes the 2-agonists good candidates or use as asthma relievers (or rescue inhalers) during acute attacks. However, this pro le also makes the 2-agonists poor candidates or control o nocturnal asthma and or prevention o attacks, although they can be used prophylactically be ore exposure to a known trigger such as exercise. Several newer agents, formoterol (and its enantiomerically pure orm arformoterol, approved only

β

γ

αs

Phos p hod ie s te ra s e

GTP ATP

cAMP

AMP Theophylline

PKA Bronchodila tion

or treatment o COPD), salmeterol, vilanterol, indacaterol, and olodaterol (the latter three approved only or treatment o COPD), are known as long-acting beta-agonists (LABAs). The LABAs were engineered with lipophilic side chains that resist degradation. As such, these agents have a 12- to 24-hour duration o action, making them good candidates or prevention o bronchoconstriction. Although ormoterol and salmeterol are reasonable asthma controllers, these agents do not treat the underlying inf ammation. In act, regular use o ormoterol or salmeterol may be associated with an increase in asthma deaths. The exact mechanism or this observation is unknown, although it may occur because long-acting -agonists can improve the chronic symptoms o asthma without a ecting the underlying risk o a severe asthma exacerbation. Because patients may eel better on long-acting -agonists, they may receive lower doses o inhaled corticosteroids or no inhaled corticosteroids at all. Since inhaled corticosteroids reduce the risk o asthma exacerbation (see below), the reduction or withdrawal o inhaled corticosteroids may place patients at increased risk o asthma hospitalization and atal asthma attack. For this reason, a US Food and Drug Administration (FDA) advisory committee has recommended that ormoterol and salmeterol should be used only in combination with an inhaled corticosteroid. Because salmeterol has a slower onset o action than albuterol, it should not be used or acute asthma f ares. Formoterol does have a rapid onset o action and can be used as a rescue inhaler, although it is not yet approved or this indication in the United States. One strategy has been to combine ormoterol with an inhaled corticosteroid (budesonide) or use as needed in patients with mild asthma. Every time the patient uses this combination, the ormoterol is available to provide acute relie o symptoms, and the patient also receives a dose o the inhaled corticosteroid to help quell the underlying inf ammation.

Anticholinergics

Anticholinergic agents were the rst medications used to treat asthma in Western medicine. As early as 1896, Stedman’s Twentieth Century Practice of Modern Medical Science suggested that asthma attacks could be treated by smoking “asthma cigarettes” containing stramonium extracted rom the plant Datura stramonium. The active ingredients

886 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

in stramonium were anticholinergic belladonna alkaloids. To this day, asthma exacerbations that are not responsive to inhaled 2-adrenergic agonists, or where inhaled -agonists are contraindicated (such as in patients with cardiac ischemia or arrhythmia), can be treated with inhaled ipratropium bromide . Ipratropium bromide is a quaternary ammonium salt derived rom atropine . Because inhaled atropine is highly absorbed across the respiratory epithelium, it causes many systemic anticholinergic e ects, including tachycardia, nausea, dry mouth, constipation, and urinary retention. Unlike atropine, ipratropium is not signi cantly absorbed, and these adverse systemic e ects are minimized. Nonetheless, inhaled ipratropium can cause dry mouth and gastrointestinal upset through its deposition in the mouth and inadvertent oral absorption, and i nebulized ipratropium is inadvertently delivered to the eye, it can produce mydriasis (pupillary dilation) and increase intraocular pressure, resulting in angle-closure glaucoma. Tiotropium (as well as the newer agents umeclidinium and aclidinium) is a long-acting anticholinergic agent that is used in the treatment o chronic obstructive pulmonary disease (COPD; Box 48-1). Like ipratropium, these long-acting anticholinergic agents are quaternary ammonium salts that produce ew systemic e ects because they are not systemically absorbed upon inhalation. Moreover, aclidinium is hydrolyzed rapidly in plasma, urther reducing systematic exposure. Antimuscarinic agents are competitive antagonists at muscarinic acetylcholine receptors. O the our muscarinic receptor subtypes expressed in the lung (M1, M2, M3, and M4), the excitatory M3 receptor is the most important in mediating smooth muscle contraction and mucus gland secretion in the airway. Ipratropium and the long-acting anticholinergic agents antagonize the e ect o endogenous acetylcholine at M3 receptors, leading to bronchorelaxation and decreased mucus secretion. Tiotropium, umeclidinium, and aclidinium have a long duration o action, which enables once-daily dosing, largely because o their slow dissociation rom M3 receptors. Ipratropium and the long-acting antimuscarinic agents are used mainly to treat COPD, where the major reversible bronchoconstrictive component is mediated by cholinergic neural tone. In chronic asthma, cholinergic stimulation has only a secondary role in causing bronchoconstriction, although increased vagal stimulation at night may be an important contributor to nighttime symptoms. No anticholinergic agent is approved by the FDA or asthma, but studies have suggested therapeutic uses or ipratropium in the treatment o acute asthma exacerbations and as rescue therapy in the subset o patients who cannot tolerate -adrenergic agonists or or whom therapy with sympathomimetics is contraindicated due to ischemic heart disease or tachyarrhythmia.

Methylxanthines and Phosphodiesterase Inhibitors

Two methylxanthines, theophylline and aminophylline , are occasionally used in asthma treatment. The mechanism o action o these drugs is complex, but their primary bronchodilatory e ect appears to be due to nonspeci c inhibition o phosphodiesterase isoenzymes. Inhibition o phosphodiesterase types III and IV prevents cAMP degradation in airway smooth muscle cells, leading to smooth muscle relaxation by

the cellular and molecular mechanisms detailed previously (i.e., decreased intracellular calcium, increased membrane potassium conductance, and decreased myosin light chain phosphorylation). As shown in Figure 48-5, the bronchodilatory e ect o methylxanthines results rom perturbation o the same pathway that is initiated by 2-agonists, although methylxanthines act downstream o 2-adrenergic receptor stimulation. Methylxanthines also inhibit phosphodiesterase (PDE) isoenzymes in inf ammatory cells. Inhibition o phosphodiesterase type IV in T lymphocytes and eosinophils has an immunomodulatory and anti-inf ammatory e ect. By this mechanism, theophylline can control chronic asthma more e ectively than would be expected on the basis o its bronchodilatory e ect alone. Some o the adverse e ects o methylxanthines, including cardiac arrhythmias, nausea, and vomiting, are also mediated by phosphodiesterase inhibition, although the responsible isoenzymes remain to be elucidated. Theophylline is a structural relative o ca eine , di ering only by a single methyl group, and both ca eine and theophylline are adenosine receptor antagonists. Adenosine receptors are expressed on airway smooth muscle cells and mast cells, and antagonism o these receptors could play a role in preventing both bronchoconstriction and inf ammation. In act, ca eine (as co ee) has been used to treat asthma. However, experiments with speci c adenosine receptor antagonists that do not inhibit phosphodiesterase have shown little bronchodilation, suggesting that phosphodiesterase inhibition is the primary mechanism o action o methylxanthines in asthma. Nonetheless, adenosine receptor antagonism is responsible or many secondary e ects o theophylline, including increased ventilation during hypoxia, improved endurance o diaphragmatic muscles, and decreased adenosine-stimulated mediator release rom mast cells. In addition, some adverse e ects o theophylline, such as tachycardia, psychomotor agitation, gastric acid secretion, and diuresis, are mediated through adenosine receptor antagonism. Because methylxanthines are nonselective and have multiple mechanisms o action, they cause multiple adverse e ects and have a relatively narrow therapeutic index. Moreover, there is signi cant variation in the metabolism o theophylline by the P450 isoenzyme CYP3A, and theophylline use is susceptible to drug–drug interactions with CYP3A inhibitors such as cimetidine and the azole anti ungals. At supratherapeutic levels, theophylline produces nausea, diarrhea, vomiting, headache, irritability, and insomnia. At even higher doses, seizures, toxic encephalopathy, hyperthermia, brain damage, hyperglycemia, hypokalemia, hypotension, cardiac arrhythmias, and death can occur. For this reason, the role o theophylline in the treatment o chronic asthma has diminished. Theophylline is still used occasionally with routine monitoring o plasma drug levels when -adrenergic agonists and corticosteroids are ine ective or contraindicated. A phosphodiesterase type IV inhibitor, rof umilast, has recently been approved or use in severe COPD (see Box 48-1), where it is associated with a small improvement in pulmonary unction and a reduced likelihood o symptom exacerbation. Un ortunately, as with theophylline, o target inhibition o PDE IV in the brain results in nausea, vomiting, and weight loss. Research is now ocused on the

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

development o PDE IV inhibitors with better inhaled ormulations and ewer adverse e ects.

Magnesium

Magnesium ions inhibit calcium transport into smooth muscle cells and can inter ere with intracellular phosphorylation reactions that induce smooth muscle contraction. For this reason, magnesium sul ate is commonly used as a tocolytic to inhibit uterine contraction and delay preterm labor. Magnesium has similar e ects on airway smooth muscle and has been used experimentally in acute asthma exacerbations. Although the results o clinical studies have been variable, two meta-analyses have suggested a bene t to using magnesium sul ate in patients with severe asthma exacerbations presenting to the emergency department. Magnesium was not used in the introductory case, but it would have been a reasonable therapeutic option at the time o Mr. Y’s visit to the emergency department.

Anti-Inf ammatory Agents As detailed above, allergic inf ammation o the airways orms the pathophysiologic basis or asthma. To control persistent asthma and prevent exacerbations o acute asthma, treatment o all but the mildest orms o the disease should generally include anti-inf ammatory agents. Corticosteroids have long been mainstays o asthma treatment, although the pro ound adverse e ects o systemically administered corticosteroids remained problematic until the development o inhaled ormulations. Three additional classes o drugs with anti-inf ammatory mechanisms o action are also used or the treatment o asthma: cromolyns, leukotriene pathway modi ers, and a humanized monoclonal anti-IgE antibody.

Corticosteroids

Inhaled corticosteroids are the chie preventive treatment or the vast majority o patients with asthma. Because inhaled corticosteroids produce higher local drug concentrations in the airway than an equivalent dose o systemically administered corticosteroids, a lower overall dose can be administered, reducing the likelihood o signi cant systemic e ects. Corticosteroids bind to the intracellular glucocorticoid receptor. The steroid–receptor complex translocates to the nucleus, where it binds to glucocorticoid response elements (GREs) in DNA, altering the transcription o dozens o genes. In general, corticosteroids increase the transcription o genes coding or the 2-adrenergic receptor and a number o anti-inf ammatory proteins such as IL-10, IL-12, and IL-1 receptor antagonist (IL-1Ra). Corticosteroids decrease the transcription o genes coding or many proinf ammatory (and other) proteins; examples include IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, IL-13, IL-15, TNF- , GM-CSF, SCF, endothelial adhesion molecules, chemokines, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX), phospholipase A2, endothelin-1, and NK1-2 receptor. As described above, IL-4 is important in inducing B-cell production o IgE, while IL-5 is an important recruiter o eosinophils (Fig. 48-3). There ore, inhibition o IL-4 and IL-5 markedly reduces the inf ammatory response in asthma. Moreover, corticosteroids induce apoptosis in a number o inf ammatory cells, particularly eosinophils and TH2 lymphocytes.

887

Corticosteroids do not directly a ect mast cells, probably because most mast cell mediators are pre ormed; however, mast cells are indirectly inhibited over time as the overall inf ammatory response is muted. Corticosteroids reduce the number o inf ammatory cells in the airways and decrease the damage to airway epithelium. Vascular permeability is also reduced, leading to resolution o airway edema. In addition, although steroids do not directly a ect the contractile unction o airway smooth muscle, over time, the reduced inf ammation leads to a reduction in airway hyperresponsiveness. The net result is that corticosteroids reverse many o the eatures o asthma. Un ortunately, steroids merely suppress the inf ammatory cascade and do not cure asthma, so they must be taken chronically. In addition, steroids cannot reverse airway remodeling caused by long-standing, poorly controlled asthma. Nonetheless, because the e ects o these agents are so ar-reaching, inhaled corticosteroids constitute the most important drug class or most patients with asthma. Most systemic e ects can be mitigated, i not eliminated, by delivering corticosteroids directly to the airway (i.e., by inhalation). Although all corticosteroids are active in asthma when given systemically, substitution at the 17 position increases topical absorption and allows such drugs to be active when given by inhalation (see Fig. 29-7). The currently available inhaled steroids include beclomethasone , triamcinolone , f uticasone , budesonide , f unisolide , mometasone , and ciclesonide . Even though only 10–20% o the administered dose is delivered to the airways by inhalation (the rest is deposited in the oropharynx and swallowed, unless the mouth is rinsed a ter using the inhaler), a much higher airway concentration o drug is produced than would occur with a similar dose administered systemically. Compared to systemic dosing, inhaled delivery allows a 100- old decrease in the dose required to achieve a similar anti-inf ammatory e ect. In addition, the newer steroids (all but beclomethasone and triamcinolone) are subject to rst-pass metabolism in the liver, such that much o the inadvertently swallowed dose does not reach the systemic circulation. Ciclesonide, the most recently approved inhaled corticosteroid, is an ester prodrug that is converted to its active compound, desisobutyrylciclesonide, by carboxyesterases and cholinesterases expressed in the upper and lower airway epithelium, thereby urther limiting local oropharyngeal and systemic adverse e ects. The combination o lower dose and rst-pass metabolism in the liver limits the incidence o adverse e ects o inhaled corticosteroids. At su ciently high doses, however, enough drug is absorbed through the gastrointestinal tract and pulmonary epithelium to cause systemic e ects with prolonged use, including osteopenia or osteoporosis in adults and delayed growth in children. In addition, inhaled steroids can cause local adverse e ects, such as oropharyngeal candidiasis rom deposition into the oropharynx and hoarseness due to deposition into the larynx. These e ects can be prevented by using a large-volume spacer to capture large droplets o steroid that would be deposited in the oropharynx and by rinsing the mouth a ter use. Sometimes, however, inhaled corticosteroids are inadequate and systemic corticosteroids such as prednisone must be used as either a short “burst” or acute exacerbations or as long-term therapy when asthma cannot be controlled with other medication. For example, systemic steroids were

888 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

necessary to control Mr. Y’s symptoms during and a ter his asthma exacerbation. Systemic corticosteroids have a more widespread anti-inf ammatory e ect than inhaled corticosteroids. However, they also have a much more substantial adverse e ect pro le, as discussed in Chapter 29, Pharmacology o the Adrenal Cortex. For this reason, the use o systemic corticosteroids is typically limited to asthmatic patients with severe acute or chronic disease that cannot be otherwise controlled.

Cromolyns

Roger Altounyan was a physician with a predictable asthmatic response to guinea pig dander. In the 1960s, Dr. Altounyan tested a series o synthetic compounds based on a traditional Egyptian olk remedy or their ability to decrease his response to guinea pig dander extracts. These tests resulted in his discovery o a novel class o compounds, o which two—cromolyn (also known as disodium cromoglycate ) and nedocromil—have since entered clinical practice. Studies showed that cromolyn inhibits the immediate allergic response to an antigen challenge but does not relieve an allergic response once it has been initiated. Further studies ound that cromolyn decreases the activity o mast cells, preventing release o their inf ammatory mediators upon antigen challenge. For this reason, cromolyn is commonly viewed as a “mast cell stabilizing agent.” This view is somewhat simplistic, however, as cromolyn also inhibits the release o mediators rom eosinophils, neutrophils, monocytes, macrophages, and lymphocytes. The underlying molecular mechanism o action has not been ully elucidated but may involve inhibition o chloride transport, which in turn a ects calcium gating and prevents mediator release rom intracellular granules. Because it prevents the acute allergic response in susceptible patients, cromolyn has ound a role as a prophylactic therapy in patients with allergic asthma associated with speci c triggers. It has also been use ul in patients with exercise-induced asthma, as it can be taken immediately prior to exercise. Clinical experience has shown that cromolyn is more e ective in children and young adults than in older patients. Cromolyn has a better sa ety pro le than any other asthma medication, largely due to its low systemic absorption. Cromolyn is administered by inhalation; less than 10% o the drug that reaches the lower airway is systemically absorbed, and less than 1% o the drug that reaches the gastrointestinal tract is absorbed. Un ortunately, cromolyn’s clinical utility is limited because it is less e ective than inhaled corticosteroids, particularly in cases o moderate and severe asthma, and it must be taken our times daily.

Leukotriene Pathway-Modifying Agents

The central role o leukotrienes in the pathogenesis o asthma suggests that inhibiting steps in the leukotriene pathway could serve as a treatment or the disease. 5-Lipoxygenase catalyzes the conversion o arachidonic acid to leukotriene A4. Inhibition o 5-lipoxygenase by zileuton reduces the biosynthesis o LTA4 and its active derivatives, the cysteinyl leukotrienes (Fig. 48-4). Downstream, montelukast and zaf rlukast inhibit binding o LTC4, LTD4, and LTE4 to the cysteinyl leukotriene receptor (CysLT1) (Fig. 48-4). Finally, inhibition o the protein that activates

5-lipoxygenase (5-lipoxygenase activating protein, or FLAP) is being actively explored, although no currently approved agents work by this mechanism. The leukotriene pathway inhibitors have two major clinical e ects. In patients with moderate or severe asthma who have pulmonary unction impairment at baseline, zileuton, montelukast, and za rlukast produce an immediate, albeit small, improvement in lung unction. This e ect is likely due to antagonism o the abnormally constricted bronchial tone that results rom cysteinyl leukotriene stimulation o CysLT1 receptors at baseline. With chronic administration, the leukotriene-modi ying agents reduce the requency o exacerbations and improve control o asthma—as evidenced by ewer symptoms and less requent use o inhaled -agonists—even in patients who have mild asthma and only episodic symptoms. Nonetheless, compared to the e ect o inhaled corticosteroids, the e ect o leukotriene pathway modi ers on lung unction and symptom control is limited. Because the leukotriene pathway is just one o several processes responsible or the inf ammatory response in asthma, it is not surprising that leukotriene pathway modi ers are less e ective than inhaled corticosteroids, which a ect multiple inf ammatory pathways and there ore have broader anti-inf ammatory e ects. Leukotriene-modi ying agents are particularly use ul or treating the e ects o aspirin-exacerbated respiratory disease (or aspirin-sensitive asthma). This condition is thought to result rom the stimulation o the leukotriene pathway that results when synthesis o prostaglandin E2 (PGE2), which down-regulates the 5-lipoxygenase pathway, is reduced. Aspirin and other NSAIDs inhibit the cyclooxygenase pathway and decrease the synthesis o prostaglandins, including PGE2. Patients with aspirin-sensitive asthma have an exaggerated leukotriene response to aspirin, and inhibition o the leukotriene pathway by leukotriene-modi ying agents is an e ective treatment. Unlike many drugs used in the treatment o asthma, the leukotriene-modi ying agents are all available as oral tablets rather than inhaled ormulations. Although inhaled ormulations generally decrease adverse e ects by delivering the drug to the target organ directly, there are several advantages o the orally administered leukotrienes. First, many patients, particularly children, nd it easier to take a tablet than use an inhaler, so adherence is requently better. Second, because inhalers are o ten used improperly, there is a higher likelihood that the intended dose o a tablet is delivered. Finally, because orally delivered drugs are absorbed systemically, the drugs can be used to treat other coexisting allergic diseases, such as allergic rhinitis, that are also responsive to leukotriene pathway inhibition. All three leukotriene-modi ying agents are well tolerated and have ew extrapulmonary e ects, particularly compared to oral corticosteroids. Zileuton has a 4% incidence o hepatotoxicity, so periodic liver unction testing is required. The leukotriene receptor antagonists are considered generally sa e but have been associated with Churg-Strauss syndrome on rare occasions. Churg-Strauss syndrome is a serious granulomatous vasculitis a ecting the small arteries and veins o the lungs, heart, kidneys, pancreas, spleen, and skin. Because Churg-Strauss syndrome is independently associated with asthma and eosinophilia, it is not clear whether the reported reactions represent a distinct

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

e ect o the drug or an unmasking o the preexisting syndrome due to the reduction in corticosteroid use allowed by the addition o a leukotriene receptor antagonist to the therapeutic regimen.

Anti-IgE Antibodies

The prominence o IgE-mediated allergic responses in asthma suggests that inactivation or removal o IgE antibodies rom the circulation would mitigate the acute response to an inhaled allergen. Omalizumab is a humanized mouse monoclonal antibody that binds to the high-a nity IgE-receptor (Fc RI) binding domain on human IgE. Omalizumab both decreases the quantity o circulating IgE and blocks the remaining IgE rom binding to mast cell Fc RI (Fig. 48-3). Because omalizumab does not cross-link Fc RI-bound IgE, the drug does not typically induce anaphylaxis. Furthermore, omalizumab a ects both the early- and late-phase asthmatic responses to challenge by an inhaled allergen, since mast cells, basophils, and dendritic cells down-regulate the Fc RI receptor in response to the lower levels o circulating IgE. Receptor down-regulation reduces stimulation o TH2 lymphocytes and decreases the late-phase asthmatic response beyond the decrease that could be expected rom removal o the circulating IgE alone. These mechanisms decrease the requency o asthma exacerbations in patients treated with omalizumab. Because it is an antibody, omalizumab must be administered subcutaneously every 2–4 weeks. Although its high cost and the inconvenience o parenteral administration have limited the use o omalizumab to severe cases o asthma, the drug also reduces the dose o corticosteroids needed or disease control and decreases the requency o exacerbations in moderate asthma (as in the case o Mr. Y). Despite the act that omalizumab is a humanized antibody in which 95% o the original mouse amino acid sequence has been replaced by the corresponding human sequence, the drug is recognized as an antigen and triggers an immune response on rare occasions, so patients must be monitored closely or several hours a ter administration.

Drug Delivery Many adverse e ects o drugs used to treat asthma, especially the corticosteroids and -agonists, can be minimized by delivery o the drug directly to the airway. There are three principal delivery systems or inhaled drugs: metered-dose inhalers , dry powder inhalers , and nebulizers . In a metereddose inhaler, a compressed gas propels a xed dose o drug out o the device upon activation o the canister. In the past, a chlorof uorocarbon (CFC) such as Freon® was used as the propellant. However, because o the environmental e ects o CFCs on the ozone layer, these gases have been replaced by hydrof uoroalkane (HFA) propellants. Although the canisters are easy to use, they do require coordination between inhalation and actuation o the device and requently require a 10-second breath hold, making them potentially challenging to use or young children and the elderly. This is not the case or dry powder inhalers, where the act o inspiration creates turbulent f ow within the device that aerosolizes and scatters a dry powder. Some patients nd dry powder inhalers easier to use than metered-dose inhalers, but others nd the powder irritating or nd that they cannot generate a su cient inspiratory orce to activate the device. Nebulizers

889

pass a compressed gas such as compressed air or oxygen through a liquid ormulation o the medication to convert it into a mist that is then inhaled. Although nebulizers are not as portable as the other delivery devices, they can be used in a hospital or home setting or treatment o acute asthmatic exacerbations and are easier to use or the delivery o inhaled medication. For example, in ants who cannot use metereddose inhalers can be treated via nebulizer. Nebulizers are also use ul in cases o acute exacerbation, where breathlessness can limit the patient’s ability to per orm a long breath hold.

Clinical Management of Asthma Treatment o asthma should be based on the severity o disease. Current guidelines rom the National Institutes o Health state that patients should use the smallest dose o medication needed or adequate control o symptoms. As a practical matter, this means adjusting the dose o medication to achieve adequate control and then reducing it to the lowest e ective dose. A stepwise care approach has been advocated to acilitate the ambulatory treatment o asthma. This approach separates asthma into two domains: (1) impairment, a measure o ongoing asthma symptoms, and (2) risk, a measure o the requency and severity o exacerbations. Patients are classi ed into one o our clinical categories based on their impairment and risk (Table 48-1). For example, patients with mild intermittent asthma have the ollowing characteristics: no chronic impairment in lung unction, symptoms occurring no more than twice a week and nocturnal awakenings due to asthma no more than twice a month, in requent use o their rescue medication, and zero or one asthma exacerbations requiring systemic corticosteroids a year. Such patients can o ten be satis actorily managed with inhaled -agonists as needed or relie o symptoms or be ore exposure to known asthma triggers, and they require little or no ongoing controller medication. Patients with more requent or severe symptoms, or with impairment in lung unction, should be treated with regular preventive therapy, such as inhaled corticosteroids at escalating doses depending on severity o symptoms. Other medications, such as long-acting -agonists or leukotriene-modi ying agents, may be added to improve control. Combination agents that include an inhaled corticosteroid and a long-acting inhaled -agonist (such as the f uticasone/salmeterol ormulation ultimately given to Mr. Y) can improve adherence by reducing the number o inhalers needed. As in Mr. Y’s case, asthma management also involves avoiding environmental exposures known to provoke airway inf ammation. For example, eliminating environmental tobacco smoke reduces symptoms and the requency o asthma attacks in children whose parents or caregivers are cigarette smokers, and allergen reduction is an important component o patient education to maintain control o asthma symptoms.

CONCLUSION AND FUTURE DIRECTIONS The increasing incidence o asthma entails a signi cant burden o disability, economic cost, and death. Nonetheless, biomedical research has uncovered key eatures o

890 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

TABLE 48-1 Clinical Management of Asthma SEVERITY OF ASTHMA

CLINICAL CHARACTERISTICS

SHORT-TERM RELIEF

LONG-TERM CONTROL

Mild intermittent (Step 1)

Symptoms 2 times/week Nocturnal awakenings 2 times/month Exacerbations brie Lung unction normal between exacerbations Limited peak f ow variability

Short-acting -agonist as needed or symptoms or prior to expected exposures

No medications necessary

Mild persistent (Step 2)

Symptoms 2 times/week Nocturnal awakenings 2 times/month Exacerbations brie and may a ect activity Lung unction normal when asymptomatic Peak f ow decreased 20–30% when symptomatic

Short-acting -agonist as needed or symptoms

Pre erred: inhaled low-dose corticosteroid Alternative: leukotriene pathway modi er, mast cell stabilizer, or theophylline

Moderate persistent (Step 3)

Daily symptoms Nocturnal awakenings 1 time/week Frequent exacerbations lasting days, a ecting activity Lung unction 60–80% o predicted Peak f ow variability 30%

Short-acting -agonist as needed or symptoms

Pre erred: low- to medium-dose inhaled steroid and long-acting inhaled -agonist Alternatives: Medium-dose inhaled steroid alone; or low- to medium-dose inhaled steroid plus sustained-release theophylline; or low- to medium-dose inhaled steroid plus leukotriene pathway modi er

Severe persistent (Step 4)

Continual symptoms Limited activity Frequent nocturnal awakenings Frequent, severe exacerbations Lung unction 60% o predicted Peak f ow variability 30%

Short-acting -agonist as needed or symptoms

Pre erred: high-dose inhaled corticosteroid and long-acting inhaled -agonist Oral corticosteroids i needed Addition o more controllers has not been studied adequately

asthma pathophysiology that can be exploited or pharmacologic management o the disease. At its core, asthma is a disease caused by an aberrant inf ammatory response in the airways that leads to airway hyperresponsiveness and bronchoconstriction. There is no cure or asthma, but a therapeutic approach that treats both aspects o asthma, by using anti-inf ammatory medications and bronchodilators, along with the avoidance o known triggers, can be success ul in achieving long-term clinical control and enabling success ul management o the disease in most patients. As our understanding o the pathophysiology o asthma has improved, new targets or therapeutic intervention have become available. In general, research has ocused on three areas: improving existing therapies by altering the ratio o bene t to adverse e ect, devising new targeted therapies, and attempting to prevent or reverse permanent airway remodeling in long-standing asthma. One example o the rst approach is the development o novel inhaled corticosteroids with reduced systemic e ects, such as selective glucocorticoid receptor modulators that retain anti-inf ammatory activity while minimizing adverse e ects. Recent research on new targeted therapies has involved urther characterization o asthma on a molecular and phenotypic basis. Using both gene expression data and clinical characteristics, researchers have ound a substantial amount o heterogeneity in the disease, with subsets o populations having di erent immunologic eatures. For example, while many asthmatic patients display a TH2 response as described

above in “Pathophysiology o Asthma,” others show a much less active TH2 response. This has opened the possibility o using biomarkers to identi y the aberrant pathways in individual patients and targeting therapy or each patient that is most likely to be bene cial. Inhibitors o inf ammatory cytokines are under development as potential therapeutics to prevent airway remodeling in asthma. One example is the anti-IL-5 monoclonal antibody mepolizumab, which reduces the number o circulating and airway eosinophils in patients with asthma and appears to reduce the requency o asthma exacerbations in a rare subgroup o patients with prednisone-dependent asthma and sputum eosinophilia. However, mepolizumab has shown no e cacy in the general population o patients with asthma, suggesting that reducing eosinophils alone may not signi cantly a ect the disease in the majority o patients. A second example is pitrakinra, a variant o IL-4 that blocks binding o IL-4 and IL-13 to the IL-4 receptor alpha. This drug has shown promise in early-stage clinical studies. Lebrikizumab, an anti-IL-13 antibody, has also shown promise in early-stage clinical trials, particularly in patients with elevated levels o the biomarker periostin, which appears to signal an aberrant TH2 pathway (Fig. 48-6). Finally, TNF- (see Chapter 46, Pharmacology o Immunosuppression) is a cytokine that is up-regulated in asthma and that recruits neutrophils and eosinophils to the airways. The TNF pathway inhibitors etanercept (a recombinant usion protein that inhibits TNF- ) and inf iximab (an anti-TNF-

C h a p t e r 4 8 Integrative Inf ammation Pharmacology: Asthma

891

IL-17-s pe cific Ab: Secukinumab

CD4 IL-17A IL-17F Alle rge n Ne utrophil

TH17 ce ll

IL-13-s pe cific Abs : Lebrikizumab Tralokinumab Anrukinzumab

IL-4 va ria nt: Pitrakinra De ndritic ce ll (a ntige n-pre s e nting ce ll)

CD4

IgE-s pe cific Ab: Omalizumab

CD4 IL-13

IL-4

TH 0 ce ll (na ive T-ce ll)

P la s ma ce ll

TH 2 ce ll

IL-5

IL-5-s pe cific Abs : Mepolizumab Reslizumab Benralizumab

IL-9-s pe cific Ab: MEDI-528

CD4 IL-9

Eos inophil

TH 9 ce ll

Ma s t ce ll

FIGURE 48-6. Targeted therapy for asthma cytokines. Much current research in asthma ocuses on developing drugs that inhibit cytokines mediating the pathogenesis o asthma. These include variants o IL-4 such as pitrakinra; anti-IL-13 antibodies such as lebrikizumab, tralokinumab, and anrukinzumab; and anti-IL-5 antibodies such as mepolizumab, reslizumab, and benralizumab. Omalizumab, an approved monoclonal antibody, targets IgE be ore it can cross-link Fc receptors on mast cells.

monoclonal antibody) have shown promising results in early clinical studies.

Suggested Reading Barnes PJ. The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest 2008;118:3546–3556. (Reviews the role o cytokines in the chronic asthmatic reaction and suggests targets or new drug development.) Fanta CH. Asthma. N Engl J Med 2009;360:1002–1014. (Discusses the clinical management o asthma, ocusing on commonly prescribed therapeutics.) Guidelines or the diagnosis and management o asthma (EPR-3). http:// www.nhlbi.nih.gov/health-pro/guidelines/current/asthma-guidelines. (This is the most recent set o practice guidelines or the diagnosis and treatment o asthma rom the expert panel convened by the National Heart, Lung, and Blood Institute o the National Institutes o Health.)

Locksley RM. Asthma and allergic inf ammation. Cell 2010;140:777–783. (Reviews the dysregulated interactions between airway epithelia and innate immune cells that initiate and maintain asthma.) Pelaia G, Vatrella A, Maselli R. The potential o biologics or the treatment o asthma. Nat Rev Drug Discov 2012;11:958–972. (Describes approaches to individualized therapy or asthma and e orts to develop drugs targeting speci c inf ammatory pathways.) Rhen T, Cidlowski JA. Antiinf ammatory action o glucocorticoids— new mechanisms or old drugs. N Engl J Med 2005;353:1711–1723. (Discusses the molecular mechanisms by which glucocorticoids act and e orts to develop novel glucocorticoids with improved adverse e ect pro les.) Vestbo J, Hurd SS, Agustí AG, et al. Global strategy or the diagnosis, management, and prevention o chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2013;187:347–365. (Describes approaches or the diagnosis and treatment o chronic obstructive pulmonary disease.)

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894 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

MS S U c rys taa ls l

m nos odi mo diium u ura raa te te (MS U)

Monoo cy Mo cyte te

P ha h g oc ocyt ytos ytos tos o s is 2 of M of MS SU NALP NALP P -33 3 inffl in fla m fla mma ma s o me a cti tivva tio tion

49

propr o IL-1β β

Integrative Inf ammation Pharmacology: Gout

Ca s pa s e

IL-1β

4 IL-1 IL β re lee a s e

8 Cytokine re le a s e (e .g., g , ILLEh 1 βrin ) J . Arm s t ro n g a n d Lloyd B. Klicks t e in IL-1 re ce p tto r INTRODUCTION NTRODUCTION NTRO O &C CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 895–8 889 95 95– 5 89 8896 96 96 Manag Ma M Man Mana aana n ge ge me m nt of Chronic Gout: Agents That Lower Plasma 5PURINE Ura U raaate Concent ntra rati tion o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899 PHYS PH YSIO IOL OLOGY GY Y OF F PU NE M METAB METABOL ET OLIISM . . . . . . . . . . . . . . . . . . . 8895 955 Ag nts That Decrease Uriic Ac Agen Acid id Syn y thesiss . . . . . . . . . . . . 8999 PATHOPHYSIOLOGY PH PHY HYSIOLO YSIOL L LOG OF GOUT UT T . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 8 Agents That Increase Uric Acid Excretionn . . . . . . . . . . . . . 900 PHARMACOLOGIC P PH HA ARMACOL M COL OLO LO LOGIC L OG OGIC C CL CLASSES C ASSES AND A AG AN AGENTS . . . . . . . . . . . . .7. . .Ne 898 N utrophil u l Agents Ag That Enhanc ncee Ur Uric ic Acid cid Metabolism m . . . . . . . . . . . 900 Management Ma M a na ggeeem me nntt of of A Acute Ac c uttee G Goout: ut: Supp ut S ressors of Leukocyte koc re cruitme tme ntLUSI aSION nd d AND CONC U ON N FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 901 Recruitment R e c ru ruiittm me nt aan and nd A Activation Ac c tivvaaatttiion . . . . . . . . . . . . . . . . . . . . . . . . . .a89 898 8 c cumu ccu cc m la tion mu mula Nons N No onns nste s te tero erroi oiid idal ddaall A Anti-Inf ntii Inf Inf aam amm mmatory mm ma m y Drugs (NSAIDs) NSA ) . . . . . . 898 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .901 withi w with h in joint joiinntt Colchicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 898 S igna i na n a l tra tra ns dduction u cti tion Glucocorticoids . . . . . .6. . .P roinfla mma tor y. . . . . 899

a nd ge g ne a ctiva ctiva ttion ion

INTRODUCTION

m dia tor re le a s e me ( IL 8)

Gout is a uniquely human disease. Most mammals possess uricase, an enzyme that metabolizes purine breakdown products into a reely water-soluble substance, allantoin. Humans, in contrast, excrete most purines as sparingly soluble uric acid. High plasma levels o uric acid can lead to deposition o uric acid crystals in joints, most requently the f rst metatarsophalangeal joint (great toe). Acute attacks o gout cause intense pain but typically occur in requently. A number o rational therapies exist or the treatment o gout. These therapies are broadly divided into two groups: those that treat acute gout attacks and those that prevent recurrent attacks. Drugs that suppress the immune response to crystal deposition or limit the extent o in ammation may be used or both indications, although they are more commonly used to treat acute attacks. Agents that reduce uric acid synthesis or increase the renal excretion o uric acid prevent monosodium urate crystal ormation and are use ul or prevention o recurrent attacks. These pharmacologic interventions provide e ective therapy or most cases o gout.

PHYSIOLOGY OF PURINE METABOLISM Gout is caused by imbalances in purine metabolism. To understand the cause and treatment o gout, it is necessary to recall the principles o nucleotide biochemistry. Although pyrimidines such as cytosine, thymidine, and uracil are straight orward or the body to metabolize and excrete, it is a challenge to metabolize purines (most notably the

nucleotides guanine and adenine). The intermediates o purine metabolism are toxic to some cells, necessitating tight regulation o purine synthesis and degradation. Furthermore, the f nal breakdown product o purine metabolism is uric acid, which is barely soluble in blood or urine. Increased plasma levels o uric acid are the strongest risk actor or gout, although, or poorly understood reasons, not everyone with high plasma uric acid levels develops gout. Purines are synthesized via two general pathways: de novo synthesis and the salvage pathway (Fig. 49-1). The f rst step in the de novo pathway is the reaction o phosphoribosyl pyrophosphate (PRPP, a ribose sugar with two pyrophosphates attached) with glutamine. PRPP provides the ribose sugar as one precursor or the nascent nucleotide. Hydrolysis o the pyrophosphate in a later step makes the de novo pathway irreversible. Glutamine is the precursor or inosine monophosphate (IMP), a precursor that is common to adenine and guanine biosynthesis. The reaction o PRPP with glutamine is catalyzed by the enzyme amidophosphoribosyltrans erase (amidoPRT). AmidoPRT is activated allosterically by high levels o PRPP; PRPP is thus both a substrate and an activator o amidoPRT. In general, the cellular level of PRPP is the most important determinant of de novo purine synthesis. High PRPP levels result in enhanced de novo purine synthesis, whereas low PRPP levels decrease the rate o synthesis. The salvage pathway is the second important route to purine synthesis. The f rst step in the salvage pathway is catalyzed by the key regulatory enzyme hypoxanthine-guanine phosphoribosyltrans erase (HGPRT). HGPRT trans ers PRPP to either hypoxanthine or guanine, resulting in the ormation 895

896 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY

Mr. J , a 53-year-o ld m an , awaken s o ne m orn in g w ith e xcru cia tin g pa in in his great toe. Even the weight o the bed sheet is enough to m ake him want to scream ; he is unable to put on a sock or shoe. Worried i d that th t som ething terrible has occurred, Mr. J rushes to his doctor. Based on the history and physical f ndings, the physician diagnoses an acute attack o gout. The physician prescribes a course o high-dose ibuproen, which im proves his sym ptom s in the f rst day and relieves the pain a ter 3 days. Mr. J is then well until 5 years later, when the sym ptom s recur, and he treats him sel with ibupro en success ully. Subsequently, Mr. J learns to anticipate the attacks, which over the next 10 years slowly increase in requency until they occur once weekly. He uses ibupro en at the f rst hint o pain. Th e m o rn in g a te r o n e o h is a tta cks b e g in s , Mr. J g o e s to h is d o cto r b e ca u s e th e p a in is n o t re lie ve d a d e q u a te ly w ith ib u p ro e n . Fo cu s e d e xa m in a tio n re ve a ls a s w o lle n , re d , a n d wa rm le t kn e e , rig h t m id o o t, a n d rig h t f rs t m e ta ta rs o p h a la n g e a l jo in t. Th e re a re 0.5-cm m o b ile n o d u le s n e a r th e o le cra n o n b ila te ra lly a n d a n o th e r a t th e in e rio r p o le o th e rig h t p a te lla . Th e re s t o th e e xa m is u n re m a rka b le . Th e p h ys icia n a s p ira te s Mr. J ’s le t kn e e , re ve a lin g a clo u d y ye llo w u id th a t, o n m icro s co p ic e xa m in a tio n , co n ta in s n u m e ro u s le u ko cyte s .

Diet

Ade nine Gua nine

De gra da tion S a lva ge

ATP GTP

De novo s ynthe s is

P RP P + Gluta mine Amido PRT

HGPRT

Hypoxa nthine Xanthine oxidase Allopurinol Xa nthine Xanthine oxidase Uric a cid Uricase

Re na l e xcre tion Probenecid Sulfinpyrazone

Alla ntoin

FIGURE 49-1. Purine metabolism. Purines are synthesized via de novo synthesis or via the salvage pathway. The de novo pathway utilizes the amino acid glutamine and phosphoribosyl pyrophosphate (PRPP) in a reaction catalyzed by amidophosphoribosyltrans erase (amidoPRT). In the salvage pathway, hypoxanthine-guanine phosphoribosyltrans erase (HGPRT) phosphorylates and ribosylates dietary adenine and guanine, orming the purine nucleotides (ATP and GTP) used or DNA and RNA synthesis. Degradation converts purines and purine nucleotides to hypoxanthine, and xanthine oxidase converts hypoxanthine to xanthine and ultimately to uric acid, which is excreted by the kidneys or gastrointestinal tract (not shown). Pharmacologic interventions that reduce plasma urate levels include reducing urate synthesis (allopurinol and its metabolite oxypurinol), increasing urate excretion (probenecid and sul npyrazone), or converting urate to the more soluble allantoin (uricase).

Ab u n d a n t b lu e a n d ye llo w n e e d le -s h a p e d m icro s co p ic crys ta ls a re s e e n w ith th e u s e o a p o la rizin g f lte r w ith a re d co m p e n s a to r, s o m e o th e m in tra ce llu la r. An x-ra y o th e le t kn e e is n o rm a l e xce p t o r th e p re s e n ce o a n e u s io n ; a f lm o th e rig h t o o t s h o w s a b o ny e ro s io n o th e d is ta l f rs t m e ta ta rs a l. Th e cu ltu re o th e jo in t u id is n e ga tive . Mr. J is tre a te d w ith h ig h -d o s e p re d n is o n e o n th e f rs t d a y, o llo w e d b y a ta p e rin g d o s e ove r th e n e xt 10 d a ys . His co n d itio n im p rove s ra p id ly. Th re e w e e ks la te r, h e re tu rn s to h is p hys icia n w h ile e e lin g w e ll. He is g ive n a p re s crip tio n o r a llo p u rin o l to ta ke o n a lo n g -te rm b a s is a n d o n e o r co lch icin e to ta ke d u rin g th e f rs t 6 m o n th s o a llo p u rin o l th e ra p y.

Questions 1 . Why was ibupro en e ective or most o Mr. J ’s acute attacks o pain? 2 . How does prednisone reduce the inf ammatory response during an acute attack o gout? 3 . How does allopurinol act? Will it alter the requency o Mr. J ’s pain ul attacks? 4 . Why was Mr. J prescribed colchicine during the rst 6 months o treatment with allopurinol?

of IMP or guanosine monophosphate (GMP), respectively. Nucleotide interconversions can then yield adenosine triphosphate (ATP) and guanosine 5 -triphosphate (GTP). Increased activity of the salvage pathway has two important consequences. First, increased scavenging activity depletes cells of PRPP, thus decreasing the rate of de novo purine synthesis. Second, the salvage pathway leads to the generation of more ATP and GTP. Increased levels of these nucleotides inhibit amidoPRT in a feedback manner, also resulting in decreased de novo purine synthesis. Although purines can be synthesized by these two interrelated pathways, degradation occurs via a convergent mechanism (Fig. 49-1). Adenosine monophosphate (AMP) is deaminated, dephosphorylated, and deribosylated, forming hypoxanthine. GMP is also deaminated, dephosphorylated, and deribosylated, forming hypoxanthine. Hypoxanthine, which is moderately soluble, is oxidized to xanthine. Thus, xanthine is the common product of purine metabolism. A further oxidation step converts xanthine to uric acid. The enzyme xanthine oxidase catalyzes both the oxidation of hypoxanthine to xanthine and the oxidation of xanthine to uric acid. Crosstalk between the de novo and salvage pathways is important for overall regulation of purine metabolism. The de novo pathway is the most important generator of purine breakdown products. High de novo pathway activity increases purine turnover, resulting in higher plasma uric acid concentrations. In contrast, increased salvage pathway activity leads to decreased de novo synthesis and reduced plasma uric acid levels. The importance of crosstalk in purine metabolism is demonstrated by several inherited enzyme disorders. Certain

C h a p t e r 4 9 Integrative Inf ammation Pharmacology: Gout

genetic polymorphisms that increase PRPP synthase activity lead to increased intracellular levels o PRPP; because PRPP activates amidoPRT, high levels o PRPP cause increased de novo purine synthesis, leading to increased turnover and degradation o purines and increased plasma levels o uric acid. Similarly, genetic def ciencies o HGPRT (the critical enzyme in the salvage pathway) lead to decreased salvage pathway activity and increased de novo purine synthesis and degradation, resulting in increased uric acid levels. The inherited absence o HGPRT results in Lesch-Nyhan syndrome , a devastating disorder characterized by sel -mutilation, mental retardation, and hyperuricemia. Partial de ects in HGPRT (e.g., polymorphisms in the HGPRT gene that lead to decreased HGPRT synthesis or activity) are thought to explain some cases o hereditary gout. Uric acid is eliminated by the kidney (65%) and gastrointestinal tract (35%). Uric acid is f ltered and secreted by the kidney by the same mechanisms that process other organic anions. Approximately 90% o f ltered uric acid is reabsorbed. The major mediator o uric acid reabsorption is urate transporter 1 (URAT1), a member o the organic anion transporter amily (SLC22A12) that is expressed in the kidney proximal tubule (see Chapter 5, Drug Transporters). Recent genetic association studies have suggested that polymorphisms in URAT1 may predispose to the development o gout. Renal excretion is important or the maintenance o normal plasma uric acid levels, and kidney ailure o ten leads to high plasma urate levels.

PATHOPHYSIOLOGY OF GOUT The likelihood o developing gout correlates strongly with increased plasma uric acid levels. Uric acid is a weak acid (pKa 5.6); at physiologic pH, 99% o plasma uric acid is in the ionized, urate orm. The normal urate concentration in human plasma is 4–6 mg/dL, re ecting a balance o urate synthesis, breakdown, and excretion. Urate is sparingly soluble: the plasma becomes saturated i urate levels exceed 6.8 mg/dL. A plasma level over 7.0 mg/dL or men or 6.0 mg/dL or women is classif ed clinically as hyperuricemia. The gender di erence may be attributable to di erences in urate excretion between women and men. Any variable that decreases the solubility of urate can promote urate crystal deposition. Gout occurs most commonly in peripheral joints. Urate is less soluble at lower temperatures, which may explain the peripheral distribution o urate crystal deposition. Also, joint synovial uid is more acidic than blood, avoring crystal ormation. Nevertheless, a complete explanation or the pattern o joint involvement in gout remains elusive. The pathogenesis o gout is thought to re ect deposition o urate crystals in the periarticular f brous tissue o synovial joints a ter years o hyperuricemia. However, it is also possible to develop gout without hyperuricemia (e.g., due to an immune response to urate or to pre erential deposition o urate in synovial uid). The natural history o gout has our stages (Table 49-1). First, asymptomatic hyperuricemia develops because o either increased purine breakdown or decreased urate excretion. Most cases o hyperuricemia never develop into gout, and there is no indication or treatment o hyperuricemia in the absence o gout. It is, however, important to determine the cause o marked hyperuricemia: such causes can include

897

TABLE 49-1 Natural History of Gout PHARMACOLOGIC INTERVENTION

STAGE

FEATURES

1. Asymptomatic hyperuricemia

Plasma urate 6.0 mg/dL in women, 7.0 mg/dL in men

None

2. Acute gout

Acute arthritis Typically rst metatarsophalangeal joint Excruciating pain

NSAIDs Colchicine Glucocorticoids

3. Intercritical phase

Asymptomatic hyperuricemia 10% may never have another acute attack

None

4. Chronic gout

Hyperuricemia Development o tophi Recurrent attacks o acute gout

Allopurinol Probenecid Sul npyrazone

The degree o hyperuricemia correlates with the likelihood o developing gout; however, developing gout without hyperuricemia is possible. No pharmacologic intervention is indicated or asymptomatic hyperuricemia, but the cause should be investigated.

lymphoma (increased purine turnover) and kidney ailure (decreased urate excretion). For patients with symptomatic gout, the second phase involves an acute attack o arthritis or, less requently, renal colic due to a monosodium urate stone. The arthritis is typically mani ested as a rapid onset o acute pain in a single joint, as occurred in Mr. J. More than 50% o patients with gout have their f rst attack in the f rst metatarsophalangeal joint (pain at this site is re erred to as podagra ), and most patients with recurrent, symptomatic gout have podagra at some point. Without treatment, an acute attack o gout may last or days to weeks but generally resolves spontaneously. It is not understood what causes the periodic onset o gout attacks, or why these attacks resolve spontaneously. The end o an attack leads to the third, intercritical phase, characterized by hyperuricemia without acute symptomatic gout. Some individuals will experience only one acute attack o gout and remain in the intercritical phase or long periods or even or the remainder o their lives. Five years a ter his initial attack, Mr. J developed chronic, recurrent attacks o gout, the ourth phase. Typically, these attacks become polyarticular and more severe. Chronically high levels o plasma uric acid can also lead to deposition o urate crystals around synovial joints or at sites o tissue damage, called tophi. Mr. J’s mobile olecranon and patellar nodules are tophi. Juxta-articular tophi may eventually destroy the synovial lining and cartilage. Recent research has begun to elucidate the cellular and molecular mechanisms responsible or the in ammatory events that are initiated by deposition o urate crystals (Fig. 49-2). Interestingly, these mechanisms may represent a pathologic mani estation o a normal physiologic pathway in which uric acid released rom injured and dying cells unctions as a “danger signal” that initiates an in ammatory response, leading to tissue repair and host de enses.

898 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY Toll-like re ce ptor MS U crys ta ls

1 Re cognition of monos odium ura te (MS U)

Monocyte

P ha gocytos is 2 of MS U NALP -3 3 infla mma s ome a ctiva tion

pro-IL-1β

Caspase IL-1β 4 IL-1β re le a s e 8 Cytokine re le a s e (e .g., IL-1β)

PHARMACOLOGIC CLASSES AND AGENTS There are two main strategies or the treatment o gout: (1) management o acute attacks o gouty arthritis and (2) long-term management o chronic gout. Although some o the same drugs are used in treating acute and chronic gout, the aims o therapy di er in the two cases. The goal o acute gouty arthritis management is to control pain using drugs that limit joint in ammation. In contrast, therapy o the chronic disease aims to modi y purine metabolism to achieve normal concentrations o plasma urate. Thus, pharmacologic agents or the treatment o chronic gout either decrease the production o urate or increase the renal clearance o urate.

Management o Acute Gout: Suppressors o Leukocyte Recruitment and Activation Nonsteroidal Anti-Inf ammatory Drugs (NSAIDs)

IL-1 re ce ptor 5

S igna l tra ns duction a nd ge ne a ctiva tion

7 Ne utrophil re cruitme nt a nd a ccumula tion within joint 6 P roinfla mma tory me dia tor re le a s e (e .g., IL-8)

Endothe lium

FIGURE 49-2. Mechanisms o the inf ammatory response to urate crystals. In an acute attack o gout, monosodium urate (MSU) binds to toll-like receptors (TLRs) on monocytes (1). TLR activation initiates phagocytosis o MSU crystals (2), with subsequent assembly o intracellular inf ammatory response enzymes re erred to as the NALP-3 inf ammasome (3). Assembly o the NALP-3 inf ammasome activates caspase-1, an enzyme that cleaves inactive pro-IL-1 to the active cytokine IL-1 (4). IL-1 is released into the extracellular space, where it binds to receptors expressed on endothelial cells. Subsequent endothelial cell activation (5) leads to release o chemotactic actors such as IL-8 (6) that recruit neutrophils (7). Proinf ammatory mediators released by activated endothelial cells and neutrophils complete a positive eedback loop o urther IL-1 release, endothelial activation, and neutrophil recruitment (8).

In this model, pathologic urate crystals activate monocytes and synoviocytes by binding to toll-like receptors (TLRs), which are transmembrane signaling proteins that initiate an innate immune response. In monocytes, urate crystal binding and phagocytosis initiate assembly o an intracellular protein complex re erred to as the NALP-3 inf ammasome. Assembly o the in ammasome activates caspase-1, a proteolytic enzyme that cleaves inactive pro-IL-1 to active IL-1 . IL-1 is a potent cytokine that initiates a cascade o immune responses, including endothelial cell activation and increased neutrophil transmigration to the site o in ammation. Importantly, IL-1 up-regulates its own transcription and may provide positive eedback to ampli y the initial innate immune response. Clinical trials o IL-1 antagonists and IL-1 receptor antagonists or the treatment o acute gout have shown some clinical benef t, but none o these drugs are approved by the US Food and Drug Administration (FDA) or use in the treatment o gout.

Metabolites o arachidonic acid play an important role in the in ammatory response to urate crystals in the joint. NSAIDs inhibit cyclooxygenase (COX) and thereby inhibit prostaglandin and thromboxane synthesis (see Chapter 43, Pharmacology o Eicosanoids). These drugs were e ective or most o Mr. J’s acute attacks o gout; his pain responded well to ibupro en. Clinically, indomethacin is one o the NSAIDs used most o ten to treat acute attacks o gout. The choice o an NSAID or colchicine (see discussion below) or treatment o acute gout is generally based on the adverse e ect prof le, since the agents have similar e f cacy. The serious adverse e ects o NSAIDs include bleeding, salt and water retention, and renal insu f ciency. COX-2 selective inhibitors are potentially use ul or the management o acute gout attacks because they may be associated with a lower risk o gastrointestinal bleeding, although concerns about adverse cardiovascular e ects limit their long-term use.

Colchicine

Colchicine binds to tubulin, inhibiting its polymerization

and preventing the ormation o microtubules. Colchicine inhibits cell division because microtubules are critical or the alignment and separation o chromosomes during mitosis (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance). Microtubules are also essential in intracellular tra f cking. In an acutely inamed joint, colchicine limits the in ammatory response by inhibiting neutrophil activation. The mechanisms o neutrophil inhibition by colchicine include (1) decreased tra f cking o phagocytosed particles to lysosomes; (2) decreased release o chemotactic actor; (3) decreased motility and adhesion o neutrophils; and (4) decreased tyrosine phosphorylation o neutrophil proteins, with a resulting decrease in leukotriene B4 synthesis. Colchicine can also be administered in low doses as a prophylactic therapy or chronic gout to inhibit the occurrence o acute attacks. Drugs that alter urate homeostasis are o ten co-administered initially with colchicine to avoid precipitating an acute attack o gouty arthritis (see discussion below). Colchicine causes several important adverse e ects. Colchicine inhibits the turnover o epithelial cells in the gastrointestinal (GI) tract, and diarrhea is a common complication o moderate or high doses o the drug. Colchicine is myelosuppressive, particularly in high doses or in combination

C h a p t e r 4 9 Integrative Inf ammation Pharmacology: Gout Cyclosporine Tacrolimus Verapamil

ATP

Cyclosporine Tacrolimus

GTP

O

O

Colchicine MDR

Excre tion in bile

GFR

N

Excre tion in urine

Xanthine oxidase

N

HN

899

Hypoxa nthine

N H

H N

Xanthine oxidase HN

N

HN O

N H

O

N H

O

Xa nthine

O N H

N H

Uric a cid

FIGURE 49-3. Important drug interactions involving colchicine. Cyclosporine and tacrolimus (immunosuppressant drugs requently prescribed a ter organ transplantation) and verapamil (a Ca 2 channel blocker used to treat hypertension and some cardiac arrhythmias) each inhibit the activity o the multidrug-resistance (MDR) protein responsible or the hepatic excretion o colchicine. Cyclosporine and tacrolimus are also nephrotoxic, acting to reduce the glomerular ltration rate (GFR); this adverse e ect can compromise the renal excretion o colchicine. There ore, co-administration o colchicine with cyclosporine, tacrolimus, verapamil, or other inhibitors o hepatic MDR proteins can lead to systemic colchicine toxicity at the usual therapeutic doses; this systemic toxicity is not accompanied by the usual dose-limiting toxicity o diarrhea, because the drug is not redelivered to the GI tract via enterohepatic recirculation.

with other myelosuppressive agents such as ganciclovir or azathioprine. Colchicine undergoes extensive enterohepatic recirculation, and drug secretion into the bile is mediated by the liver multidrug-resistance (MDR) protein. Repeated delivery o colchicine rom the liver to the GI tract (i.e., enterohepatic recirculation) likely explains why diarrhea is a common adverse e ect o the drug. Drugs that inhibit the liver MDR protein, such as cyclosporine and verapamil, can signif cantly increase the raction o a colchicine dose that is delivered to (and remains in) the systemic circulation (Fig. 49-3). By this mechanism, such drugs can cause systemic colchicine toxicity that may not be accompanied by diarrhea because the GI exposure to colchicine is decreased. Accordingly, the dose o colchicine should be lowered when the drug is administered concurrently with any drug known to inhibit MDR activity.

Glucocorticoids

Glucocorticoids have power ul anti-in ammatory and immunosuppressive e ects (see Chapter 29, Pharmacology o the Adrenal Cortex). Glucocorticoids inhibit numerous steps in the in ammatory response during an acute attack o gout. Because they have widespread adverse e ects when administered systemically, glucocorticoids are primarily reserved or use in the treatment o acute polyarticular gout, such as the most recent attack described or Mr. J, or when there are contraindications to other e ective therapies, such as renal insu f ciency. When an acute attack o gout occurs in a single joint and is unresponsive to NSAIDs or colchicine, depot preparations o prednisolone or another glucocorticoid can be injected directly into the joint to yield high local drug levels at the site o in ammation.

Management of Chronic Gout: Agents That Lower Plasma Urate Concentration Agents That Decrease Uric Acid Synthesis

Allopurinol is an example o a drug designed to inhibit a well-

understood biochemical pathway. Allopurinol is a structural analogue o xanthine. By inhibiting xanthine oxidase,

OH

OH N

N N

N H

Allopurinol

Xanthine oxidase

N HO

N N

N H

Oxypurinol

FIGURE 49-4. Mechanism of allopurinol action. Allopurinol is a structural analogue o hypoxanthine. Oxidation o allopurinol yields oxypurinol, a noncompetitive inhibitor o xanthine oxidase. (Although allopurinol is a competitive inhibitor o xanthine oxidase, oxypurinol is the more important inhibitor because o its much longer elimination hal -li e.) Inhibition o xanthine oxidase decreases the production o uric acid by inhibiting two steps in its synthesis. The increased plasma levels o xanthine and hypoxanthine are tolerated because these metabolites are more soluble than uric acid.

allopurinol decreases the concentration o uric acid in the blood (Fig. 49-4). Because o its close structural similarity to xanthine, allopurinol also acts as a substrate or xanthine oxidase. The oxidized orm o allopurinol, known as oxypurinol, inhibits xanthine oxidase by preventing molybdenum in the active site o the enzyme rom interconverting between the 4 and 6 oxidation states, essentially “ reezing” the enzyme. Recall that xanthine oxidase is important or two sequential steps in purine degradation—oxidation o hypoxanthine to xanthine and oxidation o xanthine to uric acid. There ore, inhibiting xanthine oxidase results in increased plasma levels o hypoxanthine and xanthine (see Fig. 49-1). Unlike uric acid, hypoxanthine and xanthine are moderately soluble in blood and can be f ltered by the kidney without crystal deposition. Allopurinol is used in the treatment o chronic gout, especially in cases caused by increased purine degradation. It should not be administered during an acute attack o gout because disruption o urate homeostasis can potentially worsen or precipitate acute attacks o gouty arthritis. There ore, an NSAID or colchicine is o ten co-administered during the f rst 4–6 months o allopurinol therapy to reduce the chance o precipitating an acute attack o gout. This was the concern that prompted Mr. J’s doctor to co-administer colchicine during his f rst 6 months o allopurinol therapy. Because allopurinol inhibits purine degradation, caution should be used when a patient is taking other purine analogues. For example, azathioprine and its active orm 6-mercaptopurine (see Chapter 39) are anticancer and immunosuppressive drugs that contain a purine backbone, and 6-mercaptopurine is metabolized by xanthine oxidase (Fig. 49-5). Inhibition o xanthine oxidase by allopurinol can result in toxic levels o co-administered mercaptopurine or azathioprine because o decreased degradation o the

900 PRINCIPLES OF INFLAMMATION AND IMMUNE PHARMACOLOGY NO 2

N N

S

S

S N

N

HN

N

Azathioprine

6-Mercaptopurine

H N

HN O

N H

N

N H

N

Xanthine oxidase

O N H

N H

6-Thiouric a cid

Excre tion OH

OH N

N N

N H

Allopurinol

Xanthine oxidase HO

N

N N

N H

Oxypurinol

FIGURE 49-5. Interaction between 6-mercaptopurine and allopurinol. 6-Mercaptopurine and azathioprine (a prodrug) are metabolized and eliminated from the body via the same pathways as other purines. Allopurinol and its metabolite, oxypurinol, inhibit xanthine oxidase, thereby inhibiting the breakdown of 6-mercaptopurine. Decreased degradation causes plasma levels of 6-mercaptopurine to rise. When co-administering 6-mercaptopurine and allopurinol (e.g., in cancer chemotherapy), the dose of 6-mercaptopurine should be substantially reduced.

latter drugs. There ore, the dose o mercaptopurine or azathioprine should be reduced by approximately 75% when allopurinol is co-administered. In some cases, switching rom azathioprine to a nonpurine immunosuppressive drug, such as mycophenolic acid (see Chapter 46, Pharmacology o Immunosuppression), is another option. Although allopurinol is generally well tolerated, several important adverse e ects should be considered when prescribing this agent. A small percentage o patients taking allopurinol may develop a hypersensitivity reaction characterized by a rash that, in rare instances, can progress to Stevens-Johnson syndrome. For this reason, all patients who develop a cutaneous reaction to allopurinol should discontinue the drug. Rarely, allopurinol may also cause leukopenia, eosinophilia, and/or hepatic necrosis. Febuxostat is a nonpurine small-molecule inhibitor o xanthine oxidase approved or the treatment o chronic gout. In a large clinical trial, ebuxostat was as e ective as allopurinol in preventing recurrent ares o gout. Unlike allopurinol, ebuxostat undergoes extensive hepatic metabolism, and it may not require dose adjustment in renal insu f ciency. Because o its nonpurine structure, ebuxostat might not be associated with development o cutaneous reactions. As with allopurinol, initiation o ebuxostat therapy should be accompanied by a suppressive medication such as colchicine in order to reduce the risk o gout ares in the f rst several months a ter initiation o urate-lowering therapy.

Agents That Increase Uric Acid Excretion

Because the kidney reabsorbs a substantial amount o f ltered uric acid, pharmacologic agents that block tubular reabsorption increase uric acid excretion. Such drugs are called uricosuric agents . Probenecid was one o the f rst drugs used to increase urate excretion. Individuals lacking the URAT1 anion transporter protein have very low serum uric acid levels and do not respond to uricosuric agents, including probenecid, indicating

that URAT1 is the molecular target or this class o drugs. Probenecid is not specif c or URAT1; it also inhibits other transporters, including some o the renal organic anion transporters (OATs) responsible or penicillin secretion. Decades ago, when penicillin was in limited supply, it was co-administered with probenecid to prolong the hal -li e o the antibiotic and decrease the penicillin dose required to achieve therapeutic levels o the drug. In patients with gout, probenecid is use ul or the treatment o chronic hyperuricemia. Probenecid shi ts the balance between renal excretion and endogenous production o urate, thereby lowering plasma urate levels. Uric acid levels lower than 6.0–6.5 mg/dL support dissolution o urate crystals, thereby reversing the process o crystal deposition in synovial joints. However, increasing renal urate excretion can predispose to ormation o urate stones in the kidney or ureter. The likelihood o this complication can be diminished by recommending that patients increase their uid intake and make their urine less acidic, commonly by co-administration o oral calcium citrate or sodium bicarbonate: uric acid has a pKa o 5.6, and it remains predominantly in the more soluble neutral orm i the urine pH is above 6.0. Because probenecid inhibits the secretion o many organic anions, the dose o other drugs excreted by this pathway should be reduced when probenecid is co-administered. Low-dose aspirin may antagonize probenecid action; the mechanism o this antagonism is unknown. Sulf npyrazone is a uricosuric agent that acts by the same mechanism as probenecid. It is more potent than probenecid, and it is e ective in mild to moderate renal insu f ciency. In addition to acting as a uricosuric, sulf npyrazone has antiplatelet e ects; it should there ore be used with caution in patients taking other antiplatelet agents or anticoagulants. Benzbromarone is a uricosuric agent with a mechanism o action similar to that o probenecid and sulf npyrazone. Benzbromarone may have greater uricosuric e f cacy than probenecid and sulf npyrazone, particularly in patients with impaired renal unction. However, the requent incidence o hepatotoxicity has limited widespread use o the drug, and it is currently not available in the United States. Losartan is an angiotensin II receptor antagonist (see Chapter 22, Pharmacology o Vascular Tone) that has a modest uricosuric e ect. Losartan may be a logical therapeutic choice in patients with both hypertension and gout, although no controlled studies have been per ormed to prove that losartan reduces the incidence o acute gout attacks.

Agents That Enhance Uric Acid Metabolism

Most mammals other than humans express the enzyme uricase. This enzyme oxidizes uric acid to allantoin, a compound that is easily excreted by the kidney (Fig. 49-1). In cancer chemotherapy, the rapid lysis o tumor cells can liberate ree nucleotides and greatly increase plasma urate levels. By this mechanism, tumor lysis syndrome can lead to massive renal injury. Exogenous uricase can be co-administered with cancer chemotherapy to reduce plasma urate levels rapidly and thereby to prevent renal damage. Allopurinol can also be used to prevent this component o tumor lysis syndrome. Currently, uricase is available in Europe as a protein purif ed rom the ungus Aspergillus f avus. A recombinant version o the Aspergillus uricase, rasburicase , is available in the United States. A small percentage o patients have allergic reactions to the oreign protein and antidrug antibodies

C h a p t e r 4 9 Integrative Inf ammation Pharmacology: Gout

are common. Pegloticase , a pegylated ormulation o recombinant porcine uricase, is approved or the treatment o gout re ractory to conventional therapy.

CONCLUSION AND FUTURE DIRECTIONS Gout can be thought o as a disorder o purine metabolism and excretion. An imbalance between urate synthesis and excretion leads to hyperuricemia; in some individuals, hyperuricemia progresses to gout. Acute therapeutic interventions are aimed at symptomatic treatment o gout attacks; these treatments interrupt inf ammatory pathways by inhibiting neutrophil and monocyte activation. Treatments or chronic gout lower plasma urate levels by reestablishing the balance between urate synthesis and excretion. Allopurinol and ebuxostat inhibit urate synthesis; probenecid increases renal urate excretion. Recombinant uricase rapidly decreases plasma urate levels by converting uric acid to allantoin, thereby preventing the adverse renal consequences o tumor lysis syndrome. New therapies are under development or the treatment o both acute and chronic gout. For example, IL-1 antagonists such as anakinra, canakinumab, and rilonacept are being studied or the treatment o acute gout f ares unresponsive to standard

901

therapies or or patients in whom standard therapies are contraindicated. Lesinurad is an investigational agent that inhibits URAT1 and OAT4, thereby limiting urate resorption. Disclosure

Lloyd B. Klickstein is an employee and stockholder o Novartis, Inc., which manu actures or distributes drugs discussed in this chapter, including canakinumab.

Suggested Reading Crittenden DB, Pillinger MH. New therapies or gout. Annu Rev Med 2013;64:325–337. (Provides clinical and mechanistic details on ebuxostat, URAT-1 inhibitors, and uricases.) Khanna PP, Gladue HS, Singh MK, et al. Treatment o acute gout: a systematic review. Semin Arthritis Rheum 2014;44:31–38. (Clinical review suggesting similar e cacy o NSAIDs, corticosteroids, and colchicine in the treatment o acute gout.) Kingsbury SR, Conaghan PG, McDermott MF. The role o the NLRP3 inf ammasome in gout. J Inf amm Res 2011;4:39–49. (Detailed review o uric acid-induced inf ammation and inf ammasome biology.) Neogi T. Clinical practice. Gout. N Engl J Med 2011;364:443–452. (Clinical practice review o gout.) Punzi L, Scanu A, Ramonda R, Oliviero F. Gout as an autoinf ammatory disease: new mechanisms or more appropriate treatment targets. Autoimmun Rev 2012;12:66–71. (Reviews advances in gout pathophysiology, including the role o IL-1 and development o IL-1 antagonists.)

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Ge no to xi xicc N ng e noo to No to xic x ic m c hanis ms me mee c hanis Ce ycle yc leC. Gre e n Lallucra , Sha ra hmsR. Arm s t ro n g , a n d J o s h u a M . Ga la n t e r DNA A a d ducts Infla mma tionn DNA re pa ir DN Diffe re ntia tion C h romo Chro m some bre a ka ge, Immunoo s upp Im u ppre s sion Apoptos is fus ion,, dee le ti fu tion on, R e a ccti tivve oxyge n s pe cie s miss -se - gre ga tiion, c tiiva ivaa tio ionn Re ce ptor a ct ctiv ttiion nondisjuncti nond n o nd ond n d is isju isjunction junc ncti ttion on n INTRODUCTION TI & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epige 5 906 CARCINOG ENIC ICIT ITY AND CH HRO RONI N C TOXICOLOGY . . . . . . . . . . . 911 E905 ne ttic sile n ci nci ccing ing n OGEN Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 912 ACUTE AND SUBCHR HRON O IC TOXICOLOGY TOX O ICOLOGY . . . . . . . . . . . . . . . . . . 905 9005 Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9133 Carbon Monoxide M e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905 Aff atox a toxi toxins n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 914 Cy Cyanide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907 97 90 Ge nomi m. . c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Alte Al lte ree d s iign igna g a lniic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914 gn gna Arseni Lead L e adG . . no . . .mi 907 d ma ge da t nns duction tra ction Workpplaace Exposures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 915 Food ood Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908 Asbestos, Silica, Dusts, and Metalls . . . . . . . . . . . . . . . . . . . 91 Asbes 915 Toxic Plants and nnd Fun Fun ungi gi. . . . . . .Hype . . . . .rmuta . . . . bility . . . . . . . . . . . . . . 909 r.muta Chlorinated Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 9166 nomi no Acids and Bases. . . . . . . . . . Ge . . . nomic . . .m. .c. .ins . . .ta. bility . . . . . . . . . . . . . . 909 Air Po Poll llut ll utio ut ionn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916 io Los prolife Pesticides . . . . . . . . . . . Lo . . .s.s. .o.f.ppr . . . . . .r.a.ttio .ion . n. . control . . . . . . . . . . . . 90 9099 Re s is ta nce to a p opp tos is CONCLUSION AND ND FUT UTUR URE E DI D RECTIONS . . . . . . . . . . . . . . . . . . 917 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .917

INTRODUCTION

Canc e r

Environmental toxicology is the study o deleterious e ects o

physical, chemical, or microbiological agents present in air, water, ood, or other media. In this context, airborne exposures include those received by cigarette smokers and people working in various industries, as well as those received by all o us rom pollutants. Many o the principles and mechanisms pertinent to drug toxicity, discussed in Chapter 6, Drug Toxicity, apply also to nondrug toxicants. In particular, the tenet o dose–response explains why low-level exposures to ubiquitous chemicals are typically harmless, while increasing levels o exposure con er increased risks o harm. In the United States and elsewhere, actions by regulatory agencies such as the US Food and Drug Administration (FDA), the Occupational Sa ety and Health Administration, and the Environmental Protection Agency have resulted in oods, workplaces, and environments that are signif cantly sa er than they were in the mid-twentieth century and earlier. Nonetheless, accidental poisonings, ood poisoning, cigarette smoking, excessive consumption o alcoholic beverages, and the legacy o occupational overexposures to asbestos, silica, and other occupational carcinogens continue to be responsible or considerable burdens o disease. Similarly, although gross overexposures o children and others to the toxic metal lead are becoming less common in much o the world (due primarily to the removal o tetraethyl lead rom gasoline and the reduced use o lead-based pigments), environmental sources o lead remain, and children def cient in iron and calcium in particular are at risk o lead-induced neurobehavioral disease. Worldwide, the prevalence o , and protections against, toxic agents vary widely, both across

and within countries, such that the health o children, workers, and others may be considerably compromised. This is especially the case in groups su ering rom malnutrition, chronic in ections, and other insults that, per se and in concert with toxic exposures, harm health.

ACUTE AND SUBCHRONIC TOXICOLOGY Numerous substances can cause serious acute illness and death. This section describes some common causes o acute and subchronic poisoning, the mechanisms by which they act, and, as appropriate, treatments.

Carbon Monoxide The combustion o any organic material produces carbon monoxide (CO) gas and other products o incomplete combustion. Insu f ciently vented home-heating urnaces, wood stoves, and other combustion sources can result in accumulation o CO in indoor air to toxic concentrations. The propane generator in the introductory case was not su f ciently ventilated, causing carbon monoxide to reach lethal concentrations. In the United States, some 15,000 emergency department visits and 500 deaths annually are caused by overexposures to CO. These deaths are exclusive o f rerelated deaths, many o which are also caused in part by elevated concentrations o CO. CO causes tissue hypoxia by binding more than 200old more tightly to the heme iron in hemoglobin than does O2, thereby reducing the transport o oxygen in the blood (Fig. 50-1). In addition, carboxyhemoglobin (COHb) shi ts the dissociation curve or oxyhemoglobin (OHb) to the le t, 905

906 ENVIRONMENTAL TOXICOLOGY

Th e W a m ily is ru n n in g o u t o m o n e y. Mr. W h a s lo s t h is jo b , a n d Ms . W’s h o u r s h a ve b e e n cu t b a ck. A t e r m o n th s o tryin g to m a ke e n d s m e e t, Mr. W d e cid e s to s to p p a yin g th e e le ctricity b ill. He b o rro w s a p ro p a n e g e n e ra to r ro m a rie n d a n d s e ts it u p in th e g a ra g e a tta ch e d to th e h o u s e . Th a t e ve n in g , Mr. W, h is w i e , a n d th e ir te e n a g e s o n d in e a t h o m e . Ms . W e e ls a s i s h e is co m in g d o w n w ith th e f u , Mr. W h a s a h e a d a ch e , a n d th e s o n e e ls irrita b le . All th re e re tire e a rly to b e d .

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FIGURE 50-1. Mechanism of carbon monoxide poisoning. A. The ligandbinding site o hemoglobin is a errous heme that can reversibly bind oxygen. Carbon monoxide prevents oxygen binding by orming a bond to errous heme that is signif cantly stronger than the heme–oxygen bond (shorter bold line). B. Carbon monoxide inter eres strongly with oxygen transport because it both prevents oxygen binding and increases the a f nity o heme or oxygen. Under normal conditions (blue line), hemoglobin is 85% saturated with oxygen in the alveoli (where the partial pressure o oxygen is approximately 90 torr). At tissue partial pressures (40 torr), normal hemoglobin is 60% saturated with O2. Thus, under normal conditions, 25% o the heme sites deliver their oxygen to the tissues. When 50% o oxygen binding sites are occupied by carbon monoxide (red line), hemoglobin oxygen saturation can be no more than 50% at a partial pressure o 90 torr. At tissue partial pressures (40 torr), the hemoglobin oxygen saturation is still greater than 35%, indicating that less than 15% o the heme sites can deliver their oxygen to the tissues.

Th e n e xt m o rn in g , th e s o n is a b s e n t ro m s ch o o l, a n d Ms . W a ils to a rrive a t w o rk. Frie n d s ca ll, b u t th e p hon e goes un ans wered . Th e police a re n oti ed ; th ey a rrive , b re a k in , a n d n d a ll th re e d e a d in th e ir b e d s .

Questions 1 . What toxic agent(s) could have caused the amily members’ deaths? 2 . Which routine laboratory test(s) could conf rm the likely cause o death?

impeding the dissociation o O2. CO also binds to cytochromes and to myoglobin in heart and skeletal muscle; this bound CO can serve as an internal reservoir o CO as COHb concentrations in the blood decrease. Binding o CO to myoglobin in the myocardium inter eres with oxidative phosphorylation, thus depriving heart muscle o energy. Patients with cardiac disorders are particularly susceptible to acute CO poisoning, and survivors o moderate to severe CO poisoning appear to be at increased risk o cardiac death years a ter the incident. Because the initial symptoms o CO poisoning are nonspecif c, including headache, dizziness, and irritability, an accurate diagnosis is sometimes elusive. The W amily did not react to the presence o carbon monoxide because it is odorless and nonirritating and because the symptoms they experienced shortly be ore sleep would not have been a cause o alarm. Had their house been equipped with a carbon monoxide detector, their deaths would almost certainly have been prevented. Measurement o COHb is straight orward, and concentrations higher than about 3% in nonsmokers and 5–10% in smokers indicate an unusual exposure. (Note that pO2, the partial pressure o oxygen, is likely to be normal in a CO-poisoned patient.) Signs and symptoms o acute poisoning track approximately with COHb concentrations, with severe headache, vomiting, and visual disturbances at 30–40% COHb and collapse and convulsions at 50–60% COHb. Death is likely at 70% COHb and possible at lower concentrations. Survivors o severe CO poisoning are at risk o brain damage; areas o the brain with high oxygen demand are most likely to be impaired, although the mechanisms and outcomes o CO poisoning di er rom those o simple hypoxia. CO-induced neuropathy is due to marked cerebral vasodilation, mitochondrial dys unction, cell death rom apoptosis, and, upon reoxygenation, reper usion injury. The hal -li e o COHb is approximately 5 hours in room air and decreases to about 90 minutes in a 100% O2 environment at normal pressure. Hyperbaric oxygen therapy (3 atmospheres, 100% O2) can reduce the hal -li e to about 20 minutes and appears to protect against long-term brain damage by improving energy metabolism, minimizing lipid peroxidation, and decreasing neutrophil adherence. A rule o thumb is that victims with COHb concentrations above 25% (or above 15% in pregnant women) should receive hyperbaric oxygen therapy. However, COHb concentrations alone are only approximate indicators o risk, and use o hyperbaric oxygen therapy is pre erred when available.

C h a p t e r 5 0 Environmental Toxicology 907

Cyanide The cyanide ion (C N ) is a highly toxic and requently lethal poison. It may be inhaled, ingested, or absorbed through the skin rom sources as diverse as hydrogen cyanide gas, cyanide salts, apricot pits, peach pits, cherry pits, cassava, f re smoke, and vapors rom industrial metal plating operations. Cyanide is also a metabolite o nitriles and nitroprusside. Cyanide binds to the erric iron in the heme a3/CuB center o cytochrome c oxidase, thereby blocking aerobic respiration and preventing cellular use o oxygen. This causes a shi t to anaerobic metabolism and a resulting metabolic acidosis. As with CO poisoning, cyanide poisoning damages tissues with high oxygen demand, such as the brain and heart. Signs and symptoms o cyanide poisoning depend on dose and route o exposure and are somewhat nonspecif c: headache, con usion, altered mental status, hypertension (early) or hypotension (late), nausea, and other symptoms are all possible. Pallor or cyanosis is not present (assuming that there is no co-exposure to carbon monoxide). Unless cyanide exposure is reported or witnessed, or is likely to have occurred given the patient’s occupation or recent activities, diagnosis may be di f cult. Sometimes, an odor o bitter almonds may be noted. Because cyanide is cleared rapidly rom the blood, and because o technical challenges, measurements o cyanide in the blood may be both time-consuming and misleading. Moreover, some endogenous production o cyanide occurs in healthy individuals, and smokers’ blood contains elevated cyanide concentrations. There is some debate about the blood concentrations o cyanide deemed toxic or potentially lethal, but 1 mg/L (39 mol/L) is typically regarded as a potentially toxic level. Treatment or acute cyanide poisoning may include decontamination, supportive therapy, and administration o an antidote. Decontamination may entail simply the removal o contaminated clothes, and care should be taken to avoid inadvertent exposure o responders to the cyanide-containing material. Supportive therapy, including supplemental oxygen, should aim to avoid organ ailure and may be needed to address toxicities such as coma, lactic acidosis, hypotension, and respiratory ailure. The traditional antidotal treatment or acute cyanide poisoning in the United States is a cyanide antidote “kit” (CAK) that contains amyl nitrite , sodium nitrite , and sodium thiosulfate . The nitrites act by oxidizing hemoglobin to methemoglobin to provide a substrate that can compete with heme a3 in cytochrome c oxidase or cyanide molecules. Amyl nitrite is usually given by inhalation and acts (and is cleared rom the bloodstream) rapidly, while sodium nitrite is administered intravenously and has a longer duration o action. The methemoglobin-bound cyanide is oxidized to the relatively nontoxic thiocyanate by the enzyme rhodanese (also known as transsulfurase) and excreted in urine. Sodium thiosul ate provides a ready source o sul ur or the detoxication reaction and enhances cyanide metabolism. Importantly, use o the CAK may pose signif cant risk to the patient, since a substantial raction o hemoglobin must be converted to methemoglobin to compete e ectively or the cyanide ion. Smoke inhalation victims (who have high levels o exposure to carbon monoxide) are sometimes presumed to have been poisoned by cyanide gas. Such patients may already be su ering rom hypoxia be ore CAK treatment is begun. Exacerbation o hypoxia by orcing conversion o hemoglobin to methemoglobin may be detrimental

to such patients. CAK should also be avoided in pregnant women and in ants, who may carry etal hemoglobin and have immature methemoglobin reductase activity. Furthermore, the CAK may cause severe hypotension and lead to cardiovascular collapse. Concerns regarding possible terrorist use o cyanide led to the approval in 2006 by the FDA o an alternative antidote, hydroxocobalamin (Cyanokit®). This member o the vitamin B12 amily is an endogenous compound that was already in use at lower doses or treatment o vitamin B12 def ciency. The mechanism o action o this drug di ers rom that o the compounds in the CAK: the cobalt moiety in hydroxocobalamin has high a f nity or cyanide and competes directly with the erric iron in cytochrome c oxidase or cyanide, orming nontoxic cyanocobalamin that is excreted in the urine. Hydroxocobalamin is generally well tolerated, but anaphylactic reactions are possible. The compound also causes urine to have a bright red color or about a week and may discolor the skin at the site o injection. Inter erence with spectrophotometric tests and assays or oxyhemoglobin, carboxyhemoglobin, and methemoglobin may also occur.

Lead Lead is ubiquitous in the environment because o its persistence, its ormerly widespread and unnecessary use as a gasoline additive, and its use in pigments, paints, plumbing, solder, and other products. Lead is toxic to the central nervous system, making exposure a particular concern or etuses and children up to the age o about 7 years. Young children are also at risk because they are more likely than adults to ingest lead-contaminated paint dust and other non ood materials. Despite a f ve old decrease in the exposure to lead in the United States and elsewhere since the mid-twentieth century, children today may still be at risk o developing lead-induced neurocognitive def cits. This is especially true or children who live near active, poorly controlled lead mines or smelters or in countries where leaded uels are, or recently were, used. (Leaded gasoline was not banned in China until 2000, or example.) Lead-glazed clay cookware and solder remain common in some areas, and some o this lead contaminates ood and water. Exposures to lead that present no overt symptoms may nonetheless be toxic, and testing young children’s blood lead levels is essential. Although the hal -li e o lead in so t tissues is relatively short, its hal -li e in bone is more than 20 years; a substantial exposure in early childhood can result in elevated bone lead levels or decades. Lead disrupts the blood–brain barrier, allowing both lead and other potential neurotoxins to reach the CNS. There, lead can block voltage-dependent calcium channels, interere with neurotransmitter unction, and, most importantly, inter ere with cell–cell interactions in the brain; the latter e ect causes permanent changes in neuronal circuitry. Overt lead encephalopathy, which is rare in the United States today, results in lethargy, vomiting, irritability, and dizziness and can progress to altered mental status, coma, and death. Low- and moderate-level exposures to young children are believed to result in IQ def cits o two to our points or every 10 g/dL increase in blood lead concentration. Whether some blood lead levels are so low as to present essentially no risk o neurobehavioral def cit is a matter o debate and ongoing research.

908 ENVIRONMENTAL TOXICOLOGY

Lead inter eres with the synthesis o hemoglobin at multiple steps and thereby causes microcytic, hypochromic anemia. Specif cally, lead inhibits delta-aminolevulinic acid dehydratase (ALA-D), which catalyzes the synthesis o porphobilinogen, a heme precursor. Lead also inhibits the incorporation o iron into the porphyrin ring. In the kidney, lead causes both reversible and irreversible toxicity. Lead can inter ere reversibly with energy production in proximal tubular cells by inter ering with mitochondrial unction, resulting in decreased energy-dependent reabsorption o ions, glucose, and amino acids. Chronic exposure to lead results in interstitial nephritis, with the eventual development o f brosis and chronic kidney disease. When indicated clinically, body burdens o metals such as lead, mercury, or cadmium can be reduced using electron donors such as an amine, hydroxide, carboxylate, or mercaptan to orm metal–ligand complexes . A chelator, which in Greek means “claw,” is a multidentate structure with multiple binding sites (Fig. 50-2). Binding o the metal at multiple sites shi ts the equilibrium constant in avor o metal ligation. High-a f nity metal–ligand binding is critical because the chelator must compete with tissue macromolecules or binding. In addition, the chelator should be nontoxic and water-soluble, and the complex should be readily cleared. Finally, an ideal chelator should have a low binding a f nity or endogenous ions such as calcium. To prevent the depletion o tissue calcium, many chelators are administered as calcium complexes. The target metal is then exchanged or calcium, and the body’s calcium stores are not depleted. The most important heavy metal chelators are edetate disodium (the calcium, disodium complex o EDTA), which can be used to bind lead; dimercaprol (also known as British anti-Lewisite or BAL), which binds gold, arsenic, lead, and mercury to its two thiol groups; and succimer (2,3-dimercaptosuccinic acid), which has supplanted dimercaprol or the removal o lead, cadmium, mercury, and arsenic. Deferoxamine is used or the removal o toxic levels o iron, such as would occur in accidental overdoses o iron-containing supplements or in patients with trans usion-dependent anemias. Deferasirox is an orally bioavailable iron chelator that may supplant de eroxamine or many conditions associated with chronic iron overload. Removal o copper, typically in patients with Wilson’s disease, is accomplished with penicillamine or, or patients who do not tolerate penicillamine, trientine .

A

B

H2 N

L L

M

N H2

L L

Na + O -

SH OH

O -Na +

N

N

Ca 2+

O

Dimercaprol

O

Calcium disodium edetate (EDTA)

Le a d comple x

OH SH

O

Penicillamine

Coppe r comple x

O

OH H N

N OH

NH2

O

O-

O-

O

Me rcury comple x

H2 N

NH2

Cu +2

C HS

N

O

N

2

O

Deferoxamine

Iron comple x

FIGURE 50-2. Heavy metal chelators. A. A ligand (L) is a compound con-

Food Contaminants

taining a Lewis base (such as amine, thiol, hydroxyl, or carboxylate groups) that can orm a complex with a metal (M). B. A chelator is a multidentate ligand, that is, a ligand that can bind to a metal through multiple atoms, as in this example o a tetra-amino ligand bound to copper (Cu2 ) via its our amine groups. C. The structures o dimercaprol, calcium EDTA, penicillamine, and de eroxamine are shown; the atoms that orm bonds with the metal are identif ed in blue. Three-dimensional structures o the mercury complex o dimercaprol, the lead complex o EDTA, the copper complex o penicillamine, and the iron complex o de eroxamine are also shown. Here, the heavy metal is highlighted in red. For simplicity, hydrogen atoms are not shown.

An estimated one in our Americans experience signif cant food-borne illnesses each year. The mechanisms o ood poisoning involve either in ection, which typically mani ests one to several days a ter exposure, or intoxication rom a pre ormed microbial or algal toxin, with symptoms occurring within a ew hours o exposure. In ectious ood poisoning is typically caused by species o Salmonella, Listeria, Cryptosporidium, or Campylobacter. Less common but quite virulent are poisonings by enteropathogenic Escherichia coli, which can cause sometimes- atal hemorrhagic colitis and hemolytic uremic syndrome (HUS), likely through the uptake o pathologic bacterial proteins by host cells. Food intoxication is o ten caused by toxins elaborated by Staphylococcus aureus or Bacillus cereus or by marine algal toxins ingested via sea ood. S. aureus produces a variety o toxins; the staphylococcal enterotoxins (SE) induce emesis

by stimulating receptors in the abdominal viscera. Highprotein oods, such as meats, cold cuts, and egg and dairy products, are contaminated with S. aureus upon improper ood handling a ter cooking, ollowed by poor re rigeration. B. cereus, a common contaminant o cooked rice, produces several toxins that cause vomiting and diarrhea. O particular concern is the production o cereulide , a small, cyclic peptide that stimulates intestinal 5-HT3 receptors, resulting in emesis. The peptide is heat-stable to 259°F or up to 90 minutes, so reheating o contaminated cooked rice will typically not prevent intoxication. Most algal toxins are neurotoxic and heat-stable, so, again, cooking leaves the toxins intact. Algal saxitoxins are a group o approximately 20 heterocyclic guanidine derivatives that bind with high a f nity to the voltage-gated sodium

C h a p t e r 5 0 Environmental Toxicology 909

channel, thus inhibiting neuronal activity and causing tingling and numbness, loss o motor control, drowsiness, incoherence, and, with su f cient doses (greater than about 1 mg), respiratory paralysis. Many ood-borne illnesses appear to be caused by pathogens that are not yet characterized. Moreover, novel pathogens can emerge because o changing ecologies or technologies or can arise via trans er o mobile virulence actors such as bacteriophages.

Toxic Plants and Fungi Acute illness can also be caused by mistaken ingestion o non ood items, such as poisonous mushrooms collected by amateur mycologists or any number o poisonous plants. The highly toxic “death cap” mushroom, or instance, Amanita phalloides, produces numerous cyclopeptide toxins that are not destroyed by cooking or drying, have no distinctive taste, and are taken up by hepatocytes. The amatoxins bind tightly to RNA polymerase II, substantially slowing RNA and protein synthesis and leading to hepatocyte necrosis. The somewhat less toxic phallotoxins and virotoxins interere with F- and G-actins in the cytoskeleton. Consumption o Amanita species or their relatives can thus cause severe liver dys unction, hepatic and renal ailure, and death. Initial symptoms o poisoning, such as abdominal pain, nausea, severe vomiting and diarrhea, ever, and tachycardia, may occur 6–24 hours a ter consumption o the mushrooms. Hepatic and renal unction may deteriorate even while the initial symptoms abate, leading to jaundice, hepatic encephalopathy, and ulminant liver ailure; death may occur 4–9 days a ter consumption. There is no specif c antidote. An anticholinergic syndrome may be caused by deliberate or accidental ingestion o jimson weed, a plant belonging to the Datura amily. All parts o the plant are toxic, but the seeds and leaves, in particular, contain atropine, scopolamine, and hyoscyamine. These compounds are rapidly absorbed and produce anticholinergic symptoms such as mydriasis, dry, ushed skin, agitation, tachycardia, hyperthermia, and hallucinations. The mnemonic or anticholinergic e ects, “blind as a bat, dry as a bone, red as a beet, mad as a hatter, and hot as a hare,” is applicable to jimson weed poisoning. Some plants in the amilies Umbelliferae (such as parsley, parsnip, dill, celery, and giant hogweed), Rutaceae (such as limes and lemons), and Moraceae (such as f gs) contain psoralen isomers ( urocoumarins) in leaves, stems, or sap that can be absorbed into the skin a ter contact. Subsequent exposure to ultraviolet (UV) A radiation o wavelength 320 nm (generally via sunlight) can excite the urocoumarins, resulting in epidermal tissue damage. Within 2 days, burning, redness, and blistering are observed in areas o contact with the plant and light; a ter healing, hyperpigmentation may persist or months. The response is greater with increasing plant contact, humidity, and duration and intensity o radiation exposure. This nonallergic phytophototoxic mechanism is the basis o psoralen UV-A (PUVA) therapy or eczema and other dermatologic disorders.

Acids and Bases Strong acids, alkalis (caustic agents), oxidants, and reducing agents damage tissue by altering the structure o proteins, lipids, carbohydrates, and nucleic acids so severely that

cellular integrity is lost. These substances, such as potassium hydroxide in drain cleaners and sul uric acid in car batteries, produce chemical burns by hydrolyzing, oxidizing, or reducing biological macromolecules or by denaturing proteins. High concentrations o detergents can also cause nonspecif c tissue damage by disrupting and dissolving the plasma membrane o cells. Although some o these agents may target particular macromolecules, direct tissue-damaging agents tend to be relatively nonspecif c. Thus, the systems most commonly a ected are those most exposed to the environment. Skin and eyes are requently a ected by splashes or spills. The respiratory system is a ected when toxic gases or vapors are inhaled, and the digestive system is a ected by accidental or deliberate ingestion o toxic substances. Many agents can cause damage to deep tissues a ter breaking through the barrier ormed by the skin. Other agents are able to pass through the skin while causing relatively little local damage but destroy deeper tissues such as muscle or bone. For example, hydrof uoric acid (HF; ound in, among other products, grout cleaner) causes milder skin burns than an equivalent amount o hydrochloric acid (HCl). However, once HF reaches deeper tissue, it destroys the calcif ed matrix o bone. In addition to the direct e ects o the acid, the release o calcium stored in bone can cause li e-threatening cardiac arrhythmias. For this reason, HF can be more dangerous than an equivalent amount o HCl. Three characteristics determine the extent o tissue damage: the compound’s identity, its concentration/strength, and its bu ering capacity, or its ability to resist change in pH or redox potential. As mentioned above, HF is more injurious than an equivalent amount o HCl. In general, a stronger acid or base (measured by pH) or oxidant or reductant (measured by redox potential) will cause more damage than an equivalent compound at a more physiologic pH or redox potential. A solution o 10 2 M sodium hydroxide in water has a pH o 12 but has low capacity to cause tissue damage because it has a small bu ering capacity and is rapidly neutralized by body tissue. In contrast, a bu ered solution o pH 12, such as that ound in wet ready-set concrete [made with bu ered Ca(OH)2], can cause more serious alkali burns because tissues cannot readily neutralize the material’s extreme pH.

Pesticides Pesticides include insecticides, herbicides, rodenticides, and other compounds designed to kill unwanted organisms in the environment. By their nature, pesticides—o which there are hundreds (vastly more natural than synthetic)—are biologically active; however, the degree o their specif city toward target organisms varies, and many o these compounds cause toxicity in humans and other nontarget organisms. Some o the more common acute poisonings involve organophosphate and pyrethroid insecticides and rodenticides. Organophosphate insecticides , derived rom phosphoric or thiophosphoric acid, include parathion, malathion, diazinon, enthion, chlorpyri os , and many other chemicals. These widely used compounds are acetylcholinesterase (AChE) inhibitors due to their ability to phosphorylate AChE at its esteratic active site (Fig. 50-3). Inhibition o AChE, and consequent accumulation o acetylcholine at cholinergic junctions in nerve tissue and e ector organs, produces acute muscarinic, nicotinic, and central nervous system (CNS) e ects such as bronchoconstriction, increased bronchial

910 ENVIRONMENTAL TOXICOLOGY A O

O

R1

P

R1

R3 R2 Orga nophos phona te

N H

O

R2

Ca rba ma te

B O P

N

O

F

S a rin

O

O

O

P

P

P

CN

O

F

Ta bun

O

O

N

S

S oma n

VX

O

C

NO 2

S P

O

S O

O

O

P O

P a ra thion

O O

S

O Ma la thion

O

D

H N

R1

X

O

HO

O P

O

Ace tylcholine s te ra s e a ctive s ite (s e rine )

OR 2

R1

N+

Orga nophos pha te

N

P O

OR 2

Orga nophos pha te -bound pra lidoxime 1

2

O

O

O H N

3

H N

O

R1 O

P OH

OR 2

4

O

O

P

OH

R1

Afte r a ging

O HOR 2

O

P

N+

OR 2

N

OH

R1

Orga nophos pha te -bound a ce tylcholine s te ra s e

Pralidoxime

FIGURE 50-3. Structures and mechanisms of acetylcholinesterase inhibitors. A. Structures o typical acetylcholinesterase inhibitors, an organophosphonate on the le t and a carbamate on the right. B. Structures o the principal nerve gases sarin, tabun, soman, and VX, which are potent inhibitors o human acetylcholinesterase. C. Structures o the organophosphate insecticides parathion and malathion. The thiophosphate bonds between sul ur and phosphorus are oxidized more e f ciently by arthropod oxygenases than by mammalian oxygenases, so the compounds are less toxic to humans than the structurally related nerve gases. D. Organophosphates attack the serine active site in acetylcholinesterase, orming a stable phosphorus–oxygen bond (1). Pralidoxime abstracts the organophosphate rom serine, restoring active acetylcholinesterase (2). Organophosphate-bound pralidoxime is unstable and spontaneously regenerates pralidoxime (3). Organophosphate-bound acetylcholinesterase can lose an alkoxy group, in a process called aging. The end product o aging is more stable and cannot be detoxif ed by pralidoxime (not shown).

secretions, salivation, lacrimation, sweating, nausea, vomiting, diarrhea, and miosis (muscarinic signs), as well as twitching, fasciculations, muscle weakness, cyanosis, and elevated blood pressure (nicotinic signs). CNS effects can include anxiety, restlessness, confusion, and headache. Symptoms usually occur within minutes or hours of exposure and resolve within a few days in nonlethal poisonings. Toxic exposures may occur by inhalation, ingestion, or dermal contact, depending on the product formulation and

manner of use or misuse. Toxic secondary exposures have occasionally occurred in people coming into close contact with the victim of direct exposure; for example, emergency responders and emergency department staff have suffered organophosphate toxicity after contacting—or simply being near—contaminated clothing, skin, secretions, or gastric contents. Because the common organophosphate insecticides are metabolized and excreted relatively rapidly, the toxins do

C h a p t e r 5 0 Environmental Toxicology 911

not accumulate in the body. However, the toxic e ect may increase a ter repeated exposure because recovery o cholinesterase activity, either by dissociation o the phosphorylated AChE or de novo synthesis o the enzyme, is slow in the absence o treatment. Because the organophosphate insecticides are pre erentially toxif ed by arthropod cholinesterases and/or pre erentially detoxif ed by mammalian carboxyesterases, these compounds are more toxic to arthropods than to humans, an example o selective toxicity—although toxicity to humans exists as well. Acute treatment or organophosphate poisoning involves restoration o the active site o the enzyme. While the administration o anticholinergic agents such as atropine can block the e ect o excess acetylcholine at muscarinic receptors, it cannot restore the enzymatic unction o AChE. As noted in Chapter 10, Cholinergic Pharmacology, pralidoxime can acilitate the hydrolysis o the serine–phosphate bond between the organophosphate and AChE, but this antidote must be administered be ore “aging” renders organophosphate inhibition essentially irreversible (Fig. 50-3). Pyrethroid insecticides , such as permethrin, deltamethrin, cypermethrin, and cyf uthrin, are semisynthetic chemicals that are structurally related to the naturally occurring pyrethrins ound in chrysanthemum owers. The pyrethroids (and pyrethrins) have very high a f nity or voltage-gated sodium channels, and, while they do not alter activation o sodium currents by membrane depolarization, they signif cantly delay termination o the action potential. Pyrethroids are common agricultural pesticides and are also ound in some household products, including anti-lice shampoos. Two classes o pyrethroids have been def ned based on activity determined largely in laboratory experiments. Type I pyrethroids do not contain a cyano group, produce shorter duration sodium tail currents and repetitive discharges, and cause a tremor (T) syndrome in mammals that can include f ne tremor, increased response to stimuli, and hyperthermia. Type II pyrethroids usually contain a cyano group, produce a longer duration sodium tail current and stimulus-dependent nerve depolarization and block, and cause a choreoathetosiswith-salivation syndrome (CS) that may include sinuous writhing (choreoathetosis) and salivation, coarse tremor, clonic seizures, and hypothermia. A ew pyrethroids elicit intermediate syndromes. As in laboratory animals, T and CS signs are seen in people with large acute exposures to pyrethroids, as may occur during agricultural use o these insecticides. Pyrethroids are o ten ormulated with a synergist, such as piperonyl butoxide, that inhibits insect cytochrome P450 enzymes (and thus pyrethroid metabolism) and increases pyrethroid toxicity. Pyrethroid toxicity is relatively low in humans, but a small number o case reports o death in asthmatics exposed to pyrethroid-containing dog shampoos suggests a potential or exacerbation o asthma. Occupational exposure to pyrethroids o ten involves both inhalation and dermal exposure, since the insecticides are typically sprayed and workers may be caught in the dri t. Absorption is rapid across the lung but very slow across the skin. Common symptoms include paresthesias (most requently acial skin), dizziness, headache, blurred vision, nasal and laryngeal irritation, and shortness o breath. It is not clear to what extent other chemicals in the insecticidal ormulation, such as petroleum hydrocarbons, contribute to these symptoms.

CARCINOGENICITY AND CHRONIC TOXICOLOGY Environmental exposures are major causes o cancer. Consistent with the important role o environmental actors in carcinogenesis, the children o immigrants tend to develop cancers typical o their new, rather than ancestral, environs. Dietary actors di er according to locales and cultures, so that exposures to both procarcinogens and anticarcinogens in ood are o ten di erent in adult immigrants relative to their o spring. Some o these environmental actors are, or work in concert with, carcinogenic viruses and other microorganisms, the prevalence and types o which vary remarkably rom region to region. Thus, the prevalence o many types o cancer varies substantially among (and o ten within) countries. Carcinogenic exposures (Table 50-1 ) include tobacco, alcoholic beverages, diet, chronic in ections, radiation (ionizing and nonionizing), and occupational exposures to specif c f bers, dusts, and chemicals. Carcinogenesis due to the toxic by-products o oxygen and other endogenous or unavoidable

TABLE 50-1 Some Environmental Exposures Known to Cause Cancer EXPOSURE

TYPES OF CANCER

Acquired immunode ciency syndrome (AIDS) due to human immunode ciency virus (HIV)

Kaposi’s sarcoma, non-Hodgkin’s lymphoma, Hodgkin’s disease, invasive cervical cancer

Af atoxins (in diet)

Liver cancer

Alcoholic beverages

Oral, pharyngeal, laryngeal, esophageal, liver, colorectal, and emale breast cancer

Arsenic (in water and in workplace air)

Lung, skin, and bladder cancer

Asbestos

Lung cancer, mesothelioma

Helicobacter pylori

Stomach cancer

Hepatitis B and C viruses

Liver cancer

Human T-lymphotropic virus type I

T-cell leukemia, T-cell lymphoma

Ionizing radiation

Leukemia, skin cancer, cancer o internal organs

Tobacco, smokeless

Oral cancer

Tobacco smoke

Cancer o lung, oro-, naso-, and hypopharynx, nasal cavity and paranasal sinuses, larynx, oral cavity, esophagus (adenocarcinoma and squamous cell carcinoma), stomach, colorectum, liver, pancreas, uterine cervix, ovary (mucinous), bladder, kidney (body and pelvis), and ureter; acute myelogenous leukemia

Ultraviolet radiation

Skin cancer

912 ENVIRONMENTAL TOXICOLOGY

causes (such as spontaneous errors in DNA replication and repair) also accounts or a presumably sizable share o cancers that arise in humans and all other animals. All aerobic organisms, including bacteria, have developed de enses against oxidative and other damage to DNA, and these deenses work to counter at least low-level exposures to endogenous and many exogenous mutagens and carcinogens. As outlined in Figure 50-4, carcinogens vary widely in their modes o action. Many organic chemical carcinogens are not genotoxic per se, but only via one or more electrophilic metabolites that orm addition products—adducts — with one or more bases o DNA. These adducts can cause mutations, some o which lead ultimately to tumors. Interestingly, some such electrophiles have very short hal -lives and so are mutagenic only in the organ, such as the liver or kidney, in which they are ormed; others are stable enough to migrate to other tissues and organs, increasing risks o cancers at these distal sites. Carcinogenic metals can be directly toxic, or toxic via metabolism such as methylation, and can

Ca rcinoge n e xpos ure Excre tion

Me ta bolis m Ge ne s Genotoxic mechanisms DNA adducts Chromos ome brea kage , fusion, de le tion, mis -s egre gation, nondis junction

Ce ll cycle DNA re pa ir Diffe re ntia tion Apoptos is

Ge nomic da ma ge

Nongenotoxic mechanisms Infla mma tion Immunos uppres sion Rea ctive oxyge n specie s Receptor a ctiva tion Epigene tic s ile ncing

Alte re d s igna l tra ns duction Hype rmuta bility Ge nomic ins ta bility Los s of prolife ra tion control Re s is ta nce to a poptos is

Cancer

FIGURE 50-4. Overview of genotoxic and nongenotoxic effects of carcinogens. When chemical carcinogens are internalized by cells, they are o ten metabolized, and the resulting metabolic products are either excreted or retained. Retained carcinogens or their metabolic products can directly or indirectly a ect the regulation and expression o genes involved in cell-cycle control, DNA repair, cell di erentiation, and apoptosis. Some carcinogens act by genotoxic mechanisms, such as orming DNA adducts or inducing chromosome breakage, usion, deletion, mis-segregation, and nondisjunction. Others act by nongenotoxic mechanisms such as induction o inf ammation, immunosuppression, ormation o reactive oxygen species (ROS), activation o receptors such as aryl hydrocarbon receptor (AhR) or estrogen receptor (ER), and epigenetic silencing. Together, these genotoxic and nongenotoxic mechanisms can alter signal transduction pathways, thus leading to hypermutability, genomic instability, loss o proli eration control, and resistance to apoptosis—some o the characteristic eatures o cancer cells.

alter chromosomal structure via hypermethylation o DNA and deacetylation o histones. Carcinogenic viruses and the carcinogenic bacterium, Helicobacter pylori, may act via many mechanisms, including induction o in ammation, itsel a risk actor or cancer. Worldwide, chronic in ections contribute to an estimated 15% o all cancers. Carcinogenesis occurs via progressive stages broadly characterized as tumor initiation, promotion, and progression (Fig. 50-5). The sequence involves multiple rounds o stochastic mutations and selection, notably in proto-oncogenes and tumor suppressor genes. In requent mutations in other genes and cancer pathways are also involved, and determining which mutations are “cancer drivers” and which are mere passengers is a subject o active research. The evolution rom a normal cell to a clinically apparent tumor typically occurs over decades, so that cancer risk increases with age or the majority o cancers. Cigarette smokers, or example, develop lung cancer on average 30 years a ter f rst exposures. This explains why people who success ully quit smoking (a notoriously di f cult task) reduce but do not eliminate their increased cancer risks relative to li elong nonsmokers. Cancer deaths in dogs, cats, and laboratory rodents also occur largely in old age—and occur despite the animals’ lack o deliberate exposures to exogenous chemical carcinogens. Exceptions to long latencies include cancers o childhood and acute myelogenous leukemias that develop secondary to treatment o another cancer with alkylating agents: such leukemias may arise in as short a time as 2–5 years a ter therapy.

Tobacco It is di f cult to overstate the toxicity o tobacco. Worldwide, tobacco kills 5 million people annually. Cigarette smoke is the most signif cant cause o cancer known: 30% o cancer deaths in developed countries are caused by cigarettes, and the burden o deaths due to cigarettes in developing nations is expected to rise in proportion to increasing prevalence o cigarette use. Smoking also causes nonmalignant pulmonary disease (such as COPD) and increases smokers’ risks o cardiovascular disease and death, such that about hal o all people addicted to tobacco die o tobacco-related diseases. The carcinogenicity o cigarette smoke is due to the combined actions o at least 60 carcinogens and countless ree radicals. Among the ormer are two “tobacco-specif c” (that is, derived rom nicotine) nitrosamines: 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) and N -nitrosonornicotine (NNN). Other carcinogenic components o cigarette smoke include polycyclic aromatic hydrocarbons (PAHs), aromatic amines, benzene, aldehydes and other volatile organic compounds, and various metals. Benzo[a]pyrene (Fig. 50-6) is among the carcinogenic PAHs in tobacco smoke and is also believed to account, in part, or the carcinogenicity o soots and coal tars. The important carcinogens and other toxins in tobacco smoke appear to be both in the solid “tar” phase o the smoke and in the gases and vapors. Thus, “low tar” cigarettes are apparently no less potent as carcinogens or causes o cardiovascular disease than are “regular” cigarettes. Smokeless tobacco, which is variously “dipped,” used as snu , or chewed (alone, or with betel quid or other substances), contains signif cant concentrations o carcinogenic nitrosamines (and nicotine) and causes oral cancer as well as gum disease. The raction o oral cancer that is attributable

C h a p t e r 5 0 Environmental Toxicology 913 A Tumor initia tion BP

Me n+ (e .g., Ni2+)

O

OH

Be nzo[a ]pyre ne

OH OH

Be nzo[a ]pyre ne -4,5-e poxide

NER

De coy

Muta tions in ca nce rs us ce ptibility ge ne s S

Alte ra tions in p53 or RAS

Gluta thione

OH

B Tumor promotion CYP 1A1, CYP 1B1, CYP 1A2

TCDD

AhR

O OH

O

Be nzo[a ]pyre ne -7,8-e poxide

ARNT

Glucurona te

Conjuga te d products (nonca rcinoge nic)

AhR ARNT XRE

Extra ce llula r s igna lling

P rolife ra tion

O

Apoptos is

Ce ll cycle

PAI1

GEF

TNF

Cyclin B2

MT-II

COT

HS P 40

NEK2

HEF1

KRAS 2

HO OH

Be nzo[a ]pyre ne -7,8-diol9,10-e poxide (ca rcinoge n)

FIGURE 50-6. Metabolism of benzo[a]pyrene. Benzo[a]pyrene is metaboFIGURE 50-5. Tumor initiation and tumor promotion. Genotoxic carcinogens can induce damage in tumor suppressor genes or oncogenes in various ways, some o which contribute to the trans ormation o normal cells into tumor cells: this is known as tumor initiation. Some chemical carcinogens can also promote the outgrowth o trans ormed cell clones: this is termed tumor promotion. A. Tumor initiation typically occurs via mutations. For example, the benzo[a]pyrene (BP)–DNA adduct can cause mutations in cancer-susceptibility genes such as p53 or RAS. The potency o such adducts can be increased due to inhibition o nucleotide excision repair (NER) by metals such as nickel (Ni2 ) or as a result o NER actor immobilization at repair-resistant DNA-adduct sites, also known as decoy adducts. B. Chemical compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can serve as tumor promoters through aryl hydrocarbon receptor (AhR)mediated signal transduction. Binding o TCDD to AhR leads to activation and translocation o the complex into the nucleus. A ter heterodimerization with the AhR nuclear translocator (ARNT), the complex binds to xenobioticresponsive elements (XREs) and induces the expression o a variety o di erent genes involved in carcinogen metabolism, including cytochrome P450 (CYP) iso orms 1A1, 1B1, and 1A2. Binding o the AhR–ARNT complex to XREs also changes the expression pattern o actors involved in cellular growth and di erentiation, such as plasminogen-activator inhibitor type 1 (PAI1), metallothionein II (MT-II), human enhancer o f lamentation 1 (HEF1), guanine nucleotide exchange actor (GEF), COT (a serine/threonine protein kinase), and K-RAS (KRAS2). Pro-apoptosis actors such as tumor necrosis actor (TNF) and heat shock protein 40 (HSP40) are down-regulated, and cell-cycle genes can either be up-regulated (such as cyclin B2) or downregulated (such as NEK2, another serine/threonine protein kinase).

lized into several products (not all shown). Epoxidation at carbons 4 and 5, ollowed by conjugation with glutathione or glucuronate, leads to nontoxic derivatives that are readily excreted. In contrast, oxidation at the “bay region” generates the proximate carcinogen benzo[a]pyrene-7,8-diol-9,10epoxide, which goes on to orm a repair-resistant adduct with guanine. Subsequent DNA replication, in the presence o this “bulky” polycyclic aromatic adduct, leads to G to T base pair transversions, including in the cancer genes p53 and RAS.

to smokeless tobacco in any given population depends on the prevalence o the habit, the potency o the local tobacco products (such as tobacco in betel quid and tobacco-areca nut mixtures), and competing causes o oral cancer. Thus, in the United States, smokeless tobacco accounts or 7% o oral cancer cases, while in India, more than 50% o oral cancers, in both men and women, are attributable to smokeless tobacco.

Ethanol Excessive consumption o ethyl alcohol is a common and complex problem. Binge drinking occurs in a sizable minority o adolescents and young adults, at least in some cultures. In adults with coronary artery disease, binge drinking can cause myocardial ischemia and angina. Acutely, alcohol is a sedative and causes psychomotor retardation. A sizable raction o morbidity and mortality rom alcohol intoxication results rom injuries su ered (and inf icted) while impaired.

914 ENVIRONMENTAL TOXICOLOGY

Chronic excess drinking results in atty liver disease in almost 100% o such consumers. Some 30% o those a icted go on to develop f brosis; 10–20% progress to hepatic cirrhosis; and a signif cant raction o those with cirrhosis develop and die o liver cancer (hepatocellular carcinoma). As expected, risk o liver disease increases with increasing dose-rates o alcohol: on average, chronic drinkers increase their risk o hepatocellular carcinoma by two old; this risk increases to six old or people drinking f ve drinks or more per day. Ethanol metabolism produces several reactive species, including acetaldehyde, hydroxyl radicals, superoxide anions, and hydrogen peroxide. Acetaldehyde in particular is genotoxic and is believed to be the proximate carcinogen in some cases o alcohol-associated cancer. Chronic excess drinking up-regulates the production o CYP2E1, which not only generates acetaldehyde rom ethanol but also trans orms various nitrosamines and polycyclic aromatic hydrocarbons into their proximate genotoxic metabolites. Although acetaldehyde can be urther metabolized to nontoxic acetate, the requisite enzymes, aldehyde dehydrogenases, may be inherently impaired or inactive in certain groups o people (notably East Asians), leading to substantially increased cancer risks rom alcoholic beverages. Ethanol is a teratogen as well; it causes etal alcohol syndrome (FAS), which is characterized by retardation o cranio acial growth, both pre- and postnatally, and neurocognitive impairment. General physical growth is also retarded in children with FAS, a ecting boys more severely than girls. Children with FAS present with a range o disabilities, presumably due to both the amount and the timing o maternal alcohol ingestion. With regard to the latter, women may drink immoderately in the early stages o pregnancies o which they are as yet unaware. Thus, the simplest prevention strategy (elimination o exposure) may not always be practical. FAS can be generated in laboratory rats and mice, and mechanistic studies using animal models have led to several working hypotheses. The acial abnormalities o FAS are believed to be due to apoptosis o neural crest cells during gastrulation or neurulation. Embryonic exposure to alcohol can result in reduced production o retinoic acid, and retinoic acid is essential or normal morphogenesis. Other postulated mechanisms involve ethanol-induced ree-radical ormation, altered gene expression, disruption o lipid bilayers in cell membranes, and inter erence with the activity o growth actors. Excess drinking also increases the risks o pancreatitis, hemorrhagic stroke, and heart ailure. The pathophysiology o alcoholic cardiomyopathy is complex and appears to involve cell death and pathologic changes in myocyte unction. Light to moderate ingestion o alcohol, on the other hand, appears to protect against cardiovascular disease. Red wine is believed by some to be particularly protective, perhaps because it contains not only ethanol but also resveratrol and other polyphenols; various cardioprotective mechanisms have been hypothesized, including improvements in endothelial unction and e ects on hemostasis. However, to the extent that evidence o the benef ts o moderate drinking derives rom observational rather than experimental studies, one must be mind ul o the possibility that moderate drinkers are, by genetics or other habits or actors, less susceptible to cardiovascular disease in general, so that the association

with moderate drinking and health protection is con ounded rather than causal. I so, then encouraging nondrinkers to start may do them no good.

Af atoxins In 1960, a mysterious disease killed more than 100,000 armed turkeys in England; other birds and arm animals also succumbed. The deaths were linked to specif c batches o peanut meal, and, eventually, to secondary metabolites o the mold Aspergillus f avus: these compounds are termed af atoxins. The most important orm, a atoxin B1 (AFB1, where “B” denotes blue uorescence under ultraviolet light), causes both acute liver toxicity and liver cancer (hepatocellular carcinoma) in myriad mammalian and other species. The proximate metabolite is an unstable, exocyclic epoxide that reacts with DNA, orming a potent mutagenic adduct on the N-7 position o guanine. Minuscule concentrations o AFB1 in the eed o laboratory rodents induce liver tumors. Co-administration o drugs that induce glutathione S-trans erases, such as the antihelminthic oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol3-thione], renders rodents resistant to AFB1-tumorigenesis. Trials to determine whether such chemoprevention works in humans are ongoing. As suggested above, various a atoxin metabolites are nontoxic, including the glutathione conjugate and a hydrolysis product that binds to lysine residues on proteins such as serum albumin. A atoxin adducts and other biomarkers in blood and urine re ect a person’s exposures to dietary a latoxins over the prior 2–3 months and, in tropical regions where a atoxin contamination is endemic and rural diets contain ew oods, over many years. Epidemiological studies using these markers in populations in A rica and China have demonstrated that a atoxin causes hepatocellular carcinoma, both directly and acting synergistically with liver damage due to hepatitis B virus (HBV). Both vaccination against HBV and reduction in a atoxin exposure reduce the risk o liver cancer, which worldwide accounts or approximately 500,000 deaths annually.

Arsenic In several regions o the world, such as parts o Bangladesh, Taiwan, West Bengal, Chile, Argentina, and the United States, the groundwater contains naturally high concentrations o inorganic arsenic (up to thousands o micrograms per liter, g/L). Some o this water is tapped by underground wells and, inadequately treated, serves as drinking water. Sa er water supplies may sometimes be ound and utilized, but various constraints have resulted in hundreds o thousands o people developing chronic arsenic poisoning— arsenicosis—and millions being at risk o arsenic-induced cancers. Arsenicosis is characterized by skin lesions, peripheral vascular disease, cerebrovascular disease, cardiovascular disease, and other chronic conditions. Arsenic-induced skin lesions and peripheral vascular diseases are well recognized and include abnormal pigmentation, keratoses, black oot disease, and Raynaud’s syndrome o f ngers and toes. Both hyper- and hypopigmentation may result, typically on the soles o the eet, the palms, and the torso. Hyperkeratosis also occurs on the soles and palms. Epidemiologic study suggests that these skin lesions may develop at lower arsenic concentrations (tens o g/L) than do other arsenic-induced

C h a p t e r 5 0 Environmental Toxicology 915

toxicities. Black oot disease used to be endemic in southwestern Taiwan, where arsenic was present at high concentration in artesian wells, and reached its highest incidence in the late 1950s be ore the introduction o tap water rom sa er sources. The disease has a typical progression: the f rst signs are preclinical peripheral vascular disease, ollowed by progressive discoloration o the skin rom the toes toward the ankles. Feelings o numbness or coldness develop in the legs, ollowed by intermittent claudication, and eventually gangrene, ulceration, and surgical or spontaneous amputation. It is not clear why black oot disease is not seen in other regions with high, chronic oral exposures to arsenic. Epidemiologic studies have indicated associations between exposure to high concentrations o arsenic (hundreds o g/L) and various cardiovascular diseases such as hypertension and ischemic heart disease and between urinary levels o arsenic and levels o circulating markers o in ammation and endothelial damage, such as soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion molecule-1 (sVCAM-1), both o which correlate with cardiovascular disease risk. In addition, studies o ApoEknockout mice (which are vulnerable to development o atherosclerosis) exposed to inorganic arsenic support an association between this environmental contaminant and cardiovascular disease. Mechanisms remain to be elucidated, however, as does the degree o cardiovascular risk posed by drinking water containing lesser concentrations o arsenic. Inorganic arsenic is a recognized human carcinogen that is causally associated with cancers o the skin, bladder, and lung. Associations with other cancers (e.g., liver, prostate) are less certain. The links to cancer were established in communities with extensive exposure to arsenic in drinking water, particularly in Taiwan and Chile, with clear dose–response patterns. Skin cancers may, but do not always, arise at sites o nonmalignant keratotic lesions and tend to be nonmelanomas. Interestingly, no adequate animal models have been identif ed or arsenic-induced cancers. However, experimental models have been used (and human cohorts have been observed) to shed light on the potential carcinogenic mechanisms: arsenic has indirect genotoxicity, e ects on cell-cycle control, and the ability to cause oxidative damage, and it inter eres with DNA repair or methylation. Other actors, such as nutritional status, genetic polymorphisms, and co-exposure to other toxins, may also in uence the risk o arsenic-induced cancer.

Workplace Exposures Various occupational exposures increase workers’ risks o developing cancer and other diseases. As a general matter, exposure levels in industry are much higher than those in the general environment. To the extent that important and harm ul occupational exposures have been characterized and minimized, the toll o occupational carcinogenesis has decreased. Needless to say, en orcement or even presence o occupational exposure limits is not guaranteed, and groups o workers in certain industries or nations continue to be at excess risk o developing one or more orms o cancer. An eighteenth-century English surgeon, Percivall Pott, was among the f rst to recognize occupational carcinogenesis, deducing that “lodgment o soot in the rugae o the scrotum” caused scrotal cancer in young men employed as chimney sweeps (who typically worked naked, to avoid soiling their clothes). In the nineteenth and twentieth centuries,

industrial workers overexposed (1) to benzene were ound to develop bone marrow disease, including aplastic anemia and acute myelogenous leukemia; (2) to 2-naphthylamine in dyemaking were at high risk or bladder cancer; (3) to various metals were susceptible to lung cancer; and (4) to asbestos developed lung cancer and mesothelioma. Other occupational carcinogens (including specif c chemicals, industries, and industrial processes) were also identif ed.

Asbestos, Silica, Dusts, and Metals

Numerous cases o occupational lung injury are (or were) caused by inhalation o f bers or dusts—such as asbestos, crystalline silica, talc, and coal dust—and various metals. Asbestos is carcinogenic to the lung and mesothelium a ter long-term exposure to respirable f bers o specif c dimensions. The ormerly widespread use o asbestos-containing products in shipbuilding, construction, textiles, and other industries caused perhaps 200,000 cancer deaths in industrialized countries; because o latency, such deaths continue to occur. Current occupational exposures to asbestos are problematic in parts o India and elsewhere in Asia. Asbestos and cigarette smoking act synergistically, such that the risk o lung cancer (although, interestingly, not o mesothelioma, which is not caused by smoking) due to co-exposure is much larger than the risk rom either actor alone. The toxic and carcinogenic potency o asbestos f bers and f ber types varies with their dimensions, sur ace chemistry, and biopersistence. The mechanisms by which asbestos f bers damage the lung or pleura involve production o reactive oxygen and nitrogen species by macrophages attempting to destroy the f bers. Asbestos also causes severe nonmalignant respiratory disease, asbestosis , characterized by f brotic lesions in the lung parenchyma that impair gas exchange. Black lung, or coal worker’s pneumoconiosis (CWP), is another nonmalignant (but potentially atal) f brotic lung disease induced by excessive exposure to coal dust. The simple orm o CWP may not markedly impair respiration and may a ect only small areas o the lung, whereas progressive CWP can develop and worsen even in the absence o continued exposure, leading to severe emphysema. Interestingly, coal dust does not appear to increase the risk o lung cancer. Although worker exposures to coal dust have been limited by US regulations in recent decades and underground mining is less common than in the past, thousands o coal miners in other countries, especially China, are at risk or CWP and related illnesses. Occupational exposures to metals such as arsenic, cadmium, chromium (VI), and nickel increase workers’ risks o cancers o the lung and, in some cases, nasal cavity and paranasal sinuses. A large number o mechanisms have been identif ed, both genetic and epigenetic. Overexposures to certain metals can also cause nonmalignant disease. Chronic exposure to cadmium, or example, causes kidney disease. Abnormal renal unction, characterized by proteinuria and decreased glomerular f ltration rate (GFR), was f rst reported in cadmium workers in 1950 and has been conf rmed in numerous investigations. The proteinuria consists o low-molecular-weight proteins such as 2-microglobulin, retinol binding protein, lysozyme, and immunoglobulin light chains; these proteins are normally f ltered in the glomerulus and reabsorbed in the proximal tubule. Cadmium-exposed workers also have a higher rate o kidney stone ormation, perhaps due to disruption o calcium

916 ENVIRONMENTAL TOXICOLOGY

metabolism as a consequence o renal damage. Renal tubular dys unction appears only a ter a threshold concentration o cadmium is reached in the renal cortex. The threshold varies among individuals but has been estimated to be approximately 200 g/g wet weight. Several studies o the prevalence o proteinuria in worker populations suggest that inhalation exposure in excess o about 0.03 mg/m3 or 30 years is associated with increased risk o tubular dys unction. Un ortunately, removal rom exposure does not necessarily halt disease in workers with cadmium-induced kidney damage; progressive decreases in GFR may occur and endstage renal disease may develop. Progression o disease may depend on both the body burden o cadmium and the severity o proteinuria at last exposure. Unless renal damage is signif cant, urinary cadmium concentration re ects the body burden o the metal. Although renal damage is clearly due to accumulation o cadmium in the kidney, the molecular mechanism responsible or this damage is unclear. Metallothionein may be involved; this cadmium-binding protein, which is synthesized in the liver and kidney, appears both to acilitate transport o cadmium to the kidney and to promote retention o cadmium in the kidney.

Chlorinated Hydrocarbons

Low-molecular-weight chlorinated hydrocarbons are widely used in industrial and other settings. Vinyl chloride, or example, is a gas used to make the plastic polyvinylchloride (PVC). Vinyl chloride gas is neither irritating nor acutely toxic (except at extremely high, narcotizing concentrations), and PVC workers were initially exposed to quite high concentrations. In the 1970s, exposures to vinyl chloride were ound to cause angiosarcoma, a rare orm o liver cancer, in both laboratory rats and workers; strict workplace exposure limits have since been imposed in most settings. Carcinogenesis is due to the epoxide metabolite o vinyl chloride. Some 98% o the DNA adducts ormed rom vinyl chloride epoxide are benign, but the other 2% are highly mutagenic etheno adducts with guanine and cytosine. Interestingly, these adducts are the same as those ormed rom everyday oxidative stress and lipid peroxidation. The ethenoguanine and ethenocytosine adducts are normally eliminated by base excision repair (see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance), but at su f ciently high rates o DNA damage, repair ails to be 100% e ective. Thus, high-level exposures to vinyl chloride and similar genotoxins are demonstrably carcinogenic, while low-level exposures may not be. For example, laboratory rats exposed to low doses o vinyl chloride develop precancerous changes (altered hepatic oci) at rates indistinguishable rom those seen in unexposed laboratory controls. Trichloroethylene (TCE) and tetrachloroethylene (perchloroethylene) are solvents used or degreasing and dry cleaning. Essentially all humans are exposed to trace concentrations o TCE and perchloroethylene in ambient air. Exposures to high concentrations o TCE cause kidney tumors, but moderate- and low-level exposures apparently do not. This is because at low doses, trichloroethylene is converted to nontoxic metabolites that are readily eliminated, whereas at high doses, the detoxif cation pathway is saturated and a second metabolic pathway becomes operative. The latter pathway orms a nephrotoxic metabolite, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and the

subsequent kidney damage appears to be a necessary precursor to TCE-induced kidney tumors. Nontoxic exposures to trichloroethylene up-regulate genes associated with stress, DCVC metabolism, cell proli eration and repair, and apoptosis, a ording protection against renal tubular damage. Perchloroethylene does not appear to cause tumors in people, probably because virtually all o it is eliminated without metabolic activation.

Air Pollution Toxicity due to ambient air pollution depends on both the types and concentrations o pollutants. As with other environmental exposures, air pollution takes much o its toll in regions lacking adequate environmental protection or resources. Combustion o uels is an important source o air pollution; in most cities and suburbs, exhaust rom gasolineand diesel-powered vehicles is the largest source o pollutants. New and recently manu actured motor vehicles burn much more cleanly than vehicles made prior to the 1970s, but the number o vehicles in use continues to grow, and tailpipe emissions are, o course, close to ground level, limiting dilution into cleaner air. Combustion o low-quality uels indoors is not uncommon in some settings. For example, wood, so t coal, charcoal, or dried cow dung are burned or cooking and heating in poorly ventilated homes in some regions o A rica, Asia, and elsewhere. Measurements indicate indoor pollutant levels that exceed outdoor concentrations by two orders o magnitude. As a result o these residential exposures, women and children in particular are at risk o developing chronic bronchitis, dyspnea, and, eventually, interstitial lung disease. Moreover, the carcinogenic potency o so t coal smoke is 1,000 times greater than that o cigarette smoke (in a mouse skin-tumor assay). Women in China who use so t coal indoors have extraordinarily high body burdens o benzo[a]pyrene-adducted guanine, and their rates o death due to lung cancer are eight times higher than the national average or women. Combustion generates thousands o chemicals, some o which depend on the material burned and others o which are inherent to combustion. These include carbon monoxide, organic irritants such as ormaldehyde and acrolein, nitrogen oxides, sul ur dioxide, ammonia, hydrogen cyanide, and hydrogen uoride, among other potentially toxic substances. Semi-volatile and nonvolatile chemicals also orm in abundance and adsorb to the particle phase o smoke. Metals present in the combusted material are, o course, not destroyed by combustion and so may add to the acute and chronic toxicity o inhaled smoke. Polluted air may become unusually acidic under certain meteorological and chemical conditions, and inhalation o acidic aerosols can induce bronchoconstriction and reduce the e f cacy o mucociliary clearance. The action o ultraviolet radiation (sunlight) on reactive hydrocarbons and nitrogen oxides results in the ormation o smog, containing signif cant concentrations o oxidizing chemicals such as ozone, peroxides, and peroxyacetyl nitrate. Acute and subchronic exposures to toxic levels o such oxidants can cause respiratory tract in ammation and irritation, sloughing o epithelium, and loss o cilia. Chronic overexposures can result in pulmonary f brosis or chronic obstructive pulmonary disease, perhaps via altered metabolism o collagen and elastin.

C h a p t e r 5 0 Environmental Toxicology 917

The pulmonary e ects o air pollutants depend in part on their water solubility. Sul ur dioxide, or example, dissolves readily in the mucous membranes o the upper airways and so does not typically reach the lung. Dissolution o the gas is not instantaneous, however, so that exercising or otherwise hyperventilating permits some o the inhaled sul ur dioxide to reach the lower respiratory tract, where, at su f cient concentrations, it can induce bronchoconstriction. Asthmatics are particularly sensitive to this e ect.

CONCLUSION AND FUTURE DIRECTIONS Much o the treatment or toxic exposures ocuses on the acutely poisoned patient. Much o the morbidity associated with environmental actors, however, is caused by chronic exposures and may be clinically apparent only years or decades a ter the initial insult. In act, there is generally no specif c treatment or injury caused by chronic toxic exposures, and treatment modalities or cancers are independent o the underlying causes. In theory, cancers and other chronic diseases caused by habits such as tobacco smoking and excessive drinking are entirely preventable, and although progress has been made in this regard, more remains to be done, although complete eradication o these threats seems unrealistic. Occupational exposures are well controlled in most o the developed world but remain problematic in industrializing nations. Epidemiologic evidence indicates that some specif c oods—such as Chinese-style salted f sh (containing high concentrations o the carcinogen dimethylnitrosamine) and oods contaminated with a atoxins—increase people’s risk o cancer, and that consumption o ruits and vegetables in general decreases risks o cancer, but the specif c dietary components or characteristics that modi y risk remain areas o active research. Obesity (and perhaps sedentary li estyle) is an increasingly important risk actor or cancer, presumably in combination with environmental exposures or other actors. Environmental exposures typically involve complex mixtures o only partially characterized chemicals. Traditional toxicological testing o individual chemicals or simple mixtures may yield results o incomplete or uncertain relevance. Additional in ormation may be generated via microarray technologies and other tools o genomics, proteomics, and metabolomics applied to toxicological inquiry. Our microbiomes—that is, the microorganisms within us that, in toto, outnumber our own cells ten to one—presumably a ect our responses to environmental exposures in many ways yet to be elucidated. More broadly, basic, mechanistic, and applied research is expected to continue to unravel the interconnections between and among genetic, environmental, and random actors involved in disease causation, with the hope that sa er environments will lead to healthier lives.

Suggested Reading Busl KM, Greer DM. Hypoxic-ischemic brain injury: pathophysiology, neuropathology and mechanisms. NeuroRehabilitation 2010;26:5–13. (Reviews the pathophysiologic and molecular basis o hypoxic and cytotoxic brain injury.)

Clower JH, Hampson NB, Iqbal S, Yip FY. Recipients o hyperbaric oxygen treatment or carbon monoxide poisoning and exposure circumstances. Am J Emerg Med 2012;30:846–851. (Reviews data regarding 864 carbon monoxide-poisoned patients and makes recommendations or prevention and treatment.) Gordon SB, Bruce NG, Grigg J, et al. Respiratory risks rom household air pollution in low and middle income countries. Lancet Respir Med 2014;2:823–860. (Reviews evidence or association between household air pollution and in ections, cancers, and chronic diseases o the respiratory system.) Hall AH, Saiers J, Baud F. Which cyanide antidote? Crit Rev Toxicol 2009;39:541–552. (Reviews mechanisms, clinical e f cacy, sa ety and tolerability, and supporting toxicology or antidotes to cyanide poisoning in use in the United States and elsewhere.) Hecht SS. Progress and challenges in selected areas o tobacco carcinogenesis. Chem Res Toxicol 2008;21:160–171. (Review by a major researcher in the f eld.) International Agency or Cancer Research (IARC). Continuing series o monographs. http://monographs.iarc. r/. (As part o ongoing e orts since 1971, IARC convenes panels o experts charged with evaluating published evidence relevant to the determination o the established, probable, or possible carcinogenic e ects o various chemical, biological, and physical agents and exposures. To date, some 116 substances and exposures have been characterized by IARC as carcinogenic to humans.) Klaassen CD, ed. Casarett & Doull’s toxicology: the basic science o poisons. 8th ed. New York: McGraw-Hill; 2013. (A comprehensive textbook o toxicology, this resource provides a solid oundation or the understanding o toxicology. It includes sections on general principles, toxicokinetics, nonspecif c toxicity, organ-specif c toxicity, toxic agents, environmental toxicology, and applications o toxicology, including a chapter on clinical toxicology.) Lang CH, Frost RA, Summer AD, Vary TC. Molecular mechanisms responsible or alcohol-induced myopathy in skeletal muscle and heart. Int J Biochem Cell Biol 2005;37:2180–2195. (Reviews cellular and molecular mechanisms by which alcohol impairs skeletal and cardiac muscle unction, with special emphasis on alterations in signaling pathways that regulate protein synthesis.) Luch A. Nature and nurture—lessons rom chemical carcinogenesis. Nat Rev Cancer 2005;5:113–125. (Reviews mechanisms o chemical carcinogenesis.) Pogribny IP, Rusyn I. Environmental toxicants, epigenetics, and cancer. Adv Exp Med Biol 2013;754:215–232. (Reviews epigenetic changes caused by environmental carcinogens.) Schuhmacher-Wolz U, Dieter HH, Klein D, Schneider K. Oral exposure to inorganic arsenic: evaluation o its carcinogenic and non-carcinogenic e ects. Crit Rev Toxicol 2009;39:271–298. (Emphasizes f ndings with respect to risk o disease ollowing relatively low exposures to arsenic.) Seitz HK, Stickel F. Risk actors and mechanisms o hepatocarcinogenesis with special emphasis on alcohol and oxidative stress. Biol Chem 2006;387:349–360. (Review by major researchers in the f eld.) States JC, Srivastava S, Chen Y, Barchowsky A. Arsenic and cardiovascular disease. Toxicol Sci 2009;107:312–323. (Reviews epidemiologic and experimental data.) Tauxe RV. Emerging oodborne pathogens. Int J Food Microbiol 2002;78:31–41. (Overview o common sources o ood poisoning.) Toxnet. http://toxnet.nlm.nih.gov/. (This government resource, sponsored by the National Library o Medicine, contains a vast database o both toxic substances and articles in the f eld o toxicology.) Tzipori S, Sheoran A, Akiyoshi D, Donohue-Rol e A, Trachtman H. Antibody therapy in the management o shiga toxin-induced hemolytic uremic syndrome. Clin Microbiol Rev 2004;17:926–941. (Reviews the structure and mechanism o action o shiga toxins produced by E. coli O157:H7 and other enteropathic bacteria, the mani estations and treatment o hemolytic uremic syndrome, and the potential utility o antibody therapy.) Wogan GN, Hecht SS, Felton JS, Conney AH, Loeb LA. Environmental and chemical carcinogenesis. Semin Cancer Biol 2004;14:473–486. (Review by major researchers in the f eld.)

V III Fundamentals of Drug Development and Regulation

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Drug Discovery and Preclinical Development

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H INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919–9 919 9 919–920 FPPROCESS Fl ow oRO w S . . . . . . . . . . . . . . . . .Fl Flo THE TH ED DRU DR DISCO OVERY VERYFlo PROCE ROCESS . .o. .w. . . . 9921 21 IUG DISCOVERY J re aDrug cto ctor torD r ess . . . . . . . . . . . . re Comp Co mpou ound n -Centered tered Design Des . . .a. ctor . . . . . . 922 9 Natural and Synttheti ticc Co Comp mpou ound ndss . . . . . . . . . . . . . . . . . . . . 922 Analogues of Natural Ligands . . . . . . . . . . . . . . . . . . . . . . . . 92 9222 Target-Centered Drug Design . . . . . . . . . . . . . . . . . . . . . . . . . . 92 9222 High Hi gh-T Thr hrou ough ghpu putt Sc Scre reen eniingg . . . . . . . . . . . . . . . . . . . . . . . . . 924 Combinatorial Chemistry. y . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924 Structure-Based Drug Design . . . . . . . . . . . . . . . . . . . . . . . . 925 Lead Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926 PHASES OF DRUG DEVELOPMENT. DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . 926 926

INTRODUCTION Over the past decade, the US Food and Drug Administration (FDA) has approved approximately 260 new therapeutics, including 220 new molecular entities (small molecules) and 42 biotherapeutics (generally, recombinant protein products). Many such therapies have enabled treatments or diseases that were previously untreatable. Others have yielded expanded treatment options because they are more e f cacious and/or less toxic than previously available treatments. In the f ght against in ectious diseases, or example, pharmaceutical and biotechnology companies, university laboratories, and others have continued to develop new agents to treat diseases that have become resistant to existing treatments. With the availability o new technologies such as next-generation sequencing and novel protein engineering strategies, it is anticipated that important new classes o therapeutics will continue to be discovered and developed in the coming decades. The development o a new drug is di f cult and costly. Very ew molecules that reach the development stage are ultimately approved as drugs: o 10,000 chemical compounds considered promising rom the results o initial screening assays, ewer than 10 make it to clinical trials and only 2 are eventually approved. Furthermore, the costs associated with discovering and developing a new drug are estimated to be slightly over $1.2 billion, with some esti-

KEYDISCIP CIPLINES CIPLINE PLINE LINES N S IN DRU DRUG DISCOVERYAND DEVELOPMENT. . 92 9277 Floo w . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927 Fl Flow Discov Disco oovver eryy Ch Chemistry KD a ctor t Bi Discovery D veryreBiology: Biochemic ical al Ass ssayys, Cellular l Assays, As and Animal Models . . . . . . . . . . . . . . . . . . 928 Abso Ab s rpption, Distributiion on,, Me Meta tabo boli lism, and Excreti tion (ADME (AD A E). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928 L Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929 Development Chemistry: Chemical Synt nthe hesi siss , Scale-Up, and Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . 930 Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931 CONC CO NCLU LUSI SION ON AND FUT UTUR URE E DI DIRE RECT CTIO IONS NS . . . . . . . . . . . . . . . . . . 93 9322 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .932

mates suggesting costs o up to $5 billion. Although the development o new drugs is a risky venture, success ul drugs can be quite prof table or those willing to take such risks. The biggest commercial successes, such as Abilify® , have annual sales o more than $6 billion each. Increased attention has recently been ocused on the inability o the biomedical research community to produce innovative new therapies. The challenges involved in drug discovery and development were highlighted (along with potential solutions) in the 2004 report o the FDA Critical Path Initiatives (see “Suggested Reading”). This report noted that both the National Institutes o Health (NIH) budget and pharmaceutical company research and development spending approximately doubled over a 10-year period beginning in 1993. The added investment did not increase the rate o development o new medicines, however, as evidenced by a decline in major drug and biological product submissions to the FDA. While several potential solutions have been o ered to address this issue, it is important to note that a joint report by the FDA and the Association o American Medical Colleges highlighted the critical role o physicianscientists in improving the e ectiveness o drug discovery and development. This chapter describes the phases o drug discovery and development and the scientif c disciplines that are involved in these phases. Drug discovery spans the period rom the identif cation o a potential therapeutic target to the selection 919

920 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

Dru g d is co ve ry p ro g ra m s o te n s ta rt w ith a w e ll-ch a ra cte rize d b io ch e m ica l o r m o le cu la r ta rg e t th a t is kn o w n to m e d ia te a d is e a s e o in te re s t. Dru g d is co ve ry th e n in vo lve s th e p u rs u it o a ch e m ica l co m p o u n d (s m a ll m o le cu le ) o r e n g in e e re d m a cro m o le cu le (g e n e ra lly, a p ro te in ) d e s ig n e d to m o d u la te th e p a th wa y o in te re s t. Th e o llo w in g ca s e p rovid e s a n e xa m p le o a n a lte rn a tive a p p ro a ch , in w h ich a n e xte n s ive g e n o m ics a n d b io in o rm a tics p ro g ra m le d to th e id e n tif ca tio n o a n ove l p ro te in (o s te o p ro te g e rin ) a n d th e d is cove ry o a n e w p a th wa y im p o rta n t in b o n e m e ta b o lis m . Th e s e b a s ic s cie n ce d is co ve rie s th e n le d to th e d e ve lo p m e n t a n d co m m e rcia liza tio n o a n o ve l p ro te in th e ra p e u tic th a t is cu rre n tly u s e d to tre a t o s te o p o ro s is a n d b o n e d e s tru ctio n a s s o cia te d w ith ca n ce r m e ta s ta s is . In th e e a rly 1990s , re s e a rch e rs a t Am g e n e n ga g e d in a n e xte n s ive g e n o m ics p ro g ra m d e s ig n e d to id e n ti y n o ve l g e n e s a n d p ro te in s in th e a n ticip a tio n th a t n e w b io lo g ic p a th w a ys a n d p o te n tia l th e ra p e u tic ta rg e ts w o u ld b e id e n tif e d . Fu ll-le n g th s e q u e n ce s w e re g e n e ra te d o r s p e cif c g e n e s o in te re s t a n d w e re o ve re xp re s s e d in th e live rs o m ice . Th e s e tra n s g e n ic m ice w e re th e n u s e d in a p h e n o typ ic s cre e n to id e n ti y b io ch e m ica l, h e m a to lo g ic, ra d io g ra p h ic, a n d h is to lo g ic d i e re n ce s ro m w ild -typ e co n tro l m ice . Prio rity wa s g ive n to g e n e s e n co d in g s e cre te d p ro te in s a n d g e n e s e n co d in g n e w m e m b e rs o a m ilie s o p ro te in s a lre a d y kn o w n to h a ve im p o rta n t ro le s in d is e a s e p a th wa ys . Th ro u g h th is p ro ce s s , in ve s tig a to rs id e n tif e d o s te o p ro te g e rin (OPG), a n e w p ro te in w ith s e q u e n ce h o m o lo g y to m e m b e rs o th e tu m o r n e cro s is a cto r re ce p to r (TNFR) a m ily. Mice o ve re xp re s s in g OPG h a d a s trikin g b o n e p h e n o typ e , w ith m a rke d in cre a s e s in b o n e th a t f lle d th e m e d u lla ry ca vity. Th is in cre a s e in b o n e d e n s ity w a s a ttrib u te d to a m a rke d re d u ctio n in o s te o cla s t n u m b e r. In 1995, Am g e n p a te n t f lin g s id e n tif e d OPG a s a n im p o rta n t re g u la to r o b o n e m e ta b o lis m . S ta rtin g ro m th is d is co ve ry o OPG, in ve s tig a to rs ro m Am g e n a n d o th e r la b o ra to rie s e lu cid a te d a n o ve l a n d critica l p a th wa y in o s te o cla s t b io lo g y, in clu d in g th e d is co ve ry o re ce p to r a ctiva to r o NF- B (RANK) a n d its liga n d (RANKL). In itia l s tra te g ie s to co n ve rt th is b io lo g ic u n d e rs ta n d in g in to a u s e u l th e ra p e u tic o cu s e d o n OPG, in clu d in g a u s io n p ro te in o OPG co m b in e d w ith a h u m a n im m u n o g lo b u lin G1 (Ig G1) Fc re g io n . In a n im a l m o d e ls , th is u s io n p ro te in wa s 20 0 tim e s m o re a ctive th a n u ll-le n g th OPG, a n d , o llo w in g s a e ty te s tin g in a n im a ls , it e n te re d clin ica l tria ls in 1998. Th e in itia l Ph a s e 1 s tu d y d e m o n s tra te d

d o s e -re la te d re d u ctio n s in b o n e tu rn o ve r m a rke rs a n d p ro vid e d p ro o o co n ce p t th a t m o d u la tio n o th e RANKL p a th wa y co u ld h a ve b e n e f cia l e e cts o n th e h u m a n s ke le to n . Fu rth e r o p tim iza tio n e o rts le d to th e d e ve lo p m e n t o a s im ila r m o le cu le , d e rive d ro m a m a m m a lia n ce ll lin e , w ith in cre a s e d ta rg e t a f n ity a n d a lo n g e r h a l -li e . Th is m o le cu le (AMGN0007) e n te re d clin ica l d e ve lo p m e n t; h o w e ve r, its d e ve lo p m e n t wa s h a lte d b a s e d o n th e in d u ctio n o a n im m u n e re s p o n s e to OPG in a clin ica l tria l s u b je ct. Be ca u s e o co n ce rn s re ga rd in g th e p o te n tia l o r a n OPG co n s tru ct to in d u ce a n im m u n e re s p o n s e a n d n e u tra lize e n d o g e n o u s OPG, e o rts to d e ve lo p a n OPG-b a s e d d ru g w e re d is co n tin u e d . Am g e n h a d n o t lim ite d its re s e a rch p ro g ra m to a n OPG-b a s e d th e ra p e u tic b u t h a d a ls o ca p ita lize d o n th e s ig n if ca n t p ro g re s s th a t h a d b e e n m a d e in th e a re a o m o n o clo n a l a n tib o d y g e n e ra tio n te ch n o lo g y to in itia te d e ve lo p m e n t o a u lly h u m a n m o n o clo n a l a n tib o d y ta rg e tin g RANKL. Th e s e e o rts le d to th e id e n tif ca tio n o AMG 162 (la te r kn o w n a s d e n o s u m a b ). Be ca u s e d e n o s u m a b wa s n o t a ctive in ro d e n t s p e cie s , s tu d ie s in n o n h u m a n p rim a te s w e re e s s e n tia l to d e m o n s tra te th a t d e n o s u m a b h a d b e n e f cia l e e cts o n th e s ke le to n . S tu d ie s in n o n h u m a n p rim a te s w e re a ls o u s e d to ch a ra cte rize th e toxicity p ro f le o d e n o s u m a b , a te r w h ich th e d ru g e n te re d clin ica l tria ls o r th e in d ica tio n o o s te o p o ro s is in 20 01. In th e Ph a s e 1 clin ica l p ro g ra m , d e n o s u m a b d e m o n s tra te d lo n g -la s tin g re d u ctio n s in b o n e tu rn o ve r m a rke rs . Ph a s e 2 s tu d ie s d e m o n s tra te d b o th in cre a s e s in b o n e m in e ra l d e n s ity a n d re d u ctio n s in b o n e tu rn ove r m a rke rs a n d w e re u s e d to d e f n e th e d o s e le ve l to s tu d y in th e Ph a s e 3 p ro g ra m . Th e Ph a s e 3 s tu d ie s d e m o n s tra te d ro b u s t re d u ctio n o ra ctu re ris k a n d , in 2010, le d to th e a p p rova l o d e n o s u m a b a s a n a g e n t to tre a t o s te o p o ro s is (Pro lia ®) in th e Un ite d S ta te s . It wa s h yp o th e s ize d th a t RANKL is a n im p o rta n t m e d ia to r o o s te o cla s tic a ctivity n o t o n ly in o s te o p o ro s is b u t a ls o in m e ta s ta tic b o n e d is e a s e . Va rio u s ro d e n t m o d e ls o s ke le ta l m e ta s ta s is w e re o u n d to h a ve in cre a s e s in s tro m a l RANKL, a n d RANKL e xp re s s io n h a d b e e n o b s e rve d in s o m e tu m o r typ e s . No n clin ica l s tu d ie s co n d u cte d b y Am g e n a n d o th e rs e s ta b lis h e d th a t RANKL in h ib itio n ca n d e cre a s e o s te o cla s t n u m b e r a n d a ctivity, lim it th e d e ve lo p m e n t o lytic b o n e le s io n s , a n d re d u ce s ke le ta l tu m o r b u rd e n . Ba s e d o n th e s e p o s itive f n d in g s ro m p re clin ica l s tu d ie s , a clin ica l d e ve lo p m e n t p ro g ra m w a s in itia te d a n d u ltim a te ly le d , a ls o in 2010, to th e a p p ro va l o d e n o s u m a b (Xg e va ® ) o r th e p re ve n tio n o s ke le ta l-re la te d e ve n ts in p a tie n ts w ith s o lid tu m o rs .

C h a p t e r 5 1 Drug Discovery and Preclinical Development

Questions 1 . The case describes an approach that f rst seeks to identi y novel proteins, then determines whether the protein has a biologically meaning ul role in a particular pathway(s), and f nally develops a use ul therapeutic that modulates the novel pathway. What are the advantages and disadvantages o such an approach compared to a program that starts with a validated biologic target? Which approach would you expect to be more likely to result in an approved therapeutic agent?

o molecules or testing in humans. Drug development is generally def ned as the period rom the preclinical studies that support initial clinical trials through approval o the drug by regulatory authorities. The process o drug discovery and development is complex, requiring contributions rom many otherwise disparate scientif c disciplines (Fig. 51-1).

THE DRUG DISCOVERY PROCESS The term drug discovery re ers to the process by which pharmaceutical, biotechnology, academic, and government laboratories identi y or screen compounds to f nd potentially active

2 . A ter the elucidation o the OPG–RANK–RANKL pathway, what types o protein constructs were engineered to modulate the pathway? What were the advantages and disadvantages o these di erent protein constructs? 3 . What role did animal studies play in a discovery approach that was centered in genomics and bioin ormatics? 4 . What were the key data that would represent proo o concept that manipulation o the OPG–RANK–RANKL pathway could have benef cial e ects in patients?

therapeutic agents. The discussion below is ocused on the discovery o low-molecular-weight, chemically synthesized compounds. Many o the described concepts and principles are similar or the discovery o biotherapeutics (monoclonal antibodies and related molecules, oligonucleotide-based molecules, and others) but, generally, many ewer such molecules are interrogated during the screening process. Screening consists o testing many compounds in assays relevant to the disease in question: a compound that passes such a screen is called a hit. I the compound or its structural derivatives continue to show promise a ter urther biological and chemical characterization, it becomes a lead. Drug discovery

Drug discovery P ha s e

Ta rge t-ce nte re d Compound-ce nte re d

921

Drug development

Le a d optimiza tion

P re clinica l de ve lopme nt

P ha s e 1

P ha s e 2

P ha s e 3

Dis cove ry che mis try Dis cove ry biology

ADME

Toxicology

Ta rge t ide ntifica tion

As s a y de ve lopme nt a nd s cre e ning In vitro me ta bolis m

Anima l mode ls of dis e a s e

Me ta bolis m

P ha rma cokine tics (a nima l)

(huma n)

Drug–drug inte ra ctions Development Carcinogenesis and reproduction

S cre e ning

P re clinica l

GLP toxicology

De ve lopme nt che mis try

Me dica l

S a fe ty

Effica cy

Expos ure

Dos e s e le ction

IND

Re gis tra tion tria ls

NDA

FIGURE 51-1. Sequence and phases of drug discovery and development. The important points to note are the general sequence o activities and the considerable overlap o unctions with time. The process is highly interactive among multiple disciplines in an attempt to obtain the drug molecule with the greatest e f cacy, least serious adverse e ects, and greatest sa ety. The clinical trials and regulatory approval phases are described in Chapter 52. The entire process rom hit to drug approval can take 8–12 years and cost more than $1 billion. IND, investigational new drug application; NDA, new drug application; ADME, absorption, distribution, metabolism, excretion; GLP, good laboratory practices.

922 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

should ideally be cost-e ective, producing hits that have a high likelihood o conversion to leads and eventually to success ul drugs (Fig. 51-1). Two basic strategies are used to identi y hits. In a compound-centered approach, a compound is identif ed by one o several methods (described below), and its biological prof le is explored. I the compound displays desirable pharmacologic activity, it is ref ned and developed urther. In a target-centered approach, which is now the more common mode, the putative drug target is identif ed f rst. The potential target could be a receptor thought to be involved in a disease process, a critical enzyme, or another biologically important molecule in the disease pathway. Once the target is identif ed, researchers search or compounds that interact with the target as agonists, antagonists, or modulators. The search may be systematic, using in ormation about the structure o the target as a starting point (structural biologybased approach or simply structure-based approach), or it may take a shotgun approach, in which all the compounds in a large library o substances, synthesized via combinatorial chemistry, are tested in a high-speed automated assay. A ter any o these approaches identif es a hit, the hit is then o ten modif ed with the aid o specif c knowledge about its target. For example, such knowledge can be used to design a high-throughput screen that will test the biologic activity o compounds generated by chemical modif cations o the original hit.

Compound-Centered Drug Design Natural and Synthetic Compounds

Traditionally, drugs were discovered using a compound-centered approach. Many o the earliest drugs discovered were natural products isolated rom plants, molds, or other organisms. O ten, the discoveries were made serendipitously. For example, penicillin (see Chapter 35, Pharmacology o Bacterial and Mycobacterial In ections: Cell Wall Synthesis) was discovered when Alexander Fleming observed that spores o the contaminant mold Penicillium notatum inhibited bacterial growth in a petri dish. Other natural products that have been trans ormed into success ul drugs include paclitaxel, a chemotherapeutic agent derived rom the Pacif c yew tree; morphine , an opioid analgesic obtained rom the opium poppy, which has also been trans ormed into the drug oxycodone in a ew synthetic steps; halichondrin, derived rom the marine sponge Halichondria okadai, which was used as the lead or the synthesis o the anticancer drug eribulin (Halaven®); streptokinase , a thrombolytic agent obtained rom streptococcal bacteria; and cyclosporine , an immunosuppressive agent obtained rom a ungus. In recent years, there has also been a resurgence o interest in the use o cytotoxic natural products as “warheads” in antibody– drug conjugates (ADCs). Table 51-1 shows the structures o several drugs obtained rom natural products. There are several advantages to examining natural products as a source or potential drugs. First, natural products have a reasonable likelihood o biological activity. Second, it may be easier to isolate a compound rom its natural source than to synthesize a compound de novo, especially i the structure o the compound is complex or requires di f cult synthetic manipulations. Paclitaxel, or example, has a complex structure that contains our used rings, one o which contains eight carbons. A chemical synthesis o the

compound took over 50 steps to complete and had a total yield o less than 1%. Third, it may be easible to use the natural compound as a starting point or synthetic f ne-tuning (i.e., to orm a semisynthetic product). Natural products also have disadvantages: it o ten takes signif cant e ort to isolate a natural product, without a guarantee o success, and, even i it is ound to be pharmacologically active, a natural product can be expensive to isolate and modi y. Also, since the in vivo mechanism o action is o ten unknown or many natural products, establishing screening systems and identi ying clinically easible sa ety monitoring tools or these highly potent compounds present signif cant challenges in the current environment o targeted pharmaceutical therapies. Synthetic compounds are now requently used to search or new drugs. Researchers can construct a library consisting o thousands o compounds with di ering structural characteristics, tailored or a particular type o investigation. A library could, or example, consist o numerous compounds that have a phenylalanine–proline bond or that are likely agonists or antagonists o a particular class o receptors.

Analogues of Natural Ligands

An alternative compound-centered approach uses the natural ligand (o ten an agonist) o a receptor as the starting point or drug development. For example, because lack o dopamine in the nigrostriatal pathways is associated with Parkinson’s disease (see Chapter 14, Pharmacology o Dopaminergic Neurotransmission), one o the f rst e ective treatments involved administering the drug levodopa (L-DOPA), a metabolic precursor o dopamine. Insulin was developed in much the same way; once it was discovered that the signs and symptoms o diabetes were caused by low insulin levels, insulin was administered exogenously as an e ective treatment. The natural agonist or a receptor can also serve as a skeleton on which chemical modif cations can be made. These changes can alter the compound’s binding a f nity, physiologic e ect (such as converting an agonist into an antagonist; see Chapter 1, Drug–Receptor Interactions), distribution, metabolism, or pharmacokinetics. This approach was employed in the development o cimetidine (see Chapter 44, Histamine Pharmacology), an H2 receptor antagonist. Starting with histamine, researchers made successive modif cations in the structural skeleton to synthesize an antagonist with high a f nity or the receptor and decreased toxicity. Similarly, modif ed insulins with di erent pharmacokinetic properties are now used to treat patients with diabetes. Modi ying a small-molecule agonist has a relatively high likelihood o success. Because the natural agonist is biologically active, chemical derivatives o that compound are also likely to be biologically active. Problems may also arise. Dopamine ormed rom exogenous L-DOPA can bind to receptors in areas o the brain other than the nigrostriatal pathways and cause hallucinations. Moreover, many disease processes are not mediated by the interaction o a smallmolecule agonist and its receptor. Many targets or drug molecules, such as voltage-gated ion channels or intracellular signaling proteins, have no endogenous small-molecule agonists and hence are not amenable to the analogue approach.

Target-Centered Drug Design In a target-centered approach to drug discovery and design, researchers use a biochemical or molecular target known to

C h a p t e r 5 1 Drug Discovery and Preclinical Development

923

TABLE 51-1 Examples of Natural Products Used as Drugs, Their Clinical Uses, and Sources Natural Product

Clinical Use, Source, and Chapter Reference

Drug

Antimalarial Artemisia annua (sweet wormwood) Chapter 37

H H O

O

O

O H

H

O

O

H

H

O

O

O

H

O

HO

O

O

Arte mis inin O

Artesunate

Antiarrhythmic, cardiac inotrope Digitalis lanata (white oxglove), Digitalis purpurea (purple oxglove), numerous other plants Chapters 24, 25

O OH

H OH

H H

O

HO

O

O

O

OH

O

O

OH

OH

Digoxin O O

HN

Anticancer antibody–drug conjugate (ADC) Maytenus species (sta vine amily) Chapter 40

O O

HN

O

O OH

OH

H O

O

O

N

O

N

H O S

N

O

N O

Cl

O N O

O

Cl

O

H N

MAb

O

O

O

O

Ma yta ns ine

Trastuzumab emtansine

N

Analgesic Papaver somniferum (poppy plant) Chapter 18

N OH H

O

HO

H

OH

O

Morphine

O

H

Oxycodone

Anticancer microtubule inhibitor Taxus brevifolia (Pacif c yew tree) Chapter 39

O O

O

NH

O

OH

O O OH

OH O

O

O

H O O O

Paclitaxel Portions o structures in blue and bold indicate semisynthetic additions to the natural product.

924 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

be essential in the disease o interest (a “validated” target) to search or hits. This approach has several advantages. First, i the target has been associated with a disease process, a hit that success ully interacts with the target has a relatively high likelihood o use ul pharmacologic activity. Second, because the target is known, it may be easier to devise assays capable o isolating the e ect o potential hits on the target. This is especially true or disease processes too complex to observe in cell or tissue preparations. For example, although a potential drug’s e ect on the process o atherosclerosis may be di f cult to measure rapidly, it is relatively easy to measure whether the drug inhibits an enzyme known to be involved in the pathogenesis o atherosclerosis, such as HMG-CoA reductase (see Chapter 20, Pharmacology o Cholesterol and Lipoprotein Metabolism). As knowledge o the pathophysiology o disease processes has increased, target-centered approaches to drug discovery have become increasingly success ul, and most new drugs have been discovered using target-centered methods. HIV protease inhibitors, such as ritonavir, are notable examples o a small-molecule drug class discovered using a target-centered approach (see Chapter 38, Pharmacology o Viral In ections). In a complementary target-centered approach, dissection o the underlying biological pathway has allowed the development o macromolecules, including antibodies, as novel pharmaceuticals to interrupt the pathway (Box 51-1).

Second, a robust assay that leads to rapid identif cation o true hits must be developed. The assay may be as simple as detecting the binding o drug candidates to a receptor (see Chapter 2, Pharmacodynamics), or it may be more sophisticated, involving complicated biochemical or cell-based biological readouts. The library is then “run through” the assay, and any hits giving a positive signal are examined more closely. An assay per ormed in a 96- or 384-well plate allows researchers to screen many compounds simultaneously. In addition, once a library o compounds has been assembled, the same library can be run through many di erent assays. The quality o the results is dependent on the quality o the assay and the compounds in the library, so a poorly designed assay or a limited library may result in alse hits or may miss viable candidates. In practice, because high-throughput screening places a premium on rapid assays, alse positives and alse negatives are not uncommon and require ollow up “validation” o potential hits. In addition, hits in a primary assay will o ten be “counter-screened” in second-tier assays designed to rule out binding o a hit to other related or unrelated targets. Even when a true hit is ound, it will most likely need to be structurally ref ned to increase its binding a f nity or to change its pharmacologic or pharmacokinetic properties (specif city, solubility, stability, kinetics, etc.); this process is called hit-to-lead development.

High-Throughput Screening

One important ref nement in the process o high-throughput screening has been the introduction o combinatorial chemistry. In a strategy analogous to that used by nature to construct a wide variety o proteins rom a relatively small number (approximately 20) o amino acids, combinatorial chemistry uses a relatively small number o precursor molecules to generate a large number o chemical compounds. Researchers are not limited to natural substances; instead, they

The simplest target-centered approach involves rapidly screening many molecules using an assay based on the drug target. High-throughput screening uses a target-based assay and robotic automation to test many thousands o compounds in a ew days. Two aspects are critical in this approach. First, a large library o compounds must be available or screening.

Combinatorial Chemistry

B O X 5 1 - 1 Ma cro m o le cu la r Bio lo g ics a n d Th e ra p e u tics Increasingly, pharmaceutical and biotechnology companies are turning toward large molecules such as peptides , peptidomimetics , proteins , antisense oligonucleotides , and monoclonal antibodies . The pharmacologic properties and clinical utility o these therapies are described in Chapter 54, Protein Therapeutics. The approach to the discovery and development o these molecules can di er signif cantly rom that or small molecules. Consider, or example, the development o agents or the treatment o diseases related to an insu f ciency or lack o an endogenous compound, such as insulin or diabetes, erythropoietin or anemia, or a coagulation actor (factor VIII or factor IX) or an inherited coagulopathy. In these situations, re erred to as replacement therapy, y it is not necessary to per orm extensive screening o a large number o molecules to determine whether the endogenous molecule needs to be modif ed. There ore, these agents may rapidly move into development and human testing. Natural or modif ed macromolecules are increasingly used not only to replace but also to modulate physiologic processes, and engineered macromolecules such as antibodies are being used in the treatment o disease (Table 51-2). In the case o antibodies,

the drug discovery and development process may involve modif cations that increase the a f nity or specif city o the antibody or the desired molecular target or that “humanize” the antibody in order to minimize its immunogenic potential. Because these types o molecules must typically be administered parenterally, the need to screen or acceptable pharmacokinetic properties is lessened. Furthermore, the required discovery biology and animal toxicity testing may not be as extensive because toxicities or biotherapeutics are generally related to “hyperpharmacology” and there is o ten less risk o “o -target” toxicity (see Chapter 6, Drug Toxicity). Manu acturing a biologic product is much more expensive and more technically challenging than manu acturing a chemically synthesized molecule. Major challenges in development o a biologic product are to develop a system capable o producing the desired macromolecule in a bacterium, yeast, or mammalian cell and then to isolate the compound in pure orm rom the large mixture o metabolic products that o ten result rom the synthesis. Faith ully reproducing the complex procedures involved in macromolecule synthesis and purif cation makes the preparation o generic biologic drugs a substantial challenge. ■

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TABLE 51-2 Examples of Macromolecular Therapies NAME

INDICATION

MOLECULAR CATEGORY

ORIGIN

Antivenin

Snake bite

Antibody

Equine or cell culture

Erythropoietin

Anemia

Growth factor

Bacteria (recombinant human)

Heparin

Anticoagulant

Glycosaminoglycan

Porcine or bovine

Human growth hormone

Growth retardation

Hormone

Bacteria (recombinant human)

Insulin

Diabetes

Hormone

Bacteria (recombinant human)

Parathyroid hormone

Osteoporosis

Hormone

Bacteria (recombinant human)

Streptokinase

Thrombolysis

Protein

Streptococcus

Trastuzumab

Cancer

Antibody

Chinese hamster ovary cell culture (humanized monoclonal antibody)

generally use a group o precursors with common unctional groups and divergent side chains. For example, a researcher starting with three sets o 30 precursor building blocks can create 27,000 (30 30 30) di erent compounds in two synthetic steps (Fig. 51-2). One could theoretically create each compound individually in its own reaction well, but in practice, it is o ten easier to synthesize the molecules on a solid support phase such as a polystyrene bead. In a parallel

O O

O

Br

Br

O

O O

O

O

H N

H N H2 N

H2 N

O

O O

N H

N

OH

OH O

O

N

OH

O

N H

OH

FIGURE 51-2. Diversity through combinatorial chemistry. Combinatorial chemistry uses simple building blocks to produce a complex library of compounds. In this example, the functionalized skeleton (black) has multiple sites of attachment. Two building blocks (blue) combine with the functionalized skeleton to produce a wide variety of products. In this example, two different side groups for each of the two building blocks results in four (22) possible products (highlighted in blue boxes). Combinatorial chemistry libraries use several building blocks, each with up to 20 or more different side groups, and can produce thousands of complex molecules using the same basic chemistry.

synthesis , the beads are split so that thousands are reacted

at once and then successively recombined and split to undergo successive reactions. This strategy drastically reduces the number o reactions in the synthesis (30 at a time instead o 27,000 at a time, in the previous example). However, the challenge then becomes sorting the beads in order to know which compound has been synthesized on each bead. Researchers have solved this problem by tagging each bead with a unique chemical code, such as a ribonucleotide sequence, during each reaction. To identi y a bead that bears a success ul hit compound, the tag is cleaved, amplif ed by standard methods, and sequenced. The code then reveals which reactions the bead has been subjected to, and consequently the identity o the success ul compound. Large chemical libraries can be synthesized in this manner and then screened in high-throughput assays or activity, sometimes with the compounds still attached to the beads. The use o combinatorial chemistry and high-throughput screening is termed a shotgun approach, because researchers test a wide range o compounds blindly against a single target. This approach can also be modif ed to search or a particular result by using biased libraries or di erent types o targets. For example, researchers have synthesized large libraries o compounds that are more likely to interact with G protein-coupled receptors, proteolytic enzymes, kinases, or ion channels, based on the structural characteristics o each type o target.

Structure-Based Drug Design

Another target-centered approach is termed structure-based drug design or rational drug design. In this approach, a drug candidate is discovered using the three-dimensional structure o the target obtained through nuclear magnetic resonance (NMR) or x-ray crystallography. In theory, researchers could identi y the active site within the structure o the target, use modeling algorithms to study the shape o the active site, and design a candidate drug molecule to f t into the active site. More commonly, though, the target is co-crystallized with a substrate analogue or receptor ligand (agonist or antagonist) in order to identi y the structure o the active site. The structure o the analogue is then modif ed to increase the molecule’s a f nity, as was done in the case o the antiviral ritonavir. Alternatively, researchers can

926 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

ref ne the structure o a new compound that binds to the target in a screening assay. By iteratively improving the f t o the prototypic molecule in the active site o the target, the binding a f nity is increased. There are several advantages to a structure-based drug design approach. The ref ned hit (also called lead) compounds are o ten extremely potent, with binding a f nities in the nanomolar range. Moreover, only a limited number o candidates need to be tested because there is a high likelihood that one or more o the designed compounds will bind the target. In addition, iterative modif cation o the compound is relatively straight orward because it is known which parts o the molecule are critical or binding to the active site o the target. Thus, in comparison to a structure-blind approach, ewer analogues are prepared in a structure-based approach, but each analogue has a higher likelihood o activity. One disadvantage to this approach is that the modif ed compounds are o ten more di f cult to synthesize because the molecular design demands specif c unctionalities in specif c locations o the molecule. Another disadvantage is that obtaining a crystal structure o the target can be di f cult, especially or membrane-bound proteins. O ten, other methods o drug design yield hits long be ore the target can be crystallized. However, even i the initial hit compound results rom another method, that hit can o ten be ref ned into a lead using a structure-based design approach. As structure-based drug design gains easibility, more drugs will be produced using structural in ormation about the target even i the initial hits are discovered through other methods. Rational drug design has been critical or the development o HIV protease inhibitors such as ritonavir; structure-based methods have also been used to develop a second class o antiviral drugs, the neuraminidase inhibitors (see Chapter 38), as well as many tyrosine kinase inhibitors (TKIs) that are used widely in anticancer drug therapies (see Chapter 40, Pharmacology o Cancer: Signal Transduction).

Lead Optimization The early drug discovery process typically identif es a promising group o lead molecules that appear to interact with the target in a desirable way. For these promising molecules, however, many o the critical physical, chemical, biological, pharmacologic, pharmacokinetic and sa ety properties that are important attributes o an e ective drug have not been identif ed at the time o lead identif cation. Lead optimization is the stage o drug discovery where these properties are characterized and ref ned, with the ultimate goal o selecting a single molecule to enter into clinical testing and ormal drug development. In practice, most lead compounds have one or more characteristics (e.g., low solubility, low oral bioavailability, complex metabolism, o -target promiscuity) that make them poor candidates or clinical use. Using the data generated in lead optimization, it is o ten possible to modi y the structure o the molecule to overcome these def ciencies. A variety o actors may cause a molecule to be terminated at the lead optimization stage. These include: ■

■

Failure to demonstrate e f cacy in a rigorous animal model o human disease Failure to attain adequate systemic exposures a ter oral administration (low bioavailability)

■

■

■ ■

■

Extensive or complex metabolism within the body, resulting in the generation o potentially dangerous reactive metabolites Extremely low solubility that prevents the preparation o a suitable ormulation or dosing Negative e ects in preliminary sa ety evaluation studies In vitro evidence that the molecule may damage DNA (genotoxicity) Extremely di f cult chemical synthesis that cannot be scaled up in a cost-e ective manner

PHASES OF DRUG DEVELOPMENT The outcome o the lead optimization process is the selection o a molecule suitable or testing in humans. At this point, the molecule moves rom drug discovery to drug development. Early drug development consists o preclinical activities designed to support clinical trials and clinical drug development. The initial preclinical phase o drug development includes the ollowing activities: ■

■

■

Manu acture, ormulation, and packaging o a su f cient amount o high-quality drug material or both def nitive animal sa ety testing and clinical trial use Animal toxicology and pharmacokinetic studies to def ne sa e-use conditions o initial drug administration in humans Preparation o regulatory documents and submissions to regulatory authorities; these activities are described in more detail in Chapter 52, Clinical Drug Evaluation and Regulatory Approval.

Initial planning or clinical drug development proceeds concurrently with preclinical drug development. Key initial activities include def ning outcome objectives or the clinical trial, selection o clinical trial investigators, and development o clinical trial protocols. The initial regulatory f lings must include detailed protocols to allow regulators to assess the risk–benef t relationships o the proposed clinical investigation. In recent years, there have been e orts to standardize this process internationally in order to streamline global drug development and approval (Box 51-2). Clinical development o a drug candidate re ers to a wide range o studies conducted in humans. As described in more detail in Chapter 52, these studies are most commonly but somewhat arbitrarily divided into three phases, with the ultimate goal o providing a rigorous evaluation o the sa ety and e f cacy o the candidate molecule. Clinical studies may be conducted in various patient populations and disease states. The number, duration, and complexity o the required clinical trials depend on the nature o the proposed disease indication or the drug. For example, an assessment o the ability o a drug candidate to lower blood pressure in hypertensive patients may require only a ew weeks o dosing, whereas the ability o a drug candidate to reduce the risk o racture in a patient with osteoporosis may require 2 years o drug administration. Although evaluating the e ects o the candidate molecule in humans is the primary ocus o the drug development phase, extensive activities must also be completed by various scientif c disciplines to support both these clinical trials and the ultimate regulatory approval o the drug. These activities are described in the ollowing section, and they must be care ully coordinated in order or drug development to proceed as e ectively as possible.

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B O X 5 1 - 2 In te rn a tio n a l Co n fe re n ce o n Ha rm o n iza tio n The International Con erence on Harmonization (ICH) brings together regulatory authorities and pharmaceutical industry experts rom J apan, Europe, and the United States. The mission o the con erence is to reach consensus on the scientif c and technical aspects o drug development. The stated objective o the project is to create: . . . a more economical use o human, animal and material resources, and the elimination o unnecessary delay in the global development and availability o new medicines whilst maintaining sa eguards on quality, sa ety and e f cacy, and regulatory obligations to protect public health. The project is divided into our topic areas, including: 1. Quality – related to assuring the chemical qualities o the product 2. Sa ety – relating to sa ety testing in animals 3. E f cacy – relating to clinical studies in human subjects 4. Multidisciplinary – relating to topics involving multiple aspects o drug development Each o the topic areas is articulated through a set o guidance documents. Be ore the existence o the ICH, it was not uncommon or di erent political jurisdictions (United States, Europe, J apan) to have di erent and con icting regulatory requirements regarding preclinical and clinical drug development. Thus, a drug development “package” satis ying the requirements o one jurisdiction might not satis y the requirements o another. As a result, a pharmaceutical company could spend years designing and completing a package to satis y the requirements or one jurisdiction, only to f nd that another jurisdiction required additional, new, or reconf gured drug development activities.

KEY DISCIPLINES IN DRUG DISCOVERY AND DEVELOPMENT Having discussed the overall process o drug discovery and development, we now turn to the undamental tools— rom basic chemistry and biology to manu acturing and ormulation—that are crucial in the discovery and development o new therapeutic agents.

Discovery Chemistry Chemists and biologists work hand in hand in the early phases o drug discovery. In compound-centered drug design, medicinal chemists begin the discovery process by preparing the molecules to be tested in biologic and pharmacologic assays. In target-centered design, the process begins with the identif cation o potential drug targets against which chemists then design and prepare the molecules or testing. Thus, in both approaches, there is close interaction and collaboration between chemists and biologists. Initially, the amount o a drug candidate needed to run a simple screening assay is small—typically, less than 1 mg.

The ICH is meant to uni y and clari y drug development regulatory requirements across regulatory jurisdictions. The preclinical sa ety guidelines o the ICH cover a range o topics, including: 1. Carcinogenicity – addressing the potential or the pharmaceutical to cause tumors 2. Genotoxicity – addressing the potential or damage o genetic material 3. Toxicokinetics and pharmacokinetics – addressing the need to characterize the ADME properties in animal species 4. Toxicity testing – addressing both acute and chronic toxicity in animals 5. Reproductive toxicity – addressing the potential o the molecule to impair ertility or cause developmental de ects 6. Biotechnology products – addressing actors specif c to preclinical studies or biotherapeutics 7. Pharmacology – addressing studies per ormed to characterize the acute e ects on organ systems 8. Immunotoxicology – addressing studies per ormed to understand the impact on the structure and unction o the immune system 9. Anticancer products – addressing special considerations or the preclinical sa ety assessment o anticancer drugs 10. Phototoxicity – addressing evaluation methods or determining whether a drug may cause toxicity a ter absorbing ultraviolet or visible light In addition, a key multidisciplinary guidance describes when the above studies should be conducted in relationship to the clinical trials and product registration. This document also provides guidance on specialized topics such as understanding toxicity in juvenile animals. ■

This is important because synthesizing or isolating even small amounts o a compound can be expensive, at least until the synthesis can be ref ned. Once a lead is identif ed, gram quantities are needed to carry out biological, toxicological, and chemical characterization studies. Kilogram quantities are required when a drug enters clinical trials, and i a drug is approved, plants need to manu acture material on a scale su f cient to meet expected clinical use. Quality and documentation o the specif cations o the manu acturing process must be maintained throughout the scale-up (see Chapter 52). Chemical characterization re ers to the chemical properties o the drug candidate, including physical characteristics such as melting point, crystal orm, and solubility, as well as purity and stability. The physical and chemical characteristics o a drug candidate are critical or determining how the drug could best be administered and stored (Table 51-3). The compound’s chemical structure is commonly elucidated using a range o techniques, including mass spectrometry, which gives the compound’s molecular weight; elemental analysis, which determines its atomic composition; NMR, which elucidates the types and connectivity patterns o atoms within

928 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

TABLE 51-3 Information Obtained in Chemical Characterization Studies TYPE OF ASSAY

EXPERIMENTAL TECHNIQUE

CLINICAL IMPLICATIONS

Characterization, structure

Elemental analysis, mass spectrometry, NMR spectroscopy, IR spectroscopy, x-ray crystallography

Atomic composition, molecular weight, isomeric purity, compound structure

Impurities

HPLC, GC, mass spectrometry

Possible adverse reactions rom impurities, toxicology

Partition coe f cient

Octanol/water partition

Pharmacokinetics, including absorption, distribution, metabolism, and excretion; tissue distribution

Solubility

Solubility in various solvents

Pharmacokinetics, including absorption, distribution, metabolism, and excretion; ormulations

Stability

Stability measurements under di erent conditions (heat, cold, humidity, light)

Shel li e, degradation products

NMR, nuclear magnetic resonance; IR, in rared; HPLC, high-per ormance liquid chromatography; GC, gas chromatography.

the molecule; and x-ray crystallography, which determines its three-dimensional structure. It is also important to distinguish among various isomers o the same compound, because biologic activity is o ten stereoisomer-selective. For example, propranolol (see Chapter 11, Adrenergic Pharmacology) is a mixture o ( ) and ( ) stereoisomers, but only the ( ) isomer acts as a -adrenergic receptor antagonist. Chemists also characterize physical properties o the molecule that are used in developing the ormulation, such as the pKa o an acidic or basic drug (see below). In addition, the drug’s solubility is measured in a variety o solvents, especially water, to provide in ormation on the molecule’s likely oral bioavailability and possible hepatic metabolism. The partition coe f cient describes the distribution o a molecule between an aqueous solvent, analogous to blood, and a hydrophobic solvent, analogous to the plasma membrane. Finally, the compound’s stability over time and its impurity prof les must be determined.

Discovery Biology: Biochemical Assays, Cellular Assays, and Animal Models The goal o discovery biology is to determine whether a molecule is likely to be e ective in a particular disease state. E ectiveness may be assessed at the biochemical, cell, tissue, organ, and organism levels. I undesirable biologic properties are ound, it may be possible to modi y the structure o the molecule so as to improve its pharmacologic prof le. In general, biochemical and cell-based assays are used early in the drug discovery process, while more complex organ and whole-animal studies are used in the lead optimization phase to characterize the pharmacologic properties o the molecule. Biochemical assays evaluate the mechanism o action o the drug candidate at a molecular level. Receptor binding assays measure both the binding a f nity and selectivity o the molecule or the target receptor. Enzyme activity assays measure the ability o the drug to inhibit the activity o a target enzyme. Selectivity or the desired target is critically important in the design and testing o lead molecules. Development o these assays is o ten a costly and rate-limiting step in the drug discovery process, since assay development requires identif cation and synthesis o key reagents and extensive optimization and validation o the assay.

In cellular or biologic assays , researchers aim to determine whether the lead molecule(s) acts appropriately in an environment that more closely approximates its in vivo use. For example, i the drug is designed to act in the cytoplasm, then it is essential to determine whether the drug can cross the plasma membrane. Early identif cation o potential sa ety problems may be assessed by incubating the lead molecule with a variety o receptors, ion channels, tissues, or cells, including induced pluripotent stem cells (iPS) or human embryonic stem cells (hES), either o which may be di erentiated by culture techniques to cardiomyocytes or neuron-like cells to assess potential sa ety issues in those organ systems. Drug-induced changes in complex patterns o gene expression can be assessed using gene-array chips capable o measuring mRNA levels or thousands o genes simultaneously. Finally, at the highest level o complexity, the e ects o the drug candidate on whole organisms are established. Ideally, animal models are used that mirror the critical aspects o human pathophysiology or the target disease. For example, cancer chemotherapeutic agents can be tested in nude (T-cell-de icient) mice that have had human tumor cells implanted subcutaneously. Similarly, drugs or the treatment o postmenopausal osteoporosis can be tested in rats that have been ovariectomized to mimic the postmenopausal state. Table 51-4 describes some o the many animal models used by pharmaceutical researchers.

Absorption, Distribution, Metabolism, and Excretion (ADME) Studies characterizing the ate o a molecule a ter its administration are critical to understand the potential e ectiveness as well as the sa ety o that molecule. Such studies collectively describe the ADME (absorption, distribution, metabolism, and excretion) prof le o the molecule. These studies are initially conducted using in silico and in vitro methods as well as animal studies, and more def nitive in ormation is obtained during clinical drug development. The basic principles investigated in the course o these studies are described in Chapters 3 (Pharmacokinetics) and 4 (Drug Metabolism). The systemic exposure o a drug candidate in animals is typically determined in pharmacokinetic studies, in which the concentration o drug in the systemic circulation is

C h a p t e r 5 1 Drug Discovery and Preclinical Development

TABLE 51-4 Examples o E f cacy Models Used in Drug Discovery DRUG EXAMPLE

DISEASE

ANIMAL MODEL

Cancer

Tumor xenografts in nude mice

Cisplatin

Diabetes

Genetically predisposed rodents (Zucker Diabetic Fatty Rat)

Insulin Metformin Thiazolidinediones

Hypercholesterolemia

Genetically hypercholesterolemic rats/mice Diet-induced hypercholesterolemia

Statins

Obesity

db/db and ob/ob rats

Orlistat Rimonabant Sibutramine

Postmenopausal osteoporosis

Ovariectomized rats

Bisphosphonates SERMs (raloxifene) Teriparatide

Rheumatoid arthritis

Collagen-induced arthritis

Anti-TNF antibodies

SERM, selective estrogen receptor modulator; TNF, tumor necrosis factor.

measured at various time points a ter administration. Important parameters include the maximal level o systemic exposure (Cmax), the time a ter drug administration at which the maximal systemic exposure occurs (Tmax), the overall systemic exposure during a treatment interval (area under the time-concentration curve [AUC]), and the length o time over which the drug remains in the circulation (hal -li e or T1/2). These parameters are measured or di erent administered dose levels and are also evaluated or acute (singledose) and chronic (repeat-dose) administrations. The tissues to which the drug distributes and the routes o excretion o the drug are typically measured by administering radiolabeled drug and then measuring the radioactivity levels in the di erent organs and body uids. As described in Chapter 4, metabolism or biotrans ormation re ers to the processes by which biochemical reactions alter drugs within the body. As drug discovery and development proceeds, there is a continual accrual o data to understand these processes or a candidate drug. Initial studies are o ten conducted in vitro, using animal and human liver microsomes (cellular ractions containing smooth endoplasmic reticulum) or hepatocytes as the source o the drugmetabolizing enzymes. Measured parameters include the metabolic stability o the drug and its ability to inhibit or induce important drug-metabolizing enzymes and cellular drug transporters. The latter studies help to assess the potential o the molecule to cause metabolic drug–drug interactions. Later in drug development, studies are conducted to characterize the metabolic ate o the candidate drug in both animals and humans. In addition, ormal drug–drug interaction clinical studies are per ormed to determine whether the candidate drug is likely to a ect the metabolism o other drugs that are already in clinical use or the indicated disease state.

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Toxicology Animal toxicity studies are conducted to determine the conditions (doses, dosing regimen, route o administration) under which it is sa e to initiate clinical trials with the drug candidate and ultimately to market the drug in the intended patient population. Studies o increasing duration and complexity are completed as the molecule proceeds through clinical drug development. The animal toxicity testing program is customized based on the desired therapeutic goal. For example, a drug designed to be used acutely in a critical care setting would require only short-term animal studies, whereas an agent intended or chronic use would require studies encompassing nearly the li etime o the animal. Because these animal toxicity studies are critical or an accurate assessment o the potential risks to clinical trial subjects rom administration o the drug candidate, they are governed by a complex set o regulations. To ensure the quality o the study data, pivotal toxicology studies that directly support a clinical trial must be conducted under regulations called the Good Laboratory Practices (GLP). Many drug discovery organizations will per orm an initial, limited assessment o the molecule’s toxicity during lead optimization. At this stage, the toxicity assays may involve in vitro assays designed to assess cytotoxicity or screen or o target receptor binding, in silico and in vitro studies designed to assess the potential o the molecule to alter DNA (genotoxicity testing), in vitro and in vivo studies o the potential o the molecule to a ect the cardiovascular system (cardiovascular pharmacology testing), and studies o the toxicity o the molecule in short-term animal studies. These studies may provide insights into the nature and mechanisms o potential toxic e ects o the molecule. Unacceptable target-organ toxicity ( unctional and/or histopathologic) may be a requent source o molecule termination at this phase o drug development. As a molecule proceeds into the testing required or clinical trial authorizations, a more comprehensive set o toxicity studies is conducted. Some o the most important sa ety data derive rom repeat-dose toxicity studies . In general, these studies are conducted in both a rodent (e.g., rat or mouse) and a nonrodent (e.g., dog or monkey) species. For smallmolecule drugs, the species are chosen most o ten based on the similarity o the number and identity o metabolites produced during ex vivo exposure o the drug to human and animal liver microsomes and/or hepatocytes. Toxicity species or biotherapeutics are chosen based on demonstration o the pharmacologic responsiveness o the species to the drug. Animals in these studies are administered various dose levels o the molecule or periods o time that depend on the duration o the proposed clinical trial (e.g., 2 weeks to 1 year). Repeat-dose toxicity studies evaluate body weight, clinical signs, and clinical laboratory parameters (hematology, clinical chemistry, and urinalysis). Histologic evaluation o all organ systems is also per ormed. Sa ety pharmacology studies are employed to assess potential adverse drug e ects on the central nervous, cardiovascular, and respiratory systems. Genotoxicity is thoroughly assessed by evaluating drug e ects on mutational status (nucleotide sequence changes) and clastogenicity (chromosomal damage). Animal studies are also per ormed to characterize e ects on ertility, reproduction, and development and to study the ability o the drug to induce tumors in animal models. I unique human metabolites are identif ed that are not produced by the selected toxicity species, the potential toxicity o those metabolites must be assessed in a separate toxicology study.

930 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

In sum, the results o these comprehensive animal studies identi y the potential toxicities that could occur upon administration o the drug to humans and assess the systemic exposures and durations o treatment that could potentially be related to those adverse e ects. An additional desired outcome is to identi y clinically translatable biomarkers that would enable monitoring o patients or early detection o the toxicities that have been identif ed in the animal studies.

Development Chemistry: Chemical Synthesis, Scale-Up, and Manufacturing An e ective chemical synthesis must satis y several requirements. Ideally, it should require ew synthetic steps. Each

additional step in a synthesis increases the possibility o impurities, decreases the yield (the amount o material obtained at the end o the synthesis), and increases the cost. I multiple isomers o a compound are possible products o the synthesis, then a synthesis that produces only the target isomer is pre erable. Finally, the synthesis should be amenable to scale-up. Two techniques, retrosynthetic analysis and convergent synthesis , aid in the establishment o an e ective synthetic scheme. In a retrosynthetic analysis, key steps are developed by examining important structural elements in the f nal product and f guring out how specif c reactions could lead to the product (Fig. 51-3A). This procedure is per ormed iteratively so that a complex f nal molecule is reduced to

A

H

O

OH

H

O S imple

Comple x

OH B

Cl

NH2

F O

1. KNO 3 , H2 S O 4 2. Cs 2 CO 3 , is opropa nol

Cl

O

Cl

N N

Cl

O2N

S

Na H DMF/DMS O

Cl

O

Cl 3. 4-pyridine boronic a cid 4. P tO 2 , H2 5. (Boc)2 O

O

S

N H

N N

Cl

O

NH Cl

6. P d(OAc)2 , xa niphos 7. TFA

O

S

N H

N N

N H

O

O Ceritinib

FIGURE 51-3. Retrosynthetic analysis and convergent synthesis of a complex molecule. A. A retrosynthetic analysis o a complex molecule, such as the illustrated bicyclic compound, allows the identif cation o simple starting materials such as cyclohexene. Analysis o the structural element (blue) demonstrates the creative process required to envision how a complex structure could be deconstructed into its component parts. The structure in the blue box illustrates the thinking required when deconstructing a molecule. These simple starting materials can then be combined in a series o steps to create the complex molecule. B. Retrosynthetic analysis and subsequent convergent synthesis were used to synthesize the ALKpathway inhibitor ceritinib (see Chapter 40).

C h a p t e r 5 1 Drug Discovery and Preclinical Development

simpler intermediates, as was done in the convergent synthesis o the ALK pathway inhibitor ceritinib (Fig. 51-3B). Flow chemistry is a new technology that has greatly simplif ed process chemistry practices. Each ow reactor contains reagents and catalysts to e ect a single chemical reaction. As the starting material ows through the f rst reactor, it is trans ormed to the desired end product and is then ready to ow into the next reactor, poised or the next chemical transormation. This technology can be applied to a linear synthesis or a convergent synthesis; in a convergent synthesis, two or more individual parts o a molecule are synthesized separately, and the parts are assembled only near the end o the synthesis (Fig. 51-4; see also Fig. 51-3B). Convergent synthesis increases the overall yield o the synthesis by reducing the number o linear steps required and allows the synthesis o each key component o the f nal product to be optimized individually. Retrosynthetic analysis and convergent synthesis are complementary and are o ten employed together in planning the chemical synthesis o a compound. Flow chemistry can also be developed in a discovery-chemistry setting and then trans erred to the process-chemistry setting. For early drug development, the goal o development chemistry is to generate enough product to meet the demands o chemical and biological characterization, particularly or animal toxicology and ormulation studies. As the scale o the need increases, the synthesis strategy must evolve. For example, a chemical synthesis o ten starts out using available raw materials, which may include expensive specialty chemicals. However, as the scale o the synthesis increases, these reagents must be replaced with cheaper (and/or sa er) alternatives. Furthermore, in an early synthetic scheme, each intermediate is typically isolated, purif ed, and characterized to ensure that each sequential step o the synthesis is e ective. However, as chemists gain more experience with the synthesis, multiple steps may be combined without isolating intermediates or puri ying the products o each reaction, in a so-called one-pot synthesis . Once the synthesis strategy o a drug candidate has been ully developed, process chemists must then adapt the

Line a r flow s ynthe s is A

Flow re a ctor

B

Flow re a ctor

C

Flow re a ctor

G

Flow re a ctor

D

Conve rge nt flow s ynthe s is E

Flow re a ctor

F

Flow re a ctor

H I

Flow re a ctor

J

Flow re a ctor

K

Flow re a ctor

L

FIGURE 51-4. Flow chemistry. Each f ow reactor contains reagents and catalysts to e ect a single chemical reaction. Flow chemistry can be applied to linear synthesis or convergent synthesis o the desired compound. In a linear synthesis, each component is added sequentially. In a convergent synthesis, each component is assembled separately and then combined in the last step. The convergent synthesis approach generally results in higher yields. The arrows indicate sequential synthetic reactions.

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synthesis or large-scale commercial manu acturing. This process must be initiated be ore a drug is approved, because the approval process requires that several batches o the drug be success ully manu actured, ormulated (see below), and rigorously tested or quality and stability. A pharmaceutical company must also be prepared to meet market demands immediately a ter approval, which means that a manu acturing process must be established be ore the commercial launch o the drug. The process chemist must also ensure that the synthesis is sa e and meets environmental regulations or emissions and disposal o waste. This may preclude the use o certain solvents that are commonly used by synthetic chemists in small-scale syntheses.

Formulation Drugs must be manu actured in a orm that can be administered to animals and humans in a measured dose. The type o ormulation depends on the intended route o administration (Table 51-5). Enteral formulations , which include oral, sublingual, and rectal dosage orms, are designed to be absorbed across portions o the digestive tract. Parenteral formulations include intravenous, intramuscular, and subcutaneous injections; transdermal patches; and inhaled agents. The pre erred route o administration is determined by many variables, including the drug’s stability and its pharmacokinetic properties o absorption, distribution, metabolism (including f rst-pass metabolism), and excretion. Oral dosage orms are avored or drugs that are relatively stable in the digestive tract, are not rapidly metabolized in the liver, have high oral bioavailability, and do not require an immediate action. Parenteral dosage orms are pre erred or drugs that must be ast-acting and are more reliably absorbed by nonenteral than by enteral routes. Macromolecules, which generally have little or no oral bioavailability, are typically administered via injection (see Chapter 54, Protein Therapeutics). Most drugs are administered orally in either tablet or capsule orm. In addition to the measured dose o the drug, most tablets contain binders , which keep the components together, and stabilizers , which enhance the drug’s shel li e. For acid-sensitive drugs, it is o ten possible to coat the tablet with an enteric coating that is acid-resistant but dissolves in the intestine. Formulation chemists can also manipulate the rate at which the tablet or capsule dissolves or releases its contents, thus enabling “sustained-release” ormulations in which the drug is released slowly over the course o hours (see Chapter 55, Drug Delivery Modalities). Drugs ormulated into liposomes are also being increasingly used as mechanisms or controlling drug release in vivo. Doxil® is a liposome-encapsulated orm o doxorubicin used to treat Kaposi’s sarcoma. A drug’s absorption prof le and f rst-pass metabolism are typically not issues or drugs delivered intravenously. However, the drug must be dissolved in a vehicle, usually water. Moreover, the solution must be made isotonic with plasma by adding osmotically active compounds such as saline, dextrose, or mannitol, so that the solution does not cause hemolysis. The solution must also be sterile or intravenous injection. Finally, a drug is o ten less stable in solution than as a solid, so ormulation chemists must test its stability in solution. I the drug is unstable, it may be prepared as a lyophilized powder that can be dissolved in water or bu er immediately be ore administration.

932 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

TABLE 51-5 Advantages and Disadvantages of Common Formulations FORMULATION

ADVANTAGES

DISADVANTAGES

EXAMPLES

Oral

Ease o administration

Slow absorption First-pass metabolism Reduced bioavailability

Acetaminophen Oxycodone Pravastatin

Sublingual

Rapid action No f rst-pass metabolism

Few drugs are absorbed by this route

Nitroglycerin

Rectal

Rapid action No f rst-pass metabolism

Uncom ortable

Morphine

Intravenous

Rapid action High bioavailability Can control dose easily

Risk o in ection Uncom ortable Must be administered by trained personnel

Lidocaine Morphine Tissue plasminogen activator

Intramuscular

Sustained release possible

Uncom ortable Adverse reaction possible

Meperidine Growth hormone

Subcutaneous

Slow action

Poor adherence

Insulin

Transdermal

Sustained release No f rst-pass metabolism

Poor absorption Slow action

Estrogen Nicotine (patch)

Inhalation

Large sur ace area or absorption Convenience (no injection)

Inconvenience (device)

Albuterol Glucocorticoids (asthma)

Enteral

Parenteral

CONCLUSION AND FUTURE DIRECTIONS The discovery and development o new drugs is a complex, interdisciplinary process that o ten requires 10 or more years and up to or exceeding a billion dollars. Researchers start by searching or a biologically active compound. This may involve a compound-centered approach or a target-centered approach. New pharmacologic targets are currently being identif ed by gene sequencing, by analysis o genetic actors that predispose to disease, by gene knockout experiments in laboratory animals, and by other techniques. In addition, in ormation about genetic polymorphisms may enable the products o specif c, mutant genes to be the targets o new drugs (see Chapter 7, Pharmacogenomics). Methods to assess potential sa ety are also rapidly evolving and signif cant emphasis is being placed on developing in vitro methods that model complex physiologic systems (e.g., organ-on-a-chip) more accurately than current cell-based assays. In silico systems, already used to provide in ormation regarding the potential o a chemical to cause genotoxicity, continue to evolve and be implemented to make predictions regarding a variety o toxicologic outcomes.

Acknowledgment We thank the late Armen H. Tashjian, Jr. or his invaluable contributions to this chapter in the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy.

Suggested Reading Cook D, Brown D, Alexander R, et al. Lessons learned rom the ate o AstraZeneca’s drug pipeline: a f ve dimensional ramework. Nat Rev Drug

Discov 2014;13:419–431. (One major pharmaceutical company’s insight ul analysis o their research and development productivity, including a discussion o key technical determinants o project success.) Drews J. Drug discovery: a historical perspective. Science 2000;287: 1960–1964. (Historical description o the major methods o drug discovery.) International Con erence on Harmonization: guidance on nonclinical sa ety studies or the conduct o human clinical trials and marketing authorization or pharmaceuticals 2009. http://www.ich.org/f leadmin/Public _Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Step4 /M3_R2__Guideline.pd . (Describes the types o animal studies required by regulatory authorities to support clinical testing and registration o pharmaceuticals.) Lacey D, Boyle W, Simonet W, et al. Bench to bedside: elucidation o the OPG–RANK–RANKL pathway and the development o denosumab. Nat Rev Drug Discov 2012;11:401–419. (Describes the drug discovery and development approach or denosumab.) Medina-Franco JL, Giulianotti MA, Welmaker GS, Houghten RA. Shi ting rom the single to the multitarget paradigm in drug discovery. Drug Discov Today 2013;18:495–501. (Many drugs are e ective because they interact with multiple targets. This article reviews alterations to the drug discovery process to allow identif cation o compounds that interact avorably with multiple targets.) Pritchard JF, Jurima-Romet M, Reimer ML, Mortimer E, Rol e B, Cayen MN. Making better drugs: decision gates in nonclinical drug development. Nat Rev Drug Discov 2003;2:542–553. (Explores the key scientif c questions that are addressed during drug discovery and preclinical development.) Sams-Dodd F. Strategies to optimize the validity o disease models in the drug discovery process. Drug Discov Today 2006;11:355–363. (Discusses how to optimize animal models o human disease to allow selection o better drug candidates.) U.S. Food and Drug Administration, U.S. Department o Health and Human Services. Innovation or stagnation: challenge and opportunity on the critical path to new medical products. March 2004. http://www. da.gov/oc /initiatives/criticalpath/whitepaper.pd . (Discusses current challenges and opportunities in the development o new drugs, biologic products, and medical devices.)

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Controlled clinical trials provide the scientif c and legal basis by which regulatory authorities around the world evaluate new prescription drugs and approve them or sale. In the United States, the regulatory review o drugs and devices is the responsibility o the US Food and Drug Administration (FDA). Over the past 50 years, improved methods or largescale clinical studies have precipitated a shi t toward evidence-based medicine. The increased emphasis on clinical trials to appropriately assess the sa ety and e f cacy o new drugs has resulted in a dramatic rise in the costs associated with drug development. According to the Pharmaceutical Manu acturers o America (PhRMA), the overall drug development process rom discovery to approval o a new drug takes an average o 10–15 years and an estimated cost o $1 to $2 billion; the clinical development phase o this work is typically in the range o 6 to 7 years. Moreover, only about 1 in 10 drugs that enters the clinic or testing ultimately receives regulatory approval and is marketed. Given the tremendous cost and duration o clinical drug development, it is imperative that every e ort is made to plan care ully and

a

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Mee ch chan a isms to Expedite Development o Therapeutics e or Serious Condi ond tionss . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942 DRUG APPROVAL PROCESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942 FDA Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942 FDA FD A Ap Appr proval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943 Appr Ap proval in Other Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 9433 Expanded Accesss of a n Inve Invest stiggational Drug or an Approved Drug with Restricted Distrib ibutiionn . . . . . . . . . . . . 94 9433 Drug Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943 Drug Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943 Additional Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 944 REGULATORY ASPECTS OF DRUG PRODUCTION AND QUALITY CONTROL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 944 GENERIC DRUGS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 944 NONPRESCRIPTION DRUGS AND SUPPLEMENTS . . . . . . . . . . 944 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 945 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .945 P

Patent Paten ON & CASE ON . . . . . . . . . . . . . . . . . Patent . . . . . . . . . . 933–934 Le ga Le gINTRODUCTIO al appl ap pppl pliliicat ic atio tionn gran gr ante ted t d HIST HI STOR ORY Y OF U US S FO FOOD O AND D DRUG G LA LAW W . . . . . . . . . . . . . . . . . . . . 935 ETHICS IN CLINICAL DRUG INVESTIG GAT ATIO I N . . . . . . . . . . . . . . . 936 DRUG EVALUATION AND CLINICAL DEVELOPME DEVELOPMENT MENT. . . . . . . . . 936 Authorizations to Initiate Clinical Trials . . . . . . . . . . . . . . . . . . 93 9366 Clinical Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 9377 Targ Ta rget et Pro rodductt Prof le . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937 Development o a Clinical Triall . . . . . . . . . . . . . . . . . . . . . . . 937 Phase 1 Studiess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939 Phase 2 Studiess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939 Phase 3 Studiess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939 Clinical Pharmacologyy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940 Pediatric Studiess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940 Challenges in the Development o Drugs to Treat Rare Diseasess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940 Adaptive Trial Designss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941 Success ul Drug Development: Design and Execution. . . 941

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execute e ectively. Drug development programs must be well designed not only to appropriately demonstrate sa ety and clinical e f cacy but also, through the use o appropriate biomarkers, pharmacodynamic markers, and sa ety monitoring, to allow or the early discontinuation o development o drugs that are destined to ail. This is an exciting and challenging time to be involved in drug development. Major advances in the biological sciences have yielded a much greater understanding o the molecular basis o many diseases and provide the opportunity to have an unprecedented impact on the alleviation o human su ering. However, translating these scientif c advances into new and more e ective therapies or human diseases has proven to be daunting. In the 10 years between 1994 and 2003, there was an average o 33.6 new drugs approved per year by the FDA; in the ollowing 10 years up to 2013, there has been an average o slightly more than 26 approvals per year, suggesting a relative stagnation in the rate o new drugs being approved and made available to patients. To close this apparent gap between the acceleration o innovative basic-science discoveries and the stagnating rate o approvals o innovative new therapies will require diligent clinical drug development programs that 933

Ph Phas asee 4

934 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

Fo r m o s t o the latte r h al o the twen tieth ce n tu ry, a d va n ce s in th e p h a rm a co lo g ic tre a tm e n t o m a lig n a n cie s re lie d p rim a rily o n th e u s e o cyto toxic a g e n ts th a t ta rg e t va rio u s a s p e cts o ce llu la r via b ility a n d p ro li e ra tio n , w ith o n ly a n a rro w w in d o w b e tw e e n th e d o s e s n e ce s s a ry o r th e killin g o tu m o r ce lls a n d th o s e th a t kill n o rm a l ce lls (i.e ., th e th e ra p e u tic w in d o w ). In th e 1970s a n d 1980s , s tu d ie s b y s cie n tis ts s u ch a s Mich a e l Bis h o p a n d Ha ro ld Va rm u s le d to th e id e n tif ca tio n o re trovira l o n co g e n e s , w h ich a re m u ta te d o rm s o n o rm a l ce llu la r g e n e s th a t co n tro l ce ll via b ility, d i e re n tia tio n , a n d p ro li e ra tio n . Ma ny o th e s e o n co g e n e s w e re s h o w n to e n co d e m u ta te d p ro te in kin a s e s invo lve d in th e p a th o g e n e s is o h u m a n m a lig n a n cie s . Ch ro n ic m ye lo g e n o u s le u ke m ia (CML) is o n e s u ch m a lig n a n cy th a t is a irly w e ll u n d e rs to o d a t th e m o le cu la r le ve l. CML h a s b e e n d e m o n s tra te d to d e p e n d o n a ch ro m o s o m a l tra n s lo ca tio n , th e s o -ca lle d Ph ila d e lp h ia ch ro m o s o m e , ch a ra cte rize d b y a re cip ro ca l tra n s lo ca tio n b e tw e e n th e lo n g a rm s o ch ro m o s o m e s 9 a n d 22, w h ich le a d s to th e re a rra n g e m e n t a n d d ys re g u la tio n o a p a rticu la r tyro s in e kin a s e ca lle d c-a b l. Th e s e f n d in g s s e t th e s ta g e o r a n e xtra o rd in a rily s u cce s s u l co lla b o ra tio n b e tw e e n Bria n Dru ke r, a n a ca d e m ic o n co lo g is t a t Ore g o n He a lth Scie n ce s Un ive rs ity, a n d Nick Lyd o n , a p h a rm a ce u tica l re s e a rch e r a t Nova rtis . Dru ke r h a d a re s e a rch o cu s o n tyro s in e kin a s e b io lo gy w ith a n e m p h a s is o n f n d in g a n e e ctive tre a tm e nt o r CML b y ta rg e tin g th e c-a b l tyro s in e kin a s e , a n d Lyd o n h a d a re s e a rch o cu s o n id e n ti yin g s p e cif c in h ib ito rs o p ro te in tyro s in e kin a s e s . Dru ke r a n d Lyd o n id e n tif e d a s m a ll m o le cu le , co d e n a m e d S TI-571 (imatinib), th a t e e ctive ly in h ib ite d c-a b l a s w e ll a s a t le a s t tw o o th e r tyro s in e kin a s e s , c-kit a n d p la te le t-d e rive d g ro w th a cto r re ce p to r B. S tu d ie s in ce ll cu ltu re d e m o n s tra te d s e le ctive toxicity o STI-571 in ce lls co n ta in in g d ys re g u la te d c-a b l, a n d p re clin ica l s tu d ie s in th e a p p ro p ria te a n im a l m o d e ls co n f rm e d th is a ctivity. Pre clin ica l to xico lo g y s tu d ie s in ra ts , d o g s , a n d m o n ke ys d e s crib e d th e h e m a to lo g ica l, re n a l, a n d h e p a to b ilia ry to xicity o im a tin ib . A p h a s e 1 s tu d y in 83 CML p a tie n ts s h o w e d th a t o ra l d o s in g in th e ra n g e ro m 25 to 1,0 0 0 m g /d a y d id n o t ca u s e d o s e -lim itin g to xicity. Ad d itio n a lly, th e s tu d y d e m o n s tra te d th a t im a tin ib h a d e xce lle n t o ra l b io a va ila b ility a n d a p h a rm a co kin e tic p ro f le s u ch th a t o n ce -d a ily o ra l d o s in g co u ld

include rigorous, well-controlled trials, integrated clinical development plans, the use of novel statistical methods, adaptive clinical trial designs, and the inclusion of novel pharmacodynamic and other biomarkers at the various stages of drug development. Coordinated teams of experts will be needed to integrate these clinical development programs with the related processes of drug discovery, preclinical development, regulatory approval, and ultimately patient treatment. In addition,

a ch ie ve s u s ta in e d p la s m a le ve ls a t co n ce n tra tio n s th a t h a d b e e n s u f cie n t to in h ib it c-a b l in p re clin ica l m o d e ls . Th re e o p e n -la b e l, s in g le -a rm p h a s e 2 s tu d ie s in 1,027 CML p a tie n ts a t va rio u s s ta g e s o d is e a s e p ro g re s s io n a ll s h o w e d m a rke d im a tin ib a ctivity, a s a s s e s s e d b y h ig h cyto g e n e tic re s p o n s e ra te s a n d h e m a to lo g ica l re s p o n s e ra te s , w ith le s s toxicity th a n th a t typ ica lly o b s e rve d w ith th e a va ila b le s ta n d a rd o ca re (in te r e ro n -a lp h a ). Ba s e d o n th e m a rke d a ctivity o im a tin ib in p a tie n ts w ith a d va n ce d d is e a s e a n d in p a tie n ts w h o h a d a ile d f rs t-lin e th e ra p y w ith in te r e ro n -a lp h a , im a tin ib re ce ive d a cce le ra te d a p p rova l b y th e FDA in Ma y 20 01, a te r o n ly 3 m o n th s o re vie w . Th is re p re s e n te d o n e o th e a s te s t re vie w s e ve r p e ro rm e d b y th e FDA a n d a ls o m a rke d th e a p p ro va l o th e f rs t s e le ctive ly ta rg e te d ca n ce r th e ra p y (i.e ., th e ra p y d ire cte d a t a ta rg e t th a t is s p e cif ca lly d ys re g u la te d in CML ce lls co m p a re d to n o rm a l ce lls ). Acce le ra te d a p p ro va l wa s g ra n te d ra th e r th a n u ll a p p rova l s in ce cyto g e n e tic re s p o n s e s a n d h e m a to lo g ica l re s p o n s e s a re s u rro g a te clin ica l e n d p o in ts th o u g h t to p re d ict clin ica l b e n e f t w ith a re a s o n a b le like lih o o d b u t a re n o t a n u ltim a te clin ica l e n d p o in t s u ch a s s u rviva l. Un d e r a cce le ra te d a p p ro va l, th e s p o n s o r (in th is ca s e , Nova rtis ) wa s re q u ire d to co n d u ct p o s t-a p p rova l s tu d ie s to ve ri y a n d co n f rm th e clin ica l b e n e f t o im a tin ib . Nova rtis th e n co n d u cte d a n d s u b m itte d o r a p p ro va l a ra n d o m ize d p h a s e 3 s tu d y co m p a rin g im a tin ib to co m b in a tio n th e ra p y w ith in te r e ro n -a lp h a a n d cyta ra b in e in p a tie n ts w ith n e w ly d ia g n o s e d CML, w ith a p rim a ry e n d p o in t o ove ra ll s u rviva l. Nova rtis a ls o co m m itte d to p e r o rm p h a s e 1 a n d p h a s e 2 s tu d ie s o im a tin ib in ch ild re n . Ba s e d o n lo n g -te rm o llo w-u p o p a tie n ts ro m th e e a rlie r p h a s e 2 tria ls a s w e ll a s n e w d a ta ro m th e p h a s e 3 tria l a n d th e p e d ia tric tria ls , im a tin ib u ltim a te ly re ce ive d u ll a p p rova l o r a ll s ta g e s o CML in b o th a d u lts a n d ch ild re n .

Questions 1 . What ethical standards govern the relationship between physicians and patients in clinical research? 2 . What are the critical elements to be considered in developing a clinical trial protocol? 3 . What data do the FDA review when considering approval of a new drug?

major health authorities such as the FDA have implemented programs to work more effectively with industry in drug development while preserving their remit to protect the public health. Drug discovery and development remains a lengthy, high-risk, and complex process. It has been estimated that of every 5,000 to 10,000 chemically synthesized molecules that are screened as potential drugs, only one becomes an

C h a p t e r 5 2 Clinical Drug Evaluation and Regulatory Approval 935

approved drug. The previous chapter (Chapter 51, Drug Discovery and Preclinical Development) outlines the preclinical phase o drug development rom target identif cation to candidate selection. This chapter describes the process by which new candidate drug molecules are evaluated in clinical trials and approved or marketing and sale in the United States.

HISTORY OF US FOOD AND DRUG LAW Drug development, testing, and approval is a lengthy process, the major milestones o which are shown in Figure 52-1. Achievement o each o these milestones requires the cooperation o researchers, clinicians, patients, pharmaceutical or biotechnology companies, and government regulators. The development o a new drug or biotherapeutic is a highly regulated process that has evolved considerably during the last century. Several public health crises have led to the development o current laws and regulations, including: 1. The public outcry over the unsanitary and unsa e conditions o the meatpacking industry: this resulted in the Pure Food and Drugs Act in 1906, which prohibited interstate commerce in adulterated and misbranded ood and drugs. 2. The death o more than 100 people a ter consuming “Strep-Elixir,” an untested product containing a sul onamide and a chemical analogue o anti reeze: this resulted in the passage o the Food, Drug, and Cosmetic Act in 1938, which, among other things, required approval o all new drugs by the FDA prior to marketing. Sponsors submitted an application or approval and, unless the FDA determined that the drug was unsa e within 180 days, the drug could be marketed. 3. The discovery that thalidomide, used to treat morning sickness, caused birth de ects in large numbers o babies born in Europe: this resulted in passage o the Kefauver-Harris

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addition to sa ety prior to drug approval and mandated reporting o adverse events. Additionally, the Amendments stated that patients must provide in ormed consent to participate in clinical trials, gave the FDA authority to regulate prescription drug advertising, and introduced good manu acturing practice standards. 4. In response to widely publicized sa ety issues with COX-2 inhibitors and several other drugs, Congress passed the FDA Amendments Act (FDAAA) o 2007. One aspect o the FDAAA provides enhanced authority to the FDA to manage the sa ety o approved drugs. In particular, the FDA has ocused on the implementation o Risk Evaluation and Mitigation Strategies (REMS) or selected new drugs as well as drugs that are already approved. The objective o REMS is to put in place measures to ensure that a drug or biologic product is dispensed and utilized in such a manner as to ensure that its benef ts outweigh its risks. 5. The FDA Safety and Innovation Act (FDASIA) of 2012 expanded FDA authority in several ways. It reauthorized prescription drug and medical device user ees and implemented generic drug and biosimilar biologic products user ees. These ees are mandated by law and are used, in part, to support the FDA’s review o marketing applications. FDASIA also introduced a new mechanism to expedite development and review o promising new drugs or serious and li e-threatening diseases. These drugs are given Breakthrough Therapy designation. This mechanism builds on past FDA programs to assist sponsors in drug development. Additionally, FDASIA expands the FDA’s ability to obtain patient input into the drug development and review process and, recognizing the increasing globalization o drug supply and sourcing ( or both f nished product and active ingredients), it expands FDA authority to ensure the sa ety and availability o this supply.

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s

m e n o c a C o r

m p

s e e l i t

n e

n

va e t

a

G

Patent granted

a

Patent application

P

Le ga l

a

r

x

i

b

c

l

p

i

e

s

r

e

Develop manufacturing Develop QA/QC program, GMP practices

Ma nufa cturing

e

P

e

s

t

d

r

i

a

a

t

Phase 1 trials

t

IND filed

Clinica l

d

p

l

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a

E

p

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Toxicology studies

Toxicology

FIGURE 52-1. Life cycle of drug approval. The li e cycle o approval or a new drug is complex, ranging rom 8 to 15 years or completion. Drug discovery, discussed in Chapter 51, produces a new drug molecule. The f rst patents are usually f led at this stage and are granted several years later. The drug development process requires that biological characterization and toxicology studies in animals are conducted be ore an IND can be f led. In turn, an IND is required or the start o clinical trials. At the conclusion o success ul clinical development, a drug company f les a NDA/BLA, which is reviewed by the FDA. Once a drug is approved, it must be monitored or sa ety or the remainder o its li espan (postmarketing surveillance). The f rst o the drug’s patents expires 20 years a ter its application. ANDA, Abbreviated New Drug Application; FDA, US Food and Drug Administration; GMP, Good Manu acturing Practice; IND, Investigational New Drug application; NDA/BLA, New Drug Application/Biologics License Application; QA/QC, quality assurance and quality control.

936 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

In the United States, the FDA Center or Drug Evaluation and Research (CDER) and the FDA Center or Biologics Evaluation and Research (CBER) are responsible or regulating the development and approval o new medicines.

ETHICS IN CLINICAL DRUG INVESTIGATION The development o new therapeutics to treat human diseases requires research to be conducted on human subjects, either normal volunteers (typically in phase 1 trials) or in patients with the disease or which the new treatment is being investigated. Any time research is conducted in humans, it is essential that every e ort is made to protect their sa ety. Regulatory agencies around the world have codif ed standards o ethical behavior or all parties involved in clinical research, including clinicians, pharmaceutical companies, and medical institutions. The ethical relationship is governed by the notion that clinical trial research represents a partnership between investigator (physician) and subject (volunteer or patient). Four major ethical principles, established by the International Conference on Harmonization and the Declaration of Helsinki, support this partnership. These principles are as ollows: ■ ■

■

■

The trial must minimize the risks or participants. Provisions must be made or the overall care o the patient. The investigator is responsible or terminating the trial when the risks become incompatible with the goals o the trial. Adverse events must be reported immediately to an ethics or sa ety committee.

Investigators must obtain subjects’ informed consent. Inormed consent is not just a signed document but rather a process in which patients (1) are made aware o the potential risks and benef ts o the trial and (2) must make an in ormed decision to participate voluntarily in a clinical study. For patients with poor prognoses and or normal volunteers, in ormed consent encompasses the understanding that the research likely will not benef t them but may benef t uture patients. At the institutional level, the FDA relies on independent Institutional Review Boards (IRBs ) or Independent Ethics Committees (IECs ) to ensure the rights and wel are o those participating in clinical trials. FDA regulations mandate that clinical study protocols be reviewed or legal and ethical issues by an IRB/IEC. These regulations give IRBs/IECs the authority to approve, require modif cation o , or disapprove research on human subjects. Specif cally, the IRB/IEC must determine whether the proposed research: ■ ■

■ ■ ■

Minimizes potential risk to human subjects Poses risks that are reasonable relative to the anticipated benef t and potential scientif c gain o the research Includes equitable selection o subjects Provides or an e ective in ormed consent process Contains sa eguards or vulnerable populations, such as children and the mentally disabled

IRB/IEC oversight and approval begins be ore the commencement o human trials and continues or the duration o clinical trials. The membership o an IRB/IEC consists o f ve or more experts and laypersons rom various backgrounds. Federal regulations stipulate that IRB membership must include at least one member whose primary expertise is in a scientif c area, one member whose primary expertise is

in a nonscientif c area, and one member who is not a f liated with the institution overseeing the clinical research protocol. In addition, the other members’ qualif cations must be such that the IRB is able to evaluate research protocols in terms o institutional requirements, applicable law, standards o pro essional practice, and community attitudes. Thus, many IRBs include clergy, social workers, and attorneys as well as physicians, scientists, and other health care pro essionals. Clinical trials must be appropriately designed and rigorously executed in order to optimize the ratio o benef t to risk and to satis actorily answer the scientif c questions under study. Scientif c clinical trial design must include appropriate control or comparator arm(s), randomization and blinding, and sample size, among other elements (see below). Some institutions have a scientif c review committee that must approve all protocols involving human subjects to ensure that the protocol is appropriately designed to answer the questions being asked. To urther assure that the f ndings o clinical trials are accurate and credible and that the rights o clinical trial subjects are protected, regulatory agencies require that clinical trials leading to the approval o new drugs be conducted according to good clinical practices (GCP). Guidelines or GCP have been developed by the International Con erence on Harmonization to provide a standard or the design, conduct, recording o f ndings, monitoring o data, analysis, auditing, and reporting o results o clinical trials.

DRUG EVALUATION AND CLINICAL DEVELOPMENT The investigation o a new drug candidate comprises several phases, beginning with preclinical evaluation and typically proceeding through phase 3 clinical studies. At the conclusion o this process, the FDA may consider the molecule or approval as a new drug.

Authorizations to Initiate Clinical Trials Preclinical research and development establishes the poten-

tial e f cacy and sa ety o a compound or use in human trials. During this stage o testing, described in Chapter 51, a compound is studied to determine its biological actions, chemical properties, and metabolism, and a process is developed or its synthesis and purif cation. A major ocus o preclinical testing is determining whether the molecule has an acceptable sa ety prof le in animals prior to initiating testing in humans. The International Con erence on Harmonization has established requirements or the animal studies used to support di erent types o clinical trials. The primary studies used to support clinical drug development are animal toxicity studies and investigations on the absorption, distribution, metabolism, and excretion (ADME) o the compound. As described in Chapter 51, the duration o animal studies is determined by the length o the clinical trials to be undertaken. For this and other reasons, it is essential that there is close coordination among the preclinical and clinical scientists on the drug development team. Many potential drug candidates either do not proceed to human trials or are removed rom clinical testing due to adverse sa ety f ndings in animal studies. The preclinical research phase is also an important time to explore potentially important pharmacodynamic markers and other biomarkers that could help acilitate clinical development. The mechanism or seeking approval to initiate clinical trials in the United States is the submission o an Investigational

C h a p t e r 5 2 Clinical Drug Evaluation and Regulatory Approval 937

New Drug application (IND) to the FDA. The IND contains

data rom the preclinical studies, data rom prior clinical investigations (i available), the proposed protocol or human trials, and other background in ormation. The IND also contains a document re erred to as the Investigator’s Brochure (IB). The IB is provided to regulators, clinical investigators, and IRBs/ IECs; it represents a summary o all available in ormation on the investigational drug and may be several hundred pages in length. The IND must also contain in ormation on characterization, manu acture, and quality o the drug. INDs can be submitted by commercial sponsors with the ultimate goal o obtaining approval or marketing and sale o a new drug product or by individual investigators and academic centers. The latter are typically re erred to as Investigator Sponsored INDs . The IND is a “living document” and, at a minimum, is updated annually. The FDA must review the IND within 30 days and decide whether human trials may begin. Figure 52-2 is a owchart representing the process used by the FDA to review an IND.

Applicant (drug sponsor)

The areas o review include a chemistry review , a pharmacology/toxicology review , and a medical review . I the IND review does not identi y any sa ety concerns, the IND is considered open or active a ter the 30-day wait period. I the review reveals the potential or unreasonable risk to participants, the FDA contacts the sponsor, and a clinical hold is issued, preventing initiation o human studies. The sponsor must address any issues in question be ore the clinical hold is li ted. A clinical hold may be issued at any time during clinical drug development; this can be based on issues such as new f ndings rom animal studies, clinical data indicating an unacceptable risk prof le, or a f nding that a sponsor did not accurately disclose the risk o the study to investigators or subjects.

Clinical Development Given the time, cost, and risks associated with clinical drug development, it is imperative to plan care ully and execute meticulously. The goals o clinical drug development include: ■ ■

■

IND

■

Me dica l

Che mis try

S pons or s ubmits ne w da ta

Compre he ns ive re vie w

S afety a ccepta ble for s tudy to proce ed?

P ha rma cology/ toxicology

These goals are accomplished through the conduct o clinical trials. Each clinical trial must be designed to answer specif c questions. In turn, each trial should be part o an integrated development plan leading to the ultimate demonstration o sa ety and e f cacy in well-controlled trials.

Target Product Prof le NO

Clinical hold decision

YES

S tudy s ta rts

Assessment o the dose–response prof le Assessment o the toxicity prof le or a given dosing regimen Assessment o pharmacokinetic/pharmacodynamic relationships Establishment o the sa ety and e f cacy prof le in wellcontrolled studies in well-def ned patient populations

Notify s pons or

Comple te re vie ws

Notify s pons or of re vie w re s ults

FIGURE 52-2. Process of Investigational New Drug (IND) review. When an IND is f led by a sponsor, the FDA has 30 days to review the application. The ow diagram shows the process o internal review. Various disciplines within the FDA review the sponsor’s data package. These reviews culminate in a decision by the FDA as to whether the proposed clinical protocol is acceptable to proceed. I the sa ety is not deemed acceptable, the sponsor is notif ed that the IND is on clinical hold and trials may not proceed. The sponsor may submit additional data to support the sa ety o the proposed trial and a subsequent round o sa ety review is initiated. I the sa ety is deemed acceptable, the study may proceed a ter the 30-day review period. The FDA then completes its reviews and may provide the sponsor with additional comments on issues that may need to be addressed in later stages o drug development. Colored boxes correspond to actions by the drug sponsor; white boxes correspond to actions by the FDA.

The target product prof le (TPP) articulates the goal o the clinical development program. The key elements o the target product prof le include the primary indication, target patient population, route o administration, pharmaceutical ormulation, dosing schedule, e f cacy assessments, expected primary endpoint in pivotal trial(s), expected sa ety prof le, and key product characteristics, including those that may allow di erentiation compared to products already available. As the development plan un olds, data rom clinical trials and product development in orm the TPP and, as a result, aspects o the TPP are likely to evolve. However, it is important to understand what the minimally acceptable target product prof le is and to have the discipline to terminate development as quickly and responsibly as possible i it becomes clear that the minimally acceptable TPP will not be attainable.

Development o a Clinical Trial

By def nition, clinical trials involve studies o human subjects. The subjects may be normal volunteers or patients with specif c diseases; the trials may be interventional (i.e., patients receive therapies and/or undergo tests or procedures) or observational (e.g., a study o the natural history o a disease). No matter what the situation, the clinical investigator has an ethical responsibility to the subjects to ensure that all elements o a trial are optimally designed to maximize what will be learned. The key elements or consideration when developing any clinical trial are detailed in Table 52-1.

938 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

TABLE 52-1 Elements of a Clinical Trial Design 1. 2. 3. 4.

5. 6.

7.

8.

9.

Title o study Study hypothesis(es) including methods to test hypothesis(es) Study objectives Study design • Indicate any proposed substudies and their design aspects. • Indicate whether interim analyses are planned and their objectives (e.g., early dose selection, utility, stopping or success). Study rationale • Include how study f ts into overall product development plan. Study population • Speci y all inclusion and exclusion criteria. • Geographical considerations. • Special regional regulatory considerations. • Does trial design f t with usual medical practice in the region? Sample size • Calculated sample size, number o sites required, and number o subjects per site. • Parameters used to determine sample size estimate, including the detectable di erence and a power statement. Enrollment period • Total projected time or enrollment period, including detailed timing or various periods (e.g., screening). • Are there any requirements or site enrollment rate? Study duration • Speci y both the study duration and the duration o treatment.

Trial protocols must be structured to provide reliable answers to specif c questions. Each test and procedure in the study should have a clearly def ned purpose that f ts into the integrated development plan. Among the most important issues to consider are: ■

Determination o the appropriate balance or the inclusion and exclusion criteria. It is o ten desirable to have a relatively homogeneous patient population to allow or better planning and interpretation o study results. However, this needs to be balanced by the understanding that patients and diseases are requently heterogeneous. By restricting study participation too severely, one runs the risk o developing a trial that will be di f cult to enroll and the results o which may be applicable only to a narrow subset o patients with a particular disease, when, in act, a broader population could potentially benef t. Furthermore, product labeling typically re ects only those patients with characteristics matching those in the clinical trials in which sa ety and e f cacy were demonstrated.

It is not uncommon or regulatory authorities to raise the ollowing issues during discussion and review o trials designed to support marketing approval: ■

■

■

■

Which prospectively def ned outcome variables are easible to measure and are scientif cally valid Whether a control group is easible and what comparator drugs, i any, need to be used in control-group subjects The ease with which subjects and investigators may be blinded (see the ollowing discussion) The numbers o participating trial sites and subjects

When developing a clinical trial protocol, one must assess chance, bias, and other con ounding actors that might a ect the trial and incorporate measures to address these issues.

10. Randomization (i any) • Speci y randomization scheme or ratio. 11. Rationale or dose selection 12. Study medications (or product) • Speci y all (test and comparator) medications to be used and how they will be obtained. 13. Study medication administration • Speci y how and when medication will be administered (i titration or other dose modif cation is permitted or to be used also, explain). 14. Pharmacokinetic/pharmacodynamic measures • Speci y pharmacokinetic/pharmacodynamic measures, including any special procedures involved. 15. E f cacy measures • Speci y details o all primary and secondary e f cacy measures, including any special procedures. • I measure is a detailed scale or questionnaire, provide it in an appendix. 16. Sa ety measures • Speci y all sa ety measures, including specif c clinical laboratory tests. • Identi y any other procedures or measurements to be done. 17. Statistical analysis plan • Speci y key statistical methods planned to test the proposed hypotheses and/or study objectives; sample size calculations and assumptions.

The likelihood o obtaining alse-positive or alse-negative outcomes can be minimized by appropriately powering a study to assure an adequate sample size. Subject bias can o ten be countered by providing a placebo control, an inert substance with the same appearance as the drug under investigation. Observer bias can be countered by blinding, usually by coding the drug and placebo so that their identities are masked; thus, the investigators cannot know which treatment is being administered to a study subject. When the identity o the intervention is unknown to both subject and observer, the study is called a double-blind study. The natural uctuations and spontaneous remissions o many diseases also con ound clinical trials. A crossover design, in which each study group is given the test drug alternately with placebo, can protect against the misinterpretation o results due to natural variation in the disease process. The presence o risk actors or comorbid diseases and their treatment, known or unknown, represents another major con ounder o clinical trials. Care ul medical histories and randomization of subjects can counter some o the e ects o these risk actors. Stratif cation between study arms based on known clinically important covariates, and/or prospectively def ning in the statistical analysis plan how corrections will be made or imbalances in clinically important covariates, can also help to minimize the impact o potentially conounding variables. In addition to the strategies mentioned above—use o placebo controls, blinded studies, crossover design, and randomization—a large sample size can help to minimize the e ect o these actors. Phase 3 trials, the key studies that typically orm the primary basis or regulatory approval, are o ten re erred to as pivotal trials and are usually randomized, well-controlled studies. Finally, it is essential to ensure that the required schedule o tests is easible in the practice settings in which the trial will be conducted. This may be accomplished by extensive

C h a p t e r 5 2 Clinical Drug Evaluation and Regulatory Approval 939

TABLE 52-2 Clinical Drug Testing in Humans PHASE

NUMBER OF SUBJ ECTS

LENGTH OF PHASE

PURPOSE

Phase 1

20–100

Several months

Safety, pharmacokinetics, and pharmacodynamics

Phase 2

Up to several hundred

Several months to 2 years

Effectiveness, safety, dose ranging

Phase 3

Several hundred to several thousand

1–4 years

Safety, dosage, effectiveness

discussions with physicians, nurses, and study coordinators who are being considered to conduct the trial. Central laboratories may be employed, especially or conduct o novel tests or evaluation o disease-associated biomarkers. Additionally, central laboratories are commonly used when conducting multicenter phase 2 and phase 3 trials to better ensure standardization o data among study sites. Once the IND is active and an IRB or IEC approves the study protocol, clinical studies proceed in three phases. Table 52-2 summarizes the typical number o subjects, length o time required, and purpose o each phase o clinical trials, although these can vary considerably based on multiple actors.

Phase 1 Studies

Phase 1 studies are primarily intended to establish the sa ety and tolerability o a drug, including determination o the maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT). Single-dose trials in phase 1A o ten precede repeatdose trials in phase 1B. To protect patient sa ety, phase 1 trials are typically dose escalation trials. Subjects are divided into dosing groups, or cohorts. The initial cohort is administered a dose o the study drug that is anticipated to have little to no e ect; subsequent subject cohorts receive increasing doses o study drug until either a DLT is reached or a relevant pharmacodynamic endpoint is achieved. Alternatively, and less commonly, intrapatient dose escalation may be employed. However, this can make interpretation o toxicity di f cult i the study medication has delayed adverse e ects. Part o phase 1 investigation also involves study o the drug’s pharmacokinetic properties, including absorption, distribution, metabolism, and excretion (ADME). Although phase 1 trials ocus on sa ety, tolerability, and pharmacokinetics, pharmacodynamic assessments are increasingly being used to provide data early in drug development on the potential e ectiveness o the molecule. Phase 1 studies requently involve between 20 and 100 subjects. These may o ten be healthy normal volunteers. However, i high levels o toxicity are expected, such as with many cancer drugs, patients with the target condition may be used instead o healthy volunteers. Phase 1 studies usually involve nonblinded trials , in which subject and investigator are both aware o what is being administered. Phase 1 studies must yield su f cient in ormation about a drug’s pharmacokinetics to in orm the design o scientif cally valid phase 2 studies. For example, knowing the drug’s volume o

distribution and clearance enables study designers to determine an appropriate maintenance dose and dosing requency or phase 2 and 3 trials (see Chapter 3, Pharmacokinetics).

Phase 2 Studies

Phase 2 studies may involve up to several hundred subjects

with the medical condition o interest. Phase 2 clinical trials have multiple objectives, including the acquisition o preliminary data regarding the e ectiveness o the drug or treatment o a particular condition. Like phase 1 trials, phase 2 trials continue to monitor sa ety. Because phase 2 studies enroll more patients, they are capable o detecting less common adverse events. Phase 2 studies also evaluate dose–response and dosing regimens, which are critically important in establishing the optimum dose or doses and requency o administration o the drug. A typical phase 2 design may involve either single-blind or double-blind trials in which the drug o interest is evaluated against placebo and/or an existing therapy. The trial usually compares several dosing regimens to obtain optimum dose range and toxicity in ormation. The results o phase 2 studies are critically important in evaluating whether or not to proceed to phase 3 and, i so, establishing a phase 3 study design. Specif cally, phase 2 studies should be designed to obtain a reasonable estimate o the size o the treatment e ect o the experimental therapy; these data will then in orm the appropriate sample size or phase 3 studies. Phase 2 results can also be used to pinpoint additional data that must be collected in phase 3 trials, such as monitoring o liver unction tests i phase 2 data suggest possible hepatotoxicity. During the drug development process, IND sponsors have multiple opportunities to consult with regulatory agencies through ormal meetings. A ter the completion o phase 2 studies and be ore the initiation o phase 3 (pivotal) studies, the sponsor will typically request a meeting with the FDA to discuss the results obtained to date and to present the phase 3 program design. Given the time and expense o phase 3 clinical trials, it is critical that there is agreement between the FDA and the sponsor on the appropriate trial design(s) beore the trial is initiated. Currently, the FDA provides the option o allowing the sponsor to use the Special Protocol Assessment (SPA) process to reach ormal agreement on the design o the study that will be used to support approval provided that the study is success ul.

Phase 3 Studies

Phase 3 studies involve several hundred to several thousand

patients and are conducted at multiple sites and in settings similar to those in which the drug will ultimately be used. Phase 3 studies utilize specif c clinical endpoints as the primary endpoints o the trial to establish e f cacy o a drug. Examples o accepted clinical endpoints include survival, reduction or prevention o disease relapse, improvement in patient unctional status, or improvement in how patients eel (e.g., pain, health-related quality-o -li e assessments). Surrogate endpoints that have been validated in prior clinical trials (e.g., reduction in serum LDL cholesterol as a surrogate or clinically meaning ul improvement in cardiac outcomes) may be acceptable endpoints in phase 3 pivotal trials. Examples o surrogate endpoints include markers or decreased disease burden, such as a reduction in the plasma levels o biochemical markers (e.g., glucose), an increase in cardiac output, or a reduction in size o a tumor.

940 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

In situations o li e-threatening diseases or which no acceptable therapy is available, surrogate endpoints that are reasonably likely to predict clinical benef t (but that are not yet validated) may be used as endpoints in pivotal trials. In such instances, the FDA may use the Accelerated Approval mechanism to approve drugs that demonstrate a avorable impact on surrogate endpoints. Accelerated approval allows the drug to be developed more quickly and to be made available to patients in a timelier manner. It is important to note that “accelerated” reers to the development process, not the FDA review timeline; however, drugs being considered or accelerated approval may also be given priority review status (6 months, compared to the 10-month standard review). This approach has been used to approve drugs or the treatment o acquired immunodef ciency syndrome (AIDS) and several types o cancer, among other indications. Under accelerated approval, the sponsor is required to conduct post-approval phase 4 studies to veri y and conf rm the clinical benef t o the drug. In the introductory case, imatinib was granted accelerated approval based on the surrogate clinical endpoints o hematological and cytogenetic response rates and was then granted ull approval a ter the success ul completion o post-approval studies demonstrating increased survival compared to the current standard o care.

Clinical Pharmacology

Many pharmaceutical and biotechnology companies have developed groups dedicated to studying the clinical pharmacology o their products in development. These groups may be called by names such as Experimental Medicine, Molecular Medicine, or Clinical Pharmacology. The groups typically investigate aspects o the drug’s clinical pharmacology, including asting and ed single-dose and repeat-dose pharmacokinetics; drug–drug interactions, with a special emphasis on the role o cytochrome P450 iso orms on drug metabolism; and the impact o renal or hepatic impairment on drug metabolism. They per orm thorough QT studies to assess the impact o the drug on cardiac electrophysiological unction (see Chapter 6, Drug Toxicity). The groups make care ul assessments o immunogenicity, particularly i the drug is a protein therapeutic, and o the drug’s clinical pharmacology in pediatric patients and in specif c ethnic groups such as Asian populations. Clinical pharmacology groups also work closely with preclinical scientists to develop appropriate biomarkers to better assess the impact o the drug at the earliest stages o clinical development. Biomarker assessments may take a variety o orms, including exploration o the population o patients most likely to benef t or most likely to be susceptible to toxicity as well as pharmacodynamic markers o drug activity. The groups may attempt to correlate gene polymorphisms or expression prof les with responsiveness. These and other clinical pharmacology studies are per ormed throughout the clinical development program and are incorporated into phase 1, 2, and 3 studies.

Pediatric Studies

Both the European Union regulatory authority, called the European Medicines Agency (EMA), and the US FDA have placed increased emphasis on per orming studies o drug sa ety, pharmacokinetics, and e f cacy in pediatric populations whenever possible and appropriate. The EMA requires approval o a pediatric investigation plan, or PIP, by its pediatric committee prior to submission o a marketing application or a new drug or biologic. Similarly, under FDASIA, the FDA now has the authority and responsibility

to promulgate regulations relating to pediatric study plans (PSPs) in order to identi y the required pediatric studies early in drug development and to begin planning or these studies, o ten prior to the f ling o the NDA/BLA.

Challenges in the Development of Drugs to Treat Rare Diseases

Historically, pharmaceutical companies had typically been disinterested in developing products or diseases with small patient populations, since the cost o developing drugs or small markets was similar to that or developing drugs or larger patient populations but the resulting revenues were smaller. In an attempt to encourage development o drugs or rare diseases, Congress passed the Orphan Drug Act in 1983. The legislation o ers f nancial incentives to companies that develop drugs or orphan diseases , which are def ned as diseases that a ect ewer than 200,000 individuals in the United States. In addition, an orphan drug enjoys exclusive approval or the orphan indication or 7 years ollowing approval. This legislation has proven to be very success ul in stimulating the development o new drugs or rare diseases. Since 1983, the FDA has approved more than 460 drugs to treat orphan diseases. Examples include imiglucerase or Gaucher disease type 1, epoetin alfa or anemia associated with end-stage renal disease, imatinib or chronic myelogenous leukemia, and mipomersen and lomitapide or homozygous amilial hypercholesterolemia. Even with orphan drug legislation, the development o drugs or very rare diseases presents several special challenges. FDA regulations require the same degree o rigor in the development o such drugs as that or non-orphan indications, including the appropriate demonstration o sa ety and statistically signif cant e f cacy in well-controlled clinical trials. Designing trials or patient populations that might be as small as 5,000 patients worldwide can prove challenging. The testing required in many o these trials may be highly specialized and may best be conducted at only a small number o centers o excellence in the world. Hence, patients, and in some instances their care providers, may need to be transported to remote clinical trial sites and housed ar rom their homes and support systems or extended periods o time. Additionally, even with a very e ective drug, i the disease in question has a long natural history, enrolling a su f cient number o patients or a long enough period o time to demonstrate a statistically signif cant di erence rom the placebo control may present major easibility challenges. Furthermore, i a rare disease is heterogeneous in its clinical course, then the expected treatment e ect o the new drug may be con ounded by a less-than-solid understanding o the disease’s natural history. This, in turn, makes it di f cult to determine the sample size necessary to ensure that the trial is designed with su f cient power to observe a statistically signif cant di erence between study arms. In some instances, sponsors have conducted natural history studies in parallel with traditional early-phase interventional studies in order to better understand the natural history o the disease and thereby in orm the design o the subsequent pivotal trials. The challenges o drug development or rare diseases may gain increasing attention in the coming years as advances in the basic sciences continue to allow diseases to be better understood and def ned at the molecular level. The result o this improved understanding will likely be that a group o heterogeneous diseases, which until now has been considered as one

C h a p t e r 5 2 Clinical Drug Evaluation and Regulatory Approval 941

disease entity encompassing many patients, will be categorized into smaller, orphan-sized subgroups based on specif c molecular markers or mutations.

Adaptive Trial Designs

As discussed earlier, it usually takes many years and many millions o dollars to success ully develop a new therapeutic. In order to be as e f cient as possible, much attention has recently been given to the development o adaptive clinical trial designs. This methodology allows or modif cation o such variables as trial duration, cohort assignment, or number o patients enrolled based on examination and analysis o the accumulated data at one or more prospectively def ned points in the trial. Adaptive designs may not only be more e f cient but may also increase the chances o demonstrating an e ect o the investigational agent (i one exists). In all cases, it is important that such designs are developed in close collaboration with regulatory authorities to ensure that they will be deemed acceptable.

Successful Drug Development: Design and Execution

Success ul drug development requires not only a thought ul, well-conceived, strategic development plan but also rigorous, responsible execution. This, in turn, requires a strong organization, competent leadership, adequate resources, and a highly unctioning, multidisciplinary global team. It cannot be emphasized enough that success ul drug development requires intensive and extensive collaboration among many people in a wide array o disciplines. Table 52-3 lists many o the most important clinical study activities that require extensive planning. Care ul planning is critical since errors that either result in the need or protocol amendments or endanger the integrity o the results will cost signif cant time and money, jeopardize the results obtained, and, most importantly, have the potential to place patients at inappropriate risk. Clinical study operations include study site start-up and initiation, interim site monitoring, interim site management, and site closeout at the conclusion o the study. Each o these activities requires the success ul completion o many smaller activities, as illustrated in Table 52-3.

TABLE 52-3 Elements to Be Considered in Clinical Trial Operations: Planning and Execution Study Management • Case report orm design and printing (unless electronic data capture is employed) • Projected (or required) enrollment rates • Health economics issues • Use o independent Data Monitoring Committees (DMCs), special monitoring, or adjudication committees (include proposed charter and/or proposed procedure) • Are special laboratories needed? • Will the study use a contract research organization (CRO) or all or key parts o study conduct? • Are there any special sample handling or supply shipping concerns? • Investigator meeting planning

• • • • • • • •

Database development Data management Medical writing Clinical monitoring Medical monitoring Paper/electronic submission Publication analysis and writing Pharmacovigilance plans, including risk evaluation and mitigation strategies (REMS) • Randomization system • Central clinical laboratories/diagnostics • Clinical trial material labeling and handling

Site Start-up • • • • •

Obtain conf dentiality agreements. Obtain clinical trial agreements. Distribute study documents. Per orm qualif cation visits. Assist with ethics committee or Institutional Review Board (IRB) approvals.

• • • •

Collect study documents. Ship study drug and case report orms. Hold investigator meeting. Initiate sites (visit).

• • • • •

Review serious adverse event reporting. Assess protocol and GCP/ICH compliance. Assess adequacy o personnel and acilities. Communicate f ndings to study personnel. Typically, monitor sites every 4–6 weeks.

Interim Site Monitoring • • • • •

Review enrollment. Review signed in ormed consents. Review regulatory binder (required documents). Per orm drug accountability. Veri y data rom source documentation (CRF vs. medical record).

Interim Site Management • • • •

Track patient enrollment. Ongoing site support. Track and supply study drug. Review monitoring reports/data error requency.

• Review protocol deviations/violations. • Assess need or protocol amendments. • Support QA audits.

Site Closeout • • • •

Per orm f nal drug accountability. Ship study drug or destruction. Veri y data rom source documentation, i needed. Ensure documentation on site complete.

• Obtain copies o site f les. • In orm site regarding communicating FDA audits to sponsor. • Review record retention and publication policy.

CRF, case report orm; GCP, good clinical practices; ICH, International Con erence on Harmonization; QA, quality assurance; FDA, US Food and Drug Administration.

942 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

Mechanisms to Expedite Development of Therapeutics for Serious Conditions

Four principal mechanisms are supported by the FDA to expedite drug development or serious unmet needs. These include (1) Fast Track designation, (2) Breakthrough Therapy designation, (3) Accelerated Approval, and (4) Priority Review. Each program targets drugs intended to treat serious conditions; the quali ying criteria and advantages o the programs are di erentiated as ollows: ■

■

■

■

Fast Track designation requires nonclinical or clinical data

that demonstrate the potential to address a serious unmet medical need. Advantages include opportunities or requent FDA interactions, rolling review (allows the sponsor to submit portions o the application or approval on a rolling basis as the documents become available rather than submitting all o the many thousands o pages at one time), and eligibility or priority review. Breakthrough Therapy designation requires preliminary clinical data that indicate the drug may demonstrate substantial improvement over existing therapies in a clinically signif cant endpoint(s). Advantages include early and intensive guidance, involvement o FDA senior sta , rolling review, and eligibility or priority review. Accelerated Approval, as described in “Phase 3 Studies” above, requires that the product provides a meaning ul advantage over available therapy and demonstrates an e ect in a surrogate endpoint. Priority Review criteria stipulate that the product, i approved, would provide a signif cant improvement in sa ety or e ectiveness. The advantage o priority review is that it provides or a 6-month review period rom the time o f ling o the application with the FDA to a decision rather than the standard 10-month review period.

It is important to note that a development program can f t more than one o these expedited development mechanisms. In addition, even with such expedited development opportunities, adequate data must be provided to ulf ll the FDA’s statutory requirements or a product to demonstrate sa ety and e f cacy.

DRUG APPROVAL PROCESS FDA Review Approval o new drugs in the United States is based on the New Drug Application (NDA) in the case o a small-molecule therapeutics or the Biologics License Application (BLA) in the case o a biotherapeutic. The NDA/BLA must contain all relevant data collected by a sponsor during research and development o the proposed new drug. As such, data gathered or the IND are integrated into the NDA/BLA. The FDA mandates that every NDA/BLA must contain the ollowing sections: index, summary, chemistry, manu acturing and quality control, samples, methods validation, package and labeling, nonclinical pharmacology and toxicology, human pharmacokinetics, metabolism and bioavailability, microbiology, clinical data, sa ety update report (typically submitted 120 days a ter NDA/BLA submission), statistical in ormation, case report tabulations, case report orms, patent in ormation, patent certif cation, and other in ormation. The typical NDA/BLA submission is comprehensive and may consist o thousands o pages in multiple volumes. To acilitate the submission o these data to regulatory agencies in multiple countries, the data are presented in a

ormat re erred to as the Common Technical Document (CTD). With the passage o FDASIA in 2012, all CTDs will ultimately be required to be submitted in electronic ormat. Upon receipt o an NDA/BLA, the FDA has 60 days to assess an application’s acceptability to be f led or review. The review is organized into several categories, which may include the ollowing: medical review , biopharmaceutical review , pharmacology review , statistical review , chemistry review , and microbiology review . Within each o these groups, FDA experts review the data package submitted to the agency and provide an assessment o the sa ety and e f cacy o the proposed new drug. Figure 52-3 is a owchart representing the process used by the FDA to evaluate an NDA/BLA.

Applicant (drug sponsor)

NDA/BLA

Applica tion filea ble?

NO

Re fus e to file le tte r is s ue d

YES

Me dica l

Biopha rma ce utica l

P ha rma cology

S ta tis tica l

Che mis try

Microbiology

Advis ory committe e me e ting

S pons or re vis e s

Me e tings with s pons or

Re vie ws complete and acce ptable ? YES

NO

NO

Additiona l info or re vis ions re que s te d or s ubmitte d (a me ndme nt)

YES

La be ling re view a ccepta ble?

Inspection of sites acce ptable ? NO YES

P e nding sa tisfactory re sults Approval action

FIGURE 52-3. Process of New Drug Application/Biologics License Application (NDA/BLA) review. When a new drug application is f led, the drug sponsor provides data regarding the drug’s medical, pharmacologic, chemical, biopharmaceutical, statistical, and microbiological characteristics; these data are reviewed by separate committees at the FDA. I the review is complete and acceptable, then the drug application is reviewed or acceptable labeling (o f cial instructions or use). The manu acturing sites and sites where signif cant clinical trials were per ormed also undergo inspections and audits. Colored boxes correspond to actions by the drug sponsor; white boxes correspond to actions by the FDA.

C h a p t e r 5 2 Clinical Drug Evaluation and Regulatory Approval 943

In addition to reviews internal to the agency, the FDA may also call on an external advisory committee or input on an NDA/BLA. These committees provide medical and scientif c input and allow or consultation with outside experts in a particular f eld. Although the FDA usually incorporates advisory committee recommendations into its decisions, these external opinions are not binding. The FDA may also engage external experts as needed during the review process or specif c technical issues. During the review process, the FDA maintains ongoing communication with the sponsor regarding scientif c or other issues that arise during review. Regular correspondence (including ace-to- ace meetings i necessary) occurs between the sponsor and the agency, particularly i additional data are needed. The FDA requently asks the sponsor questions in writing, and the sponsor may submit additional data or a new analysis o previously available data to assist in addressing these questions. Substantial amounts o new in ormation are considered an amendment to the NDA/BLA and can prolong the time to approval.

FDA Approval The FDA may take one o two possible actions ollowing the review o an NDA/BLA—approval o the application or issuance o a Complete Response Letter (CRL) to the sponsor o the application. A Complete Response Letter is issued when the FDA determines that an application has def ciencies to such a degree that approval should not be granted; in response, a sponsor must noti y the FDA o its intent to resubmit the application, withdraw it, or request a hearing. In the CRL, the FDA must list all o the specif c def ciencies in the application that need to be satis actorily addressed in order or the application to be reconsidered or approval. O ten, the FDA will meet with the sponsor to discuss the steps that must be taken to secure approval. In some cases, these revisions may require signif cant new studies to be undertaken. The sponsor then must decide whether to generate new data or abandon a particular development program. I a sponsor ails to act on a CRL within 1 year o its issue, the FDA considers this lack o response to represent a request by the sponsor to withdraw the NDA/BLA.

Approval in Other Countries Be ore drugs may be sold in countries outside the United States, they must f rst be evaluated and approved by the appropriate regulatory authorities in those regions. In some countries, this may include a comprehensive review o all data, similar to the NDA/BLA review. In other countries, a more limited review may occur i the drug has already been approved in one o the major oreign markets (United States, European Union, Japan). During these reviews, a regulatory authority may require additional types o data or analyses that were not required or US approval. In addition, di erent regulatory agencies may have di erent approaches to the type and amount o data required in product labeling. In the European Union, many drugs are f rst evaluated by the European Medicines Agency (EMA) and then approved by the European Commission i approval is recommended by the EMA. In Canada, Health Canada administers the regulations embodied in the Canadian Food and Drugs Act. In Japan, approval o new drugs is granted by the Ministry of Health and Welfare . Importantly, Japanese regulatory authorities require

studies to be per ormed in ethnic Japanese patients in order to demonstrate that the pharmacokinetic and sa ety prof les observed in a Japanese population are similar to those observed in a Western population. Demonstration o e f cacy in Japanese patients may also be required.

Expanded Access of an Investigational Drug or an Approved Drug with Restricted Distribution The FDA has established several mechanisms or the “compassionate use” o investigational drugs or patients with serious or immediately li e-threatening conditions or which there are no alternative therapies. Examples o compassionate use may include when a patient ails to meet established inclusion criteria or an ongoing trial o an investigational drug; when a patient seeks access to an investigational drug a ter a pivotal study is completed to support a marketing application; or when access is sought or an approved drug whose distribution is controlled due to sa ety reasons. In all cases, three criteria must be met: (1) patients to be treated must have a serious or li e-threatening condition with no therapeutic alternatives; (2) the potential benef t must outweigh the potential risk o the drug; and (3) providing the drug will in no way inter ere with ongoing clinical development o the drug.

Drug Labeling Each region’s regulatory body has an established ormat and organization or drug labeling. A drug label must include the drug’s proprietary and chemical name, ormula and ingredients, clinical pharmacology, indications and usage, contraindications, warnings, precautions, adverse reactions, drug abuse/dependence potential, overdosage, dosage, rate and route o administration, and how the drug is supplied. In the United States, this in ormation is also known as the drug’s package insert. When a new drug approaches approval, the FDA reviews and negotiates the f nal package insert with the sponsor to ensure that the labeling is justif ed by the data submitted in the NDA/BLA. To provide more accessible and in ormative drug in ormation, in 2006, the FDA instituted the Physician Labeling Rule, which provides key in ormation important to prescribers in a standardized ormat. Regulatory agencies may use additional methods to ensure that important attributes o the drug are clearly communicated. For example, in the United States, package inserts or drugs that have certain sa ety risks include a “black box” warning, in which key sa ety in ormation is prominently displayed. In addition, the FDA may require sponsors to create Medication Guides or mandatory distribution to patients; these guides communicate critical sa ety in ormation in language that is readily understandable.

Drug Naming Another acet o drug approval involves the determination o a drug’s name. A drug is known by two principal names, the generic name and the brand name (or trade name ). A drug’s generic name is based on its chemical name and is unprotected by a trademark. The generic name is also known as the International Nonproprietary Name or INN. In contrast, a drug’s brand name re ers to the exclusive name o a substance or drug product owned by a company under trademark law.

944 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

For example, the generic name o the drug discussed in the introductory case is imatinib mesylate, while the brand name is Gleevec®.

Additional Indications Once a drug is approved, physicians and certain other healthcare pro essionals are permitted to prescribe the drug according to the various labeled doses or dosage regimens. Providers may modi y dosing and also prescribe the drug or indications other than that or which it was originally approved; this is known as “off-label” use . Physicians are also permitted to conduct investigational studies with the drug, provided that they ollow the rules o in ormed consent and obtain IRB approval or the studies. However, in many instances, the investigator may be required to f le an IND, particularly i the investigation involves a route o administration, dose, patient population, or other actor that has not been well studied and may signif cantly increase the risk associated with the use o the drug product. Pharmaceutical companies may not market the drug or any indications other than those or which it has been approved by the FDA. Current regulations prohibit pharmaceutical companies rom proactively providing any marketing materials, including scientif c articles, on the o -label use o a drug, unless such materials are requested by a physician. In order to market a drug or a new indication, a pharmaceutical company must conduct an additional program o development to prove that the drug is sa e and e f cacious or the new indication. These data are then submitted to regulatory authorities as a supplemental NDA/BLA (sNDA/sBLA) and subjected to additional review prior to the granting o approval or the new indication. Product labeling may then be modif ed accordingly.

REGULATORY ASPECTS OF DRUG PRODUCTION AND QUALITY CONTROL In addition to demonstrating a drug’s sa ety and e f cacy, manu acturers must also comply with FDA regulations or manu acturing as a requirement or drug approval and subsequent distribution. The Good Manufacturing Practice (GMP) guidelines govern quality management and control or all aspects o drug manu acturing, and the FDA has the authority to inspect manu acturing acilities in order to determine compliance. FDA regulations require sponsors to establish manu acturing process controls, product quality attributes, and quality control procedures in order to ensure a product’s purity, potency, and identity. A company must obtain prior FDA approval be ore implementing any manu acturing change that is determined by the FDA to have substantial potential to a ect the sa ety or e f cacy o a drug through alterations in its identity, strength, quality, purity, or potency. Other changes may be implemented either with or without submission o a supplemental NDA/BLA. GMP criteria apply whether a sponsor manu actures its own products or uses contract manu acturers.

GENERIC DRUGS The FDA also oversees approval o generic drugs , which the agency def nes as drugs that are comparable to innovator drugs in dosage orm, sa ety, strength, route o administration,

quality, per ormance characteristics, and intended use. Under the Drug Price Competition and Patent Term Restoration Act of 1984, also known as the Hatch-Waxman Act, a company may submit an Abbreviated New Drug Application (ANDA) be ore the patent governing the brand name drug expires. However, the company must wait or the original drug’s patent to expire be ore it can market a generic version. The f rst company to f le an ANDA has the exclusive right to market the generic drug or 180 days. ANDAs or generic drugs are not required to provide data establishing sa ety and e f cacy, because this has been established in the NDA or the innovator drug. To establish bioequivalence , which is required in the ANDA, sponsors may submit a ormulation comparison, comparative dissolution testing (where there is a known correlation between in vitro and in vivo e ects), in vivo bioequivalence testing (comparing the rate and extent o absorption o the generic with that o the re erence product), and, or nonclassically absorbed products, a head-to-head evaluation o comparative e ectiveness based on clinical endpoints. In addition, an ANDA sponsor must provide evidence that its manu acturing processes and acilities, as well as any outside testing or packaging acilities, are in compliance with ederal GMP regulations. “Generic” versions o biologic drugs, primarily proteins, present much greater challenges than generic versions o small-molecule drugs. Whereas small molecules can readily be shown to be comparable to the innovator drug as described above, this is not so easy with recombinant proteins, which usually have many post-translational modif cations. Seemingly minor changes in post-translational modif cations may result in marked di erences rom the innovator drug in sa ety and e f cacy. Changes in cell lines used to manu acture such proteins and changes in any step o the production process may alter post-translational modif cations. As a result, the precise regulatory path or the development o “biosimilars” was less than clear. The Biologics Price Competition and Innovation Act o 2009 (BPCIA), the analogue o the Hatch-Waxman act or biological products, authorized the approval pathway or biosimilar products. The Biosimilar User Fee Act, as authorized by FDASIA in 2012, provides the FDA with the resources and sta to review and approve biosimilar products. The FDA has issued several documents providing guidance on the development o biosimilars, including a Scientif c Considerations document, a Quality Considerations document, and a Questions and Answers (Q&A) document on Implementation o the BPCIA. In 2013, guidance was issued outlining ormal procedures or holding meetings with the FDA on biosimilar development. During 2014 and 2015, the FDA issued additional guidances on clinical pharmacology data, scientif c considerations, and quality considerations to support biosimilarity to a re erence product, as well as a revision to the Q&A document.

NONPRESCRIPTION DRUGS AND SUPPLEMENTS The 1951 Durham-Humphrey Amendment to the Food, Drug, and Cosmetic Act def ned prescription drugs as drugs that are unsa e or use except under pro essional supervision. In determining which drugs do not require a prescription, the FDA examines a drug’s toxicity and the acility with which

C h a p t e r 5 2 Clinical Drug Evaluation and Regulatory Approval 945

a condition may be sel -diagnosed. Because over-the-counter (OTC) drugs are sold in lower doses than their prescription counterparts and are used primarily to treat symptoms o disease, the FDA requires their labels to contain the ollowing: ■

■ ■ ■

Intended uses o the product, as well as the product’s e ects Adequate directions or use Warnings against unsa e use Adverse e ects

Although OTC products present a potential danger o misuse or misdiagnosis in the absence o physician oversight, the increased availability o these products has provided many US citizens with access to e ective and relatively inexpensive treatments. The Dietary Supplement Health and Education Act o 1994 def nes a dietary supplement as any product intended or ingestion as a supplement to the diet, including vitamins, minerals, herbs, botanicals, other plant-derived substances, amino acids, concentrates, metabolites, and constituents and extracts o these substances. The FDA oversees the sa ety, manu acturing, and health claims made by dietary supplements. The FDA does not, however, evaluate the e f cacy o supplements as it does or drugs. The FDA may restrict or halt the sale o unsa e supplements, but it must demonstrate that such supplements are unsa e be ore taking action. This occurred in February 2004, when the FDA announced a rule banning dietary supplements containing ephedrine alkaloids (ephedra ) a ter reviewing the substantial number o adverse events (including deaths) associated with these products.

CONCLUSION AND FUTURE DIRECTIONS Specif c laws and regulations have been established to provide or the development o new drugs, while at the same time ensuring privacy and sa ety or the individuals participating in clinical trials. Regulatory approval o new drugs ollows a disciplined process o preclinical and clinical studies in parallel with product characterization and manuacturing process development. Each phase o development provides critical in ormation that in orms subsequent phases o investigation. Industry, academia, and health authorities strive to balance sa ety and clinical benef t with speed o drug availability or patients in need. However, no amount o analytical, animal, and clinical trial data can provide a completely accurate prediction o the sa ety prof le o a drug a ter it is introduced to the marketplace. Thus, the FDA and drug manu acturers continue to monitor the adverse e ects, manu acturing processes, and overall sa ety o a drug or its li etime (see Chapter 53, Systematic Detection o Adverse Drug Events).

Acknowledgment We thank the late Armen H. Tashjian, Jr. or his valuable contribution to this chapter in the Second Edition o Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Adams CP, Brantner VV. Estimating the cost o new drug development: is it really 802 million dollars? Health Aff 2006;25:420–428. (Finds that developing a new drug costs between $500 million and $2 billion, depending on the indication.) Center or Drug Evaluation and Research, U.S. Food and Drug Administration, U.S. Department o Health and Human Services. The CDER handbook. Revised 03/16/98. http://www. da.gov/downloads/AboutFDA /CentersO f ces/CDER/UCM198415.pd . (Describes the processes by which the FDA evaluates and regulates drugs, including new drug evaluation and postmarketing monitoring of drug safety and effectiveness.) Cohen MH, Williams G, Johnson JR, et al. Approval summary or imatinib mesylate capsules in the treatment o chronic myelogenous leukemia. Clin Cancer Res 2002;8:935–942. (Summarizes the approval of imatinib mesylate, the drug discussed in the introductory case.) DiMasi JA, Grabowski HG. The cost o biopharmaceutical R&D: is biotech di erent? Manage Decis Econ 2007;28:469–479. (First paper to estimate costs of biopharmaceutical development compared to costs of traditional pharmaceutical development.) Dixon JR. The International Con erence on Harmonization Good Clinical Practice guideline. Qual Assur 1999;6:65–74. (Guidelines for standard design of drug development.) Food and Drug Administration Strategic Priorities 2014–2018. http:// www. da.gov/downloads/AboutFDA/ReportsManualsForms/Reports /UCM403191.pd . (A draft document for public comment that provides an overarching view of how the FDA is addressing and plans to address the public health challenges facing the United States in the next 5 years.) Kesselheim AS, Darrow JJ. Drug development and FDA approval, 1938–2013. N Engl J Med 2014;360:e39. (Interactive presentation of the major legislative and regulatory events related to the approval of new drugs by the FDA, including drug approvals.) Long G, Works J. Innovation in the biopharmaceutical pipeline: a multidimensional view. Boston, MA: Analysis Group; 2013. www.analysisgroup .com/uploadedFiles/Publishing/Articles/2012_Innovation_in_the _Biopharmaceutical_Pipeline.pd . (Descriptive information about the development of innovative medicines in multiple therapeutic areas.) Pharmaceutical Research and Manu acturers o America. 2014 biopharmaceutical research industry prof le. Washington, DC: Pharmaceutical Research and Manu acturers o America; 2014. http://www.phrma.org/sites /de ault/f les/pd /2014_PhRMA_PROFILE.pd . (Overview of the current status of the biopharmaceutical industry with respect to innovations in research and development and impact on patients and society, published by the Pharmaceutical Research and Manufacturers of America [PhRMA].) Swann JP. FDA’s origin and unctions. http://www. da.gov/AboutFDA /WhatWeDo/History/Origin/ucm124403.htm. (An excellent overview of the evolution of the FDA from its beginnings in 1848.) U.S. Food and Drug Administration, U.S. Department o Health and Human Services. Innovation or stagnation: challenge and opportunity on the critical path to new medical products. March 2004. http://www. da.gov/ScienceResearch /SpecialTopics/CriticalPathInitiative/CriticalPathOpportunitiesReports /ucm077262.htm. (An FDA report that addresses the slowdown in innovative drug development.)

53

Adve rs e outc ou tcom om me

A

Noo a dv N dvee rs rs e outc ou tcome

B

Drug ug e xpp oss ur uree

Ex s ure + Expo Outc Ou tc o me +

Expo po s ure + Ou u tc o me –

No drug e xp xpoo s ure

Expo p s ure – Outtc o me + Ou

Expo s ure – Outc o m e –

Systematic Detection of Adverse C D Drug Events J e rry Avo rn

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 946–947 INT CHALLENGES IN THE ASCERTAINM MEN ENT T OF DRUG SAFETY. . . 946 Studyy Size and General St aliz izab abil ilit ity . . . . . . . . . . . . . . . . . . . . . . . . . 946 Surr rrog ogat ate te Out Ouuttcom c omes e and Comparators . . . . . . . . . . . . . . . . . . 947 Duration and Post-Approval Studi tudies es . . . . . . . . . . . . . . . . . . . . 94 9488 PHARMACOEPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 948 Sources o Pharmacoepi p dee mi miol olog ogic ic Data at . . . . . . . . . . . . . . . . 949 Sponttaneous Reportss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949 Automated Databases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949 Patient Registries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949 Ad Hoc Studies. Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949 Study Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950 Cohort and Case-Control Studiess . . . . . . . . . . . . . . . . . . . . . 950 Evaluation o Risk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950

INTRODUCTION Because medications act by inter ering with one or more aspects o molecular and cellular unction, it is di f cult to do so without also causing an undesirable e ect either by that perturbation or by another (perhaps unexpected) drug action. Because all drugs have risks, the goal o pharmacotherapy cannot be to prescribe a risk- ree regimen. Instead, it is to ensure that the risks o drug therapy are as low as possible and are acceptable in the context o a medication’s clinical benef t. Some adverse e ects o a drug are apparent during its early development and o ten result rom the same on-target mechanism responsible or its therapeutic e ect (e.g., cytotoxic cancer chemotherapy). Even in such situations, however, it is necessary to know how those expected adverse e ects will be mani ested when the drug is in routine use—in terms o both their requency and their severity. A ter a drug has been approved or clinical use, the goal becomes detecting and quanti ying the risks as quickly and rigorously as possible. Serious or even li e-threatening adverse e ects have led to the withdrawal o widely used drugs. This has heightened the sensitivity o clinicians and patients to the growing f eld o pharmacoepidemiology—the measurement o drug e ects in large, “real-world” populations o patients. Advances in in ormatics and analytic techniques in this f eld 946

Issues in Study Design and Inter erpr pret ettat a tio ion. . . . . . . . . . . . . . . . 950 Con ounding by Indication . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 9511 Selection Bias. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 9511 The Healthy Us User er E ectt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 952 In nte terrpre rpreti ting Statistical Signif cance. . . . . . . . . . . . . . . . . . . 952 ti ADVERSE DRUG EFFECTS AND THE HEALTH CARE SYSTEM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 952 Balancing Benef ts and Risks. . . . . . . . . . . . . . . . . . . . . . . . . . . 952 Role o the FDA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953 Legal and Ethical Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953 CONCLUSION CONC CO NCLU LUSI SION ON A AND ND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 953 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .953

hold promise or enhancing our understanding o drug risks so that they can be better understood and managed, with the goal o putting a drug’s benef ts into context and guiding clinical decision making and regulatory action.

CHALLENGES IN THE ASCERTAINMENT OF DRUG SAFETY The randomized controlled trial (RCT) is the gold standard or determining the e f cacy o a drug and is the main criterion used by regulatory agencies, such as the US Food and Drug Administration (FDA), in deciding whether to approve a new medication or use. But this valuable tool also has limits, and it is important to understand those limits when assessing the benef ts and risks o a given agent.

Study Size and Generalizability Compared to the number o patients who eventually use a drug, the number o subjects in clinical trials supporting the approval o that drug is relatively modest. Approval decisions are generally made on the basis o trials that include 2,000–4,000 participants, or ewer or rare conditions. I a particular adverse event occurs just once in every 1,000 patients, it may not occur at all during clinical trials,

C h a p t e r 5 3 Systematic Detection of Adverse Drug Events

Mr. Ke e le y is a 67-ye a r-o ld m a n w ith s e ve re d e g e n e ra tive jo in t d is e a s e o b o th h ip s . He b rin g s to h is d o cto r s e ve ra l m a ga zin e a d s a n d n e w s p a p e r clip p in g s th a t d e s crib e th e n o n s te ro id a l ti-in a n ti in f a m m a to ry d ru g ro e co xib . Th e s e a d s cla im th a t ro e co xib p ro vid e s e xce lle n t re lie o a rth ritic p a in w ith a lo w e r ris k o g a s tro in te s tin a l to xicity. Ro e co xib s e le ctive ly in h ib its cyclo o xyg e n a s e -2, w h ich m e d ia te s p a in a n d in f a m m a tio n , ra th e r th a n cyclo oxyg e n a s e -1, w h ich m a in ta in s ga s tro in te s tin a l m u co s a l in te g rity a n d w h o s e in h ib itio n ca n ca u s e ga s tro in te s tin a l b le e d in g . Th is d ru g is w id e ly h a ile d a s a “ s u p e r-a s p irin ” w ith m in im a l g a s tro in te s tin a l toxicity a n d is h e a vily p ro m o te d . Mr. Ke e le y’s p hys icia n d e cid e s to p re s crib e ro e coxib , a n d th e p a tie n t re p o rts th a t it w o rks b e tte r th a n th e a ce ta m in o p h e n h e h a d p re vio u s ly b e e n u s in g . Th e p a tie n t co n tin u e s to e xp e rie n ce g o o d re lie o h is a rth ritic p a in . In th e e n s u in g m o n th s , h e d e ve lo p s m ild h yp e rte n s io n th a t is e a s ily m a n a g e d w ith a th ia zid e m e d ica tio n b u t is o th e rw is e w e ll. Nin e m o n th s a te r Mr. Ke e le y b e g in s ta kin g ro e coxib , h is w i e ca lls to re p o rt th a t h e r h u s b a n d h a s

or i it does occur, it may be di f cult or impossible to determine whether its rate o occurrence is meaning ully higher among study subjects compared to controls. One in 1,000 may seem like a rare event, but i 10 million people take a drug each year, that rate would result in 10,000 occurrences o the adverse event annually. For a li e-threatening adverse e ect such as ulminant hepatotoxicity, this could have important clinical and public health consequences. Subjects in clinical trials o new drugs are nearly always volunteers—people who have come orward to participate in medical research and have given their in ormed consent to take part in the study. There is ample evidence that such people tend to be di erent rom typical patients who will receive the drug when it is in routine use; study subjects tend to be younger, healthier, better educated, and o higher socioeconomic status. This problem is o ten exacerbated by strict exclusion criteria in preapproval study protocols. Some o these exclusions prohibit participation o patients over a given age cuto (such as 65 or 70), even i the drug is expected to be used disproportionately by the elderly. Other entry criteria may exclude patients who have important comorbidities in addition to the disease being studied (thereby also excluding those who are taking multiple other medications). While this may be the “cleanest” way to test the e f cacy o a new agent, there is growing concern that the data thus generated have limited generalizability to the populations who ultimately use these medications. Other kinds o patients may be excluded or unassailable ethical reasons, such as not allowing pregnant women or children into most preapproval drug trials. However, when such patients then take these drugs in routine care, there is little in ormation to guide their use. By def nition, clinical trials are conducted by physicians and support sta who have experience in clinical research

947

b e e n h o s p ita lize d w ith a m yo ca rd ia l in a rctio n . He s u rvive s s e ve ra l e p is o d e s o a rrhyth m ia a n d ca rd io g e n ic s h o ck a n d is d is ch a rg e d to h o m e . His p hys icia n is n o t s u rp ris e d a t th is re p o rt, s in ce th e p a tie n t wa s a n a ctive s m o ke r, h a d e le va te d s e ru m ch o le s te ro l, a n d h a s a re ce n t d ia g n o s is o hyp e rte n s io n . Tw o ye a rs a te r Mr. Ke e le y’s m yo ca rd ia l in a rctio n , ro e coxib is w ith d ra w n ro m th e m a rke t w h e n a ra n d o m ize d co n tro lle d tria l re ve a ls th a t ro e coxib n e a rly d o u b le s th e ris k o m yo ca rd ia l in a rctio n a n d s tro ke .

Questions 1 . How do regulatory agencies such as the US Food and Drug Administration assess the safety of medications before they are approved? 2 . How do physicians, patients, and the FDA learn about the adverse effects of drugs once they are in widespread use? 3 . How can observational studies be used to determine the adverse effects of drugs that are in widespread use? 4 . What issues must be considered in interpreting and acting on the results of such analyses?

and who work in settings accustomed to such activities. Their actions are guided by study protocols that o ten require close monitoring or adverse e ects as well as e f cacy. Such protocols also ensure that patients are taking the prescribed product as directed. This, too, is ar di erent rom routine care in typical settings, in which both patient adherence and the intensity o surveillance or early detection o adverse events are generally lower.

Surrogate Outcomes and Comparators It would be di f cult to postpone the approval o every new antihypertensive drug until it had been shown to reduce stroke rates, or not to allow a new statin lipid-lowering drug on the market until it had been shown to prevent myocardial in arctions. Such a requirement could delay the availability o potentially use ul new therapies, as well as urther increase their cost. As a consequence, new products may be approved on the basis o their e ect on “surrogate outcomes,” such as blood pressure or antihypertensives, hemoglobin A1c level or drugs used to manage diabetes, serum LDL cholesterol level or statins, intraocular pressure or drugs used to treat glaucoma, or biomarkers o tumor growth or oncology therapies. While such a metric can be use ul in making drug approval quicker and more e f cient, its utility depends on the association between the surrogate marker and the clinical outcome o concern. These may correlate well, but not always. For example, the antiarrhythmics encainide and f ecainide reduced the surrogate outcome o ventricular ectopy a ter myocardial in arction, but a larger study (the CAST trial) demonstrated that they actually increased mortality in such patients, despite their success in “treating” the surrogate marker. Similarly, rosiglitazone (Avandia®) was approved based on its capacity to reduce hemoglobin A1c

948 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

levels in preapproval trials. However, once the drug was in widespread use, meta-analysis o those trials ound that it increased the risk o myocardial in arction. When easible, placebos are the comparison treatment pre erred by manu acturers and the FDA or premarket trials used to determine approval. Such comparisons provide the clearest contrasts and the most straight orward statistical analysis, and there is no possibility o con usion resulting rom therapeutic or adverse events caused by an active agent used in the control group. Placebo controls also acilitate the approval o new products whose e f cacy is similar to that o existing drugs; per orming “equivalency” or “nonin eriority” studies against active therapies requires larger numbers o patients and is more demanding statistically. I it is ethically or pragmatically impossible to conduct placebocontrolled trials (e.g., with a new AIDS drug or an antibiotic or a serious bacterial in ection), then an active comparator is used. However, while the “better than placebo” comparison may be su f cient or a manu acturer to meet the FDA’s legal requirements or drug approval, the data it yields o ten all short o what the clinician, patient, or payor needs to know about a new drug’s sa ety or comparative e ectiveness. A new drug may work better than placebo, but is it better than an existing treatment the physician may choose instead? Or is it even as good? The new drug may produce a serious adverse e ect (e.g., rhabdomyolysis with a statin), but is the rate o occurrence o the e ect higher or lower than that seen with older therapies? And even i it poses a higher risk o a given adverse e ect, does the new drug also provide greater e f cacy (in this case, prevention o ischemic cardiac events)? I so, the trade-o might possibly be acceptable; i not, it would not be. But i no such comparative data exist, the question cannot even be considered.

Duration and Post-Approval Studies The duration o e f cacy trials or certain new drugs can be as short as 8–16 weeks, i the comparator is placebo and surrogate endpoints are used to meet a legal def nition o e f cacy. However, such short-term trials may yield little use ul in ormation about benef ts and risks that occur beyond this time rame. The FDA requires a minimum o 6 months o sa ety testing or a new drug that is designed or chronic use (where chronic is def ned as any period longer than 6 months), although even this duration o sa ety testing may be too short or a chronically administered medication that may be taken or many years. In approving a new drug or widespread use, the FDA may ask the manu acturer to conduct additional postmarketing studies (phase IV studies ) to address questions that were not resolved by the evidence submitted prior to approval. Sometimes, use ul new data about a drug’s benef ts and risks are obtained in this way. But until 2007, the agency had little authority to oblige a drug’s sponsor to complete these studies, since its main regulatory power, once a drug had been approved, had been conf ned to the “nuclear option” o threatening to take it o the market—an action that was o ten not possible in the absence o additional data. Each year, the agency reports how well such “postmarketing commitments” are being met by manu acturers. A report by the Government Accountability O f ce noted that up to hal o the “mandated” postmarketing sa ety studies requested by the agency had not been initiated, even years a ter the drugs

had entered widespread use. Concern about these problems was intensif ed by public concern over several prominent drug sa ety problems, particularly rofecoxib (Vioxx®). The drug had been used widely or 5 years be ore it was withdrawn rom the market ollowing a study demonstrating that it nearly doubled the risk o myocardial in arction or stroke. A 2006 report by the Institute o Medicine recommended sweeping changes in the way the FDA addresses drug sa ety (see below).

PHARMACOEPIDEMIOLOGY Pharmacoepidemiology is the study o drug outcomes as documented in observations o clinical data rom large populations o typical patients receiving routine care. To understand this approach, it is necessary to think about drug e ects in ways that are di erent rom those o conventional pharmacology (Table 53-1). This perspective considers the population as the experimental system being studied. Medications can be considered to be variables introduced into this system much as they might be studied in an individual patient, in tissue culture, or in an isolated single-cell preparation. The di erences are that, in populations, true randomization does not occur, the intervening decision making and behavior o doctors and patients can alter the drug’s e ect, outcomes are measured in terms o probabilities (or rates) o events, and the magnitude o drug experience in the analysis is much larger than that o conventional pharmacology, ranging to millions o patients and millions o person-years o exposure. The importance o pharmacoepidemiology is highlighted by a number o prominent drug withdrawals in recent years. Each o these withdrawals was preceded by severe or atal adverse e ects that had been unrecognized or underappreciated at the time o approval (Table 53-2). Using the tools o pharmacoepidemiology, it is possible to identi y adverse e ects that may be overlooked in randomized trials because those adverse e ects are uncommon, represent an increase in risk rom an already high baseline (e.g., an increase in risk o myocardial in arction or stroke in older patients), occur primarily in patient groups underrepresented in clinical trials (e.g., the elderly, children, or pregnant women), require many months or years to develop, occur primarily with coadministration o specif c other drugs, and/or occur primarily in patients with a specif c comorbidity or genotype. TABLE 53-1 Conventional Pharmacology Compared to Pharmacoepidemiology CONVENTIONAL PHARMACOLOGY

PHARMACOEPIDEMIOLOGY

Modest number o patients studied

Large populations o patients studied

Direct dose–response relationships

Def ne probabilities o benef t and risk

Focus on biology

Focus on behavior o prescribers and patients as well as biology

Outcomes over short time rame

Longer time rame o study

Rare events di f cult to study

Able to identi y rare events

C h a p t e r 5 3 Systematic Detection o Adverse Drug Events

TABLE 53-2 Important Withdrawals of Widely Used Drugs TRADE NAME (GENERIC NAME)

REASON FOR WITHDRAWAL

Duract (brom enac)

Hepatotoxicity

Posicor (mibe radil)

Hypotension, bradycardia

Fen-phen ( enf uramine/ phentermine)

Pulmonary hypertension, cardiac valvulopathy

Rezulin (troglitazone)

Hepatotoxicity

Baycol (cerivastatin)

Rhabdomyolysis

PPA (phenylpropanolamine)

Intracerebral hemorrhage

Vioxx (ro ecoxib)

Myocardial in arction, stroke

Bextra (valdecoxib)

Stevens-J ohnson syndrome, myocardial in arction

Sources of Pharmacoepidemiologic Data Once a drug is in routine use, in ormation about its adverse e ects can come rom a variety o sources. These include (1) spontaneous reports submitted to the FDA or manu acturer by physicians, other health pro essionals, or patients; (2) analysis o data sets assembled by large health care systems, government programs, or private insurers in the course o paying or prescriptions and clinical services; (3) ongoing registries o patients given a specif c medication or with a given disease; and (4) individual ad hoc studies designed to answer a specif c question. Each approach has its strengths and weaknesses, which must be considered in assessing the quality o the evidence derived rom a particular source.

Spontaneous Reports

By de ault, spontaneous reports have been one o the most heavily relied-upon sources o in ormation used by the FDA to track the adverse e ects o marketed drugs. Such reports are submitted by practitioners or patients to drug makers or to the FDA, describing an adverse event in a single patient that may have been drug related. A strength o spontaneous reports is that they are o ten the f rst signal o an e ect that was not previously suspected (e.g., cardiac valvulopathy in patients taking en uramine-type diet aids). While such reports can be use ul to generate new hypotheses, they have important limitations. First, the majority (90–99%) o drug-induced illness is never reported; this is true even or previously unknown, serious adverse e ects. The rate o reporting is in uenced heavily by the newness o a drug, by reports in the medical literature and lay media, and by other actors. Because such reports originate in undef ned populations o users, it is di f cult to learn much rom their requency—an important issue in trying to compare the rates o a particular known adverse e ect between one drug and other members o the same class. Limited availability o clinical data about the reported case can also hamper e orts to assess con ounders that may have distorted the drug–outcome relationship (see discussion below).

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Automated Databases

Automated health care utilization databases have become increasingly important or def ning associations among medications and adverse e ects. Nearly all prescriptions f lled by patients are recorded in computerized databases, o ten or billing purposes, making such databases some o the best “wired” components o the health care system. For many patients, individual clinical encounters (e.g., physician visits, hospitalizations, procedures, and diagnostic tests) are recorded or the same reason, usually with one or more associated diagnoses, in separate billing databases. Even when these services are delivered in an uncoordinated manner (as or most Medicare and Medicaid patients), the data trail produced makes it possible to measure the requency o use o a given drug in a def ned population o patients, as well as the requency o specif c outcomes (desired or undesired) in users o such drugs. I a population is relatively well def ned and stable (as may be the case in many public insurance programs and some nongovernmental health care systems), it is possible to evaluate exposures and outcomes systematically. The increasing availability o adequate clinical in ormation in such data sets (e.g., diagnosis and number and length o hospitalizations or specif c reasons) makes it possible to conduct rigorous studies o specif c drug–outcome relationships, as described below. In the past, an important concern with the use o such utilization-based databases has been the limited and o ten unvalidated nature o the diagnostic in ormation, particularly in the outpatient setting. Care is required in evaluating this diagnostic in ormation. Whereas a f lled prescription or a 30-day supply o simvastatin 30 mg is specif ed unambiguously in a pharmacy data f le, the presence (or absence) o a code or depression or drug allergy or heart ailure may represent a much wider spectrum o clinical reality. Some diagnoses can be made with certainty rom computer-based claims data, such as a hip racture repaired surgically or a hospitalization or myocardial in arction. Others may require validation o a computer-based diagnosis by reviewing the primary medical record. Fortunately, the quantity and quality o such in ormation are growing, with more opportunities each year to link data rom the text o medical notes, laboratory test results, and other elements o the patient record.

Patient Registries

For some drugs, the manu acturer is asked by the FDA to keep track o all patients (or a sample o all patients) who use the drug. This request may be made both to def ne and to prevent specif c dangerous adverse e ects (e.g., the agranulocytosis that can result rom use o the antipsychotic medication clozapine ).

Ad Hoc Studies

Some important questions in pharmacoepidemiology cannot be addressed by these methods but must instead be answered by collecting data de novo on particular groups o patients with a given disease or patients taking a particular class o medications. One example is the def nition o sudden uncontrollable somnolence (sometimes called sleep attacks) in patients taking dopamine agonists or Parkinson’s disease (PD). Such events were not systematically documented in most large clinical trials o these drugs and were not likely to be recorded as a new diagnosis in an o f ce visit.

950 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

Determining whether some drugs are more likely to cause this problem than others required interviewing a large sample o PD patients using di erent classes o medications to def ne the details o the risk and point to means o reducing it (e.g., dose reduction).

Study Strategies

Ca s e -control s tudy

= fille d pre s cription for drug X

Once a source o pharmacoepidemiologic data has been identif ed, statistical methods are used to evaluate those data and reach conclusions about the associations between a drug and possible adverse e ects. The two most common types o analyses used to evaluate these observational data are cohort studies and case-control studies. Each is designed to evaluate the likelihood that a particular adverse outcome is caused by use o a given drug.

Controls (no M.I.) Time

Cohort (follow-up) s tudy

Cohort and Case-Control Studies

In cohort studies, one identif es a group o patients exposed to a given drug (e.g., patients with arthritis treated with a particular NSAID) and a second group o patients who are as similar as possible to the exposed group but did not take the drug o interest (e.g., patients with arthritis o comparable severity who were treated with a di erent NSAID). Both groups are then ollowed over time to determine how many in each group develop an adverse e ect o interest (e.g., myocardial in arction; Fig. 53-1). While this can be done on a real-time basis, more commonly, exposure (or nonexposure) that occurred in the past is def ned rom an existing database, so that subsequent events can be analyzed retrospectively. Cohort studies make it possible to measure actual incidence rates (i.e., the likelihood o a given outcome ollowing use o a particular drug) and to track multiple outcomes. They also make it possible to restrict analyses to new users o a given medication, since “veteran” (prevalent) users are more likely to include those who have not experienced an adverse e ect (or even died) as a result o the drug exposure. Such new-user designs also parallel the structure o a clinical trial more closely. By contrast, in case-control studies, one f rst specif es the case-def ning outcome event (e.g., myocardial in arction) and identif es a group o patients who have experienced that event; these are the cases. The controls are patients in the same population who are as similar as possible to the cases but have not had the outcome o interest (e.g., patients o similar age and gender, and with similar cardiac risk actors, who have not had a myocardial in arction). One then looks back in time prior to the occurrence o the event o interest (or its nonoccurrence, or controls) to review all the medications that were taken by cases and by controls to determine whether use o a given drug was higher than expected among cases than among controls (Fig. 53-1). The case-control design is more e f cient than the cohort design i the outcome o interest is rare and one has to interview all study participants, because it is possible to ocus on a selected group o patients known to have had the outcome o interest.

Evaluation of Risk

At the most basic level, cohort and case-control studies yield data that can be seen as comprising a 2 2 table def ning the presence or absence o exposure to the drug o interest as well as the presence or absence o the adverse outcome. The data can be arranged in our cells, as shown in Figure 53-2: patients who took the drug o interest and had the outcome (A), patients who took the drug but did not have

Cases (e .g., M.I.)

Exposed (ta king drug X)

= outcome e ve nt (e .g., M.I.)

Unexposed (not taking drug X) Time

FIGURE 53-1. Schematic design of case-control and cohort studies. Top. In a case-control study, cases are identif ed as patients in a population who have experienced the outcome event o interest (e.g., myocardial in arction [M.I.]); controls are patients in the same population who are as similar as possible to the cases but have not had that outcome. All medications taken by cases and controls are then reviewed retrospectively to determine whether use o a given medication was higher among cases than among controls. Bottom. In a cohort study, two groups o patients are identif ed: those who are taking a given drug and another group who are as similar as possible to the exposed group but are not taking the drug o interest. All patients are ollowed over time to determine how many in each group develop a specif ed outcome event o interest (e.g., myocardial in arction).

the outcome (B), patients who did not take the drug but had the outcome anyway (C), and patients who did not take the drug and did not have the outcome (D). Cells A and D are concordant or the drug–outcome relationship, and cells B and C are discordant or this association. In simple terms, the product A D divided by the product B C re ects the strength o such an association. For cohort studies, this is re erred to as the relative risk; or case-control studies (provided that the case outcome is not common), this is known as the odds ratio. A relative risk (or odds ratio) o 2 means that patients using the drug are twice as likely to have the outcome as patients not using the drug; a relative risk or odds ratio o 0.5 means that users o the drug are hal as likely as nonusers to experience the outcome (i.e., the drug has a protective e ect or that outcome).

Issues in Study Design and Interpretation Although pharmacoepidemiology provides the capacity to assess drug outcomes in large populations o typical patients,

C h a p t e r 5 3 Systematic Detection o Adverse Drug Events

951

Confounding by Indication Adve rs e outcome

A

B Exposure + Outcome +

Drug e xpos ure

C No drug e xpos ure

No a dve rs e outcome

Exposure + Outcome –

D Exposure – Outcome +

Exposure – Outcome –

FIGURE 53-2. Basic analysis o data rom case-control and cohort studies. The 2 2 table is def ned by the presence or absence o exposure to the drug o interest as well as the presence or absence o the outcome o concern. Cells A to D include, respectively, patients who took the drug and had the outcome (A), patients who took the drug but did not have the outcome (B), patients who did not take the drug but had the outcome anyway (C), and patients who did not take the drug and did not have the outcome (D). In simple terms, the product A D divided by the product B C re ects the strength o the drug–outcome association. For case-control studies (provided that the case outcome is not common), this ratio is termed the odds ratio; or cohort studies, this ratio is termed the relative risk.

it should be noted that the use o one drug rather than another in routine care is determined by individual decisions—not at random, as would occur in a controlled clinical trial. This decision bias introduces the potential problem that patients given Drug A could di er systematically rom those given Drug B—and that those di erences, rather than di erences in the drugs, could lead to a higher incidence o a particular outcome. Epidemiologists and statisticians have developed several strategies to correct or this problem o con ounding, which is inherent in observational studies. The goal is to address the possibility that the drug does not cause the outcome o interest but appears associated with it because both are associated with a third con ounding actor. For example, lung cancer is more common among co ee drinkers; this is not because co ee causes lung cancer, but because co ee drinkers are more likely to be smokers. To address con ounding, researchers attempt to learn as much as possible about the characteristics o patients who use each drug regimen under study. Were the patients who were prescribed one drug older than patients given a comparator drug? Or sicker? Or more likely to be taking (or not taking) other medications that could in uence the likelihood o a given outcome? For example, in a study comparing rates o myocardial in arction in patients taking ro ecoxib (Vioxx®) with those taking celecoxib (Celebrex®), ibupro en (Motrin®), or no NSAID, one would want to know as much as possible about the patients’ history o cardiovascular disease as well as their cardiac risk actors. I these characteristics were evenly balanced across the users o the various drugs, there would not likely be a problem. However, i not ( or example, i users o ro ecoxib were more likely to be smokers than users o celecoxib, or less likely to take prophylactic doses o aspirin), this would have to be adjusted or in the analysis. Such adjustment can be accomplished by statistical techniques that include multiple regression, propensity scores, or instrumental variable methods.

In a randomized trial, subjects are assigned arbitrarily to one treatment versus another. I the study is large enough and the randomization works adequately, di erences in outcomes between subjects in the various study arms are likely to be the result o the di erent treatments they received because they were (by def nition) similar in all other respects. By contrast, in an observational study, the researcher is obliged to study outcomes in patients or whom a physician has already chosen to prescribe one drug versus another versus no drug. It is there ore necessary to move beyond the simple 2 2 ormulation described above, adjusting the observed relationships so as to control or di erences that may have existed be ore the patients took the drugs under study. For example, patients who take antihypertensive medications are likely to have more cardiovascular disease than a group o age- and sex-matched people in the same community who do not take antihypertensive medications. O course, this is not because blood pressure medicines cause heart disease; on the contrary, antihypertensive medications reduce the risk o cardiovascular disease (including heart ailure, myocardial in arction, and stroke) in such patients. But while these medications reduce the risk o heart disease, they do not reduce it to zero. Furthermore, many patients with hypertension start therapy later in li e, or do not adhere adequately to their prescribed regimens. As a result, antihypertensive medication users overall have a higher rate o heart disease than demographically identical individuals who do not take blood pressure medication. This problem is known as confounding by indication.

Selection Bias

A second problem is produced by the act that in routine care, patients’ drug use is determined by their physicians and not by a research protocol. For example, when f uoxetine (Prozac®) f rst introduced the selective serotonin reuptake inhibitor (SSRI) class o antidepressants in the late 1980s, reports emerged that depressed patients given the new drug were more likely to commit suicide than patients taking older antidepressants such as the tricyclic antidepressants (amitriptyline , nortriptyline , desipramine ). Indeed, concern persists (based on placebo-controlled randomized trials) that SSRIs may precipitate suicidal thoughts or attempts in some patients, especially adolescents and children. However, the early reports o increased risk suggest that selection bias could provide an alternative explanation or suicide in uoxetine users. Patients doing well on older antidepressants would have been less likely to be switched to the newer drug when it was f rst marketed; use o a novel medication would have occurred disproportionately more in depressed patients who were not doing well—including, perhaps, those who were continuing to consider suicide. Moreover, the lethal dose (LD50; see Chapter 2, Pharmacodynamics) or the older drugs is low because o their cardiovascular toxicity, whereas it is much more di f cult to ingest enough SSRI or a atal overdose. Thus, a physician would pre er a potentially suicidal patient to have a supply o uoxetine at home rather than a supply o tricyclic antidepressant. Whatever the underlying risk o suicide caused by either drug, these actors alone would combine to create a prof le o higher suicide rates among new uoxetine users compared to tricyclic antidepressant users in an observational assessment.

952 FUNDAMENTALS OF DRUG DEVELOPMENT AND REGULATION

The Healthy User E ect

Several epidemiologic studies o drug use and outcomes have def ned relationships that have not been borne out in randomized controlled trials. These include reduced rates o cardiac disease, incontinence, and depression in women taking postmenopausal estrogen and reduced rates o cancer and Alzheimer’s disease in patients taking statins. Such studies are o ten awed by what has been called the “healthy user” e ect. Patients who are regular users o any preventive medication appear to be di erent rom those who do not exhibit this behavior: they are more likely to visit their doctor seeking preventive therapy, or are at least open to receiving it, and their physicians are su f ciently prevention oriented to write such a prescription. Such patients are probably also more likely to engage in other health-promoting behaviors, such as tobacco avoidance, weight control, exercise, and adherence to their other prescribed drug regimens. These characteristics likely exist to an even greater extent among patients who adhere aith ully to the prescribed regimen or a prolonged period o time. Several large randomized trials have proven a similar point: patients randomized to placebo who adhere well to their dummy pill “regimen” have better outcomes (including mortality) than patients who do not adhere well to their placebo “regimen.” Because the content o the placebo could not have produced this e ect, these f ndings provide clear evidence that patients who consistently behave in a healthpromoting manner are more likely to have better clinical outcomes, apart rom any therapeutic e ect o a specif c drug in their regimen. To address this issue in observational studies, some research groups use only “active controls” as comparator groups— or example, comparing patients adherent to statin regimens with patients adherent to regimens o other preventive drugs rather than simply comparing such patients with patients who are not regular statin users.

Interpreting Statistical Signif cance

In evaluating the results o both observational studies and randomized trials, it is conventional to use a p value o 0.05 as a threshold or benchmark or statistical signif cance. This criterion is o ten mistakenly interpreted to mean that a f nding is “real” i the p value or the di erence between groups is less than that value and “not real” i it is greater than that value. However, more sophisticated readers o the literature understand that such a cut point is largely arbitrary (compared to, or example, a p value o 0.03 or 0.07), and that attention must also be paid to the magnitude o the di erence. For example, a p 0.05 di erence between a new drug and placebo may be clinically meaningless i there is only a 2% di erence in e ect size. The situation is even more critical in assessing the statistical signif cance o data about adverse events, whether rom a randomized trial or rom an observational analysis. It is use ul to recall that the p value is determined by both sample size and the magnitude o an observed di erence. Most clinical trials are powered to be large enough to detect a di erence between a study drug and its comparator in producing a clinical outcome that is relatively common (e.g., reduction in blood pressure or LDL cholesterol level). As a result, however, such studies are not likely to have adequate power to f nd a statistically signif cant di erence between groups or outcomes that are much more rare (e.g., hepatotoxicity). Adherence to a “p 0.05” standard or uncommon

adverse e ects can lead to dismissal o important risks that a study may not have been powered to detect. The solution is not to embrace all di erences in adverse e ect rates regardless o their statistical properties. Instead, it is to consider such rate di erences thought ully and to seek additional evidence to clari y worrisome relationships even i they are not “signif cant” in p-value terms. For example, when the FDA was evaluating the risk o suicidal thoughts and actions in adolescents and children taking SSRI antidepressants in placebo-controlled trials, the rates o these relatively rare outcomes were generally higher in the treated patients than in those randomized to placebo. Each individual study did not f nd a p 0.05 level o signif cance or these di erences. However, when the data rom all such trials were aggregated (in some cases, years a ter the studies were completed), it became clear that the risk across all studies was clear and consistent (and also met the conventional p 0.05 level). The opposite problem arises when considering the statistical signif cance o data rom large population-based epidemiologic studies. Here, sample size (power) is not a limitation, especially when studies employ data on several hundred thousand patients through use o an automated claims database. A 4% or 5% di erence in rates o a given e ect (either therapeutic or adverse) may achieve a p value 0.001, simply because o the huge size o the population studied. But here, even i the f nding appears to have statistical signif cance, a di erence o such small magnitude may have little or no clinical importance.

ADVERSE DRUG EFFECTS AND THE HEALTH CARE SYSTEM The series o sa ety-related withdrawals o commonly used drugs in the 1990s and early 2000s led to renewed interest in developing ways to prevent such problems, or at least to limit the number o patients exposed to risk by identi ying adverse e ects earlier. As a result, the concept o risk management has become an important theme in drug development and regulation.

Balancing Benef ts and Risks As noted above, new products are o ten not compared with existing alternatives when they are evaluated or approval, and such studies are not commonly per ormed a ter approval either. For drugs with known risks, it is there ore di f cult to know whether an adverse e ect occurs more commonly with a new drug than with another drug in the same class (e.g., gastrointestinal hemorrhage with nonselective NSAIDs, or rhabdomyolysis with statins). A higher rate o a given adverse event might be acceptable or a particular drug i it were accompanied by substantially higher e f cacy. In this case, however, the absence o head-to-head clinical trials makes it di f cult to make such an evaluation. Thus, in most instances, the individual clinician is le t to make therapeutic decisions without the data needed to make such choices rigorously. A recent development designed to remedy this problem is the movement toward comparative e ectiveness research—a program o publicly unded studies that systematically evaluate therapies against one another. This program was initiated in 2009 with a $1.1 billion ederal investment and is expected to be an important ongoing component o the research agendas o several ederal agencies.

C h a p t e r 5 3 Systematic Detection of Adverse Drug Events

The clinical use o medications is heavily in uenced by the $30 billion spent annually by the pharmaceutical industry to market its products. This expenditure is heavily “ ront-end loaded,” with vast sums spent soon a ter a drug is launched in order to maximize sales or as many years as possible while the company’s patent is still in e ect. Ironically, this means that the heaviest promotion o a drug occurs during the period in which there is least experience with its use and e ects in the population as a whole. At the time o approval, there may not be much (or even any) in ormation in the peer-reviewed literature about a drug’s e f cacy and sa ety, so promotional sources o in ormation are o ten the primary means by which physicians learn about new products. Industry critics have argued that these materials o ten emphasize therapeutic benef ts more persuasively than they communicate risk. As an alternative, several innovative programs have emerged that provide prescribers with noncommercial, publicly unded “marketing” o evidence-based data about drug benef ts, risks, and costs, known as academic detailing (e.g., see www.NaRCAD.org and www.alosa oundation.org).

Role o the FDA Just as the thalidomide tragedy o the early 1960s helped spark a wave o regulatory re orms that gave the FDA new authority to demand proo o e f cacy be ore a drug was approved, the 2004 withdrawal o ro ecoxib (Vioxx®) also led to calls or regulatory re orm, particularly in the way adverse events are detected and ollowed up. One area o vigorous debate was the FDA’s lack o clear authority to require postmarketing studies o drug risks. While the agency holds considerable sway over manu acturers during the initial drug approval process, it has had little power to compel urther study o a drug once it is on the market. Governmental reviews have demonstrated that, even when postmarketing sa ety studies are mandated at the time o approval, they are o ten not completed or even initiated (see above). This helps explain the tardiness with which important adverse e ects have been detected and acted on. Rationalizing the national response to this problem has become a key goal or public policy. In 2007, the FDA Amendments Act gave the agency the authority and responsibility to per orm its own systematic surveillance o adverse events o marketed drugs, to alter a drug’s o f cial labeling to warn o sa ety risks (such authority was previously in the hands o the manu acturer), and to compel drug companies to conduct ollow-up studies o potential sa ety concerns. That legislation also mandated and unded the creation o a nationwide “Sentinel System” to use large existing automated data sets rom a variety o health care delivery systems to conduct ongoing postmarketing drug sa ety surveillance. Within a ew years, the system was expanded to include data describing the medication use and clinical encounters o more than 100 million (anonymized) patients, making it a valuable tool or the systematic detection o adverse drug e ects ar earlier than had previously been possible.

Legal and Ethical Issues The drug sa ety controversies o recent years (see Table 53-2) have caused many in the medical pro ession, the government, and the public to ask how responsibility should be apportioned or discovering and acting on important adverse e ect data. There is a growing consensus that, in addition to greater vigilance on the part o the FDA, a drug’s manu acturer

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should also be expected to serve as “steward o its molecule,” responsible or proactive research into possible harms beyond the minimum required by law. Juries and courts have agreed with this notion; legal settlements exceeded $1 billion or cerivastatin (Baycol®) and $21 billion or dex enf uramine (Redux®), even in the absence o criminal convictions.

CONCLUSION AND FUTURE DIRECTIONS Increasing availability and detail o electronic data def ning drug use and clinical events in very large populations, coupled with advances in epidemiology and developments in the f elds o in ormatics and data processing, have made it possible to per orm sophisticated surveillance o the outcomes o routine medication use with rigor and e f ciency. These data are being made even more use ul by advanced methodological tools, such as propensity scores and instrumental variables, to improve control or con ounding in observational studies. Pharmacoepidemiologic analyses based on these developments can orm the oundation or decisions—made both at the bedside and at policy levels—based on science rather than on hunches, ear, or hype. These databases and epidemiologic tools also hold potential or def ning comparative drug e ectiveness by using the same tools to measure desired clinical outcomes across agents. Thus, observational studies make possible the head-to-head comparison o medications that is not required by the approval process but that is central to the in ormational needs o prescribers, patients, and payors. From a biological perspective, the systematic detection o adverse e ects will urther benef t rom the development o research tools to predict the toxicities o new compounds more accurately and to ag them or intensive surveillance once a drug is marketed. In addition, pharmacogenomics (see Chapter 7, Pharmacogenomics) is addressing many o these questions rom the perspective o inherited di erences in drug metabolism (pharmacokinetics) and drug responses (pharmacodynamics).

Suggested Reading Avorn J. Power ul medicines: the benef ts, risks, and costs o prescription drugs. New York: Knop ; 2005. (An examination o the interrelationships among pharmacology, clinical practice, epidemiology, industry, and drug policy.) Avorn J. The promise o pharmacoepidemiology in helping clinicians assess drug risk. Circulation 2013;128:745–748. (An assessment o how observational methods can be used to def ne medication sa ety problems, using anticoagulants as an example.) Eichler HG, Oye K, Baird LG, et al. Adaptive licensing: taking the next step in the evolution o drug approval. Clin Pharmacol Ther 2012;91:426–437. (A proposal to integrate epidemiologic assessment into the evaluation o drug e ectiveness and toxicity.) Gagne JJ, Wang SV, Rassen JA, Schneeweiss S. A modular, prospective, semi-automated drug sa ety monitoring system or use in a distributed data environment. Pharmacoepidemiol Drug Sa 2014;23:619–627. (A new method or automating analysis o large clinical data sets to detect adverse e ects more rapidly.) Psaty BM, Breckenridge AM. Mini-Sentinel and regulatory science—big data rendered f t and unctional. N Engl J Med 2014;370:2165–2167. (An overview o current programs to harness routine clinical data to monitor drug sa ety.) Schneeweiss S, Rassen JA, Glynn RJ, Avorn J, Mogun H, Brookhart MA. High-dimensional propensity score adjustment in studies o treatment e ects using health care claims data. Epidemiology 2009;20:512–522. (Description o an innovative approach to use large-scale electronic databases to study the outcomes o marketed drugs.) Strom BL, Kimmel SE, Hennessey S, eds. Textbook o pharmacoepidemiology. West Sussex, United Kingdom: Wiley-Blackwell; 2013. (A comprehensive textbook o pharmacoepidemiology.)

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54

Protein Therapeutics

Q u e n t in J . Ba c a , Be n ja m in Le a d e r, a n d David E. Go la n

INTRODUCTION N & CASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 955– 5 95 9566 CHALLENG NGES ES FOR PROTEIN N THE HERAPEUTICS S . . . . . . . . . . . . . . . 975 USES OF PROTEINS Sv IN MEDICINE 957 95 ONCLUSION AND FUTURE DIRECTIO 977 ‡ nsulin dettemir ‡I mir L Le Lev Levemir vemir emir (detemir) mir) ir). . . . . . . . . . .Insulin nsul n ana anal an alo al oogu og gguuues e Cwith slower onset of aaction ctio ct ion n and ONS . . . .Diabetes Diabbet etes ess mel mel melli ellilitu itus s ((detem Group I: Enzymess a nd Regulatory Proteins . . . . . . . 958 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97 9788 ‡Ins ulin lin gla rgine e Basssaglar Bas Basa Basag aaglar glar ggl larr ((glargine) (glargi glar gl argi gine g ne) nee ) long lo longer onger nger er durat ur atio ionn of aaction c tio ct ionn Group II: Targeted Prootte inss . . . . . . . . . . . . . . . . . . . . . . . 966 Lantu L Lantus Lant La ntu t u s ( (gl (gla gla g l a argine) i ) Grou Gr oupp II III: I: Protein Vacciness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973 Group IV: Protein Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . 975

‡Insulin zincc extended e xte ttend nded ded ded

Lente Ultralente

IInsulin li zinc i hhexameric complex c om ompl plex ex with witithh slower sl onsett ooff Diab Diabetes abetes m mellitus ellilitu el tuss acti ac tion o andd lon onge gerr du durattion of action genetic disorders. Fi th, the clinical development and FDA INTRODUCTION Pram Pr amlilint ntid idee Symlin Mechanism unknown; approval recombi time bina nant o ntprotein s yn ynthetic therapeutics peptide mayDiabetes be astermellitus, than thatin combination Proteins have the most dynamic and diverse roles o any o small-molecule drugs.occurring A study publishedwi inth2003 showed analogue of human amylin (a naturally with ins nsul ulin macromolecules in the body, catalyzing biochemical reac- that the average clinical development and approval time was neuroendocrine hormone regulating postprandial tions, constituting receptors and channels in membranes, more than 1 year aster or 33 protein therapeutics approved glucose control) providing intracellular and extracellular sca olding sup- between 1980 and 2002 than or 294 small-molecule drugs port, and transporting molecules within a cell or rom one approved during the same time period. Sixth, because proorgan to another. According to current estimates, there are teins are unique in orm and unction, companies are able approximately 19,000–20,000 protein-coding genes in the to obtain ar-reaching patent protection or protein therahuman genome, and with alternative splicing o genes and peutics. Seventh, many protein therapeutics address unmet post-translational modif cation o proteins (e.g., by cleavage, needs or rare diseases and are eligible or orphan drug desphosphorylation, acylation and glycosylation), the number ignation, which can provide priority review by the FDA and o unctionally distinct proteins is likely to be greater than extended patent protection terms. Last, the total sales o pro100,000. Viewed rom the perspective o disease mecha- tein therapeutics are continuing to trend upward. In the year nisms, these estimates pose an immense challenge to modern 2000, no protein therapeutics were represented in the top 20 medicine, as disease may result when any one o these pro- drugs sold in the United States. In 2013, protein therapeutics teins contains mutations or other abnormalities or is present comprised our o the top ten pharmaceuticals by sales in in an abnormally high or low concentration. Viewed rom the the United States and seven o the top ten pharmaceuticals perspective o therapeutics, however, these estimates repre- by sales worldwide. The last our advantages make proteins sent a tremendous opportunity in terms o harnessing protein attractive rom a f nancial perspective compared with smalltherapeutics to alleviate disease. At present, more than 206 molecule drugs. di erent proteins or peptides are approved or clinical use A relatively small number o protein therapeutics are puby the US Food and Drug Administration (FDA), and many rif ed rom their native sources, such as pancreatic enzymes more are in development. rom hog and pig pancreas and -1-proteinase inhibitor rom Protein therapeutics have several advantages over small- pooled human plasma. Instead, most therapeutic proteins molecule drugs. First, proteins o ten serve a highly specif c are now produced by recombinant DNA technology and and complex set o unctions that cannot be mimicked by purif ed rom a wide range o organisms. Production syssimple chemical compounds. Second, because the action o tems or recombinant proteins include bacteria, yeast, insect proteins is highly specif c, there is o ten less potential or cells, mammalian cells, and transgenic animals and plants. protein therapeutics to inter ere with normal biological pro- The system o choice can be dictated by the cost o produccesses and cause adverse e ects. Third, because the body tion or the modif cations o the protein (e.g., glycosylation, naturally produces many o the proteins that are used as phosphorylation, or proteolytic cleavage) that are required therapeutics, these agents are o ten well tolerated and are or biological activity. For example, bacteria do not per orm less likely to elicit immune responses. Fourth, or diseases in glycosylation reactions, and each o the other biological which a gene is mutated or deleted, protein therapeutics can systems listed above produces a di erent type or pattern o provide e ective replacement treatment without the need glycosylation. Protein glycosylation patterns can have a draor gene therapy, which is not currently available or most matic e ect on the activity, hal -li e, and immunogenicity 955

956 FRONTIERS IN PHARMACOLOGY

MR is a 55-ye a r-o ld tra ve lin g s a le s m a n w h o p re s e n ts to th e e m e rg e n cy d e p a rtm e n t o a s m a ll ru ra l h o s p ita l w ith le ts id e d ch e s t p a in a n d lig h t-h e a d e d n e s s . Th e p a in s ta rte d s u d d e n ly 1 h o u r a g o w h e n h e wa s ca rryin g a la rg e b o x. At f rs t, MR e lt a s i h e w a s g o in g to p a s s o u t, b u t th e p a in a n d lig h t-h e a d e d n e s s im p ro ve d a t re s t a n d e ve n tu a lly re s o lve d a te r 20 m in u te s . MR d e n ie s a ny o th e r s ym p to m s , a n d h e h a s n o h is to ry o m e d ica l p ro b le m s . He ta ke s n o m e d ica tio n s , h e is n o t a s m o ke r, a n d h is a th e r d ie d u n e xp e cte d ly in a ca r a ccid e n t a t a g e 53. On p h ys ica l e xa m , MR is a e b rile w ith h e a rt ra te 10 0 b e a ts /m in , b lo o d p re s s u re 150/90 m m Hg , a n d re s p ira to ry ra te 16 b re a th s / m in . His p u ls e o xim e te r d is p la ys 96% w ith oxyg e n o w in g a t 2 lite rs p e r m in u te b y n a s a l ca n n u la . He a p p e a rs to b e co m o rta b le , a n d th e re m a in d e r o h is p hys ica l e xa m is n o ta b le o n ly o r a n S 4 h e a rt s o u n d . His ECG d e m o n s tra te s s in u s ta chyca rd ia w ith n o ST s e g m e n t e le va tio n . His ch e s t x-ra y is n o rm a l. His S TAT ch e m is try p a n e l s h o w s n o rm a l s o d iu m , p o ta s s iu m , ch lo rid e , b ica rb o n a te , b lo o d u re a n itro g e n (BUN), a n d cre a tin in e le ve ls . Ca rd ia c b io m a rke rs a n d co a g u la tio n s tu d ie s a re p e n d in g . MR is g ive n a s p irin , m e to p ro lo l, a n d s u b lin g u a l n itro g lyce rin u p o n h is a rriva l in th e e m e rg e n cy d e p a rtm e n t. Wh ile w a itin g in th e e m e rg e n cy d e p a rtm e n t, MR’s tro p o n in T le ve l re tu rn s a t 1.34 n g /m L (n o rm a l, 0–0.1 n g /m L), h e a ga in d e ve lo p s ch e s t p a in , a n d a re p e a t EKG s h o w s 2-m m ST s e g m e n t d e p re s s io n in

o the recombinant protein in the body. For example, the hal -li e o native erythropoietin, a growth actor important in erythrocyte production, can be lengthened by increasing the glycosylation o the protein. Darbepoetin- is an erythropoietin analogue that is engineered to contain two additional amino acids that are substrates or N-linked glycosylation reactions. When expressed in Chinese hamster ovary cells, the analogue is synthesized with f ve rather than three N-linked carbohydrate chains; this modif cation causes the hal -li e o darbepoetin to be three times longer than that o erythropoietin. Perhaps the best example o trends in the production and use o protein therapeutics is provided by the history o insulin in the treatment o type 1 and type 2 diabetes mellitus . Untreated, type 1 diabetes is a disease that leads to severe wasting and death due to lack o the protein hormone insulin, which signals cells to per orm numerous unctions related to glucose homeostasis and intermediary metabolism. In 1922, insulin was f rst purif ed rom bovine and porcine pancreas and used as a li e-saving daily injection or patients with type 1 diabetes. At least three problems hindered the widespread use o this protein therapy: f rst, the availability o animal pancreases or purif cation o insulin; second, the cost o insulin purif cation rom animal pancreas; and third, the immunological reaction o some patients to animal insulin. These problems were addressed by isolating the human

le a d s V1–V3. At th is tim e , h e is a ls o g ive n h e p a rin , a b cixim a b , a n d clo p id o g re l, a n d h is ch e s t p a in re s o lve s . He is a d m itte d to th e h o s p ita l a n d h is clin ica l co u rs e is s ta b le ove rn ig h t. Th e n e xt d a y, h o w e ve r, MR d e ve lo p s cru s h in g s u b s te rn a l ch e s t p a in a n d d ia p h o re s is , a n d h is ECG s h o w s 4-m m ST s e g m e n t e le va tio n in le a d s V2–V4. Be ca u s e ca rd ia c ca th e te riza tio n is n o t a va ila b le a t th e re g io n a l ca rd ia c ce n te r o r a t le a s t 4 h o u rs , MR is g ive n te n e cte p la s e in th e co ro n a ry ca re u n it, a n d h is a s p irin , m e to p ro lo l, n itro g lyce rin , h e p a rin , a n d clo p id o g re l a re co n tin u e d . He s ta b ilize s o n th is re g im e n . A te r a n o th e rw is e u n e ve n t u l 5-d a y h o s p ita liza tio n , MR is tra n s e rre d to th e re g io n a l ca rd ia c ce n te r o r ca th e te riza tio n , w ith a d ia g n o s is o u n s ta b le a n g in a th a t e vo lve d in to a n ST e le va tio n m yo ca rd ia l in a rctio n . Ou tp a tie n t p la n s in clu d e ca rd ia c re h a b ilita tio n a n d tre a tm e n t w ith a s p irin , m e to p ro lo l, e n a la p ril, s p iro n o la cto n e , a n d s u b lin g u a l n itro g lyce rin a s needed.

Questions 1 . By what mechanism does tenecteplase act? 2 . How does the action of tenecteplase differ from that of heparin? 3 . By what mechanism does abciximab act? 4 . How could abciximab augment the function of clopidogrel and aspirin in this case?

insulin gene and engineering Escherichia coli to express human insulin by using recombinant DNA technology. By growing vast quantities o these bacteria, large-scale production o human insulin was achieved. The resulting insulin was abundant, inexpensive, o low immunogenicity, and ree rom other animal pancreatic substances. Recombinant insulin, approved by the FDA in 1982, was the f rst commercially available recombinant protein therapeutic and has been the major therapy or type 1 diabetes (and a major therapy or type 2 diabetes) ever since. Recombinantly produced proteins can have several urther benef ts compared with nonrecombinant proteins. First, transcription and translation o an exact human gene can lead to a higher specif c activity o the protein and a decreased chance o immunological rejection. Second, recombinant proteins are o ten produced more e f ciently and inexpensively and in potentially limitless quantity. One striking example is ound in the protein-based therapy or Gaucher’s disease, a chronic congenital disorder o lipid metabolism caused by a def ciency o the enzyme -glucocerebrosidase (also known as glucosylceramidase) that is characterized by an enlarged liver and spleen, increased skin pigmentation, and pain ul bone lesions. At f rst, -glucocerebrosidase purif ed rom human placenta was used to treat this disease, but this requires purif cation o protein rom 50,000 placentas per patient per year, which clearly places a practical limit

C h a p t e r 5 4 Protein Therapeutics

on the amount o purif ed protein available. A recombinant orm o -glucocerebrosidase was subsequently developed and introduced, which is not only available in su f cient quantities to treat many more patients with the disease but also eliminates the risk o transmissible (e.g., viral or prion) diseases associated with puri ying the protein rom human placentas. This also illustrates a third benef t o recombinant proteins over nonrecombinant proteins—the reduction o exposure to animal or human diseases. A ourth advantage is that recombinant technology allows the modif cation o a protein or the selection o a particular gene variant to improve unction or specif city. Again, recombinant -glucocerebrosidase provides an interesting example. When this protein is made recombinantly, a change o amino acid arginine-495 to histidine allows the addition o mannose residues to the protein. The mannose is recognized by endocytic carbohydrate receptors on macrophages and many other cell types, allowing the enzyme to enter these cells more e f ciently and to cleave the intracellular lipid that has accumulated in pathological amounts, which results in an improved therapeutic outcome. Last, recombinant

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technology allows the engineering and production o proteins that provide a novel unction or activity, as discussed below. The more than 30 years since the approval o recombinant insulin by the FDA have seen a remarkable expansion in the number o therapeutic applications o proteins. More than 206 unique proteins (over 160 o which are produced recombinantly) are currently approved or clinical use by the FDA, and many more are in development.

USES OF PROTEINS IN MEDICINE An appreciation o the many therapeutic uses o proteins may be acilitated by categorizing such therapies according to their mechanism o action. In this chapter, we summarize currently approved protein therapeutics using a classif cation system that is based on their pharmacologic action (Box 54-1). Examples o protein therapeutics in each category and clinical conditions in which they are used are discussed in the text, and a listing o FDA-approved protein therapies and their unctions and clinical uses is presented in Tables 54-1 through 54-5. Examples o protein-based vaccines and diagnostics, which also highlight

B O X 5 4 - 1 Fu n ctio n a l Cla s s if ca tio n o Pro te in Th e ra p e u tics Protein therapeutics in the tables are organized by unction and therapeutic application. The numbers o therapeutics per group ref ect the relative di culty associated with drug development across the various classes o protein therapeutics. Every e ort has been made to include in these tables all US Food and Drug Administration (FDA)-approved Group I and Group II proteinbased therapies. Groups III and IVpresent selected examples that highlight the use o proteins in vaccines and diagnostic agents.

to them and blocking their unction, targeting them or destruction, or stimulating a signaling pathway. This group has grown as monoclonal antibody technology has matured and will expand urther as signaling pathways and etiologies o disease are more clearly identi ed. Group IIb therapeutics deliver other compounds or proteins to a speci c site. This class has great potential to grow, as demonstrated by the breadth o the speci cally targeted Group IIa therapies.

Group I: Enzymes and Regulatory Proteins

Group III: Protein Vaccines

■

■ ■

Ia: Replacing a protein that is de cient or abnormal (Table 54-1) Ib: Augmenting an existing pathway (Table 54-2) Ic: Providing a novel unction or activity (Table 54-3)

Endocrine and metabolic disorders with de ned molecular etiologies dominate Group Ia. As more diseases are linked to deciencies o speci c proteins, this class will continue to grow. Group Ib is dominated by therapies that augment hematological and endocrine pathways and immune responses. The many inter eron and growth actor therapies in Group Ib e ectively treat disease even when their precise pharmacologic mechanism o action is unknown. Group Ic demonstrates the rational use o naturally occurring proteins to modi y the pathophysiology o human diseases. The uture growth o this class depends on understanding protein unction in human physiology as well as protein unction in other organisms.

Group II: Targeted Proteins ■ ■

IIa: Inter ering with a molecule or organism (Table 54-4) IIb: Delivering other compounds or proteins (Table 54-5)

Group IIa therapeutics use their special targeting activity to inter ere with molecules or organisms by binding speci cally

■ ■ ■

IIIa: Protecting against a deleterious oreign agent (Table 54-6) IIIb: Treating an autoimmune disease (Table 54-6) IIIc: Treating cancer (Table 54-6)

Although this is currently a small class o therapies, there is great potential or the production o recombinant vaccines that provide broad protection against in ectious agents. Similarly, individualized vaccines against cancers are likely to be in great demand. Selected examples o the more than 80 FDA-approved vaccines in Table 54-6 highlight the use o recombinant protein technology in vaccine production. Many o the FDA-approved vaccines protect against multiple in ectious agents and include synthetic, recombinant, and puri ed protein components. A complete list o FDA-approved vaccines may be ound at http://www . da.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts.

Group IV: Protein Diagnostics Protein diagnostics, o which selected examples are shown in Table 54-7, are a class that power ully a ects clinical decision making. These diagnostics use technology and therapeutics developed in other classes to answer clinical questions. This table presents primarily in vivo protein diagnostics, but in vitro protein diagnostics are also critical to medical decision making and are too numerous to address comprehensively here. ■

958 FRONTIERS IN PHARMACOLOGY

the growing importance o proteins in medicine, are provided in Tables 54-6 and 54-7.

Group I: Enzymes and Regulatory Proteins Protein therapeutics in this group unction by a classic paradigm in which a specif c endogenous protein is def cient, and the def cit is then remedied by treatment with exogenous protein. Protein therapeutics classif ed in Group Ia are used to replace a particular activity in cases o protein def ciency or abnormal protein production. These proteins are used in a range o conditions, rom providing lactase in patients lacking this gastrointestinal enzyme to replacing vital blood-clotting actors such as actor VIII and actor IX in hemophiliacs. A classic example, as mentioned above, is the use o insulin or the treatment o diabetes. Another important example is in the treatment o cystic f brosis, a common and o ten lethal genetic disorder. In this disease, de ects in the chloride channel encoded by the CFTR gene lead to abnormally thick secretions, which can (among other e ects) block pancreatic enzymes rom travelling down the pancreatic duct into the duodenum. This prevents ood rom being properly digested and results in malnutrition. Patients with cystic f brosis are o ten treated with a combination o pancreatic enzymes isolated rom pigs—including lipases, amylases, and proteases—that allow the digestion o lipids, sugars, and proteins. Patients who have had their pancreas removed or who su er rom chronic pancreatitis can also benef t rom this therapy. Other striking examples include various diseases caused by metabolic enzyme def ciencies, such as Gaucher’s disease as mentioned above, mucopolysaccharidosis, Fabry disease, and others. Additional protein therapies that replace a particular activity are listed in Table 54-1. It may sometimes be desirable to enhance the magnitude or timing o a particular normal protein activity, and protein therapeutics classif ed in Group Ib are administered to achieve this. Such protein therapeutics have been success ul in treating hematopoietic de ects; the most prominent example is recombinant erythropoietin, a protein hormone secreted by the kidney that stimulates erythrocyte production in the bone marrow. In patients with chemotherapy-induced anemia, recombinant erythropoietin is used to increase erythrocyte production and thereby ameliorate the anemia. In patients with chronic kidney disease, whose levels o endogenous erythropoietin are below normal, recombinant protein is administered to correct this def ciency. Another example is provided by the use o granulocyte or granulocytemacrophage colony-stimulating actor (G-CSF or GM-CSF, respectively) to treat patients with chemotherapy-induced neutropenia. G-CSF and GM-CSF stimulate the bone marrow to increase the number o neutrophils produced, which allows these patients to better combat microbial in ections. Similarly, patients with chronic immune thrombocytopenia can be treated with romiplostim, a thrombopoietin receptor agonist that increases platelet production and thereby prevents bleeding complications. In vitro ertilization (IVF) is another area in which Group Ib proteins are applied. Increased levels o ollicle-stimulating hormone (FSH) are normally produced by the anterior pituitary gland just be ore ovulation. These high levels o FSH can be enhanced by treatment with recombinant FSH, leading to maturation o an increased number o ollicles and to an increased number o oocytes available or IVF. Similarly, recombinant human chorionic gonadotropin (HCG) is used in

assisted reproductive technology to promote ollicle rupture, a process that must occur be ore the oocytes can be transported into the allopian tubes or ertilization. Group Ib proteins can also have li e-saving e ects on thrombosis and hemostasis. Alteplase (recombinant tissue plasminogen activator [tPA]) is used to treat li e-threatening blood clots in conditions such as coronary artery occlusion, acute ischemic stroke, and pulmonary embolism. Endogenous tPA is secreted by the endothelial cells that line blood vessels. The secreted tPA normally cleaves plasminogen to plasmin, which then degrades f brin and thereby lyses f brin-based clots. Although endogenous tPA may be present at normal or even increased levels near the site o a blood clot, administration o relatively large amounts o exogenous tPA may be required to disrupt these clots. Reteplase , a genetically modif ed orm o recombinant tPA, is used to treat acute myocardial in arction, and tenecteplase , another genetically engineered derivative o tPA, has greater specif city than tPA or binding to plasminogen and there ore causes more e f cacious lysis o f brin in blood clots. In the introductory case, MR received tenecteplase when his unstable angina evolved into an ST elevation myocardial in arction. Supraphysiologic levels o coagulation actor VIIa may catalyze thrombosis and thereby stop li e-threatening bleeding in patients with hemophilia A or B. Studies have suggested that recombinant activated protein C can improve immunoregulation and prevent excessive clotting reactions in patients with severe, li e-threatening sepsis and organ dys unction. Many other Group Ib protein therapeutics are also used or immunoregulation—chronic hepatitis B and C, Kaposi’s sarcoma , melanoma, and some types o leukemia and lymphoma have been treated with various orms o inter eron, as noted in Table 54-2. Other disease states treated with Group Ib proteins are summarized in Table 54-2. Occasionally, the activity o a particular protein is desirable even though the body does not normally express that activity. Protein therapeutics classif ed in Group Ic include oreign proteins with novel unctions and endogenous proteins that act at a novel time or place in the body. Papain, or example, is a protease purif ed rom the Carica papaya ruit. This protein is used therapeutically to degrade proteinaceous debris in wounds. Collagenase , obtained rom ermentation by Clostridium histolyticum, can be used to digest collagen in the necrotic base o wounds. The protease-mediated debridement or removal o necrotic tissue is use ul in the treatment o burns, pressure ulcers, postoperative wounds, carbuncles, and other types o wounds. Collagenase can also be used to digest subcutaneous collagen that contributes to the debilitating hand de ormity known as Dupuytren’s contracture. Recombinant human deoxyribonuclease I (DNAse1) has an interesting novel use. Normally ound inside human cells, this recombinant enzyme can be used to degrade the DNA le t over rom dying neutrophils in the respiratory tract o patients with cystic f brosis. Such DNA could otherwise orm mucus plugs that obstruct the respiratory tract and lead to pulmonary f brosis, bronchiectasis, and recurrent pneumonias. Thus, recombinant protein technology has allowed the therapeutic application o a normally intracellular enzyme in a novel extracellular environment. There are many other success ul examples o this approach to protein therapy. For instance, certain orms o acute lymphoblastic leukemia are unable to synthesize asparagine and there ore require the availability o this amino acid to survive.

C h a p t e r 5 4 Protein Therapeutics

959

TABLE 54-1 Protein Therapeutics Replacing a Protein That Is Def cient or Abnormal (Group Ia) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Endocrine Disorders (Hormone Def ciencies) ‡Insulin

Humulin Novolin

Regulates blood glucose, shi ts potassium into cells

Diabetes mellitus, diabetic ketoacidosis, hyperkalemia

‡Insulin human inhalation

Exubera (withdrawn rom market in 2008) A rezza (FDA approved in 2014)

Insulin ormulated or inhalation with aster onset o action

Diabetes mellitus

‡Insulin aspart ‡Insulin glulisine ‡Insulin lispro

Novolog (aspart) Apidra (glulisine) Humalog (lispro)

Insulin analogues with aster onset o action and shorter duration o action

Diabetes mellitus

‡Isophane insulin

NPH

Insulin protamine crystalline ormulation with somewhat slower onset o action and longer duration o action

Diabetes mellitus

‡Insulin detemir ‡Insulin glargine

Levemir (detemir) Basaglar (glargine) Lantus (glargine)

Insulin analogues with slower onset o action and longer duration o action

Diabetes mellitus

‡Insulin zinc extended

Lente Ultralente

Insulin zinc hexameric complex with slower onset o action and longer duration o action

Diabetes mellitus

Pramlintide

Symlin

Mechanism unknown; recombinant synthetic peptide analogue o human amylin (a naturally occurring neuroendocrine hormone regulating postprandial glucose control)

Diabetes mellitus, in combination with insulin

Metreleptin

Myalept

Synthetic analogue o the hormone leptin, which regulates satiety and metabolic rate

Leptin def ciency, in addition to diet, in patients with congenital generalized lipodystrophy or acquired generalized lipodystrophy

‡Growth hormone (GH), somatotropin, somatropin, somatrem

Genotropin Humatrope Norditropin NorIVitropin Nutropin Omnitrope Protropin Saizen Serostim Valtropin Zorbtive

Anabolic and anticatabolic e ector

Growth ailure due to GH def ciency or chronic renal insu f ciency, Prader-Willi syndrome, Turner syndrome

‡Mecasermin

Increlex

Recombinant insulin-like growth actor 1 (IGF-1) induces mitogenesis, chondrocyte growth, and organ growth, which combine to restore appropriate statural growth

Growth ailure in children with GH gene deletion or severe primary IGF-1 def ciency

‡Mecasermin rin abate

IPlex

Similar to mecasermin; IGF-1 bound to IGF binding protein 3 (IGFBP-3) is thought to keep the hormone inactive until it reaches its target tissues, thereby decreasing hypoglycemia-like adverse e ects

Growth ailure in children with GH gene deletion or severe primary IGF-1 def ciency

Coagulation actor

Hemophilia A

Hemostasis and Thrombosis Factor VIII

Bioclate Helixate Kogenate Novoeight Recombinate ReFacto XYNTHA

continues

960 FRONTIERS IN PHARMACOLOGY

TABLE 54-1 Protein Therapeutics Replacing a Protein That Is Def cient or Abnormal (Group Ia) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Factor VIII-Fc usion protein

Eloctate

Coagulation actor conjugated to human Fc protein to prolong circulation time and decrease dosing requency

Hemophilia A

Factor IX

Benef x Rixubis

Coagulation actor

Hemophilia B

Factor IX-Fc usion protein

Alprolix

Coagulation actor conjugated to human Fc protein to prolong circulation time and decrease dosing requency

Hemophilia B

*Factor XIII

Cori act

Coagulation actor purif ed rom human plasma

Congenital actor XIII def ciency

Factor XIII A-subunit

Tretten

Coagulation actor

Routine prophylaxis o bleeding in patients with congenital actor XIII A-subunit def ciency

*Von Willebrand actor/ coagulation actor VIII complex

Wilate

Coagulation actor complex purif ed rom human plasma

Treatment o bleeding in patients with severe von Willebrand disease

*Fibrinogen

RiaSTAP

Coagulation actor purif ed rom human plasma

Control o acute bleeding in patients with congenital f brinogen def ciency

*Prothrombin complex concentrate

Kcentra

Mixture o vitamin K-dependent coagulation actors purif ed rom pooled human plasma; includes actors II, VII, IX, and X as well as antithrombotic protein C and protein S

Urgent reversal o acquired coagulation actor def ciency induced by vitamin K antagonist (VKA; e.g., war arin)

Antithrombin III *Antithrombin III

ATryn (recombinant human antithrombin III [AT-III]) Thrombate III (human AT-III purif ed rom pooled plasma)

In a reaction catalyzed by endogenous or exogenous heparin, AT-III inactivates thrombin by orming a covalent bond between the catalytic serine residue o thrombin and an arginine-reactive site on AT-III; AT-III replacement therapy prevents inappropriate blood clot ormation

Treatment o thromboembolism and prevention o perioperative and postpartum thromboembolic events in patients with hereditary antithrombin III def ciency

*Protein C concentrate

Ceprotin

A ter activation by the thrombin–thrombomodulin complex, protein C inhibits coagulation actors Va and VIIIa

Treatment and prevention o venous thrombosis and purpura ulminans in patients with severe hereditary protein C def ciency

*C1 esterase inhibitor

Berinert Cinryze

Serine protease inhibitor purif ed rom human plasma; restores serum levels o C1 esterase inhibitor and prevents inappropriate activation o complement and coagulation pathways that can lead to bradykinin generation and increased vascular permeability

Prophylaxis against angioedema attacks in patients with hereditary angioedema (HAE)

C1 esterase inhibitor

Ruconest

Recombinant human analogue o C1 esterase inhibitor; prevents inappropriate activation o complement and coagulation pathways that can lead to bradykinin generation and increased vascular permeability

Treatment o acute attacks o hereditary angioedema (HAE)

Metabolic Enzyme Def ciencies -Glucocerebrosidase * -Glucocerebrosidase

Cerezyme Ceredase (purif ed rom pooled human placenta)

Hydrolyzes glucocerebroside to glucose and ceramide

Gaucher’s disease

Taliglucerase al a (biosimilar to glucocerebrosidase)

Elelyso

Recombinant glucocerebrosidase produced rom a plant-based expression system

Gaucher’s disease

C h a p t e r 5 4 Protein Therapeutics

961

TABLE 54-1 Protein Therapeutics Replacing a Protein That Is Def cient or Abnormal (Group Ia) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Velaglucerase al a (biosimilar to glucocerebrosidase)

Vpriv

Recombinant glucocerebrosidase

Gaucher’s disease

Alglucosidase al a

Lumizyme Myozyme

Degrades glycogen by catalyzing the hydrolysis o -1,4 and -1,6 glycosidic linkages o lysosomal glycogen

Pompe disease (glycogen storage disease type II)

Laronidase

Aldurazyme

Idursul ase

Elaprase

Iduronate-2-sul atase cleaves the terminal 2-O-sul ate moieties rom the GAGs dermatan sul ate and heparan sul ate, thereby allowing their digestion and preventing GAG accumulation

Mucopolysaccharidosis II (Hunter syndrome)

Elosul ase al a

Vimizim

N-acetylgalactosamine-6-sul atase cleaves a sul ate rom the GAG keratan sul ate, thereby allowing its digestion and preventing its accumulation in lysosomes

Mucopolysaccharidosis IV

Galsul ase

Naglazyme

N-acetylgalactosamine-4-sul atase cleaves the terminal sul ate rom the GAG dermatan sul ate, thereby allowing its digestion and preventing GAG accumulation

Mucopolysaccharidosis VI

Human -galactosidase A, Agalsidase

Fabrazyme

Enzyme that hydrolyzes globotriaosylceramide (GL-3) and other glycosphingolipids, reducing deposition o these lipids in capillary endothelium o the kidney and certain other cell types

Fabry disease; prevents accumulation o lipids that could lead to renal and cardiovascular complications

-L-iduronidase is an enzyme that digests endogenous glycosaminoglycans (GAGs) within lysosomes and thereby prevents an accumulation o GAGs that can cause cellular, tissue, and organ dys unction

Hurler and Hurler–Scheie orms o mucopolysaccharidosis I (MPS I)

Pulmonary and Gastrointestinal Tract Disorders * -1-Proteinase inhibitor

Aralast Glassia Prolastin

Inhibits elastase-mediated destruction o pulmonary tissue; purif ed rom pooled human plasma

Congenital -1-antitrypsin def ciency

*Lactase

Lactaid

Digests lactose; purif ed rom ungus Aspergillus oryzae

Gas, bloating, cramps, diarrhea due to inability to digest lactose

*Pancreatic enzymes (lipase, amylase, protease)

Arco-Lase Cotazym Creon Donnazyme Pancrease Pertzye Ultresa Viokase Zenpep Zymase

Digests ood (protein, at, and carbohydrate); purif ed rom hogs and pigs

Cystic f brosis, chronic pancreatitis, pancreatic insu f ciency, post-Billroth II gastric bypass surgery, pancreatic duct obstruction, steatorrhea, poor digestion, gas, bloating

*Adenosine deaminase

Adagen (pegademase bovine, PEG-ADA)

Metabolizes adenosine, prevents accumulation o adenosine; purif ed rom cows

Severe combined immunodef ciency disease (SCID) due to adenosine deaminase (ADA) def ciency

*Pooled immunoglobulins

Bivigam Octagam Privigen Vivaglobin

Intravenous immunoglobulin preparation

Primary immunodef ciencies and chronic immune thrombocytopenic purpura (ITP)

Immunodef ciencies

continues

962 FRONTIERS IN PHARMACOLOGY

TABLE 54-1 Protein Therapeutics Replacing a Protein That Is Def cient or Abnormal (Group Ia) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Albumarc Albumin (Human) Albuminar AlbuRx Albutein Buminate Flexbumin Plasbumin

Increases circulating plasma osmolarity, thereby restoring and maintaining circulating blood volume

Decreased production o albumin (hypoproteinemia), increased loss o albumin (nephrotic syndrome), hypovolemia, hyperbilirubinemia

Other *Human albumin

Protein-based therapies derive their specif city and unction rom their structure. Molecules ranging rom large and complex enzymes to short peptide sequences have specif c biological activity due to their amino acid-based secondary and tertiary structure. For example, somatostatin is active as either a 14- or 28-amino-acid chain, and its even shorter synthetic analogues share a characteristic hairpin loop structure that def nes their specif city and biological activity. Some very short peptide therapeutics are better thought o as small-molecule drugs, since they lack secondary and tertiary structures that def ne their biological activity. For this reason, therapeutics such as glatiramer acetate (a our-amino-acid peptide consisting o acetate with L-Glu, L-Ala, L-Tyr, L-Lys) are not addressed in this chapter. Protein therapeutics are recombinant unless otherwise stated. * Nonrecombinant. ‡ Also classed in Group Ib.

TABLE 54-2 Protein Therapeutics Augmenting an Existing Pathway (Group Ib) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Erythropoietin, epoetin al a

Epogen Procrit

Stimulates erythropoiesis

Anemia due to chronic kidney disease or chemotherapy, preoperative preparation

Darbepoetin al a

Aranesp

Modif ed erythropoietin with longer hal -li e; stimulates red blood cell production in the bone marrow

Treatment o anemia in patients with chronic kidney disease ( / dialysis)

Methoxy polyethylene glycol-epoetin beta

Mircera

Erythropoietin conjugated to methoxy polyethylene glycol (PEG) butanoic acid; stimulates erythropoiesis

Anemia associated with chronic kidney disease

Peginesatide acetate

Omontys

Synthetic peptide analogue o erythropoietin, conjugated to polyethylene glycol (PEG)

Product recalled in 2013 and US FDA New Drug Application (NDA) voluntarily withdrawn by manu acturer in 2014

Granulocyte colonystimulating actor (G-CSF), f lgrastim

Neupogen

Stimulates neutrophil proli eration, di erentiation, and migration

Neutropenia in AIDS or a ter chemotherapy or bone marrow transplantation, severe chronic neutropenia

Tbo-Filgrastim

Granix

Stimulates neutrophil proli eration, di erentiation, and migration (minor structural di erences rom f lgrastim)

Shorten the duration o neutropenia a ter chemotherapy

Peg-G-CSF, pegf lgrastim

Neulasta

Stimulates neutrophil proli eration, di erentiation, and migration

Neutropenia in AIDS or a ter chemotherapy or bone marrow transplantation, severe chronic neutropenia

Granulocyte-macrophage colony-stimulating actor (GM-CSF), sargramostim

Leukine

Stimulates proli eration and di erentiation o neutrophils, eosinophils, and monocytes

Leukopenia, myeloid reconstitution a ter bone marrow transplantation, HIV/AIDS

Interleukin-11 (IL-11), oprelvekin

Neumega

Stimulates megakaryocytopoiesis and thrombopoiesis

Prevention o severe thrombocytopenia, especially a ter myelosuppressive chemotherapy

Hematopoiesis

C h a p t e r 5 4 Protein Therapeutics

963

TABLE 54-2 Protein Therapeutics Augmenting an Existing Pathway (Group Ib) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Romiplostim

Nplate

Fc-peptide usion protein (peptibody) that acts as a thrombopoietin receptor agonist; stimulates platelet production

Treatment o thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP)

Human ollicle-stimulating hormone (FSH)

Gonal-F / Follistim

Stimulates ovulation

Assisted reproductive technology or in ertility

Human chorionic gonadotropin (HCG)

Ovidrel

Stimulates ovarian ollicle rupture and ovulation

Assisted reproductive technology or in ertility

Lutropin al a

Luveris

Recombinant human luteinizing hormone (LH) increases estradiol secretion, thereby supporting ollicle-stimulating hormone (FSH)-induced ollicular development

In ertility with LH de ciency

*ACTH (repository corticotropin)

H.P. Acthar

Mechanism unknown; immunoregulator

In antile spasms (West syndrome), multiple sclerosis relapse, dermatomyositis, systemic lupus erythematosus (SLE)

Type I alpha-inter eron, inter eron al acon-1, consensus inter eron

In ergen

Mechanism unknown; immunoregulator

Chronic hepatitis C

Inter eron alpha-2a (IFN -2a)

Ro eron-A

Mechanism unknown; immunoregulator

Hairy cell leukemia, chronic myelogenous leukemia, Kaposi’s sarcoma, chronic hepatitis C

Peginter eron al a-2a

Pegasys

Mechanism unknown; immunoregulator

Adults with chronic hepatitis C who have compensated liver disease and who have not been previously treated with inter eron alpha; used alone or in combination with ribavirin (Copegus)

Inter eron al a-2b (IFN -2b)

Intron A

Mechanism unknown; immunoregulator

Hepatitis B, melanoma, Kaposi’s sarcoma, ollicular lymphoma, hairy cell leukemia, condylomata acuminata, hepatitis C

Peginter eron al a-2b

Peg-Intron

Recombinant inter eron alpha-2b conjugated to polyethylene glycol (PEG) in order to increase hal -li e

Adults with chronic hepatitis C who have compensated liver disease and have not been treated previously with inter eron alpha

*Inter eron al a-n3 (IFN -n3)

Al eron N

Mechanism unknown; nonrecombinant human inter eron al a-n3 puri ed rom pooled human leukocytes

Condylomata acuminata (genital warts caused by human papillomavirus)

Inter eron beta-1a (rIFN- )

Avonex Rebi

Mechanism unknown; antiviral and immunoregulator

Multiple sclerosis

Inter eron beta-1b (rIFN- )

Betaseron Extavia

Mechanism unknown; antiviral and immunoregulator

Multiple sclerosis

Peginter eron beta-1a

Plegridy

Recombinant inter eron beta-1a conjugated to polyethylene glycol (PEG) in order to increase hal -li e

Multiple sclerosis

Inter eron gamma-1b (IFN- )

Actimmune

Increases inf ammatory and antimicrobial response

Chronic granulomatous disease (CGD), severe osteopetrosis

Fertility

Immunoregulation

continues

964 FRONTIERS IN PHARMACOLOGY

TABLE 54-2 Protein Therapeutics Augmenting an Existing Pathway (Group Ib) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Interleukin-2 (IL-2), epidermal thymocyte activating actor (ETAF), aldesleukin

Proleukin

Stimulates T and B cells, natural killer cells, and lymphokine-activated killer (LAK) cells

Metastatic renal cell cancer, melanoma

Tissue plasminogen activator (tPA), alteplase

Activase

Promotes brinolysis by binding brin and converting plasminogen to plasmin

Pulmonary embolism, myocardial in arction, acute ischemic stroke, occlusion o central venous access devices

Reteplase (deletion mutein o plasminogen activator [tPA])

Retavase

Contains the nonglycosylated kringle 2 and protease domains o human tPA; unctions similarly to tPA

Management o acute myocardial in arction, improvement o ventricular unction

‡Tenecteplase

TNKase

Tissue plasminogen activator with greater speci city or plasminogen conversion; has amino acid substitutions o Thr103 to Asp, Asp117 to Gln, and Ala or amino acids 296–299

Acute myocardial in arction

*Urokinase

Abbokinase

Nonrecombinant plasminogen activator derived rom human neonatal kidney cells

Pulmonary embolism

Factor VIIa

NovoSeven

Prothrombotic (activated actor VII; initiates the coagulation cascade)

Hemorrhage in patients with hemophilia A or B and inhibitors to actor VIII or actor IX

Activated protein C, drotrecogin al a

Xigris

Antithrombotic (inhibits coagulation actors Va and VIIIa), anti-inf ammatory

Severe sepsis with a high risk o death Voluntarily withdrawn rom the market in 2011 due to lack o e cacy

Thrombin (human recombinant) *Thrombin (pooled rom human plasma)

Recothrom Evithrom

Cleaves brinogen to brin and activates the coagulation cascade

Hemostasis aid in surgical situations; applied topically to accelerate coagulation

*Fibrin sealant ( brinogen and thrombin mixture)

Artiss Evarrest TachoSil

Two-component brin sealant puri ed rom pooled human plasma; when combined, the brinogen and thrombin mimic the nal stage o blood coagulation

Adheres autologous skin gra ts to surgically prepared wound beds resulting rom burns; used to control bleeding during surgery

Calcitonin-salmon

Fortical (recombinant) Miacalcin (synthetic)

Mechanism unknown; inhibits osteoclast unction

Postmenopausal osteoporosis

Human parathyroid hormone residues 1–34, teriparatide

Forteo

Markedly enhances bone ormation; administered as a once-daily injection

Severe osteoporosis

§‡Exenatide

Byetta

Incretin mimetic with actions similar to glucagon-like peptide-1 (GLP-1); increases glucose-dependent insulin secretion, suppresses glucagon secretion, slows gastric emptying, decreases appetite ( rst identi ed in saliva o the Gila monster Heloderma suspectum)

Type 2 diabetes resistant to treatment with met ormin and a sul onylurea

§‡Liraglutide

Victoza

Recombinant, acylated, and modi ed human glucagon-like peptide-1 (GLP-1) agonist with amino acid sequence homology to GLP-1 residues 7–37; increases insulin secretion

Type 2 diabetes

Hemostasis and Thrombosis

Endocrine Disorders

C h a p t e r 5 4 Protein Therapeutics

965

TABLE 54-2 Protein Therapeutics Augmenting an Existing Pathway (Group Ib) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

§‡Albiglutide

Tanzeum

GLP-1 agonist conjugated to human albumin to increase hal -li e and allow once-weekly dosing

Type 2 diabetes

§Octreotide

Sandostatin

Potent somatostatin analogue; inhibits growth hormone, glucagon, and insulin

Acromegaly, symptomatic relie o vasoactive intestinal peptide (VIP)secreting adenomas and metastatic carcinoid tumors

§Lanreotide

Somatuline depot

Cyclical somatostatin analogue ormulated or sustained release

Long-term treatment o acromegaly

Recombinant human bone morphogenic protein 2 (rhBMP-2), dibotermin al a

In use

Mechanism unknown

Spinal usion surgery, bone injury repair

Recombinant human bone morphogenic protein 7 (rhBMP-7)

Osteogenic protein-1

Mechanism unknown

Tibial racture nonunion, lumbar spinal usion

†§Gonadotropin-releasing hormone (GnRH): Goserelin Histrelin Leuprolide Na arelin Tesamorelin

Egri ta Eligard Lupaneta Lupron Supprelin LA Synarel Vantas Viadur Zoladex

Synthetic analogue o human GnRH; acts as a potent inhibitor o gonadotropin secretion when administered continuously by causing reversible down-regulation o GnRH receptors in the pituitary and desensitizing pituitary gonadotropes

Precocious puberty, endometriosis, breast cancer, prostate cancer, lipodystrophy associated with treatment or HIV in ection

Keratinocyte growth actor (KGF), pali ermin

Kepivance

Recombinant analogue o KGF; stimulates keratinocyte growth in skin, mouth, stomach, and colon

Severe oral mucositis in patients undergoing chemotherapy

Platelet-derived growth actor (PDGF), becaplermin

Regranex

Promotes wound healing by enhancing granulation tissue ormation and f broblast proli eration and di erentiation

Debridement adjunct or diabetic ulcers

*Trypsin

Granulex

Proteolysis

Decubitus ulcer, varicose ulcer, debridement o eschar, dehiscent wound, sunburn

Nesiritide

Natrecor

Recombinant B-type natriuretic peptide

Acute decompensated heart ailure

Linaclotide

Linzess

Peptide agonist o guanylate cyclase 2C; increases chloride and bicarbonate secretion into intestinal lumen and decreases constipation

Irritable bowel syndrome

Ocriplasmin

J etrea

Truncated version o human serine protease plasmin; proteolytic activity against f bronectin and laminin allows release o adherent macula and vitreous

Symptomatic vitreomacular adhesion o the eye

Teduglutide

Gattex kit

Peptide analogue o glucagon-like peptide-2 (GLP-2) with one amino acid substitution to increase hal -li e; promotes intestinal mucosal growth

Short bowel syndrome

Growth Regulation

Other

Proteins are recombinant unless otherwise stated. * Nonrecombinant. § Synthetic. ‡ Also classed in Group Ic. † Also classed in Group IIa.

966 FRONTIERS IN PHARMACOLOGY

L-Asparaginase , purif ed rom E. coli, can be used to lower

serum levels o asparagine in such patients and thereby inhibit cancer cell growth. A key component o many chemotherapy regimens is the olate analogue methotrexate , which inhibits dihydro olate reductase. Lethal concentrations o methotrexate can develop in patients with renal ailure or patients who are inadvertently overdosed with the drug. This potentially atal complication can be corrected with glucarpidase , a recombinant bacterial carboxypeptidase G2 that degrades methotrexate into inactive metabolites. Studies o the medical leech, Hirudo medicinalis, revealed that its salivary gland produces hirudin, a potent thrombin inhibitor. The gene or this protein was then identif ed, cloned, and used recombinantly to provide a new protein therapy, lepirudin, which prevents clot ormation in patients with heparin-induced thrombocytopenia. Other organisms can also be used to produce proteins that are capable o breaking up clots that have already ormed; or example, streptokinase is a plasminogen-activating protein produced by group C -hemolytic streptococci. Many more therapeutic proteins that provide a novel unction or activity are presented in Table 54-3.

Group II: Targeted Proteins The exquisite binding specif city o monoclonal antibodies and immunoadhesins can be exploited in numerous ways using recombinant DNA technology. Many protein therapeutics in Group IIa use the antigen recognition sites o immunoglobulin (Ig) molecules or the receptor-binding domains o native protein ligands to guide the immune system to destroy specif cally targeted molecules or cells. Other monoclonal antibodies and immunoadhesins neutralize molecules by simple physical blocking o a unctionally important region o the molecule. Immunoadhesins combine the receptor-binding domains o protein ligands with the Fc region o an Ig. The Fc region can target a soluble molecule or destruction because cells o the immune system can recognize the Fc region, endocytose the attached molecule, and break down the molecule chemically and enzymatically. When an immunoadhesin is bound to specif cally recognized molecules on the sur ace o a cell, the Fc region can target the cell or destruction by the immune system. Cell killing can be mediated by macrophages, by other immune cells, or by complement f xation. Several Group IIa protein therapeutics have been approved or the treatment o in ammatory diseases, such as the immunoadhesin etanercept, which is a usion between two human proteins: tumor necrosis actor (TNF) receptor and the Fc region o the human antibody protein IgG1. The TNF receptor portion o the molecule binds excess TNF in the plasma, while the Fc portion o the molecule targets the bound complex or destruction. By combining these two unctions, etanercept neutralizes the deleterious e ects o TNF (a cytokine that stimulates increased activity o the immune system) and thereby provides an e ective therapy or in ammatory arthritis and psoriasis . Another Group IIa protein that targets TNF is adalimumab. This recombinantly produced monoclonal antibody binds to TNF- and is used to neutralize the action o TNF- in a variety o in ammatory conditions including rheumatoid arthritis , psoriasis , and inf ammatory bowel disease . Adalimumab was the top selling therapeutic worldwide in 2013, with more than $11 billion in sales—a distinction that, until recently, had always been held by a small-molecule drug.

Some Group IIa proteins are used to treat in ectious diseases. Patients at high risk or severe respiratory syncytial virus (RSV) in ection, one o the leading causes o hospital admissions or pediatric respiratory illness, are given a recombinant monoclonal antibody, palivizumab, which binds to the RSV F protein and thereby directs the immunemediated clearance o the virus rom the body. En uvirtide is an example o a Group IIa protein therapeutic that is not a monoclonal antibody or an immunoadhesin. By binding to gp120/gp41—the HIV envelope protein responsible or usion o the virus with host cells—this 36-amino-acid peptide prevents the con ormational change in gp41 that is required or viral usion and thereby inhibits viral entry into the cell. Group IIa antibodies are o growing importance in oncology. For example, rituximab is a human/mouse chimeric monoclonal antibody that binds to CD20, a transmembrane protein expressed on 90% o B-cell non-Hodgkin’s lymphomas, and targets the cells or destruction by the body’s immune system. Although rituximab is most o ten used in combination with anthracycline-based chemotherapy, it is one o the ew monoclonal antibody anticancer therapies that is approved as a monotherapy. Cetuximab is a monoclonal antibody used to treat colorectal cancer and head and neck cancer; this monoclonal antibody binds epidermal growth actor receptor (EGFR) and impairs cancer cell growth and proli eration. Other recently developed Group IIa protein therapeutics are listed in Table 54-4, and many more protein therapeutics utilizing the exquisite specif city o monoclonal antibodies are in development, especially or cancer and inammatory diseases. Many important processes are modulated by cell sur ace receptors that are activated upon binding o their cognate ligands. By binding to such receptors, targeted protein therapeutics may activate cell signaling pathways and pro oundly a ect cell unction. Outcomes may range rom cell death (through the induction o apoptosis), to down-regulation o cell division, to increased cell proli eration. Although it has been di f cult to prove that a particular target-binding protein mediates an in vivo e ect through the modulation o a particular signaling pathway, in vitro evidence suggests that this type o modulation is involved in the mechanism o action o certain therapeutic proteins. For example, treatment o certain breast cancers, in which the malignant cells express the HER2/neu (also known as ERBB2) cell sur ace receptor, is enhanced by the addition o trastuzumab (an anti-HER2/neu monoclonal antibody) to the therapeutic regimen. Although trastuzumab contains an Fc region that acilitates antibodydependent cellular cytotoxicity mediated by natural killer cells, it seems unlikely that this is trastuzumab’s only mechanism o action. Other monoclonal antibodies, with similar Fc regions and abilities to target breast cancer cells, have ailed to show e f cacy in vivo. Trastuzumab, however, has been shown in vitro to induce intracellular signaling events that control the growth o breast cancer cells. It is there ore likely that a combination o mechanisms accounts or the therapeutic activity o trastuzumab, including inhibition o the phosphatidylinositol 3-kinase (PI3K) pathway, inhibition o angiogenesis, and inhibition o HER2 receptor cleavage. The complex action o trastuzumab highlights the act that, while modulation o cell physiology through simple receptor binding may play a role in the activity o some targeted therapies, the relative contribution o receptor binding to the overall e f cacy o the therapeutic may be di f cult to dissect.

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TABLE 54-3 Protein Therapeutics Providing a Novel Function or Activity (Group Ic) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Enzymatic Degradation of Macromolecules *Botulinum toxin type A

Botox Dysport Xeomin

Cleaves SNAP-25 at neuromuscular junction to disrupt SNARE complex and prevent acetylcholine release, causing f accid paralysis

Many types o dystonia, particularly cervical; cosmetic uses

*Botulinum toxin type B

Myobloc

Cleaves synaptobrevin at neuromuscular junction to disrupt SNARE complex and prevent acetylcholine release, causing f accid paralysis

Many types o dystonia, particularly cervical; cosmetic uses

*Collagenase

Collagenase Santyl

Collagenase obtained rom ermentation by Clostridium histolyticum; digests collagen in necrotic base o wounds

Debridement o chronic dermal ulcers and severely burned areas

*Collagenase

Xiaf ex

Mixture o two collagenases (AUX-I and AUX-II) obtained rom ermentation by Clostridium histolyticum; digests subcutaneous collagen

Treatment or Dupuytren’s contracture

Human deoxyribonuclease I, dornase al a

Pulmozyme

Degrades DNA in purulent pulmonary secretions

Cystic brosis; decreases respiratory tract in ections in selected patients with orced vital capacity (FVC) greater than 40% o predicted

*Hyaluronidase Hyaluronidase

Amphadase (bovine) Hydase (bovine) Vitrase (ovine) Hylenex (recombinant human)

Catalyses the hydrolysis o hyaluronic acid to increase tissue permeability and allow aster drug absorption

Used as an adjuvant to increase the absorption and dispersion o injected drugs, particularly anesthetics in ophthalmic surgery and certain imaging agents

*Papain

Accuzyme Pana l

Protease rom the Carica papaya ruit

Debridement o necrotic tissue or lique action o slough in acute and chronic lesions, such as pressure ulcers, varicose and diabetic ulcers, burns, postoperative wounds, pilonidal cyst wounds, carbuncles, and other wounds

Enzymatic Degradation of Small-Molecule Metabolites *L-asparaginase

ELSPAR

Provides exogenous asparaginase activity, removing available asparagine rom serum; puri ed rom E. coli

Acute lymphoblastic leukemia (ALL), which requires exogenous asparagine or proli eration

*Asparaginase Erwinia chrysanthemi

Erwinaze

Provides exogenous asparaginase activity, removing available asparagine rom serum; puri ed rom E. chrysanthemi

Acute lymphoblastic leukemia (ALL), which requires exogenous asparagine or proli eration

*Peg-asparaginase

Oncaspar

Provides exogenous asparaginase activity, removing available asparagine rom serum; puri ed rom E. coli and conjugated to polyethylene glycol (PEG) to decrease immunogenicity and increase hal -li e

Acute lymphoblastic leukemia (ALL), which requires exogenous asparagine or proli eration

Glucarpidase

Voraxaze

Recombinant carboxypeptidase G2 degrades methotrexate into inactive metabolites

Treatment o supratherapeutic levels o methotrexate

Rasburicase

Elitek

Catalyzes enzymatic oxidation o uric acid into an inactive, soluble metabolite (allantoin); originally isolated rom Aspergillus f avus

Pediatric patients with leukemia, lymphoma, and solid tumors who are undergoing anticancer therapy that may cause tumor lysis syndrome

continues

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TABLE 54-3 Protein Therapeutics Providing a Novel Function or Activity (Group Ic) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Pegloticase

Krystexxa

Recombinant uricase conjugated to polyethylene glycol (PEG) to increase hal -li e; metabolizes uric acid to allantoin

Chronic gout that is re ractory to conventional therapy

Hemostasis and Thrombosis Lepirudin Desirudin

Ref udan Iprivask

Recombinant hirudin, a thrombin inhibitor rom salivary gland o medicinal leech Hirudo medicinalis

Heparin-induced thrombocytopenia (HIT), prophylaxis against deep vein thrombosis in patients undergoing elective hip replacement surgery

§Bivalirudin

Angiomax

Synthetic hirudin analogue; speci cally binds both the catalytic site and the anion-binding exosite o circulating and clot-bound thrombin

Reduce blood clotting risk in coronary angioplasty and heparin-induced thrombocytopenia (HIT)

*Streptokinase

Streptase

Converts plasminogen to plasmin; produced by group C -hemolytic streptococci

Acute evolving ST elevation myocardial in arction, pulmonary embolism, deep vein thrombosis, arterial thrombosis or embolism, occlusion o arteriovenous cannula

*Anistreplase, anisoylated plasminogen streptokinase activator complex (APSAC)

Eminase

Converts plasminogen to plasmin; p-anisoyl group protects the catalytic center o the plasminogen–streptokinase complex and prevents premature deactivation, thereby providing longer duration o action than streptokinase

Thrombolysis in patients with unstable angina

Protamine

Protamine sul ate

Inactivates heparin by orming a stable 1:1 protamine:heparin complex

Heparin overdose

Proteins are recombinant unless otherwise stated. * Nonrecombinant. § Synthetic.

TABLE 54-4 Protein Therapeutics Interfering with a Molecule or Organism (Group IIa) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Bevacizumab

Avastin

Humanized monoclonal antibody (mAb) that binds all iso orms o vascular endothelial growth actor A (VEGF-A)

Colorectal cancer

Ziv-af ibercept (same unctional entity as af ibercept; see below)

Zaltrap

Recombinant usion protein with the extracellular domains o human VEGF receptors 1 and 2 used to the Fc portion o human IgG1; inhibits neovascularization

Metastatic colorectal cancer

Ramucirumab

Cyramza

Fully human mAb (IgG1) targeting vascular endothelial growth actor receptor 2 (VEGFR2)

Advanced gastric cancer

Cetuximab

Erbitux

MAb targeting epidermal growth actor receptor (EGFR)

Colorectal cancer, head and neck cancer

Panitumumab

Vectibix

MAb that competitively inhibits ligand interactions with epidermal growth actor receptor (EGFR)

Metastatic colorectal cancer

Degarelix (GnRH receptor antagonist)

Firmagon

Synthetic linear decapeptide containing seven unnatural amino acids; GnRH receptor competitive antagonist that prevents GnRH binding to pituitary receptors and thereby decreases downstream testosterone production

Advanced prostate cancer

Cancer

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TABLE 54-4 Protein Therapeutics Interfering with a Molecule or Organism (Group IIa) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Alemtuzumab

Campath

Humanized mAb directed against CD52 antigen on T and B cells

Voluntarily withdrawn by manu acturer in United States and Europe

Rituximab

Rituxan

Chimeric (human/mouse) mAb that binds CD20, a transmembrane protein ound on more than 90% o B-cell non-Hodgkin’s lymphomas; synergistic e ect with some small-molecule chemotherapeutic agents has been demonstrated in lymphoma cell lines

CD20-positive B-cell non-Hodgkin’s lymphoma, di use large B-cell CD20-positive non-Hodgkin’s lymphoma, rheumatoid arthritis, Wegener’s granulomatosis, microscopic polyangiitis

Obinutuzumab

Gazyva

Humanized mAb targeting CD20 on B cells; mediates cell death via multiple mechanisms, including engagement o immune e ector cells, activation o intracellular apoptosis pathways, and activation o the complement cascade

Chronic lymphocytic leukemia

O atumumab

Arzerra

Human mAb targeting CD20; inhibits early-stage B lymphocyte activation

Chronic lymphocytic leukemia

Trastuzumab

Herceptin

MAb that binds HER2/neu cell sur ace receptor and controls cancer cell growth

HER2-positive breast cancer; HER2-positive metastatic gastric adenocarcinoma

Pertuzumab

Perjeta

MAb that inhibits HER dimerization and thereby prevents receptor activation

HER2-positive breast cancer (used in combination with trastuzumab)

Ipilimumab

Yervoy

MAb targeting and blocking CTLA-4, thereby inhibiting CTLA-4 interactions with CD80 and CD86 and augmenting T-cell activation and proli eration

Metastatic melanoma

Denosumab

Xgeva (same unctional entity as Prolia; see below)

MAb that inhibits RANKL; inhibits osteoclast maturation and decreases bone turnover

Prevention o skeletal racture in patients with solid tumors metastatic to bone, treatment o giant cell tumor o bone

Adalimumab

Humira

Binds to TNF- and blocks its interaction with p55 and p75 cell sur ace TNF receptors, resulting in decreased levels o inf ammation markers including CRP, ESR, and IL-6

Rheumatoid arthritis, psoriasis, Crohn’s disease

Certolizumab

Cimzia

Recombinant humanized Fab antibody ragment conjugated to polyethylene glycol (PEG); binds to and neutralizes TNF-

Crohn’s disease

Etanercept

Enbrel

Dimeric usion protein composed o recombinant soluble tumor necrosis actor receptor (TNFr) linked to Fc portion o human IgG1

Moderate to severe active rheumatoid arthritis (RA) a ter ailing other therapies, moderate to severe active polyarticular juvenile RA

Golimumab

Simponi

Human IgG/ mAb that binds and neutralizes TNF-

Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis

Inf iximab

Remicade

MAb that binds and neutralizes TNF- , preventing induction o proinf ammatory cytokines

Rheumatoid arthritis, Crohn’s disease

Abatacept

Orencia

Selective costimulation modulator composed o the extracellular domain o human CTLA-4 linked to the Fc portion o human IgG1; inhibits T-cell activation by binding to CD80 and CD86, thereby blocking interaction with CD28 and inhibiting autoimmune T-cell activation

Rheumatoid arthritis (especially when re ractory to TNFinhibition)

Immunoregulation

continues

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TABLE 54-4 Protein Therapeutics Interfering with a Molecule or Organism (Group IIa) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Tocilizumab

Actemra

Recombinant humanized antihuman interleukin-6 (IL-6) receptor mAb

Moderate to severe active rheumatoid arthritis in adults who have ailed one or more anti-TNF therapy

Anakinra

Antril Kineret Synergen

Recombinant interleukin-1 receptor antagonist

Moderate to severe active rheumatoid arthritis in adults who have ailed one or more diseasemodi ying antirheumatic drug

Canakinumab

Ilaris

Recombinant human IgG1/ mAb that binds and sequesters IL-1

Cryopyrin-associated periodic syndromes (CAPS), including amilial cold autoinf ammatory syndrome (FCAS) and MuckleWells syndrome (MWS)

Rilonacept

Arcalyst

Interleukin-1 decoy receptor; dimeric usion protein consisting o the ligand-binding domains o the extracellular portions o the human interleukin-1 receptor component (IL-1RI) and IL-1 receptor accessory protein (IL-1RAcP) linked to the Fc portion o human IgG1

Cryopyrin-associated periodic syndromes (CAPS), including amilial cold autoinf ammatory syndrome (FCAS) and MuckleWells syndrome (MWS)

Siltuximab

Sylvant

Recombinant chimeric mAb targeting IL-6

Castleman’s disease (a lymphoproli erative disorder)

Ale acept

Amevive

MAb that binds CD2 on the sur ace o lymphocytes and inhibits interaction with leukocyte unctionassociated antigen 3 (LFA-3)

Voluntarily withdrawn by manu acturer in 2011

E alizumab

Raptiva

Humanized mAb directed against CD11a

Adults with chronic moderate to severe plaque psoriasis who are candidates or systemic therapy

Ustekinumab

Stelara

Human IgG/ mAb that disrupts IL-12 and IL-23 signaling by binding to their common p40 subunit

Plaque psoriasis

Natalizumab

Tysabri

Mechanism unknown: binds to the 4 subunit o 4 1 and 4 7 integrins, blocking their interactions with vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MadCAM-1), respectively

Relapsing multiple sclerosis

Vedolizumab

Entyvio

Mechanism unknown: binds to 4 7 integrin on T cells, blocking its interaction with MadCAM-1 and thus preventing adhesion o T cells to the ileal endothelium

Ulcerative colitis, Crohn’s disease

Belimumab

Benlysta

Human mAb that inhibits B-cell activating actor

Systemic lupus erythematosus

Eculizumab

Soliris

Humanized mAb that binds complement protein C5 and inhibits its cleavage to C5a and C5b, preventing the ormation o the terminal complement complex C5b-9

Paroxysmal nocturnal hemoglobinuria (PNH)

*Antithymocyte globulin (rabbit)

Thymoglobulin

Selective depletion o T cells; exact mechanism unknown

Acute kidney transplant rejection, aplastic anemia

Basiliximab

Simulect

Chimeric (human/mouse) IgG1 mAb that blocks cellular immune response in gra t rejection by binding the alpha chain o CD25 (IL-2 receptor) and thereby inhibiting the IL-2-mediated activation o lymphocytes

Prophylaxis against allogra t rejection in renal transplant patients receiving an immunosuppressive regimen including cyclosporine and corticosteroids

Transplantation

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TABLE 54-4 Protein Therapeutics Interfering with a Molecule or Organism (Group IIa) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Belatacept

Nulojix

Fusion protein composed o the extracellular domain o human CTLA-4 linked to the Fc portion o human IgG1; inhibits T-cell activation (di ers rom abatacept by two amino acids)

Prophylaxis o organ rejection in kidney transplantation

Daclizumab

Zenapax

Humanized IgG1 mAb that blocks cellular immune response in gra t rejection by binding the alpha chain o CD25 (IL-2 receptor) and thereby inhibiting the IL-2-mediated activation o lymphocytes

Prophylaxis against acute allogra t rejection in patients receiving renal transplants

Muromonab-CD3

Orthoclone OKT3

MAb that binds CD3 and blocks T-cell unction

Voluntarily withdrawn by manu acturer in 2010

*Hepatitis B immune globulin

HepaGam B

Prepared rom puri ed gamma globulins rom human plasma; binds sur ace components on hepatitis B virus and provides passive immunization; complete mechanism not understood

Prevention o hepatitis B recurrence a ter liver transplant in hepatitis B-in ected patients; postexposure prophylaxis against hepatitis B in ection

Omalizumab

Xolair

IgG mAb that inhibits IgE binding to the high-a nity IgE receptor on mast cells and basophils, decreasing activation o these cells and release o inf ammatory mediators

Adults and adolescents with moderate to severe persistent asthma who have a positive skin test or in vitro reactivity to a perennial aeroallergen and whose symptoms are inadequately controlled with inhaled corticosteroids

Palivizumab

Synagis

Humanized IgG1 mAb that binds the A antigenic site o the F protein o respiratory syncytial virus

Prevention o respiratory syncytial virus in ection in high-risk pediatric patients

En uvirtide

Fuzeon

36-Amino-acid peptide that inhibits HIV entry into host cells by binding to the HIV envelope protein gp120/gp41

Adults and children with advanced HIV in ection

Raxibacumab

Raxibacumab

MAb that targets the protective antigen o anthrax toxin to prevent intracellular entry o the anthrax lethal actor and edema actor

Prophylaxis and treatment o inhaled anthrax

Pulmonary Disorders

Infectious Diseases †

Hemostasis and Thrombosis Abciximab

ReoPro

Fab ragment o chimeric (human/mouse) mAb 7E3 that inhibits platelet aggregation by binding to the glycoprotein IIb/IIIa integrin receptor

Adjunct to aspirin and heparin or prevention o cardiac ischemia in patients undergoing percutaneous coronary intervention or patients about to undergo percutaneous coronary intervention with unstable angina not responding to medical therapy

Ecallantide

Kalbitor

Polypeptide inhibitor o plasma and tissue kallikreins, enzymes that catalyze the generation o bradykinin as part o the nal common pathway o edema ormation in hereditary angioedema (HAE)

Hereditary angioedema (HAE)

Icatibant

Firazyr

Peptidomimetic antagonist o bradykinin B2 receptor; blocks the nal common pathway o edema ormation in HAE

Hereditary angioedema (HAE)

continues

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TABLE 54-4 Protein Therapeutics Interfering with a Molecule or Organism (Group IIa) continued PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

‡Gonadotropin-releasing hormone (GnRH) receptor antagonists: Cetrorelix Ganirelix

Antagon Cetrotide Orgalutran

Suppresses premature LH surges in the early to mid- ollicular phase o the menstrual cycle

Assisted reproductive technology (controlled ovarian hyperstimulation) or in ertility

Pegvisomant

Somavert

Recombinant human growth hormone conjugated to PEG; blocks the growth hormone receptor

Acromegaly

Denosumab

Prolia (same unctional entity as Xgeva; see above)

MAb that inhibits RANKL; inhibits osteoclast maturation and decreases bone turnover

Osteoporosis

*Crotalidae polyvalent immune Fab (ovine)

Cro ab

Mixture o Fab ragments o IgG that bind and neutralize venom toxins o 10 clinically important North American Crotalidae snakes

Crotalidae envenomation (Western diamondback, Eastern diamondback, Mojave rattlesnakes, and water moccasins)

*Digoxin immune serum, Fab (ovine)

Digi ab

Monovalent ragment antigen-binding (Fab) immunoglobulin ragment obtained rom sheep immunized with a digoxin derivative

Digoxin toxicity

Af ibercept (same unctional entity as ziv-af ibercept; see above)

Eylea

Recombinant usion protein with the extracellular domains o human VEGF receptors 1 and 2 used to the Fc portion o human IgG1; inhibits neovascularization

Wet macular degeneration

Ranibizumab

Lucentis

Binds iso orms o vascular endothelial growth actor A (VEGF-A)

Neovascular age-related macular degeneration

Endocrine Disorders

Other§

Proteins are recombinant unless otherwise stated. * Nonrecombinant. ‡ Also classed in Group Ib. † Puri ed immune globulins can also be used to mitigate the acute e ects o exposure to an in ectious agent. Human immune globulins targeting botulism, cytomegalovirus, hepatitis B, rabies, tetanus, vaccinia, and varicella have been approved by the FDA. § Three additional antivenins have been approved by the FDA: antivenin immune globulin (equine)—Latrodectus mactans (black widow spider), antivenin immune globulin (equine)—Micrurus fulvius (North American coral snake), and antivenin immune F(ab’)2 (equine)—Centruroides sculpturatus (Arizona bark scorpion). CHOP, cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin® (vincristine), prednisone/prednisolone; CTLA4, cytotoxic T-lymphocyte-associated antigen 4; CVP, cyclophosphamide, vincristine, prednisone; EGFR, epidermal growth actor receptor; LFA-3, leukocyte unction-associated antigen 3; mAb, monoclonal antibody; MadCAM1, mucosal addressin cell adhesion molecule 1; TNF, tumor necrosis actor; VCAM1, vascular cell adhesion molecule-1; VEGF-A, vascular endothelial growth actor A.

One o the great challenges in drug therapy is the selective delivery o small-molecule drugs and proteins to the intended therapeutic target. The body normally uses proteins to achieve specialized transport and delivery o many di erent molecules. An active area o current research is ocused on understanding the principles o protein-based, targeted delivery o molecules, so that these principles can be applied to modern pharmacotherapy. This strategy is exploited by protein therapeutics in Group IIb (Table 54-5), such as gemtuzumab ozogamicin, which links the binding region o a monoclonal antibody directed against CD33 with calicheamicin, a small-molecule chemotherapeutic agent. By using this therapy, the toxic compound is selectively delivered to CD33-expressing acute myelogenous leukemia cells, resulting in the selective killing o these cells. Similarly, re ractory CD20-expressing non-Hodgkin’s lymphoma cells can be destroyed selectively by ibritumomab tiuxetan, a monoclonal antibody directed against CD20 and linked to a radioactive yttrium isotope (Y-90). Another example is provided by denileukin diftitox, which uses a monoclonal antibody

directed against the CD25 component o the IL-2 receptor to deliver cytocidal diphtheria toxin to T-cell lymphoma cells that express this receptor. A unique challenge in targeted, protein-based delivery o small, toxic molecules is the choice o the chemical linker that bridges the protein and the small molecule. Many approaches to linker chemistry have used unctional groups that are designed to break apart in the intracellular environment, which is mildly more acidic than the extracellular environment. However, in some cases, preclinical testing has demonstrated nonspecif c release o the cytotoxic conjugates prior to cellular uptake, causing increased systemic toxicity in animal models. Recently approved therapeutics, such as trastuzumab emtansine , demonstrate the use o more mature linker chemistry that may serve as a plat orm technology to acilitate the development o additional targeted therapies. Trastuzumab emtansine uses a heterodimeric crosslinker, abbreviated SMCC (succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate), to link the cytotoxic maytansine molecule to the targeting antibody (see Table 51-1). This crosslinker

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TABLE 54-5 Protein Therapeutics Delivering Other Compounds or Proteins (Group IIb) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Brentuximab vedotin

Adcetris

Chimeric monoclonal antibody (mAb) targeting CD30 (brentuximab) conjugated to the small-molecule antimitotic agent monomethyl auristatin E

Hodgkin’s lymphoma and systemic anaplastic large cell lymphoma

Denileukin diftitox

Ontak

Directs the cytocidal action of diphtheria toxin to cells expressing the IL-2 receptor

Persistent or recurrent cutaneous T-cell lymphoma expressing the CD25 component of the IL-2 receptor

Gemtuzumab ozogamicin

Mylotarg

Humanized anti-CD33 IgG4 kappa mAb conjugated to calicheamicin, a smallmolecule chemotherapeutic agent

Relapsed CD33-expressing acute myelogenous leukemia Voluntarily withdrawn from the US market in 2010 due to high rate of fatal toxicity seen in postapproval study

‡Ibritumomab tiuxetan

Zevalin

MAb portion recognizes CD20-expressing B cells and induces apoptosis, while the chelation site allows either imaging (In-111) or cellular damage by beta emission (Y-90)

Relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin’s lymphoma (NHL), including rituximab-refractory follicular NHL

‡Tositumomab

Bexxar

I-131 tositumomab

Bexxar I-131

MAb that binds CD20 surface antigen and stimulates apoptosis MAb coupled to radioactive iodine-131; binds CD20 surface antigen and delivers cytotoxic radiation (used after tositumomab without I-131)

CD20-expressing follicular non-Hodgkin’s lymphoma, with and without transformation, in patients whose disease is refractory to rituximab and has relapsed following chemotherapy; tositumomab and then I-131 tositumomab are used sequentially in the Bexxar treatment regimen

‡Trastuzumab emtansine

Kadcyla

Trastuzumab conjugated to the cytotoxic agent maytansine both inhibits HER2 signaling and delivers maytansine to cancer cells that overexpress the HER2 receptor

HER2 positive metastatic breast cancer

All proteins are recombinant. ‡ Also classed in Group IIa. MAb, monoclonal antibody.

binds to the cytotoxic molecule via a covalent thioester bond, which is chemically stable and is unlikely to be degraded in the circulation be ore reaching the target cells. The antibody–drug conjugate is endocytosed selectively by the target cell, and within the cell, the antibody is su f ciently degraded that the maytansine moiety is exposed and allowed to exert its cytotoxic e ect. In addition to these current examples, interesting developments are in progress that illustrate where the f eld might be heading. One active area o research involves the delivery o proteins and other macromolecules to the CNS, which is challenging owing to the highly selective blood–brain barrier (BBB). Animal experiments have demonstrated, however, that usion proteins combining a therapeutic protein with a protein that naturally has specif c penetration through the BBB can allow success ul delivery o the therapeutic protein to the CNS. For example, a ragment o the tetanus toxin protein that naturally crosses the BBB has been shown in animal experiments to deliver the enzyme superoxide dismutase (SOD) to the CNS. Alternatively, antibodies that target endogenous protein receptors in the BBB, such as the insulin receptor or the trans errin receptor, can bypass the BBB using existing transport mechanisms. These carrier antibodies can be conjugated to other proteins or small molecules to allow them to bypass the BBB. Finally, experiments in mice suggest that targeted inhibitors o the M sd2a protein could potentially be used to locally disrupt

the BBB by allowing transcytosis o protein-containing vesicles across CNS endothelial cells. These types o therapeutics could potentially be used to treat neurological disorders such as amyotrophic lateral sclerosis , in which CNS levels o SOD are reported to be low. Exciting prospects also exist or the treatment o other disorders o the CNS in which levels o a particular protein are abnormal.

Group III: Protein Vaccines As recombinant DNA technology was being developed, great strides were also being made in understanding the molecular mechanisms that allow the immune system to protect the body against in ectious diseases and cancer. Armed with this new understanding, proteins in Group III have been success ully applied as prophylactic or therapeutic vaccines. Table 54-6 provides selected examples. For humans to develop e ective immunity against oreign organisms or cancer cells, immune cells such as helper T cells must be activated. Immune cell activation is mediated by antigen-presenting cells, which display on their sur ace specif c oligopeptides that are derived rom proteins ound in oreign organisms or cancer cells. Vaccination against certain organisms such as polio or measles has most o ten been achieved by injecting heat-killed or attenuated orms o these pathogens. Un ortunately, these methods have involved a certain amount o unavoidable risk o in ection or

974 FRONTIERS IN PHARMACOLOGY

TABLE 54-6 Protein Vaccines (Group III) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Protecting Against a Deleterious Foreign Agent (Group IIIa) HBsAg

Engerix Recombivax HB

Nonin ectious protein on sur ace o hepatitis B virus

Hepatitis B vaccination

HPV vaccine

Gardasil

Quadrivalent HPV recombinant vaccine (strains 6, 11, 16, 18); contains major capsid proteins rom our HPV strains

Prevention o HPV in ection

OspA

LYMErix

Nonin ectious lipoprotein on outer sur ace o Borrelia burgdorferi

Lyme disease vaccination

Treating an Autoimmune Disease (Group IIIb) Anti-Rh IgG

Rhophylac

Neutralizes Rh antigens that could otherwise elicit anti-Rh antibodies in an Rh-negative individual

Routine antepartum and postpartum prevention o Rh(D) immunization in Rh(D)-negative women; Rh prophylaxis in case o obstetric complications or invasive procedures during pregnancy; suppression o Rh immunization in Rh(D)-negative individuals trans used with Rh(D)-positive red blood cells

Antigen-presenting cells are extracted rom the patient and then incubated ex vivo with prostatic acid phosphatase (an antigen expressed on prostate cancer cells) and GM-CSF to stimulate the cells. The activated cells are then in used into the patient, and they activate an in vivo immune response against the prostate cancer cells.

Metastatic hormone-re ractory prostate cancer

Treating Cancer (Group IIIc) Sipuleucel-T

Provenge

Selected vaccines highlight the use o recombinant protein technology in vaccine production. Vaccines or the ollowing agents or diseases are currently approved by the FDA: anthrax, acellular pertussis, BCG ( or childhood TB protection), diphtheria, hepatitis A and B, human papillomavirus types 6, 11, 16, and 18, inf uenza types A, B, and H5N1, J apanese encephalitis, Lyme disease, measles, meningococcus, mumps, plague, pneumococcus, polio, rabies, rotavirus, rubella, smallpox, tetanus, typhoid, varicella-zoster, and yellow ever (see http://www. da.gov/cber/vaccine/licvacc.htm).

adverse reaction. By specif cally injecting the appropriate immunogenic (but nonpathogenic) protein components o a microorganism, vaccines can hope ully be created that provide immunity in an individual without exposing that individual to the risks o in ection or toxic reaction. Proteins in Group IIIa are used to generate protection against in ectious diseases or toxins. One success ul example is the hepatitis B vaccine . This vaccine was created by producing recombinant hepatitis B sur ace antigen (HBsAg), a nonin ectious protein o the hepatitis B virus. When immunocompetent humans are challenged and rechallenged with this protein, signif cant immunity results in the large majority o individuals. Similarly, the nonin ectious lipoprotein (OspA) on the outer sur ace o Borrelia burgdorferi has been engineered into a vaccine or Lyme disease . A recently approved vaccine against human papillomavirus (HPV) combines the major capsid proteins rom our HPV strains that commonly cause genital warts (strains 6 and 11) and cervical cancer (strains 16 and 18). In addition to generating protection against oreign invaders, recombinant proteins can induce protection against an overactive immune system that attacks its own body or “sel .” One theory is that administration o large amounts o this sel protein causes the body’s immune system to develop tolerance to that protein by eliminating or deactivating cells that react against the sel -protein. Proteins in Group IIIb are used

to treat patients with disorders that arise rom this type o autoimmune phenomenon. Immunological acceptance o a etus during pregnancy represents a special situation with respect to vaccine use. Occasionally, a pregnant woman can reject a etus a ter she has been immunized against certain antigens carried by a etus rom a previous pregnancy. Administration o an anti-Rhesus D antigen Ig prevents the sensitization o an Rh-negative mother at the time o delivery o an Rh-positive neonate. Because the woman ails to develop antibodies directed against the etal Rh antigens, immune reactions and pregnancy loss do not occur in subsequent pregnancies, even i the new etus carries the Rh antigens. Proteins in Group IIIc include therapeutic anticancer vaccines. The f rst protein therapeutic in this class, sipuleucel-T, was approved in 2010, and promising clinical trials are underway using a variety o approaches to develop additional patient-specif c cancer vaccines. Sipuleucel-T is designed to train the immune system to detect and attack metastatic prostate cancer cells. This is accomplished by f rst isolating dendritic cells (antigen-presenting cells) rom a patient’s peripheral blood and then incubating the cells with a usion protein composed o prostatic acid phosphatase (PAP; an antigen that is present on most prostate cancer cells), conjugated to GM-CSF (which promotes maturation o the dendritic cells). The activated dendritic cells, which now recognize the PAP antigen, are then trans used back

C h a p t e r 5 4 Protein Therapeutics

into the patient, where they help direct the immune system to destroy the prostate cancer cells. In another example in development, a vaccine or B-cell non-Hodgkin’s lymphoma uses transgenic tobacco plants (Nicotiana benthamiana). Each patient with this type o lymphoma has a malignant proli eration o an antibody-producing B cell that displays a unique antibody on its sur ace. By subcloning the idiotype region o this tumor-specif c antibody and expressing the region recombinantly in tobacco plants, a tumor-specif c antigen is produced that can be used to vaccinate a patient. This process requires 6–8 weeks rom biopsy o the lymphoma to a ready-to-use, patient-specif c vaccine. As the genomes o in ectious organisms and the pathophysiology o autoimmune diseases and cancer are more ully elucidated, more recombinant proteins will undoubtedly be developed or use as vaccines.

Group IV: Protein Diagnostics Proteins in Group IV are not used to treat disease, but purif ed and recombinant proteins used or medical diagnostics (both in vivo and in vitro) are mentioned here because they are invaluable in the decision-making process that guides the treatment and management o many diseases. Table 54-7 provides selected examples. A classic example o an in vivo diagnostic is the purif ed protein derivative (PPD) test, which determines whether an individual has been exposed to antigens rom Mycobacterium tuberculosis. In this example, a nonin ectious protein component o the organism is injected under the skin o an immunocompetent individual. An active immune reaction is interpreted as evidence that the patient has been previously in ected by M. tuberculosis or exposed to the antigens o this organism. Several stimulatory protein hormones are used to diagnose endocrine disorders. Growth hormone releasing hormone (GHRH) stimulates somatotroph cells o the anterior pituitary gland to secrete growth hormone. Used as a diagnostic, GHRH can help to determine whether pituitary growth hormone secretion is de ective in patients with clinical signs o growth hormone def ciency. Similarly, the recombinant human protein secretin is used to stimulate pancreatic secretions and gastrin release and thereby aid in the diagnosis o pancreatic exocrine dys unction or gastrinoma. In patients with a history o thyroid cancer, recombinant thyroidstimulating hormone (TSH) is an important component o the surveillance methods used to detect residual thyroid cancer cells. Be ore the advent o recombinant TSH, patients with a history o thyroid cancer were required to stop taking replacement thyroid hormone in order to develop a hypothyroid state to which the anterior pituitary would respond by releasing endogenous TSH. TSH-stimulated cancer cells could then be detected by radioactive iodine uptake. Un ortunately, this method required patients to experience the adverse consequences o hypothyroidism. Use o recombinant TSH instead o endogenous TSH not only allowed patients to remain on replacement thyroid hormone but also resulted in the improved detection o residual thyroid cancer cells. Imaging agents are a broad group o protein diagnostics that can be used to help identi y the presence or localization o a pathologic condition. For example, apcitide is a technetium-labeled synthetic peptide that binds glycoprotein IIb/IIIa receptors on activated platelets and is used to image acute venous thrombosis. Capromab pendetide is

975

an indium-111-labeled anti-PSA (prostate-specif c antigen) antibody that can be used to detect prostate cancer. Proteinbased imaging agents are o ten used to detect otherwise hidden disease so it can be treated early, when treatment is most likely to succeed. Imaging agents are currently used to detect cancer, image myocardial injury, or identi y sites o occult in ection; these agents are presented in more detail in Table 54-7. There are numerous in vitro protein diagnostics, and two are presented here as examples o a much larger class. Natural and recombinant HIV antigens are essential components o common screening (enzyme immunoassay) and conf rmatory (Western blot) tests or HIV in ection. In these tests, the antigens serve as “bait” or specif c antibodies to HIV gag, pol, and env gene products that have been elicited in the course o in ection. Hepatitis C in ection is diagnosed by using recombinant hepatitis C antigens to detect antibodies directed against this virus in the serum o potentially in ected patients.

CHALLENGES FOR PROTEIN THERAPEUTICS There are by now many examples in which proteins have been used success ully in therapy. Nonetheless, potential protein therapies that have ailed ar outnumber the successes, in part owing to several important challenges that are aced in the development and use o protein therapeutics. First, protein solubility, route o administration, distribution, and stability are all actors that can hinder the success ul application o a protein therapy. Proteins are large molecules with both hydrophilic and hydrophobic properties that can make entry into cells and other compartments o the body di f cult, and the hal -li e o a therapeutic protein can be drastically a ected by proteases, protein-modi ying chemicals, or other clearance mechanisms. One example o how such challenges are being addressed is through the production o PEGylated versions o therapeutic proteins. For example, PEG-inter eron is a modif ed orm o inter eron in which the polymer polyethylene glycol (PEG) is added to prolong the absorption, decrease the renal clearance, retard the enzymatic degradation, increase the elimination hal li e, and reduce the immunogenicity o inter eron. A second challenge is that the body may mount an immune response against the therapeutic protein. In some cases, this immune response can neutralize the protein and can even cause a harm ul reaction in the patient. For example, immune responses can be generated against Group Ia therapeutic proteins used to replace a actor that has been missing since birth, as illustrated by the development o antiactor VIII antibodies (inhibitors) in patients with severe hemophilia A who are treated with recombinant human actor VIII. More commonly, however, immune responses are generated against proteins o nonhuman origin. Until recently, the widespread clinical application o monoclonal antibodies had been limited by the rapid induction o immune responses against this class o therapeutic proteins. The need or antibody therapeutics that evade immune surveillance and response has been a driving orce in the maturation o antibody production technology. Recombinant technology and other advances have allowed the development o various antibody products that are less likely to provoke an immune response than unmodif ed murine antibodies. In humanized

976 FRONTIERS IN PHARMACOLOGY

TABLE 54-7 Protein Diagnostics (Group IV) PROTEIN

TRADE NAME

FUNCTION

EXAMPLES OF CLINICAL USE

Recombinant purif ed protein derivative (DPPD)

Nonin ectious protein rom Mycobacterium tuberculosis

Diagnosis o tuberculosis exposure

§Cosyntropin (ACTH 1-24)

Cortrosyn

Fragment o ACTH that stimulates cortisol release by the adrenal cortex

Diagnosis o primary versus secondary adrenal insu f ciency

*Glucagon

GlucaGen

Pancreatic hormone that increases blood glucose by stimulating the liver to convert glycogen to glucose

Diagnostic aid to slow gastrointestinal motility in radiographic studies; reversal o hypoglycemia

‡Growth hormonereleasing hormone (GHRH)

Gere

Recombinant ragment o GHRH that stimulates growth hormone (GH) release by somatotroph cells o the pituitary gland

Diagnosis o de ective growth hormone secretion

§Secretin

ChiRhoStim (synthetic human peptide) SecreFlo (synthetic porcine peptide)

Stimulation o pancreatic secretions and gastrin

Aids in the diagnosis o pancreatic exocrine dys unction or gastrinoma; acilitates identif cation o the ampulla o Vater and accessory papilla during endoscopic retrograde cholangiopancreatography

Thyroidstimulating hormone (TSH), thyrotropin

Thyrogen

Stimulates thyroid epithelial cells or welldi erentiated thyroid cancer tissue to take up iodine and produce and secrete thyroglobulin, triiodothyronine, and thyroxine

Adjunctive diagnostic or serum thyroglobulin testing in the ollow-up o patients with well-di erentiated thyroid cancer

In Vivo Infectious Disease Diagnostics DPPD

Hormones

Imaging Agents, Cancer Capromab pendetide

ProstaScint

Imaging agent; indium-111-labeled anti-PSA antibody; recognizes intracellular PSA

Prostate cancer detection

§Indium-111octreotide

OctreoScan

Imaging agent; indium-111-labeled octreotide

Neuroendocrine tumor and lymphoma detection

Satumomab pendetide

OncoScint

Imaging agent; indium-111-labeled mAb specif c or tumor-associated glycoprotein (TAG-72)

Colon and ovarian cancer detection

Arcitumomab

CEA-scan

Imaging agent; technetium-labeled anti-CEA antibody

Colon and breast cancer detection

No etumomab

Verluma

Imaging agent; technetium-labeled antibody specif c or small cell lung cancer

Small cell lung cancer detection and staging

Imaging Agents, Other §Apcitide

Acutect

Imaging agent; technetium-labeled synthetic peptide; binds GPIIb/IIIa receptors on activated platelets

Imaging o acute venous thrombosis

Imciromab pentetate

Myoscint

Imaging agent; indium-111-labeled antibody specif c or human cardiac myosin

Detects presence and location o myocardial injury in patients with suspected myocardial in arction

Technetium anolesomab

NeutroSpec

Imaging agent; technetium-labeled anti-CD15 antibody; binds neutrophils that inf ltrate sites o in ection

Diagnostic agent (used in patients with equivocal signs and symptoms o appendicitis)

Examples of In Vitro Diagnostics HIV antigens

Enzyme immunoassay (EIA) Western blot OraQuick Uni-Gold

Detects human antibodies to HIV

Diagnosis o HIV in ection

Hepatitis C antigens

Recombinant immunoblot assay (RIBA)

Detects human antibodies to hepatitis C virus

Diagnosis o hepatitis C exposure

Protein diagnostics are recombinant unless otherwise stated. * Also classed in Group Ib. ‡ Also classed in Group Ia. § Synthetic. ACTH, adrenocorticotropic hormone; CEA, carcinoembryonic antigen; mAb, monoclonal antibody; PSA, prostate-specif c antigen.

C h a p t e r 5 4 Protein Therapeutics

antibodies, portions o the antibody that are not critical or antigen-binding specif city are replaced with human Ig sequences that con er stability and biological activity on the protein but do not provoke an anti-antibody response. Fully human antibodies can be produced using transgenic animals or phage display technologies. The f eld o cancer therapeutics illustrates the pace o advances in monoclonal antibody development. In the 1980s, most o the monoclonal cancer therapeutics were murine, although there were a ew examples o chimeric antibodies and isolated instances o humanized and human antibodies in clinical development. During the 1990s, humanized and ully human antibodies became the most common types o antibodies introduced into clinical trials. Since 2000, there has been a urther increase in the proportion o antibodies that are ully human, and the proportion o murine and chimeric antibodies introduced into clinical trials has decreased. Fully human antibodies are especially well represented in recent FDA-approved protein therapeutics. More heavily engineered protein therapies that are based on human antibodies have also been developed over the past 10–20 years. One example is the “minibody” romiplostim, which is approved or the treatment o immune thrombocytopenic purpura. This construct consists o an Fc region o a human antibody with two copies o a peptide sequence linked to each o its IgG1 heavy chains. The peptide sequence was selected to stimulate the thrombopoietin receptor, yet the sequence has no similarity to its endogenous analogue thrombopoietin. The Fc portion extends the hal -li e o romiplostim in the circulation, and the lack o sequence homology to thrombopoietin will ideally prevent the development o cross-reactive anti-thrombopoietin antibodies— a serious adverse e ect that had been seen with a PEGylated version o thrombopoietin. A third issue is that or a protein to be physiologically active, post-translational modif cations such as glycosylation, phosphorylation, and proteolytic cleavage are o ten required. These requirements may dictate the use o specif c cell types that are capable o expressing and modi ying the protein appropriately. In addition, recombinant proteins must be synthesized in a genetically engineered cell type or large-scale production. The host system must produce not only biologically active protein but also a su f cient quantity o this protein to meet clinical demand. Also, the system must allow purif cation and storage o the protein in a therapeutically active orm or extended periods o time. The protein’s stability, olding, and tendency to aggregate may be di erent in large-scale production and storage systems than in smaller scale systems used to produce the protein or animal testing and clinical trials. Some have proposed engineering host systems that co-express a chaperone or oldase with the therapeutic protein o interest, but these approaches have had limited success. Potential solutions could include the development o systems in which entire cascades o genes involved in protein olding are induced together with the therapeutic protein; the impetus or this work is the observation that plasma cells, which are natural protein production “ acilities,” use such gene cascades to produce large quantities o monoclonal antibody. Compared to bacteria and yeast, which are generally considered easy to culture, certain mammalian cell types can be more di f cult and more costly to culture. Other methods o production, such as genetically engineered

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animals and plants, could provide a production advantage. Transgenic cows, goats, and sheep have been engineered to secrete protein in their milk, and transgenic chickens that lay eggs f lled with recombinant protein are anticipated in the uture. Transgenic plants can inexpensively produce vast quantities o protein without waste or bioreactors, and potatoes can be engineered to express recombinant proteins and thereby make edible vaccines. The f rst available protein therapeutic produced in plants is taliglucerase alfa , a glucocerebrosidase used to treat Gaucher’s disease; this drug was approved by the FDA in 2012. Finally, by using uidshaking bioreactors, microliter-sized culture systems might be able to predict the success o large-scale culture systems and thereby provide substantial cost savings by ocusing investment on systems that are more likely to succeed. A ourth important challenge is the costs involved in developing protein therapies. Although switching to recombinant methodology rom laborious purif cation o placentally derived protein has allowed the production o su f cient glucocerebrosidase to treat Gaucher’s disease in many patients, the cost o the recombinant protein can be more than $100,000 per patient per year. The example o Gaucher’s disease also illustrates aspects o a f th issue associated with protein therapeutics: ethics (although these ethical issues are not exclusive to protein therapeutics). For example, the possibility o e f cacious but expensive protein therapeutics or small but severely ill patient populations, such as patients with Gaucher’s disease, can present a dilemma with respect to allocation o f nancial resources o health care systems. In addition, the def nition o illness or disease could be challenged by protein therapeutics that can “improve upon” conditions previously viewed as variants o normal. For example, the def nition o short stature may begin to change with the possibility o using growth hormone to increase the height o a child. Finally, the regulatory landscape that governs protein therapies will likely continue to have a signif cant impact on the development o new therapies and their cost. As the f eld o protein therapeutics matures and certain therapies lose patent protection, the role o ollow-on or generic protein therapies in medicine will be decided. Only in 2010 was a regulatory pathway established in the United States that addresses the development o generic versions o protein therapeutics (so-called biosimilars ), and it remains unclear how e ective this pathway will be at reducing the cost and e ort required to bring a biosimilar to market. Due to the complexity o protein manu acture and the costs and risks associated with protein therapeutic development and testing, relatively small changes in the regulatory landscape may have strong impacts on the investment in and development o protein therapeutics.

CONCLUSION AND FUTURE DIRECTIONS Medicine is entering a new era in which approaches to managing disease are at the level o the genetic and protein in ormation that underlies all biology, and protein therapeutics are playing an increasingly important role. Already, recombinant human proteins make up the majority o FDAapproved biotechnology medicines, which include monoclonal antibodies, natural inter erons, vaccines, hormones,

978 FRONTIERS IN PHARMACOLOGY

modif ed natural enzymes, and various cell therapies. The uture potential or such therapies is huge, given the thousands o proteins produced by the human body and the many thousands o proteins produced by other organisms. Furthermore, recombinant proteins not only provide alternative (or the only) treatments or particular diseases but can also be used in combination with small-molecule drugs to provide additive or synergistic benef t. Treatment o EGFRpositive colon cancer is illustrative o this point: combination therapy with the small-molecule drug irinotecan, which prevents DNA repair by inhibiting DNA topoisomerase, and the recombinant monoclonal antibody cetuximab, which binds to and inhibits the extracellular domain o EGFR, results in increased survival in patients with colorectal cancer. The therapeutic synergy between irinotecan and cetuximab may be due to the act that both drugs inhibit the same EGFR signaling pathway, with one drug (cetuximab) inhibiting the initiation o the pathway and the other drug (irinotecan) inhibiting a target downstream in the pathway. A small-molecule drug recently approved or use in the treatment o cystic f brosis may point the way to a new conceptual approach to protein therapeutics. Ivacaftor is a potentiator o the cystic f brosis transmembrane conductance regulator (CFTR) protein. This drug is indicated or the treatment o cystic f brosis in patients who have a G551D mutation in the CFTR gene. Ivaca tor acilitates increased chloride transport through the CFTR chloride channel by increasing the open channel probability o the G551D-CFTR protein. Thus, instead o replacing the abnormal (nonconducting) G551D-CFTR protein with its normal (conducting) counterpart, the drug restores normal unction to the abnormal protein. This paradigm may be increasingly employed in the coming years to treat diseases associated with the expression o abnormal proteins. A second new concept in protein therapeutics is exemplif ed by mipomersen, the f rst antisense oligonucleotide to be approved by the FDA. This agent targets the messenger RNA or apolipoprotein B100 (apoB); by binding to apoB mRNA, mipomersen inhibits translation o apoB and thereby decreases production o apoB and secretion o verylow-density lipoprotein (VLDL) particles. Mipomersen is used to treat patients with homozygous amilial hypercholesterolemia (see Chapter 20, Pharmacology o Cholesterol and Lipoprotein Metabolism). Additional antisense oligonucleotide therapeutics are in development. The early success o recombinant insulin production in the 1970s created an atmosphere o enthusiasm and hope,

which was un ortunately ollowed by an era o disappointment when the vaccine attempts, nonhumanized monoclonal antibodies, and cancer trials in the 1980s were largely unsuccess ul. Despite these setbacks, signif cant progress has been made recently. Some o the major successes with protein therapeutics are described in this chapter, and new production methods are changing the scale, cost, and even route o administration o recombinant protein therapeutics. With the large number o protein therapeutics both in current clinical use and in clinical trials or a range o disorders, one can conf dently predict that protein therapeutics will have an expanding role in medicine or years to come.

Acknowledgment We thank the late Armen H. Tashjian, Jr. or many help ul discussions in developing this chapter or the First, Second, and Third Editions o Principles o Pharmacology: The Pathophysiologic Basis o Drug Therapy. Portions o this chapter have been published as a review article (Leader B, Baca QJ, Golan DE. Protein therapeutics: a summary and pharmacological classif cation. Nat Rev Drug Discov 2008;7:21–39) and are adapted with permission.

Suggested Reading Ben-Zvi A, Lacoste B, Kur E, et al. M sd2a is critical or the ormation and unction o the blood–brain barrier. Nature 2014;509:507–511. (Identif es M sd2a as a key regulator o blood–brain barrier unction.) Keen H, Glynne A, Pickup JC, et al. Human insulin produced by recombinant DNA technology: sa ety and hypoglycaemic potency in healthy men. Lancet 1980;2:398–401. (A milestone in the use o a recombinantly produced protein therapeutic.) Mascelli MA, Zhou H, Sweet R, et al. Molecular, biologic, and pharmacokinetic properties o monoclonal antibodies: impact o these parameters on early clinical development. J Clin Pharmacol 2007;47:553–565. (Discusses trends in antibody ormulation and how specif c properties o candidate drugs guide early drug development.) Nelson AL, Dhimolea E, Reichert JM. Development trends or human monoclonal antibody therapeutics. Nat Rev Drug Discov 2010;9:767–774. (Describes the development o human monoclonal antibodies and their increasing role as protein therapeutics.) Walsh CT. Posttranslational modif cation o proteins: expanding nature’s inventory. Greenwood Village, CO: Roberts & Company; 2005. (Reviews mechanisms and biological roles o covalent modif cations o proteins.) Woodcock J, Gri f n J, Behrman R, et al. The FDA’s assessment o ollow-on protein products: a historical perspective. Nat Rev Drug Discov 2007;6:437–442. (Discusses challenges o developing protein therapeutics, including di f culties in demonstrating bioequivalence in ollow-on protein therapeutics.)

A

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55

Drug

Wa te te r o r e n zyme

Drug Delivery Modalities

Drug g in re s e r voi oirr

Drug ug d is s olve d or dis pe p rs e d in polyme r

J o s h u a D. M o s s a n d Ro b e rt La n g e r D

E P ol olym ymee r m a tr trix W Waa&teCASE r SE INT INTRODUCTION SE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 979– 9 98 9800 NOVEL USE N NOV E OF O EXIST XIIIST STING S TIN I G DELIV DELIVERY VE E ROUTES U . . . . . . . . . . . . . . . 97 979 79 Oral Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979 O Pulmonary P ulmonna n Delivve D veery ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980 Tran T nsd sder erma mall Deli liv ivve very e ryy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981 POLYMER-BASED POL AS D D AS DELIVERY ELIVE VERY SY VE SYSTEMS YSTE YS TE S . . . . . . . . . . . . . . . . . . . . 981 General Mechan G anis isms mss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981 Diffusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 9811 Drug dis s olve d Drug Dr D ug dis s olv oolve lvee d or dis pe rs e d in in p olyme l r pollyme r

INTRODUCTION

Drugs are typically administered in either pill or injection orm, with limited control over release rate and localization. More advanced drug delivery systems have recently been developed, however. The goal o these new technologies is to alter our pharmacokinetic properties: (1) absorption o the drug, including the period o time over which it is released into the systemic circulation or at its f nal site o action; (2) distribution o the drug, whether it be to the entire body or to a specif c tissue or organ system; (3) metabolism o the drug, either to be avoided entirely or used to convert a prodrug to an active orm; and (4) elimination o the drug. This chapter describes several existing and emerging delivery modalities and discusses how these modalities inuence one or more o these our properties. The f eld o drug delivery is large and encompasses many disciplines, and this discussion will highlight approaches that illustrate these properties rather than provide an exhaustive description o all ongoing practice and research. The highlighted modalities include the novel use o existing delivery routes, polymer-based delivery systems, and liposome-based delivery systems.

NOVEL USE OF EXISTING DELIVERY ROUTES Oral Delivery Oral administration o small molecules is currently the most common method o drug delivery. The main advantages o oral delivery are ease o use and relatively low cost, both o which can improve patient adherence. However, incomplete absorption, metabolism o the drug during absorption, and metabolism o the drug during the f rst pass through the liver

F S e mipe rme a bble le Wa te te r me mbra mb raa ne Chemic Ch iccal al R al Reeact Rea React ctio ct ion oon. n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9882 Solve oolvent lvent nt A nt Acti Activation ivati tion ti tio ioon n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9833 Intel ell ige ntt Delivery elligent li . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 983 Tarrgeting T ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 9844 LIPOSOME-BAS S M -BASED SOME-BAS BA BA ASED ED DELIV DEL VER ERY SYST Y TE TEMS MS . . . . . . . . . . . . . . . . . . . 984 984 84 CONCLUSION NCLUSIO L IO ON NA AND ND F FUTURE FUTU FU UTU DIRE RECTIO CTIO ONS S . . . . . . . . . . . . . . . . . . 984 98 98 Suggested u s tted Reading st Reeading R di . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .985

Wa te te r

S wolle ll n polyme l r from fr om whi hich ch d rug is be ing rebioavailability lee a s e d can decrease

Os mo moti motic ticc Os moti m tic mo de li livve ry r y co core re c on onta ta in inin ingg oorifice the drug.drug The variability o

these actors, as well as limitations in dosing requency, also a ect the ability to maintain a therapeutic drug concentration in the blood. In addition, only relatively small molecules can be used in conventional pills: the intestine generally cannot absorb large molecules intact. Intact peptide and protein drugs, such as insulin, are poorly absorbed orally because o proteolysis in the digestive tract. Recent advances and ongoing research in oral drug delivery are beginning to address these issues. Sustained or extended-release formulations can prolong plasma drug concentrations with less requent doses. In early approaches to sustained release, pill or capsule solubility was modif ed with one or more inert substances known as excipients . By ormulating the drug in an emulsion or suspension that is relatively di f cult to digest, the period o time over which the drug dissolves and is absorbed can be extended. Similar results have been achieved by coating the drug with substances such as cellulose derivatives or wax. This approach is used in a wide variety o both prescription and over-the-counter medications. Another success ul and more recent approach to sustained release oral ormulations involves an osmotic pump capsule (see below). Techniques are also being developed or delivery o larger molecules, such as proteins and DNA, in oral ormulations. Several designs make use o drug-carrying vehicles, including liposomes and microspheres. Liposomes , small vesicles with lipid bilayer membranes, are lipophilic and can be taken up by intestinal Peyer’s patches when targeted to M cells (specialized epithelial cells) with appropriate ligands. Certain types o liposomes have been moderately success ul in experimental oral vaccine delivery; their use in intravenous delivery systems is discussed below. Polyanhydride microspheres , which adhere strongly 979

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Ma rch 1988: Mr. F is 13 ye a rs o ld . His p a re n ts b e g in to n o tice th a t h e is tire d m u ch o th e tim e , d e s p ite g e ttin g p le n ty o s le e p . He ca n n o lo n g e r p a rticip a te o n h is s ch o o l’s tra ck te a m b e ca u s e h e b e co m e s e xh a u s te d in th e m id d le o ra ce s —th e s a m e ra ce s h e h a d o te n w o n le s s th a n a ye a r e a rlie r. Als o , Mr. F co m p la in s o b e in g th irs ty co n s ta n tly a n d , a s a re s u lt, co n s u m e s la rg e q u a n titie s o wa te r. Mr. F g o e s to h is a m ily p hys icia n , w h o m e a s u re s h is b lo o d g lu co s e le ve l a t 650 m g /d L (a p p roxim a te ly s ix tim e s n o rm a l le ve ls ) a n d m a ke s a n initia l d ia g nos is o typ e 1 d ia b e te s m e llitu s . Th e d ia g n o s is is co n f rm e d in th e h o s p ita l, w h e re Mr. F’s p hys icia n s s ta b ilize h is b lo o d g lu co s e a n d d e ve lo p a n in s u lin th e ra py re g im e n . He is ta u g h t h o w to d ra w a dro p o b lo o d ro m his f n ge rtip to m ea s ure his b loo d glu co s e a n d ho w to g ive h im s e l s ubcu ta ne o us in jectio n s o in s u lin . Ea ch d a y, Mr. F in je cts re co m b in a n t h u m a n in s u lin b e o re b re a k a s t a n d b e o re d in n e r. J a n u a ry 1997: Th ro u g h o u t h ig h s ch o o l a n d m o s t o co lle g e , Mr. F ra re ly m o n ito rs h is g lu co s e le ve ls a n d p u rp o s e ly ke e p s th e m h ig h e r th a n re co m m e n d e d . He wa n ts to b e a s “ n o rm a l” a s p o s s ib le , w h ich o r h im m e a n s n e ve r a llo w in g h is g lu co s e le ve l to a ll s o lo w a s to re q u ire o o d in th e m id d le o a cla s s o r a t o th e r u n u s u a l tim e s . As Mr. F b e co m e s o ld e r, h e b e g in s to a p p re cia te th a t a vo id in g th e lo n g -te rm co n s e q u e n ce s o p o o rly co n tro lle d d ia b e te s —a th e ro s cle ro s is , re tin o p a th y, n e p h ro p a th y, a n d p e rip h e ra l n e u ro p a th y, a m o n g o th e rs — is w o rth th e in co n ve n ie n ce o b e tte r co n tro l. He s w itch e s to a re g im e n invo lvin g o u r in je ctio n s a d a y a n d b e g in s ch e ckin g h is b lo o d g lu co s e o u r to f ve tim e s a d a y. Eve n tu a lly, h e s w itch e s ro m m u ltip le s u b cu ta n e o u s in je ctio n s (MS I) to co n tin u o u s s u b cu ta n e o u s in s u lin in u s io n (CS II) w ith a n in s u lin p u m p .

to the intestinal mucosal sur ace, have been shown to penetrate intestinal epithelium. A ter the microspheres are absorbed, presumably because they can stay in contact with the intestinal epithelium or long periods o time, the complex molecules carried within them can be released into the blood. Another potential approach to delivering proteins orally involves targeting the drug to the colon, which has lower levels o protease activity than the upper gastrointestinal tract. For example, microsphere delivery vehicles can be synthesized rom polymers that have enzymatically degradable azoaromatic cross-links. The colon has a relatively high concentration o azoreductases, acilitating degradation o the microspheres and protein release within the colon. Substances that transiently increase the permeability o colonic epithelium, possibly co-incorporated in the microspheres, may improve the absorption o proteins delivered to the colon. Another approach involves carrier molecules that may

Th e p u m p d e live rs a co n s ta n t b a s a l le ve l o in s u lin th a t ca n b e s u p p le m e n te d w ith b o lu s re le a s e s b e o re m e a ls , th e re b y m o re clo s e ly a p p roxim a tin g th e b o d y’s p hys io lo g ic co n tro l o b lo o d g lu co s e le ve ls . S e p te m b e r 2024: Ba ck in 1997, Mr. F u s e d h is in s u lin p u m p o r o n ly a b o u t 3 m o n th s , d e cid in g th a t th e s m a ll m a ch in e h e n e e d e d to ke e p co n s ta n tly a tta ch e d to h is b o d y wa s n o t co m p a tib le w ith h is active li estyle o r s el -im ag e. He resu m ed MSI therapy o r s e ve ra l m o re ye a rs , u n til h e b e ga n p a rticip a tin g in h u m a n tria ls o r a n e w, im p la n ta b le in s u lin d e live ry s ys te m . No w, a 2-ye a r s u p p ly o in s u lin is in co rp o ra te d in to a p o lym e r m a trix th a t ca n b e im p la n te d in th e s u b cu ta n e o u s a t o th e a b d o m e n . A d e vice in Mr. F’s w ris twa tch co n s ta n tly m e a s u re s h is g lu co s e le ve ls tra n s d e rm a lly, a n d it tra n s m its in s tru ctio n s to a m a g n e tic o s cilla to r im p la n te d n e ar th e p o lym e r d e live ry s ys te m . Th e d o s in g a d va n ta g e s o th e in s u lin p u m p a re th u s a ch ie ve d w ith o u t Mr. F e e lin g lim ite d o r tie d to a m a ch in e in a ny wa y. He s im p ly h a s th e p o lym e r s ys te m re p la ce d e ve ry 2 ye a rs a n d m a ke s m inor da ily a djus tm e nts to the p ro gram m e d delive ry p a ra m e te rs in h is w ris twa tch d e vice . Mr. F is lo o kin g o rwa rd to re ce ivin g a tra n s p la n t o p a n cre a tic b e ta ce lls , d e ve lo p e d ro m h is o w n s te m ce lls , th a t w ill cu re h is d ia b e te s .

Questions 1 . Why is oral administration of insulin not practical? 2 . Which other routes of insulin administration have been tried? 3 . Which technologies may make it possible to monitor blood glucose levels transdermally? 4 . How can polymers be used to optimize and simplify administration of some drugs?

be able to shuttle large molecules across the epithelial lining o the intestine.

Pulmonary Delivery Patients su ering rom asthma and other respiratory diseases have long been able to treat their condition by inhaling aerosols o drugs directly into their lungs: 2-adrenergic agonists, such as albuterol, and glucocorticoid analogues are widely used examples o such locally delivered drugs. In early metereddose inhaler designs, many o which are still used, the drug is delivered in liquid orm using a high velocity chlorof uorocarbon (CFC) propellant. With this technique, very little drug is reproducibly delivered to the lung—o ten less than 10%. Particles o ten accumulate in the mouth and throat, and many are immediately exhaled. Components o the immune system and macrophages in the lung can also clear some o the drug be ore it can act. In addition, many patients use their inhalers

C h a p t e r 5 5 Drug Delivery Modalities

incorrectly; common mistakes include not shaking the inhaler well enough, pressing the inhaler too early or too late during inhalation, or using an empty inhaler. Incorrect use urther reduces delivery e f ciency. Inhaler design continues to improve. Recent advances include more consistent dosing, greater ease o use via electronic breath actuation, and non-CFC propellants. The aerosol ormulations have also been improved by adjusting several properties o the particles themselves. For example, optimized particle chemistry and sur ace morphology can minimize undesirable particle–particle aggregation. Similarly, particle solubility can be modif ed to in uence the rate o therapeutic release once delivered. Dry powder aerosol clouds that reach deep into the lungs can be generated by blowing compressed air into a drug powder, breaking the powder into tiny (1–5 m) particles inside the inhaler. Devices that take advantage o these improvements have reduced both dosing requency and cost or local applications o pulmonary drug delivery in patients with asthma and cystic f brosis. The lung also o ers several potential advantages or noninvasive, systemic delivery o molecules. The large alveolar surace area, thin tissue lining, and limited numbers o proteolytic enzymes make the lung an ideal tissue or proteins and peptides to enter the bloodstream. One dry powder aerosol device has been approved or the pulmonary delivery o insulin but is not currently manu actured or marketed due to poor acceptance among patients and physicians. Insulin remains an attractive candidate or inhalation therapy, however, as do other biotherapeutics that are currently administered subcutaneously—such as growth hormone, glucagon, and 1-antitrypsin. One approach to achieving increased delivery e f ciency is the design o large, highly porous aerosol particles with very low densities. Such particles tend to aggregate less than smaller, denser particles, resulting in more e f cient aerosolization. In addition, these particles have an “aerodynamic diameter,” a parameter based on both density and actual particle dimensions, similar to conventional aerosol particles; thus, they can reach the deep parts o the lung through an airstream, despite their relatively large (5–20 m) size. Once deposited, the particles can escape clearance by alveolar macrophages, because phagocytosis o particles by macrophages diminishes with increasing particle size beyond 2–3 m. Thus, drugs can be delivered more e f ciently over longer periods o time. In one study, insulin was encapsulated in biodegradable polymer microspheres. Some o the microspheres were small and nonporous, and some were large and porous (low density), but both types had similar aerodynamic diameters. When the microspheres were delivered into the lungs, the relative bioavailability o the large, porous insulin particle was about seven times greater, and the total time o insulin release into the systemic circulation was about 24 times longer, than that o the conventional particle.

Transdermal Delivery The stratum corneum, composed o lipids and keratinocytes, is the outermost skin layer and the major barrier to transdermal transport. Small, lipophilic drugs have been success ully delivered through the skin into the systemic circulation by passive di usion at low ux rates, thereby avoiding f rstpass metabolism by the liver. Currently, passive transdermal patches are available or hormone replacement and or pharmacologic treatment o motion sickness, angina, nicotine withdrawal, hypertension, pain, and other conditions.

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In addition to providing higher bioavailability while remaining noninvasive, transdermal delivery systems are o ten associated with ewer adverse e ects than conventional oral dosage orms. For example, potential liver damage during f rst-pass metabolism is avoided when a drug is delivered by the transdermal route. Thus, more sophisticated transdermal systems are under development in an attempt to provide these advantages or drug molecules that are otherwise unable to penetrate the skin. Iontophoresis is one approach to enhancing the transport o charged, low-molecular-mass molecules through the skin. Iontophoresis involves the application o low-voltage electric pulses or long time periods; this technology is already used clinically or local applications, such as therapy or hyperhidrosis (excessive perspiration), and is under development or systemic delivery o small-molecule analgesic drugs. The use o high-voltage pulses or a short time period—on the order o milliseconds—is also being explored. In human cadaver skin, which is commonly used as a model or skin transport, such high-voltage pulses have been shown to induce temporary pores. This phenomenon, known as electroporation, will potentially allow systemic delivery o large, charged molecules, such as heparin and oligonucleotides. Microneedles are also being studied. Ultrasound enhancement o drug delivery through the skin, termed sonophoresis , is also being explored or molecules such as insulin, inter eron, and erythropoietin. Application o ultrasound to the skin results in cavitation, the ormation o tiny air-f lled spaces in lipid bilayers o the stratum corneum. The net result o cavitation is disordering o the lipid bilayers, enhancing di usivity o the drug through the skin by up to 1,000 times. Sonophoresis does not damage the skin, which typically regains its normal structure within 2 hours, and no undesirable e ects have been observed in early clinical trials. Sonophoresis can also be used to remove diagnostic samples rom the extracellular space under the stratum corneum. Experiments have been devised in which a reservoir was placed between an ultrasound transducer and a rat’s skin, and interstitial uid was extracted. Theophylline, glucose, cholesterol, urea, and calcium could be measured in the sample; the glucose measurements were su f ciently accurate to be used as a surrogate or blood glucose monitoring in diabetics. With a portable ultrasound transducer, this technique could be incorporated in a uturistic device such as the one Mr. F used in 2024.

POLYMER-BASED DELIVERY SYSTEMS General Mechanisms Polymer-based drug delivery systems gradually release drugs into their surroundings. Polymer delivery mechanisms are widely used in diverse applications such as birth control, chemotherapy, and antiarrhythmic therapy. These systems o er advantages in both controlled release and targeting o drugs and are thus the ocus o much research. Drug delivery rom a polymer-based system can be achieved via three general mechanisms: (1) di usion, (2) chemical reaction, and (3) solvent activation (Fig. 55-1).

Diffusion

Diffusion rom either a reservoir or a matrix is the most com-

mon release mechanism. In a reservoir system, the drug is contained within a polymer membrane through which it di uses over time (Fig. 55-1A). Norplant®, a long-term contraceptive system (no longer marketed in the United States), acts by this principle. Levonorgestrel, a synthetic

982 FRONTIERS IN PHARMACOLOGY A

B

C

P olyme r ba ckbone

P olyme r ma trix

P olyme r ca ps ule

Drug

Drug in re s e rvoir

Drug dis s olve d or dis pe rs e d in polyme r

D

E P olyme r ma trix

F S e mipe rme a ble me mbra ne

Wa te r Wa te r

Drug dis s olve d or dis pe rs e d in polyme r

Wa te r or e nzyme

Drug dis s olve d in polyme r

S wolle n polyme r from which drug is be ing re le a s e d

Os motic de live ry orifice

Os motic core conta ining drug

Wa te r

Drug s olution le a ve s

FIGURE 55-1. Polymer release mechanisms. In all panels except C, the simplif ed diagrams represent polymeric systems in cross section. The most common release mechanism is di usion, whereby the drug migrates rom its initial location in the polymer system to the polymer’s outer sur ace and then to the body. A, B. Di usion can occur rom a reservoir, in which a drug core is surrounded by a polymer f lm, or rom a matrix, where the drug is uni ormly distributed through the polymeric system. C, D. Drugs can also be released by chemical mechanisms such as cleavage o the drug rom a polymer backbone or hydrolytic degradation o the polymer. E. Exposure to a solvent can also activate drug release. For example, the drug can be retained in place by polymer chains; upon exposure to environmental uid, the outer polymer regions begin to swell, allowing the drug to di use outward. F. An osmotic system in the orm o a tablet with a laser-drilled hole in the polymer sur ace can provide constant drug release rates. Water di uses through the semipermeable membrane into the tablet along its osmotic gradient, swelling the osmotic core inside the tablet and orcing drug solution out through the hole. Combinations o these approaches are also possible. Release rates can be controlled by the nature o the polymeric material and the design o the system.

progestin, is stored in small silicone tubes implanted in the arm. The drug di uses slowly through the polymer capsule over the course o 5 years, providing e ective long-term contraception. (For a urther discussion o progestin action on the menstrual cycle, see Chapter 30, Pharmacology o Reproduction.) However, such reservoir systems are limited by the size o the drug molecules being delivered. Molecules larger than approximately 300 daltons (Da) are unable to di use through the polymer shell. In one common matrix system design, the drug is contained in a series o interconnecting pores within the polymer rather than in one large reservoir (Fig. 55-1B). This system is less limited by the size o the drug molecules because each pore can accommodate molecules with molecular weights o several million daltons. The rate o di usion between the pores—and thus through the matrix and out o the system— is controlled architecturally; tight constrictions and tortuous connections between pores prevent rapid release o the stored drug. One such system is used clinically to administer gonadotropin-releasing hormone (GnRH) analogues . GnRH analogues are peptide hormones that, when administered continuously, inhibit anterior pituitary gland production o gonadotropins (LH and FSH) and are use ul in the treatment o sex-hormone-dependent diseases such as prostate cancer. A major previous limitation o this therapeutic approach was the short in vivo hal -li e o GnRH analogues ollowing intramuscular injection. When the drug is incorporated into

polymer microcapsules and the capsules are injected intramuscularly, the hal -li e o GnRH is extended signif cantly, so that therapeutic concentrations are maintained over a period o 1–4 months. Drug delivery by the microcapsule system utilizes two mechanisms: f rst, the drug di uses out o the microcapsules; and second, the polymer matrix itsel degrades slowly. The second mechanism o polymer-based drug delivery involves a chemical reaction between the polymer and water (see below).

Chemical Reaction

In chemical reaction-based systems , part o the system is designed to degrade over time. Degradation can involve either a chemical or enzymatic reaction. In some designs, covalent bonds that connect the drug to a polymer are cleaved in the body by endogenous enzymes (Fig. 55-1C). Such polymer–drug complexes are typically administered intravenously, and the use o water-soluble polymers such as polyethylene glycol (PEG) increases the biological hal li e o the drug considerably. For example, PEG-Intron®, a pegylated orm o interferon- 2b, has been approved by the US Food and Drug Administration (FDA) or weekly administration; this treatment or hepatitis C in ection previously required injections three times as o ten. In the case o the intramuscular GnRH microcapsules discussed above, the polymer itsel is degraded in a reaction with water (Fig. 55-1D).

C h a p t e r 5 5 Drug Delivery Modalities

Most insoluble polymers considered or these applications exhibit bulk erosion (i.e., the entire matrix dissolves at the same rate), which results in larger pores and a more sponge-like and unstable structure. This pattern o degradation makes constant release rates di f cult to achieve and creates the potential risk o undesirable “dose dumping.” Novel polymers have been designed to overcome this problem by optimizing degradation or controlled drug delivery (i.e., through sur ace erosion). For example, a polymer with desirable erosion properties can be engineered by using hydrophobic monomers connected by anhydride bonds. The hydrophobic monomers exclude water rom the interior o the polymer matrix, eliminating bulk erosion. In contrast, the anhydride bonds are highly water reactive, allowing sur ace erosion in the aqueous environment o the body. This design allows the polymer to degrade rom the outside only (Fig. 55-2). The rate o degradation can be controlled by using a combination o monomers, one more hydrophobic than the other. The length o time over which the polymer persists is specif ed by the ratio o monomers used, and a drug that is uni ormly distributed within such a polymer matrix will be released constantly over time. Based on these principles, Gliadel® has become the f rst local controlledrelease system or an anticancer drug to receive FDA approval. A ter surgeons remove glioblastoma multi orme, an aggressive orm o brain cancer, they place up to eight small polymer–drug wa ers at the tumor site. As the polymer surace erodes over 1 month, the drug carmustine (an alkylating agent; see Chapter 39, Pharmacology o Cancer: Genome Synthesis, Stability, and Maintenance) is slowly released.

A

S urfa ce e ros ion

B

O O

Bulk e ros ion

O R

O O

O R

O O

O R

O

P olya nhydride H2 O

O

HO

O

R

OH

Bre a kdown products

FIGURE 55-2. Surface erosion using polyanhydride polymers. A. Surface erosion of degradable polymer delivery devices allows for more accurately controlled release rates and is therefore preferable to bulk erosion. B. Polyanhydrides are used to promote surface erosion. They have hydrophobic monomers that exclude water from the interior of the polymer matrix and prevent bulk erosion. However, the monomers are linked by watersoluble anhydride bonds, allowing breakdown at exposed surfaces.

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The concentration o carmustine at the tumor site is maintained at a level su f ciently high to kill many o the remaining tumor cells, while adverse e ects o systemic delivery are avoided. This treatment signif cantly prolongs the lives o patients with this cancer.

Solvent Activation

The third mechanism or polymer-based drug delivery is solvent activation, in which the solvent does not react with the polymer chemically but rather initiates drug release via swelling (Fig. 55-1E) or osmosis (Fig. 55-1F) o the system. One widely used example o such a system is an extendedrelease oral ormulation o nifedipine , a calcium channel blocker (see Chapter 22, Pharmacology o Vascular Tone). The drug is mixed with an osmotically active agent, such as a salt, and coated with a membrane that is permeable to water but not the drug. A laser is then used to drill a small hole in the capsule membrane. A ter ingestion, the constant osmotic in ux o water through the membrane orces the drug out o the pill through the hole, thereby controlling release. This delivery technique, when compared to conventional (immediate release) oral ormulations, provides patients greater relie rom ischemic events with ewer adverse e ects. Concerta®, an extended-release ormulation o methylphenidate , uses a similar system to treat children with attention-def cit hyperactivity disorder (ADHD).

Intelligent Delivery There are situations in which pulsatile delivery is desirable to mimic the body’s natural pattern o synthesizing and releasing compounds (e.g., hormones). In the case o Mr. F, the insulin pump he wore provided a constant, basal rate o insulin to maintain his blood glucose levels between meals. When Mr. F ate, he could set the pump to provide an additional bolus o insulin and thereby prevent a sudden, excessive rise in blood glucose concentration. Several innovative approaches have been taken to incorporate such versatility in polymer-based drug delivery systems, which have traditionally been designed to deliver drugs at constant or decreasing release rates. In one early design, magnetic beads were incorporated in the polymer matrix together with a 2-year supply o insulin. The system was then implanted subcutaneously in rats, where the insulin was slowly released by di usion out o the matrix, as discussed above. When an oscillating magnetic f eld was applied externally, movement o the magnetic beads within the matrix caused alternating expansion and contraction o the drug-carrying pores. The insulin could thus be e ectively squeezed out o the matrix, resulting in higher dose delivery or as long as the oscillating magnetic f eld was applied. This system signif cantly lowered blood glucose levels in the treated rats compared to control rats and may eventually become a viable method o insulin delivery. In Mr. F’s hypothetical uture, the implanted magnetic oscillator allowed him to administer a rapid bolus o insulin simply by selecting the appropriate program on his wristwatch controller, which sent the instructions to the implanted device via a radio requency signal. Other methods o increasing the rate o drug di usion rom a polymer matrix include the application o either ultrasound or electric current. Ultrasound delivered at an appropriate requency can have an e ect similar to that o the magnetic bead system. Ultrasound causes cavitation (the ormation o tiny air pockets) in the polymer, disrupting the porous architecture

984 FRONTIERS IN PHARMACOLOGY

to acilitate aster drug release. Applying an electric current to certain polymers can induce electrolysis o water at the polymer sur ace, lowering local pH and disrupting hydrogen bonding within the complex. The polymer subsequently degrades at a aster than normal rate, allowing transient release o larger drug doses. Pulsatile delivery can also be achieved in response to local environmental stimuli. For example, hydrogels (materials composed o polymers and water) can be designed to sense changes in temperature, pH, and even specif c molecules by virtue o their structure. A silicon microchip delivery system that o ers even more control over release rates has also been designed. The microchip contains up to 1,000 tiny drug reservoirs, each covered with a thin gold f lm. Applying a small external voltage to an individual implanted reservoir dissolves the gold f lm electrochemically, releasing the drug stored in that reservoir. Because the reservoirs can be loaded and opened individually, almost limitless possibilities exist or both dosing o single drugs and combining multiple drugs.

Targeting Accurate targeting allows or larger, more e ective doses to reach the tissues o interest without risking the toxic e ects o systemic delivery. The f rst variable that can be controlled is the anatomic placement o the polymer-based drug delivery system; the carmustine wa er delivery system discussed earlier makes use o this basic consideration. Other notable examples include Estring®, a vaginal ring that delivers estradiol or vaginal dryness; Vitrasert®, an eye implant that delivers ganciclovir or the treatment o cytomegalovirus retinitis in AIDS patients (see Chapter 38, Pharmacology o Viral In ections); and drug-eluting stents that deliver sirolimus , everolimus , zotarolimus , or paclitaxel or the prevention o in-stent restenosis in coronary angioplasty (see Chapter 46, Pharmacology o Immunosuppression). Many tissues are accessed practically only via the bloodstream, however, making targeted delivery more di f cult. Both passive and active targeting techniques have been developed to direct polymer-based systems to specif c tissues ollowing intravenous administration. Passive targeting exploits vascular di erences between the target tissue and other tissues to deliver drugs selectively. For example, high-molecular-mass polymer–drug complexes accumulate in some tumor tissues to a greater extent than in normal tissues because the tumor has more permeable capillary beds. There ore, rather than using lower doses o low-molecular-mass anticancer drugs, which rapidly di use through all cell membranes and distribute throughout the body, larger and more e ective doses o high-molecularmass polymer–drug conjugates can be used to target tumors. In addition, the polymer–drug conjugates can be constructed in such a way as to allow enzymatic cleavage o the drug a ter the complex has le t the bloodstream and been taken up by tumor cells (Fig. 55-1C). In one example o such a system, the anticancer drug doxorubicin (see Chapter 39) is conjugated to a water-soluble, nonimmunogenic polymer through a peptidyl linker. The polymer–drug complex accumulates in mouse melanoma tumors at concentrations up to 70 times higher than in normal tissue because o the relatively leaky microvasculature in the tumor. Once inside the tumor cells, the peptidyl linker is cleaved by lysosomal proteases, releasing the cytotoxic drug. The polymer portions o the complex either degrade or are excreted by the kidneys.

In active targeting, the polymer–drug conjugate is linked to a molecule that is recognized specif cally by cell sur ace receptors in the tissue o interest. For example, a human IgM antibody directed against a tumor-associated antigen can be used to target a polymer–doxorubicin complex to malignant tissues. Linked to the polymer with an acid-labile bond, the doxorubicin is selectively released in the acidic environment o the tumor. In another system, galactose is used to target a polymer–drug complex to the liver via the hepatocyte cell sur ace asialoglycoprotein receptor.

LIPOSOME-BASED DELIVERY SYSTEMS Drugs attached to a single polymer chain are stable structures that can remain in the circulation or long periods o time; the drug–polymer complexes discussed above in the context o tissue targeting are examples o such systems. However, these polymer chains can accommodate only small amounts o drug, thus limiting the dose per unit volume administered. The potentially high drug-carrying capacity o liposomes , small vesicles with lipid bilayer membranes, makes them an attractive option or a circulating drug delivery system. Important considerations in the design o liposomebased delivery systems include tissue targeting and protection rom the immune system. Highly specif c antibodies, analogous to those used or active targeting o polymer– drug complexes, can be used to improve tissue targeting. For example, antibodies against the HER2 proto-oncogene, implicated in the progression o breast cancer and other cancers, are being explored or tumor targeting. Similarly, antibodies against E-selectin, an endothelial-specif c surace molecule, can be used to target vascular endothelial cells. Protection o liposomes rom the immune system can be accomplished by the attachment o water-soluble polymers to the liposomal sur ace. As discussed above, moieties such as PEG increase the hydrophilicity o the structures to which they are attached; in this case, liposomes are made more hydrophilic in the blood and are there ore less liable to be taken up by the reticuloendothelial system. Because liposomes with the PEG moiety (“stealth liposomes”) have a prolonged circulation time (days), larger doses can be administered without the risk o drug toxicity. These principles have been used to develop liposomes loaded with daunorubicin and doxorubicin or the treatment o several tumors, including HIV-associated Kaposi’s sarcoma. Liposomal amphotericin B, used to treat ungal in ections, has also been approved or clinical application (see Chapter 36, Pharmacology o Fungal In ections). Liposomal cyclosporine is being studied or targeted immunosuppression a ter transplant surgery (see Chapter 46).

CONCLUSION AND FUTURE DIRECTIONS The delivery modalities described in this chapter represent selected novel approaches to optimizing absorption, distribution, metabolism, and excretion o drugs. There are several advantages o improved drug delivery: ■

Drug levels can be continuously maintained in a therapeutically desirable range. Sustained-release oral ormulations, large particles that can be inhaled, and many polymer-based designs have this desirable property.

C h a p t e r 5 5 Drug Delivery Modalities ■

■

■

Harm ul adverse e ects can be reduced by preventing transient high peak blood levels o drug. Designs that alter absorption kinetics, targeted delivery systems (e.g., antibody-conjugated polymer–drug complexes), and systems that avoid f rst-pass liver metabolism (e.g., transdermal delivery o drugs that are otherwise taken orally) achieve this goal. The total amount o drug required can be reduced, as with advanced inhaler designs. Both a decrease in the number o required dosages and a less invasive administration route contribute to improved patient adherence. Mr. F’s case illustrates the in uence o li estyle actors on patient adherence. Pharmaceuticals with short hal -lives, such as peptides and proteins, can be success ully delivered using controlledrelease polymer-based delivery systems.

Advanced drug delivery technologies also introduce new concerns that must be considered in their design. For example, each material put in the body, as well as its degradation products, must be evaluated or toxic e ects; this actor is especially important or synthetic materials such as polymers. Other potential dangers must be avoided, such as unwanted rapid release o the drug rom a system intended or sustained release. Discom ort caused by the delivery system or its insertion is another potential disadvantage: Mr. F’s insulin pump, while providing better control o his diabetes, was uncom ortable to him. Finally, advanced technology is o ten accompanied by increased cost, which can be a problem or patients, their insurance companies, and hospitals. Despite these obstacles, advanced drug delivery technologies play an increasingly valuable role in making the

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pharmacologic management o disease sa er, more e ective, and more agreeable to patients.

Suggested Reading Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 1998;85:379–385. (Review of aerodynamic diameter principles and the potential advantages and applications of large, porous inhaled particles.) Farra R, Sheppard N, McCabe L, et al. First in-human testing o a wirelessly controlled drug delivery microchip. Sci Transl Med 2012;4:122ra21. (First report of an implantable microchip-based device used to deliver parathyroid hormone.) Hrkach J, Von Ho D, Mukkaram Ali M, et al. Preclinical development and clinical translation o a PSMA-targeted docetaxel nanoparticle with a di erentiated pharmacological pro ile. Sci Transl Med 2012;4:128ra39. (Development of a polymeric nanoparticle for delivery of docetaxel.) Langer R. Drug delivery and targeting. Nature 1998;392:5–10. (Review of drug delivery techniques, with emphasis on polymer and liposome-based systems as well as novel use of delivery routes.) Langer R. Where a pill won’t reach. Sci Am 2003;288:50–57. (Broad overview of concepts in drug delivery.) Langer R, Weissleder R. Nanotechnology. JAMA 2015;313:135–136. (Reviews use of nanotechnology for therapeutics, diagnostics, and imaging.) Leong KW, Brott BC, Langer R. Bioerodible polyanhydrides as drug-carrier matrices. I: characterization, degradation, and release characteristics. J Biomed Mater Res 1985;19:941–955. (Good starting point for learning more about polymer matrix design.) Prausnitz M, Langer R. Transdermal drug delivery. Nat Biotechnol 2008;26:1261–1268. (Reviews advances in transdermal drug delivery.) Santini JT Jr, Cima MJ, Langer R. A controlled-release microchip. Nature 1999;397:335–338. (More detailed information about intelligent drug delivery using silicon microchips with arrays of drug reservoirs.)

Credit List Figure 1-1: Adapted rom an illustration (http://www.genome.gov/Glossary/resources /protein.pd ) on the National Human Genome Research Institute website: http://www.nhgri.nih.gov. Figure 1-2: Data used to render the image in panel A were deposited in the RCSB Protein Data Bank (http://www.rcsb.org/pdb, PDB ID: 1FPU) by Schindler T, Bornmann W, Pellicena P, et al. Structural mechanism or STI-571 inhibition o Abelson tyrosine kinase. Science 2000;289:1938–1942, Figure 1. Panels B and C were adapted with permission rom Schindler et al. (ibid., Figures 1 and 2). Figure 2-7A: Adapted with permission rom Stephenson RP. A modif cation o receptor theory. Brit J Pharmacol 1956;11:379–393, Figure 10. Figure 2-7B: Data used to generate the dose– response curves or morphine and buprenorphine were published in Cowan A, Lewis JW, Mac arlane IR. Agonist and antagonist properties o buprenorphine, a new antinociceptive agent. Brit J Pharmacol 1977;60:537–545.

Public Domain. Archived at https://en.wikipedia.org /wiki/Active_transport Figure 5-4B: Adapted with permission rom Hundal HS, Taylor PM. Amino acid transporters: gate keepers o nutrient exchange and regulators o nutrient signaling. Am J Physiol Endocrinol Metab 2009;296:E603–E613, Figure 2, and Sweet DH, Bush KT, Nigam SK. The organic anion transporter amily: rom physiology to ontogeny and the clinic. Am J Physiol Renal Physiol 2001;281:F197–F205, Figure 1. Figure 6-3A: Adapted with permission rom Grattagliano I, Bon rate L, Diogo CV, et al. Biochemical mechanisms in drug-induced liver injury: certainties and doubts. World J Gastroenterol 2009;15:4865–4876, Figure 1. Figure 6-3B: Adapted with permission rom Grattagliano I, Bon rate L, Diogo CV, et al. Biochemical mechanisms in drug-induced liver injury: certainties and doubts. World J Gastroenterol 2009;15:4865–4876, Figure 2.

Figure 3-1: Adapted with permission rom Hardman JG, Limbird LE, eds. Goodman & Gilman’s the pharmacological basis of therapeutics. 10th ed. New York: McGraw-Hill; 2001:3, Figure 1-1.

Figure 7-1A: Adapted with permission rom Bertilsson L, Lou YQ, Du YL, et al. Pronounced di erences between native Chinese and Swedish populations in the polymorphic hydroxylations o debrisoquin and S-mephenytoin. Clin Pharmacol Ther 1992;51:388–397 [Erratum, Clin Pharmacol Ther 1994;55:648].

Figure 3-7: Adapted with permission rom Katzung BG, ed. Basic & clinical pharmacology. 7th ed. New York: Lange Medical Books/McGraw-Hill; 1998:38, Figure 3-2.

Figure 7-1B: Photo o the AmpliChip CYP450 array was provided by Roche Diagnostics.

Figure 4-2A: Adapted with permission rom Katzung BG, ed. Basic & clinical pharmacology (7th ed.). New York: Lange Medical Books/ McGraw-Hill; 1998:52, Figure 4-3. Figure 5-1: Adapted with permission rom Giacomini KM, Huang SM. Transporters in drug development and clinical pharmacology. Clin Pharmacol Ther 2013;94:3–9, Figure 1. Figure 5-2: Adapted with permission rom Mariana Ruiz Villarreal (Ladyo Hats). Image created February 4, 2007, “Simple di usion in cell membrane.” In Public Domain. Archived at https://en.wikipedia.org/wiki/Passive_transport Figure 5-3: Adapted with permission rom Mariana Ruiz Villarreal (Ladyo Hats). Image created February 4, 2007, “Facilitated di usion in cell membrane.” In Public Domain. Archived at https://en.wikipedia.org/wiki/Facilitated_di usion Figure 5-4A: Adapted with permission rom Mariana Ruiz Villarreal (Ladyo Hats). Image created February 23, 2007, “Sodium potassium pump.” In

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988 Credit List Figure 14-9: Adapted with permission rom Seeman P. Dopamine receptors and the dopamine hypothesis o schizophrenia. Synapse 1987;1:133–152. Figure 16-3: Adapted with permission rom Lothman EW. Pathophysiology o seizures and epilepsy in the mature and immature brain: cells, synapses and circuits. In: Dodson WE, Pellock JM, eds. Pediatric epilepsy: diagnosis and therapy. New York: Demos; 1993:1–15. Figure 16-4: Adapted with permission rom Lothman EW. The neurobiology o epilepti orm discharges. Am J EEG Technol 1993;33:93–112. Figure 16-5A: Adapted with permission rom Kandel ER, Schwartz JH, Jessell TM, eds. Principles o neural science. 4th ed. New York: McGraw-Hill; 2000:899, Figure 45-9. Figure 17-2: Adapted rom Miller KW. General anesthetics. In: Wol ME, ed. Burger’s medicinal chemistry and drug discovery, Volume 3: therapeutic agents. 5th ed. Hoboken, NJ: John Wiley & Sons; 1996: Figure 36-2. This material used by permission o John Wiley & Sons, Inc. Figure 17-6: Adapted with permission rom Eger EI. Anesthetic uptake and action. Baltimore: Williams & Wilkins; 1974: Figure 4-7. Figure 17-7: Adapted rom Eger EI. Uptake and distribution. In: Miller RD, ed. Anesthesia. 5th ed. Philadelphia: Churchill Livingstone; 2000: Figure 4-2. With permission rom Elsevier. Figure 17-9: Adapted with permission rom Eger EI. Anesthetic uptake and action. Baltimore: Williams & Wilkins; 1974: Figures 7-1 and 7-8. Figure 17-10: Adapted rom Eger EI. Uptake and distribution. In: Miller RD, ed. Anesthesia. 5th ed. Philadelphia: Churchill Livingstone; 2000: Figure 4-10. With permission rom Elsevier. Figure 17-12: Adapted with permission rom Eger EI. Anesthetic uptake and action. Baltimore: Williams & Wilkins; 1974: Figure 14-8. Figure 17-13: Adapted with permission rom Trevor AJ, Miller RD. General anesthetics. In: Katzung BG, ed. Basic & clinical pharmacology. 7th ed. New York: Lange Medical Books/McGraw-Hill; 1998:421, Figure 25-6. Figure 19-6: Adapted rom Jones RT. The pharmacology o cocaine smoking in humans. In: Chiang CN, Hawks RL, eds. NIDA research monograph 99 (research f ndings on smoking o abused substances). Washington, DC: U.S. Department o Health and Human Services; 1990:30–41. Figure 20-1: Adapted rom Larsen PR, Kronenberg HM, Melmed S, et al., eds. Williams textbook o endocrinology. 10th ed. Philadelphia: WB Saunders; 2003: Figure 34-5. With permission rom Elsevier.

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Figure 20-2: Adapted with permission rom Scapa EF, Kanno K, Cohen DE. Lipoprotein metabolism. In: Benhamou JP, Rizzetto M, Reichen J, et al, eds. The textbook o hepatology: rom basic science to clinical practice. 3rd ed. Ox ord, United Kingdom: Blackwell; 2007: Figure 2. Figure 20-6B: Adapted with permission rom Mahley RW, Ji ZS. Remnant lipoprotein metabolism: Key pathways involving cell-sur ace heparan sul ate proteoglycans and apolipoprotein E. J Lipid Res 1999;40:1–16. Figure 20-8: Adapted with permission rom Quinn MT, Parthsarathy S, Fong LG, Steinberg D.

Figure 23-2: Adapted rom Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis o disease. 6th ed. Philadelphia: WB Saunders; 1999: Figure 5-7. With permission rom Elsevier. Figure 23-3: Adapted rom Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis o disease. 6th ed. Philadelphia: WB Saunders; 1999: Figure 5-7. With permission rom Elsevier. Figure 23-11: Adapted rom Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis o disease. 6th ed. Philadelphia: WB Saunders; 1999: Figure 5-12. With permission rom Elsevier.

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990 Credit List Figure 43-2: Adapted with permission rom Serhan CS. Eicosanoids. In: Kooperman WJ, ed. Arthritis and allied conditions: a textbook of rheumatology. 14th ed. Philadelphia: Lippincott Williams & Wilkins; 1999:516, Figure 24.2. Figure 43-4: Adapted with permission rom Serhan CS. Eicosanoids. In: Kooperman WJ, ed. Arthritis and allied conditions: a textbook of rheumatology. 14th ed. Philadelphia: Lippincott Williams & Wilkins; 1999:524, Figure 24.6. Figure 44-2: Adapted with permission rom Janeway CA, Travers P, Walport M, eds. Immunobiology: the immune system in health and disease. 4th ed. New York: Garland Publishing, Inc; 1999:474, Figure 12.12. Figure 44-3: Adapted with permission rom Leurs R, Church MK, Taglialatela M. H1 antihistamines: inverse agonism, anti-in ammatory actions and cardiac e ects. Clin Exp All 2002;32:489–498, Figure 1. Figure 45-1: Adapted rom Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease. 6th ed. Philadelphia: WB Saunders Company; 1999: Figure 14-1. With permission rom Elsevier. Figure 46-7: Adapted with permission rom Fox DA. Cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition o tumor necrosis actor. Arch Intern Med 2000;160:437–444, Figure 1.

Figure 46-8: Adapted with permission rom Fox DA. Cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition o tumor necrosis actor. Arch Intern Med 2000;160:437–444, Figure 2. Figure 48-1: Adapted rom Mason RJ, Broaddus VC, Murray JF, Nadel J, eds. Murray and Nadel’s textbook of respiratory medicine. 4th ed. Philadelphia: WB Saunders Company; 2005. With permission rom Elsevier. Figure 48-3: Adapted rom Mason RJ, Broaddus VC, Murray JF, Nadel J, eds. Murray and Nadel’s textbook of respiratory medicine. 4th ed. Philadelphia: WB Saunders Company; 2005. With permission rom Elsevier. Figure 48-4: Adapted with permission rom Drazen JM. Treatment o asthma with drugs modi ying the leukotriene pathway. N Engl J Med 1999;340: 197–206, Figure 1.

carcinogenesis. Nat Rev Cancer 2005;5:113–125, Figure 3. Figure 50-5: Adapted with permission rom Luch A. Nature and nurture—lessons rom chemical carcinogenesis. Nat Rev Cancer 2005;5:113–125, Figure 4. Figure 51-3A: Adapted with permission rom Schreiber SL. Target-oriented and diversity-oriented organic synthesis in drug discovery. Science 2000;287:1964–1969. Figure 51-3B: Adapted with permission rom Marsilje TH, Pei W, Chen B, et al. Synthesis, structure-activity relationships, and in vivo e f cacy o the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloroN2-(2-isopropoxy-5-methyl-4-(piperidin 4-yl) phenyl)-N4-(2-(isopropylsul onyl)phenyl) pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials. J Med Chem 2013;56:5675–5690.

Figure 48-6: Adapted with permission rom Pelaia G, Vatrella A, Maselli R. The potential o biologics or the treatment o asthma. Nat Rev Drug Discov 2012;11:958–972, Figure 2.

Figure 52-2: Adapted rom the CDER handbook by the U.S. Food and Drug Administration, available at http://www. da.gov/.

Figure 49-2: Adapted with permission rom So A, Busso N. A magic bullet or gout? Ann Rheum Dis 2009;68:1517–1519, Figure 2.

Figure 52-3: Adapted rom the CDER handbook by the U.S. Food and Drug Administration, available at http://www. da.gov/.

Figure 50-4: Adapted with permission rom Luch A. Nature and nurture—lessons rom chemical

Table 52-2: Adapted rom http://www. da.gov / dac/special/newdrug/testtabl.html.

Index Note: Page numbers ollowed by f denote f gures; those ollowed by t denote tables; and those ollowed by b indicate boxes.

A

AA. See Alcoholics Anonymous AADC. See Aromatic L-amino acid decarboxylase Abacavir, 703f, 710, 719t Abatacept, 856, 862t, 969t Abbreviated New Drug Application (ANDA), 944 ABC. See ATP-binding cassette transporter amily ABCA1, 343 ABCA amily, 63t, 64 ABCB11, 346, 346f ABCB amily, 62, 63t ABCC amily, 63, 63t ABCDE, 488 ABCD amily, 63t, 64 ABCG2 amily, 63t, 64 Abciximab, 14, 417, 427t, 971t Abelcet, 667 Abili y®, 919 aBMD. See Areal bone mineral density Abnormal blood ow, 412 Abraxane, 742, 749t Absence seizure, 252t, 253, 255f Absorption, 27, 29–30 administration routes and rationale or, 30–31, 30t, 31t local, regional, and systemic actors in, 31–32, 32f Absorption, distribution, metabolism, and excretion (ADME), 27, 28f, 928–929 ABVD. See Doxorubicin, bleomycin, vinblastine, and dacarbazine Acamprosate, 329, 333t Acarbose, 578t ACAT. See Acetyl-CoA:cholesterol acyltrans erase Accelerated Approval, 940, 942 Acceptor site, 626–627, 626f, 627f Accessory tract pathways, 440 ACE. See Angiotensin converting enzyme Acebutolol, 60t, 160t, 161, 166t, 445, 451t ACE inhibitors, 13–14, 60t, 82, 92t, 359, 372–373, 380t, 397, 402t, 473t, 476–477, 477t, 485f, 493t, 494–495 Acetaminophen, 50, 52t, 63t, 79, 80f, 89t, 300–301, 300f, 306t, 809f, 810, 816t Acetazolamide, 375, 382t Acetic acid NSAIDs, 809f, 810, 815t Acetohexamide, 576t Acetylcholine (ACh) central nervous system and, 134–136 in cholinergic pharmacology, 127 degradation o , 127, 131–132 inhibitors o , 145t–146t in gastric acid secretion, 864–865, 866f as muscarinic cholinergic receptor agonist, 139–140, 140f, 140t at neuromuscular junction, 132, 134f, 135f as neurotransmitter, 120, 120t, 121f, 123 nicotinic acetylcholine receptor and, 8, 8f non-neuronal, 136 in peripheral nervous system, 119, 119f release o , 128–130 inhibitors o , 137, 145t single-source divergent neuronal systems and, 118f, 118t, 119

storage o , 128–130 inhibitors o , 137, 145t synaptic cle t and, 107 synthesis o , 128, 129f inhibitors o , 137, 145t Acetylcholinesterase (AChE), 14, 127, 131–132 Acetylcholinesterase inhibitors, 14, 108, 132, 136 clinical applications o , 138–139, 139t structural classes o , 137–138, 138f Acetyl CoA, 128 Acetyl-CoA:cholesterol acyltrans erase (ACAT), 339, 342 Acetylsalicylate, 301 Acetylsalicylic acid, 301 Acetyltrans erases, 635 ACh. See Acetylcholine Achalasia, 137 AChE. See Acetylcholinesterase ACh-H antiport channel, 128, 129f, 137 Acid hypersecretion, in peptic ulcer disease, 868 Acids, 909 Acid secretion. See Gastric acid secretion Acid-sensitive ion channels (ASICs), 290–291 Aclidinium, 886, 893t Acquired disorders, 412, 413t Acquired genes, or antibiotic resistance, 651 Acquired tolerance, 311 Acrivastine, 829t Acrolein, 736–737, 738f, 850 Acromegaly, 499, 503 ACS. See Acute coronary syndromes ACTH. See Adrenocorticotropic hormone Action potentials (AP) cardiac, 433–437, 435f, 436f, 436t, 437b, 437f cells and, 98 delayed rectif er channels and, 104, 105f unctions o , 98–99 local anesthetics and, 173 in phasic inhibition, 175, 175f in tonic inhibition, 174–175, 175f in nociception, 169 pharmacology o , 105 at sodium channel, 250, 251f threshold potential and, 104 threshold voltage and, 102, 103f voltage-gated channels and, 102–104, 104f Activated charcoal, 185 Activated partial thromboplastin time (aPTT), 422 Activated protein C, 958, 964t Activation, o platelets, 403, 405f Active immunization, 716 Active metabolites, 50 Active site, 5 Active state, 20 Active targeting, 984 Active transport, 28, 58, 58f Activin, 507, 545 Acute adrenal insu f ciency, 532 Acute cellular rejection, 845, 845t Acute coronary syndromes (ACS), 479, 481–482, 482f pharmacologic management o , 485f Acute humoral rejection, 845, 845t Acute lithium intoxication, 239–240

Acute mountain sickness, 375 Acute myelogenous leukemia (AML), 760 Acute rejection, 845–846, 845t Acute renal ailure, 373, 376 aminoglycosides causing, 632 Acute tubular necrosis, 80 Acute urticaria, 823 Acute withdrawal syndrome, 313 Acyclovir (ACV), 60t, 63t, 65t, 608, 609f, 696f, 703f, 702–705, 704f, 706, 706t, 718t prophylactic, 615 resistance to, 614 AD. See Alzheimer’s disease ADA. See Adenosine deaminase Adalimumab, 14, 812, 816t, 853, 860t, 966, 969t Adaptive immunity, 784, 786–790, 786t, 787f, 788f, 789f Addiction, 298 partial agonists in treatment o , 320, 321f Addison’s disease, 528–529, 535 Additivity, antimicrobial examples o , 772–773, 773f Adducts, carcinogens, 912 Ade ovir, 60t, 66t, 703f, 707–708, 719t Adenine, 622, 624f synthesis o , 724–725, 725f, 726f, 726t, 727f Adenohypophysis, 498 Adenomatous polyposis coli (APC), 755–756, 755f–756f Adenosine, 813 cardiac rhythm and, 447, 453t as neurotransmitter, 120, 120t, 121f, 123 Adenosine 5 -monophosphate-activated protein kinase (AMPK), 563 Adenosine deaminase (ADA), 724, 734, 734f, 961t Adenosine triphosphate (ATP), as neurotransmitter, 120, 120t, 121f, 123 Adenylyl cyclase activation o , 9–10, 10f in cardiac contractility, 458 muscarinic receptors and, 130 A -f bers, 169, 169t, 170f ADH. See Antidiuretic hormone ADHD. See Attention-def cit hyperactivity disorder Adhesins, ungal, 662 Adhesion, 14 Adhesion receptors, 14 ADHR. See Autosomal dominant hypophosphatemic rickets Adjuvant drugs, general anesthetics, 280 ADME. See Absorption, distribution, metabolism, and excretion ADNFLE. See Autosomal dominant nocturnal rontal lobe epilepsy ADP receptor pathway inhibitors, 415–417, 426t–427t, 485f, 486 Adrenal androgens. See Androgens Adrenal cortex case study or, 525 hormone synthesis, inhibitors o , 534, 534t, 540t overview o , 524–525, 525f Adrenaline. See Epinephrine Adrenal insu f ciency, 528–529 acute, 532

991

992 Index Adrenal medulla, 111, 112f Adrenal sex steroid, 540t Adrenarche, 536 Adrenergic unction, 150 Adrenergic neurotransmission, central, 228 pharmacologic classes and agents or, 233–240 Adrenergic pharmacology, 150 catecholamines metabolism inhibition o , 158 reuptake inhibition o , 158 storage inhibition o , 156–158, 157f synthesis inhibition o , 156 receptor agonists alpha-adrenergic, 158–159 beta-adrenergic, 159 receptor antagonists alpha-adrenergic, 159–160 beta-adrenergic, 160–161, 160t Adrenergic receptors calcium cycling and, 458, 459f, 459t neurotransmitters and, 121–122 Adrenoceptors, 153–155, 154t response regulation o , 155 Adrenocorticotropic hormone (ACTH), 500, 500t, 505, 506f, 524, 535 Adriamycin. See Doxorubicin Adverse drug events, 946, 952–953 Adverse e ects, 2, 70 AEDs. See Antiepileptic drugs A atinib, 79, 758, 765t AF-DX 117, 131t A ective disorders clinical characteristics o , 231–233 monoamine theory o depression, 233–234, 234f pathophysiology o , 231, 231b, 232b A ective attening, 216, 216b A erent neurons, 167, 168f excitation o primary, 289–291, 290f, 290t A f nity, o drug–receptor interactions, 3, 5t A-f bers, 169, 169t, 291 A atoxins, 914 A ibercept, 972t A terdepolarizations, 438–439, 439f A terload, 454, 490, 490f reduction o , 494 Agalsidase , 961t Age drug metabolism and, 53 inhaled anesthetics and, 276, 277f 2AG amilies. See 2-Arachidonylglycerol amilies Aging, 137 Agomelatine, 237, 246t Agonists ull, 7, 20, 26t inverse, 7, 20, 24 partial, 7, 20, 23–24, 23f Air pollution, 916–917 Airway immune unction o , 879, 880f smooth muscle contraction o , 877–878, 879b Airway remodeling, 882 Akathisia, 142 AKT inhibitors, 753f, 761 ALA-D. See Delta-aminolevulinic acid dehydratase Alanine racemase, 644f, 645 Al-Anon, 327 Alateen, 327 Albendazole, 687, 693t Albiglutide, 572, 576t, 965t Albuterol, 92t, 111, 159, 164t, 878, 883–885, 892t Alcohol. See also Ethanol abuse o , 310t, 322f, 323, 324f cerebellum and, 116 cocaine and, 326 metabolism inhibition, 332t neuroadaptation to, 312f–313f peptic ulcer disease and, 874 withdrawal rom, 322f Alcohol dehydrogenase, 47, 328 Alcoholics Anonymous (AA), 327 Alcohol use disorder, 318–319, 326, 328 acamprosate or, 329 selective serotonin reuptake inhibitors and, 319, 329 topiramate or, 329

Aldosterone, 534–535 hyper unction o , 535–536 hypo unction o , 535 Aldosterone receptor antagonists, 473t, 485f, 494 Ale acept, 855, 861t, 970t Alemtuzumab, 763, 769t, 855, 861t, 969t Alendronate, 592–594, 592f, 599t Al entanil, 299, 305t Alglucosidase al a, 961t Alirocumab, 355, 357t Aliskiren, 60t, 65t, 372, 373f, 380t, 397, 402t, 476 Alkaline phosphatase, 582 ALK inhibitors, 759, 766t Alkylating agents, 610f, 611, 611f, 735–737, 736f, 737f, 738f, 745t–746t, 777t, 778t or immunosuppression, 848, 850 Allan-Herndon-Dudley syndrome, 517 Allergic reactions in asthma, 881–882, 881f, 883f, 884f histamine role in, 821–823, 822f, 825 Allergic rhinitis, 823 Allergies antihistamines or, 820 penicillin, 652, 652f Allodynia, 294 Alloimmunity, 844 Allopregnanolone, 188 Allopurinol, 78, 734, 779 or gout, 896, 896f, 897t, 899–900, 899f, 900f, 902t Allostasis, 316, 329 Allyl isothiocyanate, 290 Allylamines, 662, 663f, 664f, 665, 670t Allylglycine, 189t Almotriptan, 238, 247t, 307t Alogia, 216, 216b Alogliptin, 572, 577t Alosetron, 238, 247t ALOX5, 92 Alpha-1-adrenergic agonists, 158, 163t receptor selectivity o , 463t Alpha-1-adrenergic antagonists, 397, 401t or hypertension, 473t, 475–476, 477t Alpha-1-proteinase inhibitor, 955, 961t Alpha-2-adrenergic agonists, 158–159, 163t, 302 or hypertension, 476 receptor selectivity o , 463t Alpha 2-antiplasmin, 411, 412f Alpha-adrenergic antagonists, 159–160, 164t–165t or hypertension, 477t Alpha-adrenergic receptors, 154, 154t catecholamine activity at, 155–156 Alpha cells, 561 Alpha-glucosidase inhibitors, 569t, 573, 578t Alpha-HDL, 344 Alpha-linolenic acid, 795, 796f Alpha-methyldopa, 159 Alpha-methylnorepinephrine, 159 Alpha-methyltyrosine, 156, 162t Alphaxalone, 195, 204t Alprazolam, 51t, 191, 202t clinical uses o , 192t withdrawal rom, 322f Alprenolol, 51t Alprostadil, 817t ALS. See Amyotrophic lateral sclerosis Alteplase, 424, 431t, 487, 964t Altered automaticity, 438 Altretamine, 736, 746t Aluminum hydroxide, 596, 600t, 873, 875t Alveolar capillary rate o uptake, 287 Alveolar partial pressure (Palv), 266, 267b inspired partial pressure and, 271–272, 272f tissue partial pressure and, 272–273, 273t, 274f Alveolar ventilation (Valv), 271 Alvimopan, 300 Alzheimer’s disease (AD) ACh and, 136 anatomy o , 115 cholinergic agents or, 137–139 glutamate in, 198 pedunculopontine nucleus and, 119 Amanita muscaria, 190 Amantadine, 60t, 142, 199, 205t, 215, 224t, 608, 609f, 696f, 698–700, 700f, 718t Amatoxins, 909

Ambenonium, 138, 145t AmBisome, 667 Ambrisentan, 396, 401t Amebiasis, 682–683, 683f, 684f Amide-linked LAs, 179–180, 181t–182t metabolism o , 176 structure o , 171, 171f Amidophosphoribosyltrans erase (amidoPRT), 895–897, 896f Ami ostine, 739 Amikacin, 630, 638t, 774 Amiloride, 60t, 377, 384t, 474t, 475, 509 Amine group, in local anesthetics, 172 Amino acid neurotransmitters, 119–121, 120t, 121f, 184–185, 185f Amino acids blood–brain barrier and, 123 protein structure and, 2–3, 4f Aminoacyl (A) site, 626–627, 626f, 627f Aminocandin, 668 Aminocaproic acid, 424, 432t Aminoglutethimide, 534, 534t, 540t Aminoglycoside antibiotics, 626f, 627f, 628, 630, 630t, 631f, 632f, 638t beta-lactam antibiotics combined with, 631–632, 651–652, 774 combination chemotherapy using, 772 or MDR-TB, 774 resistance to, 613t, 632 targets o , 605–606, 607f Amino penicillins, 652–653 Aminophylline, 886, 893t Aminopterin, 723 Aminoquinolines, 607 Amiodarone, 51t, 52t, 82, 420t, 444, 446, 446t, 452t, 462, 521 Amitriptyline, 51t, 52t, 143t, 163t, 236, 243t, 289, 301, 306t, 951 AML. See Acute myelogenous leukemia Amlodipine, 390–391, 398t, 476 Amobarbital, 203t clinical uses o , 194t Amodiaquine, 608, 679 Amoxapine, 143t Amoxicillin, 651–653, 657t, 865, 874 AMPA receptors, 193f, 196, 197t epilepsy and, 199 glutamate at, 291 hyperalgesia and, 199–200 seizure activity and, 259 stroke and, 199 Amphetamines, 60t abuse o , 310t, 323–325 pharmacologic treatment o , 328 in catecholamine storage inhibition, 157, 162t dopamine hypothesis and, 216 dopaminergic transmission and, 121 in serotonin storage inhibition, 234, 242t serotonin syndrome with, 158 Amphotec, 667 Amphotericin, 607, 685 Amphotericin B, 78, 80–81, 662, 664, 664f, 667–668, 672t, 774–775, 984 Ampicillin, 36, 651–653, 657t AMPK. See Adenosine 5 -monophosphateactivated protein kinase Amrinone, 459f, 464–465 Amsacrine, 625f, 740–741, 748t Amygdala, 115, 115f Amylin, 561, 566 analogues o , 569t, 573, 577t Amyl nitrate, 310t Amyl nitrite, 907 Amyotrophic lateral sclerosis (ALS), 198, 973 Anabolic steroids, war arin and, 420t Anakinra, 812, 816t, 854, 860t, 901, 970t Analgesia, 170–171, 288 Analgesic index, 267 Analgesics, 170–171 Anandamide, 293, 325 Anaphylaxis, 76, 78t antihistamines or, 825 beta-lactams causing, 652 histamine and, 823 Anastrozole, 547, 548f, 549, 556t, 777t

Index 993 ANDA. See Abbreviated New Drug Application Androgen receptors, antagonists, 548f, 551–552, 558t Androgens, 89t, 524, 536–537, 537f decreased secretion o , 548 replacement o , 555 synthesis o , 541–542, 543f Androstenedione, 536 Anemia, 833 antibiotics causing, 635 Anergy, o immune system, 788–790, 789f Anesthesia, 170–171. See also General anesthetics; Inhaled anesthetics age and, 276, 277f balanced, 280–281 dissociative, 282 dose–response curves o , 267, 268f equations or, 287 induction control o , 277, 278f stages o , 267 Ang I. See Angiotensin I Ang II. See Angiotensin II Anginal syndromes, 480, 480f Angina pectoris, 480, 480f Angioedema, 373 Angiogenesis, cancer biochemistry and, 756–757, 756t, 757f Angiogenesis inhibitors, 610–611, 762–763, 762t, 768t–769t, 778t Angiotensin converting enzyme (ACE), 13–14 inhibitors o , 13–14, 60t, 82, 92t, 359, 372–373, 380t, 397, 402t, 473t, 476–477, 477t, 485f, 493t, 494–495 Angiotensin I (Ang I), 360–361 Angiotensin II (Ang II), 362–363, 524 Angiotensin II receptor antagonists, 373–374, 381t, 402t, 477, 477t, 493t, 494–495 Angiotensinogen, 360–361 Anhedonia, 316 Anidula ungin, 668, 673t Animal models, 928 Anisoylated plasminogen streptokinase activator complex (APSAC), 968t Anistreplase, 968t ANP. See A-type natriuretic peptide Anrukinzumab, 891f Antacids, 16, 865, 869f, 873, 875t–876t antibiotic interactions with, 633 Antagonistic drugs, antimicrobial examples o , 606, 772–773, 773f Antagonists, 7, 26t competitive, 21–22, 22f noncompetitive, 22–23 nonreceptor, 21, 23 receptor, 20–21, 21f Anterior pituitary gland, 498 hypothalamic-pituitary-adrenal axis, 505–506, 506f hypothalamic-pituitary-gonadal axis, 506–507, 506f hypothalamic-pituitary-growth hormone axis, 501–504, 502f hypothalamic-pituitary-prolactin axis, 504–505, 504f hypothalamic-pituitary-thyroid axis, 504f, 505 Anthracyclines, 625f, 739f, 740, 777t, 779 Antianginal drugs, 486 Antiarrhythmic drugs, 51t action potentials and, 105 or analgesia, 307t classes o , 441, 441b class I, 441–442, 442f, 443f class IA, 442–443, 443f, 449t class IB, 443–444, 443f, 450t class IC, 443f, 444, 450t class II, 444–445, 445f, 451t class III, 445–447, 445f, 446t, 451t–452t class IV, 447, 447f, 452t local anesthetics as, 178 mechanisms o action o , 440–441 Antibiotics. See also Antitumor antibiotics bacteria cell wall biochemistry and, 641–648, 642f, 644f, 645b, 647f, 648f bacterial DNA biochemistry and, 622–627, 623f, 624f, 625f, 626f, 627f bacteriostatic and bactericidal, 771–772, 771t, 772f

case studies, 623, 642 classes o , 606, 607f cell wall synthesis, 648–655, 649f, 650f, 652f DNA replication, transcription, and translation, 627–636, 628f, 629f, 630t, 631f, 632f, 634f, 635f combination chemotherapy using examples o , 773–775 MIC and MBC o , 770–772, 771t, 772f synergy, additivity, and antagonism interactions o , 772–773, 773f un avorable combinations o , 775 drug summary tables cell wall synthesis, 656t–660t DNA replication, transcription, and translation, 637t–640t uture directions in, 636, 655 or peptic ulcer disease, 869f, 874 prophylactic, 615 resistance to antimycobacterial agents, 655 beta-lactams, 613, 613t, 651 mechanisms o , 612–614, 613t, 646, 650 Antibodies, 787 Antibody-dependent cytotoxic hypersensitivity, 76, 78t Antibody–drug conjugates, 856 Anti-CD20 mAb, 855 Anti-CD25 mAb, 855 Anti-CD52 mAb, 855 Anticholinergic drugs or asthma, 878, 879b, 885–886, 893t elderly patients and, 143b TCAs as, 236 Anticoagulants, 418–423, 419f, 419t, 420t, 421f, 428t–430t Anticoagulation inhibitors, 424, 431t Anticytokine therapy, 848f, 852–854, 852f, 853f, 861t Antidepressants, 51t, 52t, 89t. See also Tricyclic antidepressants adrenergic receptors and, 121–122 atypical, 237, 245t–246t blood–brain barrier and, 235 chronic use o , 234, 234f elderly and, 143t mechanism o action o , 235, 235f monoamine theory o depression and, 233 in pain management, 301 serotonergic raphe pathway and, 119 with substance use disorders, 329, 334f Antidigoxin antibodies, 462 Antidiuretic hormone (ADH), 360, 363f, 364, 507–509, 508f. See also Vasopressin lithium and, 240 Antiemesis, 137, 141, 211 Antiepileptic drugs (AEDs), 51t, 78, 253–254, 256t, 264t or analgesia, 307t benzodiazepines as, 192 calcium channel inhibition, 258–259 cerebellum and, 116 GABA-mediated inhibition, 259 glutamate receptor inhibition, 259–260 with mechanisms under investigation, 260 in pain management, 301–302 potassium channel inhibition, 258 sodium channel inhibition, 254–258, 257f or substance use disorders, 333t Anti-ErbB1. See Cetuximab Anti-ErbB2. See Trastuzumab Anti actor VIII antibodies, 975 Anti ungals case study, 662 classes o , 606–608, 663–668, 664f drug summary table, 670t–673t ungi membrane and cell wall biochemistry and, 661–662, 663f uture directions in, 668 in ection pathophysiology and, 662–663 prophylactic, 615 Antigen-presenting cells (APCs), 784–786, 787f, 788f, 789f Anti-HBV nucleoside and nucleotide analogues, 703f, 707–708, 719t Anti-HCV nonnucleoside RNA polymerase inhibitors, 709f, 710, 720t

Anti-HCV NS5A inhibitors, 709f, 710, 720t Anti-HCV nucleoside and nucleotide analogues, 703f, 708, 720t Antihelminthic agents, 686–687, 693t Antiherpesvirus nucleoside and nucleotide analogues, 702–705, 703f, 704f, 718t–719t Antihistamines case study, 820 classes o , 823–826, 823t, 824f, 826f drug summary table, 828t–829t elderly and, 143t reticular activating system and, 116 somnolence with, 119 TCAs as, 236 Anti-HIV drugs, 694–695, 697f classes o , 608, 609f attachment and entry inhibitors, 609f, 696f, 698, 698b, 699f, 718t integrase inhibitors, 609f, 696f, 710, 710f–711f, 721t NNRTIs. See Nonnucleoside reverse transcriptase inhibitors nucleoside and nucleotide analogues, 703f, 706–707, 706t, 707b, 719t protease inhibitors, 608, 609f, 696f, 707b, 714b, 712–714, 712f, 713f, 721t combination chemotherapy using, 707b, 775–776 Antihypertensive agents, 473–474, 473t compensatory homeostatic responses to, 474, 474f demographic actors in, 478 or hypertensive crisis, 478–479 intravascular volume reduction, 474–475, 474t monotherapy, 477–478, 477t RAAS modulation, 476–477 stepped care, 478 sympathetic tone down-regulation, 475–476 vascular smooth muscle tone modulation, 476 Anti-IgE antibodies, 878, 889, 891f, 894t Anti-IL antibodies, 890, 891f Anti-inf ammatory agents, or asthma, 887–889 Antimalarial agents classes o , 677–681, 678f, 679f drug summary table, 689t–691t resistance to, 678, 678f, 681 targets o , 607–608 Antimetabolites, 610f, 611, 611f, 723, 777t, 778t uture directions in, 618–619 or immunosuppression, 848–850, 848f, 849f, 850f purine and pyrimidine analogues, 613, 733f, 734f, 735, 735f, 744t–745t, 777t purine metabolism inhibitors, 733–734, 733f, 734f, 743t–744t resistance to, 613t ribonucleotide reductase inhibitors, 734, 744t selective targeting by, 606, 615–618, 616f, 617t thymidylate synthase inhibitors, 616f, 664, 664f, 733, 733f, 743t Antimicrobial drugs, 603. See also Antibiotics; Anti ungals; Antiparasitic drugs; Antiviral drugs case study, 604 classes o , 606–608, 607f, 609f combination chemotherapy using examples o , 773–775 MIC and MBC o , 770–772, 771t, 772f synergy, additivity, and antagonism interactions o , 772–773, 773f un avorable combinations o , 775 uture directions in, 618–619 resistance to, 612–614, 613t selective targeting by, 604–606, 604t, 615–618, 616f, 617t treatment methods using, 614–615 Antimuscarinics, 141–143, 143b, 886 elderly and, 143b, 143t Antimycobacterial drugs, 646–647, 648f, 654–655, 660t combination chemotherapy using, 771, 773–774 Antineoplastic drugs, 603 case studies, 724, 751 classes o , 608–612, 610f, 611f, 612f, 732, 777t–778t angiogenesis inhibitors, 610–611, 762–763, 762t, 768t–769t, 778t

994 Index Antineoplastic drugs (continued) DNA modif ers, 735–740, 736f, 737f, 738f, 739f, 745t–747t, 777t growth actor receptor antagonists, 611, 757–761, 765t–767t, 778t microtubule inhibitors, 610f, 611, 611f, 741–742, 741f, 748t–749t proteasome inhibitors, 611, 754f, 761–762, 768t, 778t purine and pyrimidine analogues, 613, 733f, 734f, 735, 735f, 744t–745t, 777t purine metabolism inhibitors, 733–734, 733f, 734f, 743t–744t ribonucleotide reductase inhibitors, 734, 744t signal transduction antagonists, 611, 757–761, 765t–767t, 778t thymidylate synthase inhibitors, 616f, 664, 664f, 733, 733f, 743t topoisomerase inhibitors, 610f, 611f, 628–629, 628f, 637t, 728f, 739f, 740–741, 747t–748t tumor-specif c monoclonal antibodies, 763, 769t combination chemotherapy using, 724, 777t–778t examples o , 779–780, 780f, 780t general considerations or, 776–779, 778t rationale or, 779 or re ractory or recurrent disease, 780–781 drug summary tables genome synthesis, stability, and maintenance, 743t–749t intercellular and intracellular signal transduction, 765t–769t uture directions in, 618–619, 742, 764 genome synthesis, stability, and maintenance biochemistry and, 724–732, 725f, 726f, 726t, 727f, 728f, 729f, 730f, 731f, 732f immunomodulatory agents used as, 777t, 839, 843t, 857, 863t resistance to, 612–614, 613t, 724, 778, 778t selective targeting by, 604–606, 604t, 615–618, 616f, 617t signal transduction and, 750–757, 751f, 752t, 753f, 754f, 755f, 756f, 756t, 757f treatment methods using, 614–615 Antiparasitic drugs case studies, 675, 682, 685 classes o , 606–608 drug summary table, 689t–693t uture directions, 687–688 or helminths, 686–687 or protozoa, 683–685, 684f. See also Antimalarial agents Antiparkinson agents, 224t elderly and, 143t Antiplatelet agents, 414–418, 415f, 416f, 426t Antiprotozoal agents, 683–685, 684f, 689t–692t. See also Antimalarial agents Antipsychotic agents, 51t, 217 adverse e ects o , 218 atypical, 218f, 220, 226t elderly and, 143b, 143t mechanism o action o , 217, 219f pharmacokinetics, metabolism, and drug interactions with, 219–220 typical, 217–220, 218f, 224t–225t Antiresorptive agents, 590–595, 592f, 593b Anti-Rhesus antigen Ig, 974, 974t Antisense oligonucleotides, 924b Antisense therapeutics, 13 Antispasmodics, elderly and, 143b, 143t Antistaphylococcal penicillins, 652 Antithrombin III (ATIII), 410, 411f, 960t Antithrombotic mechanisms, 404, 405f Antithymocyte globulin (ATG), 845, 855, 861t Anti-TNF agents, 14, 795, 812, 852–854, 852f, 853f, 859t–860t, 929t Antitumor antibiotics, 610f, 611f, 739f, 740 Anti-VEGF and anti-VEGFR antibodies, 762 Antivenins, 78, 925t Antiviral drugs case study, 695 classes o , 608, 609f, 697 attachment and entry inhibitors, 609f, 696f, 698, 698b, 699f, 718t

gene expression inhibitors, 696f, 700–702, 701f, 703f, 718t immune system modulators, 716–717, 722t maturation inhibitors, 696f, 712–714, 712f, 713f, 721t release inhibitors, 696f, 714–715, 715f, 721t replication inhibitors, 696f, 702–710, 703f, 704f, 706t, 707b, 709f, 710f–711f, 718t–721t uncoating inhibitors, 609f, 696f, 698–700, 700f, 718t unknown mechanisms, 716–717, 722t combination chemotherapy using, 707b, 775–776 drug summary table, 718t–722t uture directions, 717 prophylactic, 615 resistance to, 613t, 614, 697–698 viral replication and, 694–697, 696f, 697f AP. See Action potentials AP50, 267 APC. See Adenomatous polyposis coli Apcitide, 975, 976t APCs. See Antigen-presenting cells Apigenin, 60t Apixaban, 421f, 422, 430t Aplastic anemia, antibiotics causing, 635 ApoB. See Apolipoprotein B ApoB48, 339 ApoB100, 339 amilial de ective, 347 ApoB-containing lipoproteins assembly o , 338–340, 339f metabolism o , 338 intravascular, 340–341, 340f receptor-mediated clearance o , 341–342, 341f ApoB editing complex-1 (apobec-1), 338 ApoCII def ciency, 348 ApoE. See Apolipoprotein E Apolipoprotein B (apoB), 338, 338f Apolipoprotein E (apoE), 341, 341f Apolipoproteins, 338, 338f Apomorphine, dopamine hypothesis and, 216 Apoptosis, 76, 611, 723 insensitivity to, 613t, 614 Appetite, 157 Aprotinin, 424, 431t APSAC. See Anisoylated plasminogen streptokinase activator complex aPTT. See Activated partial thromboplastin time Aquaporin, 366 Aquaretics, 493t, 494 Arabinosyl trans erase, 646 Arachidonic acid, 404, 794, 882, 884f. See also Eicosanoids generation o , 795, 796f 2-Arachidonylglycerol (2AG) amilies, 293 Arcitumomab, 976t Arcuate nuclei, dopamine in, 211 Areal bone mineral density (aBMD), 587 Area postrema, 211 Ar ormoterol, 885, 892t Argatroban, 421f, 423, 430t Arginine, 503 Arginine vasopressin (AVP), 360, 363f, 364. See also Vasopressin Aripiprazole, 220, 226t Aromatase, 542 inhibitors o , 548f, 549, 556t Aromatic group, in local anesthetics, 171–172, 172f Aromatic L-amino acid decarboxylase (DOPA decarboxylase, AADC), 124, 207, 208f, 213, 214f, 228, 229f Arsenic, 914–915 Artemether, 679, 689t Artemisinin, 608, 677, 678f, 679, 679f, 681, 689t, 923t Arterial partial pressure (Part), tissue partial pressure and, 273, 273t, 274f Artesunate, 679, 689t, 923t Articaine, 179–180, 182t Asbestos, 915 Asbestosis, 915 Ascites, 371–372, 371f

Asenapine, 220, 226t ASICs. See Acid-sensitive ion channels A site. See Aminoacyl site Asoprisnil, 552, 558t L-Asparaginase, 966, 967t Aspartate, as neurotransmitter, 119–121, 120t, 121f Aspirin, 25–26, 301, 306t, 404, 414, 415f, 455, 488, 796t, 808–810, 809f, 812, 815t, 865, 868f, 884f, 900 or acute coronary syndromes, 485f, 486–487 antiplatelet e ect o , 4, 426t or chronic coronary artery disease, 484 as noncompetitive antagonist, 22–23 Aspirin-exacerbated respiratory disease, 888 Aspirin-triggered lipoxins, 813 Assay, 924 Assembly, viral, 696, 696f, 697f Astemizole, 826 Asthma, 141–142, 156–157, 159, 525 antihistamines or, 825 case study, 878 clinical management o , 889, 890t drug classes treating, 882–889, 885f drug delivery or, 889 drug summary table, 892t–894t eicosanoid pathophysiology in, 807 uture directions, 889–891, 891f pathophysiology o , 879–882, 881f, 883f, 884f physiology o airway smooth muscle contraction, 877–878, 879b immune unction, 879, 880f Astroglia, 123, 124f AT1R. See AT II receptor subtype 1 AT2R. See AT II receptor subtype 2 Atazanavir, 609f, 696f, 712, 712f, 714, 721t Atenolol, 36, 60t, 160t, 161, 166t, 401t, 445, 451t, 475 ATG. See Antithymocyte globulin Atherosclerosis, 342, 344f guidelines or reduction o , 349, 350t ATIII. See Antithrombin III AT II receptor subtype 1 (AT1 receptor, AT1R), 362 antagonists o , 373–374, 381t, 402t AT II receptor subtype 2 (AT2 receptor, AT2R), 363 Atomoxetine, 245t Atopy, 881 Atorvastatin, 12, 18, 22, 51t, 64, 66, 94, 351, 356t, 488 Atovaquone, 677, 677f, 680, 690t ATP. See Adenosine triphosphate ATP-binding cassette (ABC) transporter amily, 37, 56, 62–64, 63t ATPIII guidelines. See National Cholesterol Education Program Adult Treatment Panel III guidelines ATP-sensitive K channel, 564–565, 565f Atrasentan, 60t Atrial f brillation, 441b Atrial utter, 441b Atropa belladonna, 141 Atropine, 60t, 84, 147t, 886 autonomic ganglia and, 133 as cholinergic antagonist, 141–142 or cholinergic toxicity, 142b elderly and, 143t parasympathetic nervous system and, 112 Attachment, viral, 695, 696f, 697f Attachment inhibitors, 609f, 696f, 698, 698b, 699f, 718t Attention-def cit hyperactivity disorder (ADHD), 157, 231, 234 A-type natriuretic peptide (ANP), 363–364, 363f Atypical antidepressants, 237, 245t–246t, 334t Atypical antipsychotic agents, or schizophrenia, 218f, 220 Aura, 252 Autacoids, 794 Autoimmune reactions, 76, 78 Autoimmunity, 78, 788, 846, 847t Autolysins, 648 Automaticity, 433 altered, 438

Index 995 Autonomic ganglia blockade o , 133, 136t neurotransmission through, 132–133, 135f Autonomic nervous system, 110 acetylcholine in, 123 anatomy o , 111–112, 113f cellular organization o , 116–117, 117f cholinergic pathways in, 127 vascular tone and, 389–390, 389f Autoreceptors, 106, 134, 188 dopamine receptors, 209 serotonergic neurotransmission, 228 Autosomal dominant hypophosphatemic rickets (ADHR), 586 Autosomal dominant nocturnal rontal lobe epilepsy (ADNFLE), 136 Avanaf l, 395, 400t Avibactam, 650f, 651 Avolition, 216, 216b AVP. See Arginine vasopressin Awake states ACh in, 134 relay neurons in, 253, 255f Axitinib, 762, 762t AZA. See Azathioprine 5-Azacytidine, 735, 735f, 745t, 837–838, 841t Azathioprine (AZA), 89t, 90, 733–734, 733f, 743t, 899–900, 900f or immunosuppression, 848, 848f, 858t Azelastine, 825, 829t Azidothymidine. See Zidovudine Azilsartan, 381t Azithromycin, 13, 623, 633–634, 639t Azoles, 661, 663f, 664f, 665–667, 671t, 775 targets o , 607 AZT. See Zidovudine Aztreonam, 651–652, 654, 659t

B

B1 receptors, 291 B2 receptors, 291 Bacitracin, 643, 644f, 649, 656t Baclo en, 189t, 195, 204t Bacterial in ections case studies, 623, 642 cell wall, biochemistry o autolysins and degradation, 648 mycobacterial, 642f, 646–647, 648f peptidoglycan biosynthesis, 643–646, 644f, 645b, 647f structure and unction, 641–643, 642f DNA, biochemistry o replication and segregation, 623–624, 625f structure, 622–623, 623f, 624f transcription, 624, 626f translation, 626–627, 626f, 627f drug resistance mechanisms in, 612–614, 613t drugs targeting. See Antibiotics uture directions, 636, 655 Bactericidal drugs, 606, 631, 632f, 648, 650, 771–772, 771t, 772f Bacteriostatic drugs, 606, 771–772, 771t, 772f Bactoprenol phosphate (BP), 644f, 645 Balanced anesthesia, 280–281 Barbiturates, 78, 185 abuse o , 310t, 322–323, 322f adverse e ects o , 194, 194t clinical applications o , 193–194, 194t in cortex, 114 dependence on, 194–195 GABAA receptor modulation with, 193, 193f, 203t, 259 as GABA channel potentiator, 263t GABAergic neurotransmission and, 185 GABA receptors and, 120, 125, 187f, 189t as general anesthetic, 280, 284t metabolism o , 194 pharmacokinetics o , 194 tolerance to, 194–195 war arin and, 419t withdrawal rom, 327 Bartter syndrome, 366, 367t

Basal ganglia, 114, 115f dopamine receptors in, 209, 210f in ormation processing o , 211 Basal insulins, 570, 570t, 575t Basal nucleus o Meynert, 118f, 118t, 119, 134 Base excision repair (BER), 725, 727–728, 728f, 729f Baseline tone, 228 Bases, 909 Basic multicellular units (BMU), 582 Basiliximab, 855, 861t, 970t Basophils, 785 Bazedoxi ene, 549t, 551, 557t BBB. See Blood–brain barrier B-cell non-Hodgkin’s lymphoma, 975 B cells, 786t, 787–789 development o , 784, 785f, 835–836 specif c depletion o , 848f, 857, 861t–862t BCNU, 736, 736f BCR-Abl inhibitors, 759–760, 766t, 778t BCR-Abl tyrosine kinase, imatinib interactions with, 4–5, 5f, 12–13 BCRP. See Breast cancer resistance protein BD. See Bipolar disorder BDNF. See Brain-derived neurotrophic actor Becaplermin, 965t Beclomethasone, 532, 533f, 539t, 887, 894t Bedaquiline, 774 Beers Criteria, 143b Be oxatone, 163t, 235, 243t Behavioral tolerance, 311 Belatacept, 856, 862t, 971t Belimumab, 855–856, 862t, 970t Belladonna alkaloids, 143b Benazepril, 380t Bendamustine, 736, 746t Bendro umethiazide, 383t Benign prostatic hyperplasia (BPH), 154, 159, 547 Benperidol, 218f Benralizumab, 891f Benzbromarone, 63t, 900 Benzocaine, in dentistry, 177 Benzodiazepines, 51t abuse o , 310t, 322–323, 322f adverse e ects o , 192 or alcohol withdrawal, 327 clinical applications o , 191–192, 192t in cortex, 114 dependence on, 192 GABAA receptor modulation with, 190–191, 190f, 202t–203t, 259 as GABA channel potentiator, 263t GABAergic neurotransmission and, 185, 256t GABA receptors and, 120, 125, 187f, 189t as general anesthetic, 285t ion channel conductance and, 9 metabolism o , 192 pharmacokinetics o , 192 tolerance to, 192 withdrawal rom, 327 Benzothiazepines, 390, 398t, 437 Benztropine, 142, 143t, 215, 224t Benzylamines, 662, 663f, 664f, 665, 670t BEP. See Bleomycin, etoposide, and cisplatin Bepotastine besilate, 825, 829t BER. See Base excision repair Berberine, 63t Bestatin, 60t Beta-adrenergic agonists, 92t, 159, 164t or asthma, 878, 879b, 883–885, 885f, 889, 890t, 892t or cardiac contractility, 461, 463–464, 463t, 466t–467t Beta-adrenergic antagonists, 23, 89t, 160–161, 160t, 165t–166t or acute coronary syndromes, 485f adverse e ects o , 483 as antiarrhythmics, 444–445, 445f with calcium channel blockers, 484 or chronic coronary artery disease, 483 or heart ailure, 493t, 495 or hypertension, 473t, 475, 477t or migraine therapy, 302

o -target adverse e ects with, 74 thyroid hormone metabolism and, 521, 523t or vascular tone, 397, 401t Beta-adrenergic receptor kinase, 461 Beta-adrenergic receptors, 154–155, 154t, 878 catecholamine activity at, 155–156 desensitization o , 15, 15f tissue localization and action o , 10–11, 10t Beta-arrestin, 155 Beta-blockers. See Beta-adrenergic antagonists Beta cells, 561 Beta-endorphins, 293, 505 Beta-estradiol, 60t Beta-glucocerebrosidase, 956, 960t Beta-lactam antibiotics, 60t, 650–654, 650f, 652f, 657t–659t aminoglycosides combined with, 631–632, 651–652, 774 bacterial cell wall synthesis and, 641–642, 644f, 646, 647f combination chemotherapy using, 772–775 resistance to, 613, 613t, 651 selective targeting by, 605 Beta-lactamase, 613, 613t, 650f, 651, 653 Beta-lactamase inhibitors, 650f, 651–653, 775 Betamethasone, 81 Beta-thalassemia, 833 Betaxolol, 161, 166t Bethanechol, 112, 139–140, 140f, 140t, 146t Bevacizumab, 762, 768t, 778t, 968t Bezaf brate, 353–354 B-f bers, 169, 169t Biased agonists, 24 Biased libraries, 925 Bicalutamide, 547, 548f, 551, 558t Bicarbonate, 866. See also Sodium bicarbonate Bicuculline, 189t, 190, 202t Bier block, 177–178 Biguanides, 575t Bile acid absorption o , inhibitors o , 352, 356t secretion o , 346, 346f Bile acid sequestrants, 577t Bile acid transporters, 59, 60t–61t, 61 Bile salt export pump (BSEP), 62, 63t Biliary excretion, 37 Biliary lipid secretion, 346, 346f Bilirubin encephalopathy, 47 Bimatoprost, 817t Binders, 931–932 Binding site, 3–4, 5t o local anesthetics, 171–172 Bioavailability, 29–30, 30f Biochemical assays, 928 Bioequivalence, 944 Biogenic amine neurotransmitters, 119, 120t, 121–123, 121f, 122f Biologic assays, 928 Biological membranes, 28–29, 29f Biologics Price Competition and Innovation Act (BPCIA), 944 Biopharmaceutical review, 942, 942f Biosimilar, 944, 977 Biosimilar User Fee Act, 944 Biotrans ormation, 34 Biperiden, 142 Bipolar disorder (BD), 227, 231–233, 232b, 240 substance use disorders and, 329 Bismuth, 865, 869f, 873–874, 876t Bisoprolol, 445, 451t Bisphosphonates (BPs), 592–594, 592f, 593b, 599t, 929t Bitolterol, 884, 892t Bivalirudin, 421f, 423, 430t, 485f, 486, 968t “Black box” warning, 943 Black lung, 915 Bladder spasms, 137 Bleomycin, 82, 724, 728f, 739–741, 739f, 747t, 777t Bleomycin, etoposide, and cisplatin (BEP), 780, 780f Blepharospasm, 137 Blood–brain barrier (BBB), 29, 123–124, 124f, 613 antidepressants and, 235 levodopa and, 213

996 Index Blood ow, abnormal, 412 Blood pressure, 156 systemic, 472, 472f Blood schizonts, 675 Blood vessel physiology, 385–386, 386f B-lymphocyte stimulators, 855–856 BMU. See Basic multicellular units BNP. See B-type natriuretic peptide Boceprevir, 701–702, 701f, 718t Bone remodeling o , 581–583, 582f, 583f structure o , 580–581, 582f Bone anabolic agents, 595–596, 600t Bone mineral homeostasis uture directions or, 597–598 pathophysiology o , 587–590, 588t, 589f pharmacologic classes and agents or, 590–597, 591f, 592f, 593b physiology o , 580–587, 582f, 583f, 584t, 585f Bortezomib, 754f, 761–763, 768t, 778t Bosentan, 60t, 61t, 62, 63t, 65t, 66t, 396, 401t Bosutinib, 79, 753f, 759–760, 766t Botulinum toxin, 145t, 967t SNAREs and, 107 Botulinum toxin A, 137 BP. See Bactoprenol phosphate BPCIA. See Biologics Price Competition and Innovation Act BPH. See Benign prostatic hyperplasia BPs. See Bisphosphonates Brachial plexus block, 177 Bradycardia autonomic ganglia and, 133 vagal-mediated, 137 Bradykinin, 176, 878 vascular tone and, 389f, 390 Bradykinin receptors, 291 Brain behavioral systems, 315, 315f Brain-derived neurotrophic actor (BDNF), 233, 291 Brain reward pathway, opioids in, 320, 320f Brainstem, 114, 114f, 116 dopamine receptors in, 209 Brand name, 943–944 BRCA1 and BRCA2, 729f, 730, 742 Breakthrough Therapy, 942 Breast cancer resistance protein (BCRP), 63t, 64 Breast carcinoma, 547 Brentuximab vedotin, 856, 862t, 973t Brinzolamide, 375 Broad-spectrum antibiotics, war arin and, 420t Brodalumab, 854 Bro aromine, 163t, 235, 243t Brom enac, 949t Bromocriptine, 214, 503–504, 511t, 547, 573 Bromocriptine mesylate, 578t Brompheniramine, 143t, 828t Bronchitis, 879b Bronchoconstriction in asthma, 881, 881f histamine-mediated, 819–820 Bronchodilation, 137 Bronchodilators, 878, 879b, 883–887, 885f BSEP. See Bile salt export pump BTK inhibitors, 760, 766t B-type natriuretic peptide (BNP), 363–364, 363f, 374, 381t BuChE. See Butyrylcholinesterase Bucindolol, 95 Budesonide, 539t, 885, 887, 894t Bu ering capacity, 909 Bu uralol, 51t Bumetanide, 376, 383t, 474t, 493 Bundle o Kent, 440, 440f Bupivacaine, 176, 179, 182t as epidural anesthetic, 177 inf ltration anesthesia, 177 on-target adverse e ect with, 72 Buprenorphine, 24, 300, 305t, 309, 320, 321f, 327–328, 333t Bupropion, 78, 237, 245t, 334t or atypical depression, 232 or smoking cessation, 329 Buserelin, 549, 556t Buspirone, 238, 246t Butaclamol, 218f

Butenaf ne, 665, 670t Butoconazole, 666, 671t Butyl TMA. See Butyl trimethylammonium Butyl trimethylammonium (TMA), 23 Butyrates, 837–838 Butyrophenones, 217, 219f, 225t Butyrylcholinesterase (BuChE), 131–132

C

C1 esterase inhibitor, 960t CA. See Cocaine Anonymous Ca2 . See Calcium Cabazitaxel, 741–742, 749t Cabergoline, 503–505, 511t, 547 Cabozantinib, 759, 762, 762t, 768t CAD. See Coronary artery disease Ca eine, 886 abuse o , 310t, 325 adenosine receptors and, 123 or migraine therapy, 302 CAH. See Congenital adrenal hyperplasia CAII. See Carbonic anhydrase II CAIV. See Carbonic anhydrase IV Calci ediol, 597, 601t Calcimimetics, 596 Calcitonin, 586–587, 593b, 595, 600t Calcitonin gene-related peptide (CGRP), 291, 878 Calcitriol, 581, 596–597, 601t Calcium (Ca2 ), 601t adrenergic receptors and, 458, 459f, 459t or bone disorders, 596–597 contractile proteins and, 458–459 hormonal control o , 583–587, 584t, 585f intracellular, 386–387, 387f Nernst equilibrium potential or, 101t storage and release o , 457–458, 458f Calcium acetate, 596, 600t Calcium carbonate, 596, 600t–601t, 873, 876t Calcium channel blockers (CCBs), 51t, 61t, 89t, 258–259, 262t, 302 action potential and, 105 as antiarrhythmic drugs, 447, 447f, 452t beta-antagonists with, 483 chemical classes o , 390–391 or chronic coronary artery disease, 483–484 or hypertension, 359, 473t, 476, 477t mechanism o action, 390, 391f pharmacokinetics o , 391 sites o action o , 390, 391f or substance use disorders, 333t toxicities and contraindications o , 391–392 or vascular tone, 390, 398t Calcium channels in electrochemical transmission, 105–106, 106f N-type, 291 T-type, 253, 255f Calcium chloride, 596, 601t Calcium citrate-malate, 596–597, 601t Calcium gluconate, 596, 601t Calcium pump, 456, 458f Calcium-sensing receptor (CaSR), 367 Calcium-sensitizing agents, 461, 465, 468t Calmodulin (CaM), 387, 387f cAMP (Cyclic AMP) -adrenoceptors and, 154–155 in cardiac contractility, 458, 459f dopamine receptors and, 209 production o , 9–10, 10f protein kinase A and, 458 Campath-1 (CD52), 855 Camptothecins, 625f, 740, 777t Canadian Food and Drugs Act, 943 Canagli ozin, 572, 577t Canakinumab, 812, 816t, 854, 860t, 901, 970t Cancer, 723 case studies, 724, 751 drugs targeting. See Antineoplastic drugs eicosanoid pathophysiology in, 808 uture directions, 618–619, 742, 764 genome synthesis, stability, and maintenance biochemistry in, 724–732, 725f, 726f, 726t, 727f, 728f, 729f, 730f, 731f, 732f intercellular and intracellular signal transduction biochemistry in, 750–757, 751f, 752t, 753f, 754f, 755f, 756f, 756t, 757f

Candesartan, 381t Candida, 663 Cannabinoid receptors, 293 alcohol and, 323, 324f type-1, 325 Cannabinoids, 293, 310t, 316f, 325 Capecitabine, 733, 743t, 779 Capillary, uid f ltration o , 359, 360f Capreomycin, 774 Capromab pendetide, 975, 976t Capsid, viral, 695 Captopril, 60t, 373, 380t, 402t, 455, 476 Carbachol, 139–140, 140f, 140t, 146t Carbamazepine, 38, 50, 51t, 54, 78, 239, 247t, 250, 255–256, 256t, 261t, 296, 302, 307t, 333t, 419t Carbapenemases, 651, 653 Carbapenems, 644f, 650f, 651–652, 654, 659t resistance to, 613 targets o , 607f Carbidopa, 111 DOPA carboxylase and, 124 or Parkinson’s disease, 213–214, 214f Carbinoxamine, 143t, 828t Carbonic anhydrase, inhibitors o , 375, 382t Carbonic anhydrase II (CAII), 365–366 Carbonic anhydrase IV (CAIV), 365–366 Carbon monoxide (CO), 905–906, 906f Carboplatin, 738–739, 738f, 746t Carboprost, 817t Carboxyhemoglobin (COHb), 905–906, 906f Carboxy penicillins, 653 Carcinogenesis, 608–610 due to drug therapy, 82 environmental exposures, 911–912, 911t, 912f Carcinogens, 82, 912, 912f Cardiac action potentials, 433–437, 435f, 436f, 436t, 437b, 437f Cardiac arrhythmias, 438–440, 439f, 440f action potentials and, 105 uture directions or, 448 Cardiac compensation, 490–492, 491f, 492f Cardiac contraction calcium channel blockers and, 483 uture directions or, 465 myocytes in anatomy o , 455, 456f, 457t contraction o , 455–456, 457f physiology o , 454 regulation o , 456–459, 457t, 458f, 459f, 459t Cardiac glycosides, 461, 466t, 495 Cardiac ion currents, 448, 448t Cardiac myosin activators, 465 Cardiac neuregulins, 465 Cardiac output (CO), 271f anesthetics and changes in, 276, 276f determinants o , 490, 490f ventilation changes and, 275, 276f Cardiac remodeling, 491 Cardiac vagomimetic, 140 Cardiovascular disease (CVD), 336 channel blockers and, 105 pathophysiology o , 347–349 Cardiovascular disease, eicosanoid pathophysiology in, 808 Cardiovascular toxicity, drug-induced, 81 Carf lzomib, 762, 768t Carisoprodol, 143t Carmustine, 736, 736f, 746t, 983 Carteolol, 161, 165t Carvedilol, 51t, 66, 160t, 161, 165t, 445, 451t, 455 Case-control studies, 950, 950f, 951f Caspo ungin, 607, 668, 673t CaSR. See Calcium-sensing receptor Catalase, 685 Catalase-peroxidase, 655 Catatonic behavior, 216, 216b Catecholamines, 89t, 206 hypertensive crisis with, 235 metabolism o , 153, 153f, 208–209, 209f inhibitors o , 158 physiologic and pharmacologic e ects o , 155–156 receptors, 153–155, 154t release o , 151–153

Index 997 reuptake o , 152f, 153 inhibitors o , 158, 163t storage o , 151, 152f inhibitors o , 156–158, 157f, 162t in sympathetic signaling, 150 synthesis o , 121–122, 122f, 150–151, 152f, 206, 208f inhibitors o , 156, 162t Catechol-O-methyltrans erase (COMT), 153, 153f, 207–208, 208f, 230 inhibition o , 214f, 215 Caudate, 114, 115f CB1. See Type-1 cannabinoid receptors CB1 receptor, 293 CB2 receptor, 293 CBF. See Coronary blood ow CBG. See Corticosteroid-binding globulin CCBs. See Calcium channel blockers CCD. See Cortical collecting duct CCR5 receptor, 606, 698, 698b, 699f CD28, 788–789 CD40 ligand (CD40L), 789–790, 789f CD52. See Campath-1 CD58. See LFA-3 CDKs. See Cyclin-dependent kinases Ce aclor, 60t, 78 Ce adroxil, 60t, 653, 658t Ce azolin, 63t, 653, 658t Ce epime, 653, 659t Ce operazone, 654, 659t Ce otaxime, 653, 659t Ce otetan, 653, 654, 658t Ce oxitin, 653, 658t Ce podoxime, 653, 659t Ce taroline, 653, 659t Ce tazidime, 652–653, 659t Ce tizoxime, 60t, 63t Ce tolozane, 653 Ce triaxone, 33, 653, 659t Ce uroxime, 653, 658t Celecoxib, 63t, 78, 295, 300f, 301, 306t, 795, 811–812, 811f, 816t, 951 Celiac ganglion, 111, 113f Celiprolol, 60t, 160t Cell adhesion blockade, 848f, 856, 863t Cell-cycle nonspecif c drugs, 611, 611f Cell-cycle specif c drugs, 611, 611f Cell-mediated hypersensitivity, 78, 78t Cell membrane action potential o , 102 bacterial, inhibitors targeting stability o , 654, 660t electrical circuit model o , 100, 100f ungal, 661–662, 663f inhibitors targeting stability o , 667–668, 672t Cell proli eration, carcinogenesis and, 608–610 Cell recruitment, in in ammatory response, 791, 792f Cells, o immune system, development o , 784, 785f, 835–836 Cell sur ace adhesion receptors, 14 Cellular assays, 928 Cellular communication, 98 Cellular excitability, 98–104 Cellular immunity, 786t, 787–788, 788f Cellular organization, o nervous system, 116–119, 117f, 118f, 118t Cellular senescence, 730–731, 730f Cell wall bacterial autolysins and degradation o , 648 drugs classes targeting, 648–655, 649f, 650f, 652f mycobacterial, 642f, 646–647, 648f peptidoglycan biosynthesis in, 643–646, 644f, 645b, 647f structure and unction o , 641–643, 642f ungal, 661–662, 663f drugs classes targeting, 668, 673t Center-surround signaling, 117 Central adrenergic neurotransmission, 228 norepinephrine receptors, 230, 230t norepinephrine synthesis and regulation in, 228–230, 229f, 230f pharmacologic classes and agents or, 233–240

Central diabetes insipidus, 377 Central dopamine pathway, 209–211, 211f Central nerve blockade, 177 Central nervous system (CNS) acetylcholine and, 134–136 blood–brain barrier o , 29 cellular organization o , 116–119, 117f, 118f, 118t cholinergic pathways in, 127 general anesthetics and, 265 local anesthetics and, 167, 178 neuroanatomy o , 114–116, 114f, 115f, 116f neurotransmitters in, 119–123, 119f, 120t, 121f, 122f pain in, 288–289 Central sensitization, 169, 200, 295–296, 295f Central tolerance, 846 Cephalexin, 60t, 63t, 648, 653, 658t Cephaloridine, 60t Cephalosporins, 644f, 648, 650f, 651–654, 658t–659t resistance to, 613 targets o , 606, 607f Cephradine, 65t Cerebellar hemispheres, 115, 115f Cerebellar vermis, 115, 115f Cerebellum, 114–116, 114f, 115f Cerebral cortex, 114, 115f Cerebral hemispheres, 114–115, 114f, 115f Cerebral malaria, 675 Cerebrum, 114–115, 114f, 115f Cereulide, 908 Ceritinib, 759, 766t Cerivastatin, 60t, 949t, 953 Certolizumab, 969t Certolizumab pegol, 812, 816t, 853, 860t Cervical cancer, 974 Cervical ganglion, 111 Cervical plexus block, 177 Cestodes, 685 Cetirizine, 60t, 824–825, 829t CETP. See Cholesterol ester trans er protein Cetrorelix, 507, 549, 556t, 972t Cetuximab (Anti-ErbB1), 753f, 758–759, 765t, 778t, 966, 968t, 978 Cevimeline, 140, 146t C-f bers, 169, 169t, 170f, 291 CFR. See Coronary ow reserve CFTR. See Cystic f brosis transmembrane conductance regulator cGMP (Cyclic GMP) potentiators, 395–396, 400t production o , 9–10, 10f CGRP. See Calcitonin gene-related peptide Channel selectivity, 100–102, 100f, 101f, 101t, 102f ChAT. See Choline acetyltrans erase Chelator, 908, 908f Chelators, polyvalent cations, 668, 673t Chemical activators, 290 Chemical antagonists, 21, 23 Chemical burns, 909 Chemical gradient, 100, 101f Chemical reaction-based systems, 982–983, 982f Chemical transmission, 98–99 Chemistry review, 937, 937f, 942, 942f Chemokines, 790t, 791 Chemoprophylaxis, 615 Chemosensitive transduction receptors, 290, 290t Chemotaxis, in in ammatory response, 791 Chemotherapy. See Antimicrobial drugs; Antineoplastic drugs; Combination chemotherapy Children, anesthesia and, 276, 277f Chitin, 662 Chloral hydrate GABA physiology and, 196 war arin and, 420t Chlorambucil, 82, 736, 746t Chloramphenicol, 53, 607f, 626f, 627f, 628, 630, 630t, 634f, 635, 635f, 639t combination chemotherapy using, 773 resistance to, 635 Chlordiazepoxide, 203t clinical uses o , 192t ethanol and, 196 Chlorinated hydrocarbons, 916

Chlorine (Cl ), Nernst equilibrium potential or, 101t 2-Chloroprocaine, 178–179, 181t Chloroquine, 607–608, 675–678, 676f, 678f, 681, 689t Chlorothiazide, 383t, 474t Chlorpheniramine, 143t, 824, 824f, 828t Chlorpromazine, 143t, 217, 218f, 219f, 224t Chlorpropamide, 576t Chlorprothixene, 224t Chlorpyri os, 909–911, 910f Chlorthalidone, 376–377, 383t, 474t Cholecalci erol, 584, 597, 601t Cholesterol absorption o , 339, 339f inhibitors o , 352–353, 357t balance o , 346–347 HDL-mediated e ux o , 344 metabolism o , 336–338, 337t pharmacologic classes and agents or, 349–355, 349t, 350t reverse transport o , 342–346, 345f synthesis o , inhibitors o , 350–352, 351f, 356t Cholesterol 7 -hydroxylase (CYP7A1), 346 Cholesterol ester trans er protein (CETP), 344 Cholestyramine, 352, 356t, 419t, 519 Choline, in acetylcholine synthesis, 128, 129f Choline acetyltrans erase (ChAT), 128, 129f Cholinergic neurons, 118, 118f, 118t Cholinergic neurotransmission, 127 acetylcholine in degradation o , 131–132 storage and release o , 128–130, 129f synthesis o , 128, 129f cholinergic receptors, 131t muscarinic, 130 nicotinic, 130–131 pharmacology or, 137 acetylcholinesterase inhibitors, 137–139, 138f, 139t ACh synthesis, storage, or release inhibition, 137 receptor agonists, 139–141, 140f, 140t, 141t, 142b receptor antagonists, 141–144, 142b, 143b physiologic e ects o autonomic e ects, 132–134, 135f, 136t CNS e ects, 134–136 neuromuscular junction, 132, 134f, 135f non-neuronal cholinergic system, 136 Cholinergic receptors, 131t muscarinic, 130 agonists o , 139–140, 140f, 140t, 142b, 146t antagonists o , 141–143, 143b, 147t–148t nicotinic, 130–131 agonists o , 140–141, 141t, 142b, 147t antagonists o , 143–144, 148t–149t Cholinergic toxicity, 142b Cholinergic transmitters, o helminths, 686 Cholinesterase inhibitor poisoning, 142b Cholinesterases, 52, 131 Choreoathetosis-with-salivation syndrome (CS), 911 Chroma f n cells, 150 Chromosomal genes, or antibiotic resistance, 651 Chromosomal mutations, antibiotic resistance and, 612–614, 613t Chromosome, maintenance o , 725–731, 728f, 729f, 730f Chronic alcoholism, 326 Chronic bronchitis, 879b Chronic coronary artery disease, 479–481, 480f, 481f clinical management o , 482–485 Chronic atigue syndrome, 236 Chronic in ammation, 792 Chronic kidney disease, 462, 587, 588t, 589–590, 591f, 596 Chronic myelogenous leukemia (CML), 759 Chronic obstructive pulmonary disease (COPD), 141–142, 156, 160, 879b, 886 Chronic pain, 169 Chronic rejection, 845t, 846 Chylomicrons (CM), 336, 337t remnants, 341–342, 341f Ciclesonide, 532, 533f, 887, 894t Ciclopirox, 668, 673t

998 Index Cido ovir, 60t, 65, 65t, 66t, 703 , 705, 719t Cilastatin, 75, 654, 659t Cilazapril, 380t Cimetidine, 39, 51t, 60t, 63t, 65–66, 65t, 826, 826 , 829t, 869–870, 870 , 875t, 922 Cinacalcet, 591 , 601t Cinchonism, 678 Cingulate gyrus, 115, 115 Ciprof brate, 353–354 Cipro oxacin, 39, 52t, 60t, 71, 75, 78, 628, 628 , 637t Ciproxi an, 826 Circulatory ailure, 488–489, 489t Circumventricular organs, 211 Cirrhosis, sodium retention and, 371–372, 371 Cisapride, 54, 238 Cisatracurium, 285t Cisplatin, 60t, 63t, 65t, 66, 66t, 81, 724, 738–739, 738 , 739 , 741, 746t, 777t, 929t Citalopram, 236, 244t, 301 C-KIT inhibitors, 759–760, 766t, 778t Cl . See Chlorine Cladribine, 725 , 727 , 734 , 735, 744t Clarithromycin, 51t, 60t, 63t, 65t, 633–634, 639t, 865, 874 Class e ects, 72 Clathrin, 155 Clavulanic acid, 650 , 651, 653, 657t–658t, 775 CLC-K2, 366, 366 Clearance, 37–38, 38 Clear-core synaptic vesicles, 107 Clemastine, 143t, 828t Clevidipine, 391, 398t Clindamycin, 634 , 635, 635 , 639t, 677, 680, 690t, 775 resistance to, 633 Clinical development, 937–942, 938t, 939t, 941t Clinical endpoints, 939 Clinical hold, 937 Clinical nociceptive pain, 293–294 Clinical pharmacology, 940 Clinical trial adaptive designs, 941 development o , 937–939, 938t in humans, 939–940, 939t planning and execution, 941, 941t Clobazam, 192, 203t, 263t clinical uses o , 192t GABAA receptor and, 259 Clobenpropit, 826 Clo azimine, 774 Clomiphene, 551, 557t Clomipramine, 51t, 52t, 143t, 236, 243t Clonal deletion, 788 Clonazepam, 192, 203t, 263t clinical uses o , 192t GABAA receptor and, 259 Clonic phase, o seizure, 253, 254 Clonidine, 60t, 503 adrenergic receptors and, 121–122, 158, 163t, 302 as analgesic, 293 or hypertension, 476 hypothalamus and, 115 or substance use disorders, 309 or withdrawal symptoms, 327 Cloninger classif cation, 319 Clopidogrel, 52–53, 52t, 406, 415–417, 420t, 427t, 486, 488, 873 Clorazepate, 202t clinical uses o , 192t Clorgyline, 158, 163t Closed state, o sodium channel, 250, 251 Clostridium botulinum, 137 Clostridium di f cile, 628–629, 635, 642 Clotrimazole, 666, 671t Cloxacillin, 651–652, 657t Clozapine, 52t, 143t, 218 , 220, 226t CM. See Chylomicrons CML. See Chronic myelogenous leukemia CMV. See Cytomegalovirus CNP. See C-type natriuretic peptide CNS. See Central nervous system CNS depressant-induced withdrawal severity, 322

CNS partial pressure (PCNS), 266, 267b CNT amily. See Concentrative nucleoside transporter amily CO. See Carbon monoxide; Cardiac output Coagulation cascade, 403–404, 405 , 407, 409 Coal worker’s pneumoconiosis (CWP), 915 Coating agents, 869 , 873, 876t Cocaethylene, 326 Cocaine abuse o , 309, 310t, 317, 323–325 pharmacologic treatment o , 328 alcohol and, 326 in catecholamine reuptake inhibition, 158, 163t dopamine hypothesis and, 216 dopaminergic transmission and, 121 as local anesthetic, 158, 167, 179, 181t locus ceruleus and, 118 in mesolimbic dopamine pathway, 316 neuroadaptation to, 312 –313 reticular activating system and, 116 reuptake inhibition with, 108 route o administration and, 317–318, 318 vaccine or, 330, 330 Cocaine Anonymous (CA), 327 Coccidioides immitis, 662–663 Cockayne syndrome, 728 Codeine, 51t, 89t, 90, 298–299, 304t, 310t Cognitive unction, ACh or, 136 Cognitive impairment, 139 COHb. See Carboxyhemoglobin Cohort studies, 950, 950 , 951 Colchicine, 741 , 896, 897t, 898–899, 899 , 902t Colesevelam, 352, 356t, 573, 577t Colestipol, 352, 356t Collagenase, 958, 967t Collecting duct, 369–370, 369 Collecting duct diuretics, 377–378, 384t Collectins, 792 Colloid, 516 Colloidal bismuth, 873, 876t Colon cancer, 727 Combination chemotherapy, 614–615 antimicrobials examples o , 773–775 MIC and MBC o , 770–772, 771t, 772 synergy, additivity, and antagonism interactions o , 772–773, 773 un avorable combinations o , 775 antineoplastic drugs, 724, 777t–778t examples o , 779–780, 780 , 780t general considerations or, 776–779, 778t rationale or, 779 or re ractory or recurrent disease, 780–781 antivirals, 775–776 anti-HIV drugs, 707b case study, 771 uture directions, 781 Combination estrogen–progestin contraception, 552–553, 552 , 553 , 559t Combinatorial chemistry, 924–925, 925 Common drug targets, o antimicrobial and antineoplastic drugs, 604t, 605–606 Common lymphoid stem cell, 784, 785 Common Technical Document (CTD), 942 Competitive antagonists, 7, 21–22, 22 , 26t Competitive neuromuscular blockade, 143–144 Complement, 790, 790t Complement activation inhibition, 848 , 856–857, 863t Complex partial seizure, 251 Compound-centered drug design, 922, 923t COMT. See Catechol-O-methyltrans erase Concentration-dependent bactericidal agents, 772, 772 Concentration e ect, 278 Concentrative nucleoside transporter (CNT) amily, 60t–61t, 61 Concentric hypertrophy, 491 Conditioned opponent response, 313 Conditioned tolerance, 311, 313 Conductance, 99, 99 Conduction block, 440 Con ounding, 951 Congenital adrenal hyperplasia (CAH), 536, 537

Conivaptan, 374, 381t, 494, 507, 513t Conjugation, 612–613 Conjugation/hydrolysis reactions, 34–35, 46t, 47, 49 Contact dermatitis, 78 Context-sensitive hal -li e, 40 Contractile dys unction, 459–461, 460 etiologies o , 489–490, 489 , 490 Contractile proteins, 458–459 Contractile state, 454 Contractility, 490, 490 Contrast media, 81 Controllers, asthma, 882 Convergent signaling, 117 Convergent synthesis, 930–931, 930 COPD. See Chronic obstructive pulmonary disease Coronary artery disease (CAD), chronic, 479–481, 480 , 481 clinical management o , 482–485 Coronary blood ow (CBF), 480, 481 Coronary ow reduction, 480–481, 480 , 481 Coronary ow reserve (CFR), 480 Corpus callosum, 114, 115 , 252 Corpus luteum, 545, 546 Cortical collecting duct (CCD), 365, 369, 369 Cortical spreading depression, 297 Corticosteroid-binding globulin (CBG), 525–526 Corticosteroids, 81, 521. See also Glucocorticoids or asthma, 878, 879b, 881, 883, 885, 887–890, 890t, 894t Corticosterone, 60t Corticotropin-releasing actor (CRF), antagonist approach to withdrawal and relapse, 330, 330 Corticotropin-releasing hormone (CRH), 123, 505, 528 Cortisol, 525–527 analogues o , 529, 530 synthetic modif cations to, 529–530, 529 Cortisol secretion, 512t Cortisone, 530 Costimulation o immune system, 785, 788–790, 789 inhibition o , 848 , 856, 862t Cosyntropin, 505, 512t, 976t Cough, with ACE inhibitors, 373 Cough variant asthma, 881 Coupling, 515 Covalent bonding, 4, 5t COX-1. See Cyclooxygenase-1; Cyclooxygenase enzymes COX-2. See Cyclooxygenase-2; Cyclooxygenase enzymes Cranial nerves, 116 Craniosacral system, 112, 113 Craving, 313 Cremophor, 78 Cretinism, 517 CRH. See Corticotropin-releasing hormone Critical volume hypothesis, 281 Crizotinib, 759, 766t Cromolyn, 823, 823t, 888, 894t Cross-dependence, 320 Crossover design, 938 Cross-tolerance, 327 Crotalidae polyvalent, 972t CS. See Choreoathetosis-with-salivation syndrome CsA. See Cyclosporine CTD. See Common Technical Document CTLA-4, 789, 857 C-type natriuretic peptide (CNP), 363–364, 363 13 C-urea breath test, 868 Current, 99, 99 Cushing’s disease, 503, 505–506 Cushing’s syndrome, 529 Cushing’s ulcers, 868 Cutaneous glucocorticoids, 532–533 CVD. See Cardiovascular disease CWP. See Coal worker’s pneumoconiosis Cyanide, 907 Cyclic AMP. See cAMP Cyclic endoperoxide, 404 Cyclic GMP. See cGMP Cyclin-dependent kinases (CDKs), 731

Index 999 Cyclizine, 824f, 829t Cyclobenzaprine, 143t Cyclooxygenase-1 (COX-1), 796, 796t Cyclooxygenase-2 (COX-2), 796, 796t, 807–808 Cyclooxygenase-2 (COX-2) inhibitors, 794–795, 796t, 797f, 811–812, 811f, 816t, 868 Cyclooxygenase enzymes (COX-1 and COX-2), 300, 300f inhibitors o , 414, 415f, 426t Cyclooxygenase inhibitors, 794–795, 808–812, 809f, 811f, 815t–816t, 868, 868f Cyclooxygenase pathway, 795–799, 796f, 796t, 797f, 798f, 798t Cyclophosphamide, 82, 89t, 736–737, 736f, 738f, 745t, 777t, 831 or immunosuppression, 850, 858t Cyclopropane, 270f Cycloserine, 607f, 644f, 649, 649f, 656t Cyclosporine (CsA), 51t, 59, 60t, 61t, 63t, 65t, 66t, 78, 89t, 665, 850–851, 851f, 858t, 899, 899f, 922, 984 Cy uthrin, 911 CYP2C9. See Cytochrome P450 2C9 CYP7A1. See Cholesterol 7 -hydroxylase Cypermethrin, 911 Cyproheptadine, 143t, 824f, 828t Cys-loop super amily, 282–283 Cysteinyl leukotrienes, 799, 800f, 802, 807 in asthma, 882, 884f Cystic f brosis, 958 Cystic f brosis transmembrane conductance regulator (CFTR), 63 Cysts, o Entamoeba histolytica, 682, 683f Cytarabine, 725f, 727f, 735, 735f, 745t, 778t Cytochrome P450 2C9 (CYP2C9), 92–93, 93f Cytochrome P450 2D6 (CYP2D6), 89–90, 89t, 90f, 91f Cytochrome P450 enzymes grape ruit juice e ect, 53 induction o , 49–50, 49f, 51t–52t inhibition o , 49f, 50, 51t–52t local anesthetic metabolism and, 176, 179 Cytochrome P450 enzyme system, 34, 47 Cytochromes, 525 o malarial plasmodia, 676–677, 677f Cytokine inhibitors, 294, 812, 816t, 848f, 852–854, 852f, 853f, 861t or asthma, 890, 891f Cytokine release, 531 syndrome, 855 Cytokines, 790t, 791 Cytomegalovirus (CMV), 702–706, 708, 709f, 720t Cytoprotective roles, 798 Cytosine, 622, 624f synthesis o , 724–725, 725f, 726f, 726t, 727f Cytotoxic agents, 610–611, 610f, 611f or immunosuppression, 848–850, 848f, 849f, 850f, 858t Cytotoxic T cells (TC cells), 786t, 787–789, 788f, 879, 880f

D

D1 dopamine receptor amily, 209, 210f D2 dopamine receptor amily, 209, 210f in schizophrenia, 209 d4T. See Stavudine DA. See Dopamine Dabigatran, 421f, 423, 430t Dabra enib, 753f, 760–761, 767t Dacarbazine, 736, 746t Daclizumab, 971t DAG. See Diacylglycerol D-Ala-D-Ala ligase B (DdlB), 644f, 645 Dalbavancin, 649–650, 656t Dal opristin, 612, 634f, 635–636, 640t Dalteparin, 422, 429t Dantrolene, 279 DAP. See Diaminopimelic acid Dapagli ozin, 62, 66, 572, 577t Dapsone, 89t, 617 Daptomycin, 81, 607f, 612, 654, 660t Darbepoetin al a, 831, 837, 841t, 956, 962t Dari enacin, 142, 143t, 148t

Darunavir, 609f, 696f, 712, 712f, 714, 721t Dasabuvir, 702, 709f, 710, 720t, 766t Dasatinib, 753f, 759–760 DAT. See Dopamine transporter Daunorubicin, 63t, 739f, 740, 747t, 984 Davis, Bernard, 631, 632f Davis model, 631 Dazoxiben, 812, 818t DCT. See Distal convoluted tubule ddC. See Zalcitabine ddI. See Didanosine DdlB. See D-Ala-D-Ala ligase B Dead-end complex, 647f, 650 Debrisoquine, 60t, 89, 89t DEC. See Diethylcarbamazine Decitabine, 735, 745t, 837–838, 841t Declaration o Helsinki, 936 Decoding, 627 Decoding center, 627 Deep vein thrombosis, 553 De erasirox, 908 De eroxamine, 908 Degarelix, 549, 556t, 968t Dehydroepiandrosterone (DHEA), 536–537, 540t Delamanid, 774 Delavirdine, 51t, 63t, 708–709, 709f, 720t Delayed a terdepolarizations, 438–439, 439f Delayed rectif er voltage-gated potassium channels, 104, 105f Delayed-type hypersensitivity, 78, 78t Delta-aminolevulinic acid dehydratase (ALA-D), 908 Delta cells, 561 Deltamethrin, 911 Delusions, schizophrenia, 216, 216b Demeclocycline, 374, 507, 632–633, 638t Dementia ACh and, 136 cholinergic agents or, 137–139 Dendritic cells, 786, 786t Denileukin di titox, 763, 769t, 777t, 972, 973t Denosumab, 594–595, 599t, 969t, 972t Dense-core secretory vesicles, 107 Dense-core synaptic vesicles, 107 Dentistry, local anesthetics in, 177b Deoxycorticosterone, 188 Deoxyribonuclease I (DNAse1), 958, 967t Dependence, 309–311, 313, 315, 315f Dephosphorylase, 644f, 646 Depolarized membrane, 99 Depolarizing blockade, 137, 140–141, 141t Depot glucocorticoids, 533–534 Depot pool, 130 Depression, 157, 231–233 lithium, 239 psychotic, 233 reuptake inhibitors or, 235–237 with substance use disorders, 329 Dermatomal distribution, 113 Descending inhibitory regulation, in spinal cord, 292–293, 292f Desensitization o drug–receptor interactions, 15, 16t o GABAA, 187, 187f Des urane, 279, 284t properties o , 268t in uptake model, 273, 273t, 274f Desipramine, 51t, 90, 143t, 233, 236, 243t, 306t, 329, 334t, 951 Desirudin, 421f, 423, 430t, 968t Desloratadine, 825–826, 829t Desmethyldiazepam, 192 Desmopressin, 377, 508, 508f, 513t Desogestrel, 553, 553f, 559t Desvenla axine, 237, 245t Detergents, 909 Detoxif cation, 327 Development chemistry, 930–931, 930f, 931f Dexamethasone, 81, 529–530, 530f, 531t, 539t, 742, 763, 858t or in ammation, 808, 816t Dex en uramine, 234, 953 Dexlansoprazole, 870–873, 871f, 872b, 875t

Dexmedetomidine, 158–159, 163t Dexrazoxane, 740 Dextroamphetamine, 234, 242t Dextromethorphan, 51t, 89t, 90, 302 DGAT. See Diacylglycerol acyltrans erase DHA. See Docosahexaenoic acid DHEA. See Dehydroepiandrosterone DHFR. See Dihydro olate reductase DHOD. See Dihydroorotate dehydrogenase DHT. See Dihydrotestosterone Diabetes insipidus, 377, 507–508 Diabetes mellitus, 531, 567–569, 567t protein therapeutics or, 956 therapy or, 569–573, 569t, 570t, 574t Diabetic ketoacidosis (DKA), 568 Diacylglycerol (DAG), 154 Diacylglycerol acyltrans erase (DGAT), 339 Diaminopimelic acid (DAP), 644f, 645 Diastolic heart ailure, 459, 489 Diazepam, 191–192, 203t, 263t abuse o , 310t clinical uses o , 192t ethanol and, 196 GABAA receptor and, 259 with general anesthetic, 280, 285t or withdrawal symptoms, 322f, 327, 333t Diazinon, 909–911, 910f Diazoxide, 573–574, 578t Diclo enac, 63t, 301, 306t, 809f, 810, 815t Dicloxacillin, 642, 650, 651–652, 657t Dicumarol, 418 Dicyclomine, 143b Didanosine (ddI), 703f, 719t Diencephalon, 114–115, 114f dopamine receptors in, 209 Diet drug metabolism and, 53 peptic ulcer disease and, 874 Dietary supplement, 945 Dietary Supplement Health and Education Act, 945 Diethylcarbamazine (DEC), 687, 693t Diethyl ether, 265, 279, 284t Meyer-Overton rule and, 270f properties o , 268t in uptake model, 273, 273t, 274f, 276f Di erential unctional blockade, 173 Di use Lewy body dementia (DLB), 136, 139 Di use system o organization, 117f, 118–119, 118f, 118t Di usion, o polymer-based delivery systems, 981–982, 982f Di usion hypoxia, 278 Digitoxin, 461, 463, 466t Digoxin, 50, 59, 60t, 61t, 63t, 64, 65t, 66, 454–456, 461–463, 461f, 462t, 466t, 493t, 923t Digoxin immune serum, 972t Digoxin toxicity, 442–443 Dihydroartemisinin, 679, 689t Dihydro olate reductase (DHFR), 725, 725f, 727f Dihydro olate reductase (DHFR) inhibitors, 615, 616f, 617–618, 617t, 684 Dihydroorotate dehydrogenase (DHOD), 677, 677f Dihydropteroate synthase inhibitors, 615–618, 616f Dihydropyridines, 61t, 390–391, 398t, 437, 447, 483 Dihydrotestosterone (DHT), 541–542, 544f testosterone conversion to, 542, 544f inhibitors o , 556t Dihydroxyphenylalanine (DOPA), 150–151, 152f Diiodotyrosine (DIT), 515 Diisopropyl uorophosphate, 137, 138f, 145t Dilated cardiomyopathies, 489 Diltiazem, 39, 51t, 390–391, 398t, 437, 447, 452t, 476, 483 Dimenhydrinate, 143t, 825, 828t Dimercaprol, 908, 908f Dimethylphenylpiperazinium, 131t Dimethyltryptamine (DMT), 310t Dipeptidyl peptidase-4 (DPP-4), 567 inhibitors, 569t, 577t Diphenhydramine, 60t, 72, 143b, 143t, 820, 823t, 824, 824f, 825, 828t Dipyridamole, 414–415, 416f, 426t Direct ligand-gated conductance, 130, 132f

1000 Index Directly Observed Therapy Short Course (DOTS), 774 Direct pathway, 211, 212 Direct thrombin inhibitors, 421 , 422–423, 430t, 486 Discovery biology, 928, 929t Discovery chemistry, 927–928, 928t Disodium cromoglycate, 888, 894t Disopyramide, 60t, 443, 449t Disorganized speech, schizophrenia, 216, 216b Dissociative anesthesia, 282 Distal convoluted tubule (DCT), 365, 367–369, 367 , 368t Distribution, 27, 32, 32t o inhaled anesthetics, 269 kinetics o , 33–34, 34 , 35 o local anesthetics, 176 volume o , 32–33, 33 Disulf ram, 52t, 328–329, 332t Disulf ram-like reaction, 654 DIT. See Diiodotyrosine Diuretics. See also specif c diuretics or heart ailure, 493–494, 493t or hypertension, 473t, 474–475, 474t, 477t nonreceptor-mediated mechanisms in, 16 sites o action o , 364–365, 364 Divergent signaling, 117 Dizocilpine, 60t DKA. See Diabetic ketoacidosis DLB. See Di use Lewy body dementia DMT. See Dimethyltryptamine DNA antineoplastic agents modi ying, 735–740, 736 , 737 , 738 , 739 , 745t–747t, 777t bacterial replication and segregation o , 623–624, 625 structure o , 622–623, 623 , 624 transcription o , 624, 626 translation o , 626–627, 626 , 627 ungal, synthesis o , 663–665, 664 , 670t repair o , 725–731, 728 , 729 , 730 DNA-dependent protein kinase, 728 DNA gyrase, 624, 628 DNA ligase III, 728, 729 DNA polymerase cancer biochemistry and, 725 viral, 702. See also Polymerase inhibitors DNA polymerase , 728, 729 DNAse1. See Deoxyribonuclease I Dobutamine, 159, 164t, 464, 467t, 493t, 495 Docetaxel, 741–742, 749t, 831 Docosahexaenoic acid (DHA), 354–355, 357t, 795, 796 , 799, 803 –805 Docosanol, 716, 722t Do etilide, 65t, 446, 451t Dolutegravir, 609 , 696 , 710, 710 –711 , 721t Domagk, Gerhard, 604, 619 Domoate, 250 Donepezil, 146t acetylcholine and, 123 AChE and, 139, 139t DOPA. See Dihydroxyphenylalanine DOPA decarboxylase. See Aromatic L-amino acid decarboxylase Dopamine (DA), 206, 922. See also Catecholamines adrenoceptors or, 153–155, 154t blood–brain barrier and, 124, 124 or cardiac contractility, 463–464, 466t central pathways o , 209–211, 211 growth hormone and, 503 inactivation, 207–209, 208 , 209 metabolism o , inhibitors o , 214 , 215, 222t–223t neurotransmission with biochemistry and cell biology o , 206–207, 208 schizophrenia and, 216 as neurotransmitter, 120, 120t, 121 in nigrostriatal pathways, 211–212, 212 physiologic and pharmacologic e ects o , 156 precursors o , 213–214, 214 , 222t prolactin and, 504, 504 receptors o , 209, 210 agonists, 214–215, 222t, 573, 578t TCAs at, 236

release o , 207–209, 208 reuptake o , 207–209, 208 storage o , 207–209, 208 substantia nigra and, 118, 118 , 118t synthesis o , 121–122, 122 , 150–151, 152 Dopamine- -hydroxylase, 121, 122 , 150–151, 152 , 159, 207, 208 Dopamine dysregulation syndrome, 215 Dopamine hypothesis, 216 Dopaminergic neurons, 118, 118 , 118t in Parkinson’s disease, 212–213, 212 Dopamine transporter (DAT), 207, 230, 324 Doripenem, 654, 659t Dorsal columns, 112–113 Dorsal horn, 167, 168 descending and local inhibitory regulation in, 292–293, 292 pain transmission in, 291–292, 292 Dorsal root ganglia, 112, 112 Dorsal roots, 112, 112 Dose–response, 939 Dose–response curves, o anesthetics, 267, 268 Dose–response relationships, 18–20, 19 , 20 Dosing, 39–41, 39 , 40 , 41 DOTS. See Directly Observed Therapy Short Course Double-blind study, 938 Double-blind trials, 939 Double-strand break repair, 728–730, 728 , 729 Down-regulation, 15, 15 , 16t Doxazosin, 160, 164t, 401t, 475 Doxepin, 143t, 243t, 825, 829t Doxercalci erol, 596, 601t Doxil®. See Liposomal doxorubicin Doxorubicin (Hydroxydaunorubicin, Adriamycin), 13, 63t, 81, 740, 747t, 777t, 778t, 831, 984 Doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), 780 Doxycycline, 13, 631 , 632–633, 638t, 677, 680, 687 Doxylamine, 825 D-Phenylalanine derivatives, 576t DPP-4. See Dipeptidyl peptidase-4 Dronedarone, 65t, 447, 452t Droperidol, 218 , 225t, 280 Drospirenone, 552–553, 559t Drug approval li e cycle o , 935 process o , 942–944, 942 Drug biotrans ormation, 43 Drug delivery, 979 case study or, 980 uture directions or, 984–985 intelligent, 983–984 liposome-based, 984 oral, 979–980 polymer-based, 981–983, 982 , 983 pulmonary, 980–981 targeting, 984 transdermal, 981 Drug development, 921, 921 drug transporters and, 67, 68b–69b key disciplines in, 927–931 mechanisms to expedite, 942 phases o , 926, 927b or rare diseases, 940–941 success ul, 941, 941t Drug discovery, 919, 921 case study or, 920–921 drug transporters and, 67, 68b–69b uture directions or, 932 key disciplines in, 927–931 process or, 921–922 compound-centered approach, 922, 923t lead optimization, 926 target-centered approach, 922–926, 924b, 925t, 925 Drug–drug interactions, 35 with digoxin, 462 drug transporters and, 64–66, 65t metabolic, 53–54 pharmacodynamic, 75–76 pharmacokinetic, 75 o war arin, 418–419, 419t, 420t

Drug-eluting stent, 487 Drug evaluation, 933–935 case study or, 934 clinical trial authorization, 936–937 ethics in, 936 Drug–herb interactions, 76 Drug-induced cardiovascular toxicity, 81 Drug-induced hepatotoxicity, 79–80, 80 Drug-induced neurotoxicity, 81 Drug-induced pulmonary toxicity, 82 Drug-induced renal toxicity, 80–81 Drug-induced skeletal muscle toxicity, 81 Drug labeling, 943 Drug metabolism, 43 active and toxic metabolites, 50 conjugation/hydrolysis reactions, 46t, 47, 49 disease e ect on, 54 actors a ecting, 50–54 induction and inhibition o , 49–50, 49 , 51t–52t interactions in, 53–54 oxidation/reduction reactions, 45t, 47, 48 pathways o , 44 sites o , 43, 44 variation in enzymes o , 88–91, 89t, 90 , 91 Drug naming, 943–944 Drug overdose, 75 Drug Price Competition and Patent Term Restoration Act o 1984, 944 Drug–receptor interactions a f nity o , 3, 5t binding impact o , 5–6 mechanism o , 17–18, 19 types o , 3–4, 5t cellular regulation o , 15, 15 , 16t con ormational states and antagonists, 20–23, 21 , 22 inverse agonists, 24 partial agonists, 23–24, 23 spare receptors, 24–25, 25 major types o , 6–7, 7 , 7t membrane e ects on, 6 protein structure and, 2–3, 4 selectivity o , 6 signal processing rom, 14–15, 14 Drug receptors. See Receptors Drugs, 2 adverse e ects o , 2 con ormation and chemistry o , 2–4, 4 ion channels and, 105 receptor interactions with. See Drug–receptor interactions resistance to antimalarial agents, 678, 678 , 681 antimycobacterial agents, 655 antineoplastic drugs, 612–614, 613t, 724, 778, 778t antiviral drugs, 613t, 614, 697–698 beta-lactams, 613, 613t, 651 mechanisms o , 612–614, 613t, 646, 650 practices promoting, 614 selectivity o , molecular and cellular determinants o , 6 therapeutic e ects o , 2 Drug sa ety, 946–948 Drug-seeking, 309, 317, 317 Drugs o abuse, 308, 310t. See also Substance use disorders alcohol, 323, 323 , 324 barbiturates, 322–323, 322 benzodiazepines, 322–323, 322 ca eine, 325 cocaine and amphetamine, 323–325 inhalants, 326 marijuana, 324 , 325 methylenedioxymethamphetamine, 325 nicotine and tobacco, 323 opioids, 316 , 319–320, 320 , 321 phencyclidine, 325 Drug study duration and post-approval, 948 size and generalizability, 946 surrogate outcomes and comparators, 947–948

Index 1001 Drug toxicity, 70–71 case study or, 71 contexts o cellular mechanisms, 76, 77f drug overdose, 75 early detection and prediction o , 84–85 uture directions or, 85, 85t mechanisms o , 71–75, 72f, 73f treatment or, 83–84 Drug transport, 47–49 Drug transporters, 56, 57f clinical perspectives on, 64–67, 65t, 66t drug discovery and development and, 67, 68b–69b drug–drug interactions and, 64–66, 65t e ux, 58, 58f, 62–64, 63t uptake, 58–62, 58f, 60t–61t Drug trapping, 33, 33f Dry powder inhalers, 889 Dubin-Johnson syndrome, 63 Duloxetine, 237, 244t, 293, 296, 301, 306t Durham-Humphrey Amendment, 944–945 Dutasteride, 547, 548f, 549, 556t Dynamic instability, 731, 732f Dynorphins, 123, 293 Dysbetalipoproteinemia, 348 Dyskinesias amantadine or, 215 with levodopa, 214 Dyslipidemias, 347–349, 349t pharmacologic classes and agents or, 349–350, 349t, 350t Dysthymia, 231 Dystonia, 137, 142

E

Early-acting growth actors. See Multilineage growth actors Early a terdepolarizations, 438, 439f EAST syndrome, 367, 368t Eaton-Lambert syndrome, 137–138 Ebastine, 829t EC50. See Potency o drug Ecallantide, 971t Eccentric hypertrophy, 491 EC coupling. See Excitation-contraction coupling ECF. See Extracellular uid ECG. See Electrocardiogram Echinocandins, 607, 662, 664f, 668, 673t selective targeting by, 605 Econazole, 666, 671t ECP. See Eosinophilic cationic protein Ectopic beat, 438 Ectopic calcif cation, 586 Ectopic rhythm, 438 Eculizumab, 856–857, 863t, 970t Edema histamine causing, 820 pathophysiology o , 370–372, 371f Edematous states, 376 Edetate disodium, 908, 908f Edinger-Westphal nucleus, 112, 113f EDRF. See Endothelium-derived relaxing actor Edrophonium, 114, 137–138, 138f, 145t EEG. See Electroencephalogram E alizumab, 79, 970t E avirenz, 51t, 61t, 63t, 608, 609f, 695, 696f, 707b, 708, 709f, 720t E ective re ractory period, 441b E f cacy o drug (Emax), 19, 19f E ux proteins, 56 E ux pumps, o tetracycline-resistant microbes, 633 E ux transporters, 58, 58f, 62–64, 63t E ornithine, 685, 692t EGFR. See Epidermal growth actor receptor EGF receptor antagonists, 610, 758–759, 765t Egress, viral, 696, 696f Eicosanoids, 790–791, 790t case study, 795 drug classes targeting, 808–813, 809f, 811f, 813f drug summary table, 815t–818t uture directions, 813–814

metabolism and physiology, 794 arachidonic acid and omega-3 atty acid generation, 795, 796f cyclooxygenase pathway, 795–799, 796f, 796t, 797f, 798f, 798t epoxygenase pathway, 795, 796f, 802 inactivation o local eicosanoids, 802 in ammation schema, 802, 806, 806f, 806t isoprostanes, 796f, 802 lipoxygenase pathway, 795, 796f, 799–802, 799t, 800f, 801f, 803f–805f pathophysiology o , 806–808 Eicosapentaenoic acid (EPA), 354–355, 357t, 795, 796f, 799, 803f–805f EKG. See Electrocardiogram Elacridar, 63t, 64, 65t Elagolix, 549 Elderly patients, anticholinergic drugs and, 143b Electrical potential di erence, 99 Electrical transmission, 98 Electrocardiogram (ECG, EKG), 436–437, 437b, 437f Electrochemical gradient, 100, 101f Electrochemical transmission neurotransmitter metabolism and reuptake, 108–109 postsynaptic receptors, 107–108 sequence o , 105–107, 106f synaptic vesicle regulation, 107 Electroencephalogram (EEG) absence seizures on, 253, 255f ocal seizures on, 251–252 Electrogenic transport, 101, 101t Electron transport chain, o malarial plasmodia, 676–677, 677f inhibitors o , 679–680, 690t Electroporation, 981 Electrostatic orce, 100, 101f Eletriptan, 238, 247t, 307t Elimination hal -li e, 38 Elongation, bacterial translation, 626–627 Elongation actors, bacterial, 626–627 Elosul ase al a, 961t Eltrombopag, 839, 842t Elvitegravir, 609f, 696f, 710, 710f–711f, 721t EMA. See European Medicines Agency Emax. See E f cacy o drug Emergency contraception, 554 Emesis, 211 EMLA. See Eutectic mixture o lidocaine and prilocaine Empagli ozin, 572, 577t Emphysema, 879b Emtricitabine (FTC), 63t, 604, 615, 695, 703f, 706–707, 707b, 710, 719t ENaC. See Epithelial sodium channel Enalapril, 60t, 373, 380t, 402t, 476 Enantiomers, 74 Encainide, 444, 450t, 947 Endocannabinoid signaling, 323, 330–331 Endocrine axis, 501 Endocytosis, 28 Endogenous metabolite tra f cking, drug inter erence with, 66, 66t Endogenous pathway o lipoprotein metabolism, 340 Endometriosis, 547 Endoneurium, 172, 173f Endorphins, 123 Endothelial dys unction, 481 Endothelial injury, 411 Endothelin, 404 Endothelin-1 (ET-1), 389, 390f Endothelin receptor antagonists, 396, 401t Endothelium-derived relaxing actor (EDRF), 389 End-plate potential (EPP), 107, 132, 135f End-systolic pressure-volume relationship (ESPVR), 490, 490f Energy homeostasis, 561–563, 563t, 564f Energy repletion, 562–563, 564f En urane, 52t, 279, 284t Meyer-Overton rule and, 270f properties o , 268t in uptake model, 273, 273t En uvirtide (T-20), 609f, 696f, 698, 699f, 718t, 966, 971t

Enkephalins, 123, 293 Enoxacin, 52t Enoxaparin, 422, 429t Entacapone, 215, 223t Entamoeba histolytica, 682–683, 683f, 684f Entecavir, 703f, 707–708, 719t Enteral drug administration, 30–31, 30t Enteral ormulation, 931, 932t Enteric coating, 931 Enterohepatic circulation, 37, 62, 346 ENT amily. See Equilibrative nucleoside transporter amily Entry, viral, 695, 696f Entry inhibitors, 609f, 696f, 698, 698b, 699f, 718t Envelope, viral, 695 Environment drug metabolism and, 53 vascular tone and, 388 Environmental toxicology, 905 acute and subchronic toxicology, 905–911, 906f, 908f, 910f carcinogenicity and chronic toxicology, 911–916, 911t, 912f, 913f case study or, 906 uture directions or, 917 Enzalutamide, 547, 548f, 551, 558t Enzyme activity assays, 928 Enzymes, 12 Eosinophil-derived neurotoxin, 882 Eosinophilic cationic protein (ECP), 882, 883f Eosinophil peroxidase, 882 Eosinophils, 785, 786t in asthma, 881–882, 883f, 884f EPA. See Eicosapentaenoic acid Ephedrine, 157–158, 884 EPI. See Epinephrine Epidermal growth actor receptor (EGFR), 750–751, 751f, 752t, 753f inhibitors o , 610, 758–759, 765t Epidural anesthetic, 177 Epigenetic changes, in carcinogenesis, 608 Epigenetics, 93–94, 93f Epilepsy, 249. See also Seizures drugs or, 120 GABA signaling in, 185 glutamatergic pathways and, 199 Epinephrine (EPI), 10, 89t, 206, 823, 823t, 878, 884, 892t. See also Catecholamines adrenoceptors or, 153–155, 154t or cardiac contractility, 464, 467t with lidocaine, 168 as neurotransmitter, 120–121, 120t, 121f physiologic and pharmacologic e ects o , 155–156 synthesis o , 121–122, 122f, 150–151, 152f Epineurium, 172, 173f Epipodophyllotoxins, 625f, 740–741, 777t Epirubicin, 747t Epithelial sodium channel (ENaC), 369, 377 Epitope, 786 Eplerenone, 377, 384t, 397, 474t, 488, 536, 540t EPO. See Erythropoietin Epoetin al a, 837, 841t, 940 Epoprostenol, 396, 401t, 817t Epoxygenase pathway, 795, 796f, 802 EPP. See End-plate potential EPSPs. See Excitatory postsynaptic potentials Eptif batide, 14, 404, 417, 427t Equilibrative nucleoside transporter (ENT) amily, 60t–61t, 61 Equilibrium time constants, 287 ER. See Estrogen receptor ERCC1, 728 EREs. See Estrogen response elements Ergocalci erol, 584, 597, 601t Ergosterol inhibitors targeting synthesis o , 662, 663f, 665–667, 671t polyene binding to, 667–668 Ergot alkaloids, 238 Ergotamine, 238, 302 Ergotism, 238 Eribulin, 741, 748t, 922 Erlotinib, 65, 605, 751, 753f, 758, 765t

1002 Index Ertapenem, 654, 659t Erythema, histamine causing, 820 Erythrocytes, 830 production o , 833–834, 834f, 835t agents stimulating, 836–837, 841t Erythromycin, 51t, 54, 60t, 623, 633–634, 634f, 635f, 639t combination chemotherapy using, 773 selective targeting by, 605 Erythropoiesis, 833–834, 834f, 835t Erythropoiesis-stimulating agents (ESAs), 837, 841t Erythropoietin (EPO), 779, 833–834, 834f, 924b, 925t, 956, 958, 962t recombinant human, 837, 841t ESAs. See Erythropoiesis-stimulating agents ESBLs. See Extended-spectrum beta-lactamases Escape beat, 438 Escape rhythm, 438 Escitalopram, 236 E site. See Exit site Eslicarbazepine, 257 Esmolol, 160t, 161, 166t, 521 Esomeprazole, 74, 870–873, 871f, 872b, 875t ESPVR. See End-systolic pressure-volume relationship Essential hypertension, 472 Estazolam, 191, 202t clinical uses o , 192t Ester-linked LAs, 178–179, 181t hypersensitivity to, 178 metabolism o , 176 structure o , 171, 171f Estradiol, 984 17 -Estradiol, 63t, 542, 592f Estramustine, 737, 746t Estrogen receptor (ER), 542 antagonists, 557t Estrogen response elements (EREs), 542 Estrogens, 89t, 542, 549t decreased secretion o , 548 prolactin and, 504 replacement o , 554–555, 554t synthesis o , 541–542, 543f Estrone, 63t Eszopiclone, 191 ET-1. See Endothelin-1 Etanercept, 14, 795, 812, 816t, 852–853, 853f, 859t, 890, 966, 969t Ethacrynic acid, 376, 383t, 474t Ethambutol, 607f, 654–655, 660t, 771, 773 Ethanol, 52t, 89t abuse o , 310t cocaine and, 326 GABA physiology and, 195–196 methanol and, 54 toxicity o , 913–914 war arin and, 420t Ethinyl estradiol, 552, 552f, 559t Ethionamide, 654, 660t, 774 Ethnicity, drug metabolism and, 53 Ethosuximide, 256t, 258, 262t Ethynodiol, 553, 559t Etidronate, 592f Etodolac, 810, 815t Etomidate, 195, 204t, 280, 285t Etonogestrel, 553–554, 559t Etoposide (VP-16), 63t, 89t, 724, 740–741, 748t, 777t, 778t Etoricoxib, 812 Etravirine, 708–709, 709f, 720t European Commission, 943 European Medicines Agency (EMA), 943 Eutectic mixture o lidocaine and prilocaine (EMLA), 168, 176, 180, 182t Everolimus, 12, 487, 573–574, 578t, 753f, 761, 767t, 851, 852f, 859t, 984 Evolocumab, 355, 357t Excipients, 979 Excitability. See Cellular excitability Excitation-contraction (EC) coupling, 455 Excitatory amino acid transporter, 198 Excitatory neurotransmitters, 184–185, 185f Excitatory postsynaptic potentials (EPSPs), 106 in autonomic ganglia, 133, 135f

Excitotoxicity, 198 glutamate receptors in, 199, 199f Excretion, 27, 35–366 biliary, 37 renal, 36–37, 36f Exemestane, 547, 548f, 549, 556t Exenatide, 572, 576t, 964t Exenatide-Er, 576t Exit channel, RNA polymerase, 624 Exit (E) site, 626–627, 626f, 627f Exit tunnel, ribosome, 627 Exogenous glucagon, 575t Exogenous insulin, 575t Exogenous pathway o lipoprotein metabolism, 339 Extended-release ormulations, 979 Extended-spectrum beta-lactamases (ESBLs), 651 External advisory committee, 943 Extracellular enzymes, 13–14 Extracellular uid (ECF), 358 Extracellular potential, 99 Extractable lipids, mycobacterial, 646 Extraction ratio, 38 Extrapyramidal e ects, 217–218 Extrapyramidal symptoms, 142 Extrinsic genes, or antibiotic resistance, 651 Extrinsic pathways, 408, 409f Ezetimibe, 337, 352–353, 357t Ezogabine, 256t, 258, 262t

F

Fabry disease, 958 Facilitated di usion, 28, 123 Factor II, 408, 409f Factor IIa, 405, 408, 409f Factor IX, 408, 409f, 924b, 958, 960t Factor V, 408, 409f Factor VII, 408, 409f Factor VIIa, 958, 964t Factor VIII, 924b, 958, 959t Factor V Leiden, 412, 413t Factor X, 408, 409f Factor XII, 408, 409f Factor XIII, 960t Famciclovir, 703f, 705, 719t Familial adenomatous polyposis, 811 Familial combined hyperlipidemia (FCHL), 348 Familial de ective apoB100, 347 Familial hemiplegic migraine (FHM), 297 Familial hypercholesterolemia (FH), 347 homozygous, 352 Familial hypertriglyceridemia, 348 Familial lipoprotein lipase def ciency, 348 Famotidine, 829t, 869–870, 870f, 875t Farnesyltrans erase inhibitors (FTIs), 753f, 760 FAS. See Fetal alcohol syndrome FAS1. See Fatty acid synthetase 1 FAS2. See Fatty acid synthetase 2 Fast acetylator, 52 Fasting, 563 Fast Track, 942 Fat group (FG), 270–271, 271f Fatty acid synthetase 1 (FAS1), 646, 648f Fatty acid synthetase 2 (FAS2), 646, 648f Fc RI. See High-a f nity IgE receptors FCHL. See Familial combined hyperlipidemia FDA (US Food and Drug Administration), 933 adverse drug e ects and, 953 approval, 943 review process by, 942–943, 942f FDAAA. See FDA Amendments Act FDA Amendments Act (FDAAA), 935, 953 FDA Sa ety and Innovation Act (FDASIA), 935 FDASIA. See FDA Sa ety and Innovation Act Febuxostat, 900, 902t Fed state, 562–563, 564f Felbamate, 120, 199, 205t, 256t, 260, 263t Felodipine, 51t, 390, 398t FemA, 644f, 645 FemB, 644f, 645 Femoral nerve blocks, 177 FemX, 644f, 645 Fenamate NSAIDs, 809f, 810, 815t Fenestrae, 123, 124f

Fen uramine, 74, 81, 234 Fen uramine/phentermine, 949t Fenof brate, 352–354, 357t Fentanyl, 280, 285t, 299, 305t Fenthion, 909, 910f Fermentation pathways, o Entamoeba histolytica, 682–683, 684f Ferredoxins, 683, 684f Fesoterodine, 142, 143t, 148t Fetal alcohol spectrum disorder, 326 Fetal alcohol syndrome (FAS), 136, 914 Fetal hemoglobin (HbF), agents inducing, 837–838, 841t Fexo enadine, 60t, 63t, 65t, 74, 825–826, 829t FG. See Fat group FGF-23. See Fibroblast growth actor 23 FH. See Familial hypercholesterolemia FHM. See Familial hemiplegic migraine Fibrates, 352–354, 357t Fibrinogen, 406–407, 960t Fibrinoid arteriolar necrosis, 478 Fibrinolysis inhibitors, 424, 431t Fibrinolytic system, 412f Fibrin sealant, 964t Fibroblast growth actor 23 (FGF-23), 584t, 586 Fibrosis, 82 Fick’s law, 269–270, 287 Fidaxomicin, 607f, 612, 626f, 628, 629–630, 629f, 636, 637t 50S ribosomal subunit, 626–627, 626f, 627f antimicrobial drugs targeting, 630t, 633–636, 634f, 635f, 639t–640t Filgrastim, 79, 84, 838, 842t, 962t Finasteride, 51t, 547, 548f, 549, 556t First-generation H1-antihistamines, 823–826, 824f, 828t–829t First-order kinetics, 37, 38f First pain, 169, 170f First-pass e ect, 43 First-pass metabolism, 31 5S rRNA, 626–627, 626f, 627f FK506. See Tacrolimus FLAP. See 5-Lipoxygenase-activating protein Flavoxate, 143t Flecainide, 51t, 444, 450t, 947 Fleming, Alexander, 641 Flocculonodular lobe, 115, 115f Flow chemistry, 931, 931f Flow rate, 270 FLT3 inhibitors, 760 Fluconazole, 52t, 420t, 607, 662, 663f, 666–667, 671t Flucytosine, 616f, 663–665, 664f, 670t, 774–775 Fludarabine, 725f, 727f, 734f, 735, 744t Fludrocortisone, 529–530, 530f, 536, 540t Fluid f ltration, 359, 360f Flumazenil, 84, 187f, 203t, 280 Flunisolide, 532, 533f, 539t, 887, 894t Flunitrazepam, 196 Fluoride, 595 5-Fluorocytosine, 607 Fluoroquinolones, 74–75, 623, 628, 628f, 775 or MDR-TB, 774 5-Fluorouracil (5-FU), 664, 664f, 725f, 727f, 733, 733f, 743t, 777t, 778t, 779 resistance to, 613 selective targeting by, 606, 616f Fluoxetine, 39, 51t, 52t, 90, 108–109, 158, 228, 236, 244t, 301, 329, 334t, 420t, 951 Flupentixol, 218f Fluphenazine, 143t, 217, 218f, 219, 224t Flurazepam, 191, 203t clinical uses o , 192t Flurbipro en, 810, 815t Flutamide, 547, 548f, 551, 555, 558t, 777t Fluticasone, 532, 533f, 539t, 878, 883, 887, 889, 894t Fluvastatin, 351, 356t Fluvoxamine, 52t, 236, 244t Met. See Formylated methionine Foam cells, 342, 343f Focal seizure, 250 classif cation o , 252t pathophysiology o , 251–252, 253f Folate, 724, 725f, 726f

Index 1003 Folate metabolism inhibitors, 610f, 611f, 777t uture directions in, 618–619 or malaria plasmodia, 681, 690t–691t selective targeting and synergistic drug interactions o , 615–618, 616f, 617t Folic acid, 615, 616f. See also Folate metabolism inhibitors Folinic acid, 733 Folinic acid rescue, 618 Follicle-stimulating hormone (FSH), 500, 500t, 506–507, 506f, 958, 963t Follicular phase, 545, 546f Follicular thyroid cells, 514 Follitropin, 507, 513t Fomivirsen, 717, 722t Fondaparinux, 421f, 422, 429t Food, Drug, and Cosmetic Act, 935 Food and Drug law, history o , 935–936, 935f Food-borne illnesses, 908 Food contaminants, 908–909 Formestane, 547, 548f, 549, 556t Formoterol, 879b, 885, 892t Formulation, 931, 932t Formylated methionine ( Met), 626–627, 627f Fosamprenavir, 712, 712f, 721t Foscarnet, 708, 709f, 720t Fos omycin, 607f, 643, 644f, 648–649, 656t Fosinopril, 60t, 380t Frank-Starling law, 456, 490, 490f Frank-Starling mechanism, 490–491, 491f FRC. See Functional residual capacity Frontal cortex, dopamine receptors in, 209 Frontal lobes, 114, 115f Frovatriptan, 238, 247t FSH. See Follicle-stimulating hormone FTC. See Emtricitabine FTIs. See Farnesyltrans erase inhibitors 5-FU. See 5-Fluorouracil Full agonists, 7, 20, 26t Fulvestrant, 547, 557t Functional residual capacity (FRC), 271 Fungal in ections case study, 662 drugs targeting. See Anti ungals uture directions, 668 membrane and cell wall biochemistry o , 661–662, 663f pathophysiology o , 662–663 Fungi, toxic, 909 Fungicidal e ect, 662, 771 Fungistatic e ect, 662, 771 Furocoumarins, 909 Furosemide, 61t, 63t, 65t, 376, 383t, 455, 474t, 475, 493 GABAA receptor and, 187f Fusion, viral, 697f

G

G6PD def ciency. See Glucose-6-phosphate dehydrogenase def ciency GABA. See Gamma-aminobutyric acid GABAA channels, alcohol and, 323 GABAA-mediated chloride conductance, 187, 187f GABAA receptors, 186–188, 187f agonists, 189t, 190, 202t antagonists, 189t, 190, 202t benzodiazepines and barbiturates and, 322, 322f ethanol and, 196 modulators, 189t, 190–195, 202t–204t GABAB receptors, 188, 188f agonists, 189t, 195, 204t antagonists, 195 GABA channel potentiators, 263t or substance use disorders, 333t GABAC receptors, 188 GABAergic transmitters, o helminths, 686–687 GABA-mediated inhibition, 259 Gabapentin, 256t, 258, 262t, 289, 291, 296, 301–302, 307t, 333t GABA transaminase (GABA-T), 186 GABA transporters (GATs), 186 Gabazine, 189t, 190, 202t

Gaboxadol, 189t, 190 GAD. See Glutamic acid decarboxylase G agents, 142b G i. See Inhibitory G protein Galantamine, 139, 139t, 146t Gallbladder disease, 553 Galsul ase, 961t Gamma-aminobutyric acid (GABA), 9 in dorsal horn, 292–293, 292f in helminths, 686–687 as inhibitory signal, 250 metabolism o , 186, 186f inhibitors o , 189–190, 189t, 202t neurotransmission with, 184–185, 185f ethanol and, 195–196 pharmacologic classes and agents or, 187f, 189, 189t physiology o , 185 as neurotransmitter, 119–121, 120t, 121f receptors or, 186 ionotropic, 186–188, 187f metabotropic, 188, 188f release inhibition o , 189t synthesis inhibition o , 189t transport o , 186, 186f inhibitors o , 189–190, 189t, 202t Gamma-hydroxybutyric acid (GHB), GABA physiology and, 196 Gamma-vinyl GABA. See Vigabatrin Ganciclovir, 60t, 63t, 67, 703f, 705, 706, 706t, 719t, 984 Ganglionic blockade, 133, 135f, 136t, 137 Ganglionic blockers, 473t, 476 Ganirelix, 507, 513t, 549, 556t, 972t Gas concentration equations, 287 Gasotransmitter, 392 Gastric acid secretion, 864–867, 866f agents decreasing, 869–873, 869f, 870f, 871f, 872b, 875t agents neutralizing, 869f, 873, 875t–876t Gastrins, 864–865, 866f, 867f, 868 as neuropeptides, 123 Gastrointestinal motility modulation, 137, 238 Gastrointestinal stromal tumor (GIST), 759 Gastropathy, NSAID-induced, 808–809, 811, 865, 868, 868f, 870, 873 GATs. See GABA transporters GCP. See Good clinical practices G-CSF. See Granulocyte colony-stimulating actor Gef tinib, 92t, 753f, 758, 765t Gemcitabine, 61, 67, 735, 745t Gemf brozil, 59, 60t, 61t, 352–354, 357t Gemi oxacin, 628, 637t Gemtuzumab ozogamicin, 763, 769t, 777t, 972, 973t Gender, drug metabolism and, 53 Gene expression inhibition o , or immunosuppression, 847–848, 848f, 858t viral, 695 inhibitors o , 696f, 700–702, 701f, 703f, 718t General anesthetics, 52t, 265. See also Inhaled anesthetics adjuvant drugs, 280 in cortex, 114 equations or, 287 intravenous, 284t–285t pharmacology o , 280, 280f structures o , 281f ion channel e ects o , 282–283, 282f mechanisms o action o , 281–282, 281f structures o , 281f General growth actors. See Multilineage growth actors Generalized seizure, 250 classif cation o , 252t pathophysiology o primary, 253, 253f, 255f secondary, 252–253, 253f, 254f Generic drugs, 944 Generic name, 943–944 Gene therapy, 465 Genetic changes, in carcinogenesis, 608 Genetic disorders, 412, 413t

Genetic drug resistance, 651 mechanisms o , 612–614, 613t Genome human, synthesis, stability, and maintenance o , 724–732, 725f, 726f, 726t, 727f, 728f, 729f, 730f, 731f, 732f viral replication o , 695–696, 696f, 697f inhibitors o , 696f, 702–710, 703f, 704f, 706t, 707b, 709f, 710f–711f, 718t–721t Genomic variation and pharmacogenomics, 87 Gentamicin, 80, 630, 631f, 638t selective targeting by, 605 Geranyl-geranylation, 73 Gestodene, 553, 553f, 559t GFR. See Glomerular f ltration rate GH. See Growth hormone GHB. See Gamma-hydroxybutyric acid Ghrelin, 501–502, 502f GHRH. See Growth hormone-releasing hormone Ginkgo biloba, 76 GIST. See Gastrointestinal stromal tumor Gitelman syndrome, 367, 368t Glaucoma, 137, 375 Glibenclamide, 61t, 63t, 66t Gliclazide, 576t Glimepiride, 576t Glinides, 572 Glipizide, 576t Global equilibration, 270 Globus pallidus, 114, 115f, 211 Globus pallidus pars externa, 211 Glomerular dys unction, 372 Glomerular f ltration rate (GFR), 364, 462 Glomerulonephritis, eicosanoid pathophysiology in, 807 Glossopharyngeal nerve, 112, 113f GLP-1. See Glucagon-like peptide-1 Glucagon, 503, 561, 566, 574, 579t, 976t Glucagon-like peptide-1 (GLP-1), 562 receptor agonists, 572, 576t -(1,3)-D-Glucan, 662 -(1,6)-D-Glucan, 662 -(1,3)-D-Glucan synthase, 662 Glucarpidase, 966, 967t Glucocorticoid receptor agonists, 539t antagonists, 534, 539t type II, 526–527 Glucocorticoid response elements (GREs), 527 Glucocorticoids, 524 analogues o , 529 calcium and phosphate and, 586 cancer treatment with, 611f dosing o , 531–532 duration o action o , 530–531, 531t excess o , 529 or gout, 896, 897t, 899, 902t or immunosuppression, 845, 847–848, 858t or in ammation, 795, 808, 816t pathophysiology o , 528–529 physiology o , 525–528, 526f, 527f, 528f replacement therapy or, 531 routes o administration, 532–534, 533f structure and potency o , 529–530, 529f, 530f synthesis o , 525, 526f inhibitors o , 534, 534t, 540t Glucose, blood–brain barrier and, 123, 124f Glucose-6-phosphate dehydrogenase (G6PD) def ciency, 680 Glucose transporter (GLUT) amily, 60t–61t, 61–62 Glucosylceramidase, 956 Glucuronide conjugates, 63t GLUT2, 564 Glutamate acamprosate and, 329 local anesthetics and, 175–176 metabolism o , 186f, 196 neurotransmission with, 184–185, 185f pathophysiology and pharmacology o , 198–200, 199f, 200f physiology o , 196–198, 197f, 197t, 198t as neurotransmitter, 119–121, 120t, 121f in pain transmission, 291, 292f

1004 Index Glutamate (continued) receptors o in excitotoxicity, 199, 199f inhibitors o , 259–260, 263t–264t ionotropic, 193f, 196–197, 197t metabotropic, 197–198, 197f, 198t transporters o , 198 Glutamate-gated chloride channels, o helminths, 686–687 Glutamic acid decarboxylase (GAD), 186, 186f Glutathione, 680, 736 Glutathione and glucuronide conjugates, 63t GLUT amily. See Glucose transporter amily Glyburide, 60t, 63t, 65t, 576t Glycan polymerization, 644f, 645–646 inhibitors o , 649–650, 656t Glycine in dorsal horn, 292–293, 292f as neurotransmitter, 119–121, 120t, 121f Glycohemoglobin (HbA1c), 569 Glycoprotein IIb–IIIa (GPIIb–IIIa), 406–407, 406f antagonists, 417, 427t–428t, 485f, 486 Glycoprotein VI (GPVI), 404, 406f Glycopyrrolate, 141, 147t Glycylcyclines, 612, 626f, 627f, 628, 630t, 631f, 632–633, 639t targets o , 607f Glycylproline, 60t GM-CSF. See Granulocyte-macrophage colonystimulating actor; Granulocyte-monocyte colony-stimulating actor GMP. See Good Manu acturing Practice GnRH. See Gonadotropin-releasing hormone GnRH receptor agonists, 507 antagonists, 507 Goiter, 240, 517 Goitrogens, 519f, 520 Goldman-Hodgkin-Katz equation, 102 Golimumab, 812, 816t, 853, 860t, 969t Gonadal hormones calcium and phosphate and, 586 inhibitors o , 548–552, 548f, 549t, 550f, 551f Gonadal maturation, 512t–513t Gonadorelin, 512t, 556t Gonadotrophs, 506–507, 506f Gonadotropin-releasing hormone (GnRH), 123, 501, 965t agonists, 548–549, 548f, 556t analogues, 512t, 982 antagonists, 548–549, 548f, 556t, 972t prolactin and, 504 Gonadotropins, 506–507, 512t–513t Good clinical practices (GCP), 936 Good Manu acturing Practice (GMP), 944 Goserelin, 512t, 549, 556t, 965t Gout, 375 case study, 896 drug classes treating, 898–901, 899f, 900f drug summary table, 902t–903t uture directions, 901 pathophysiology o , 897–898, 897t, 898f purine metabolism, 895–897, 896f GPCRs. See G protein-coupled receptors GPCR-specif c protein kinases (GRKs), 155 GPIIb–IIIa. See Glycoprotein IIb–IIIa G protein-coupled receptors (GPCRs), 9–11, 9f, 10f, 26, 108 adrenoceptors as, 153 in cardiac contractility, 458 in di use system o organization, 118–119, 118f muscarinic cholinergic receptors, 127 serotonin, 230, 230t G proteins, 9, 26 catecholamine receptors and, 153 muscarinic receptors and, 130 second messengers and, 9–10, 10f GPVI. See Glycoprotein VI Graded dose–response relationships, 18–19, 19f Gra t-versus-host disease (GVHD), 844, 846 Gram-negative bacteria, 642–643, 642f Gram-positive bacteria, 642–643, 642f Grandiosity, 231–232 Grand mal seizure, 252t

Granulocyte colony-stimulating actor (G-CSF), 779, 833, 835, 958, 962t recombinant human, 831, 838, 842t Granulocyte-macrophage colony-stimulating actor (GM-CSF), 958, 962t Granulocyte-monocyte colony-stimulating actor (GM-CSF), 779, 832–833, 835 recombinant human, 838, 842t Granulocytes, 295, 784–785, 830, 958 Granulosa cells, 544, 545f Granzymes, 787 Grape ruit juice, 51t Grape ruit juice e ect, 53 Graves’ disease, 515, 518, 518f Gray baby syndrome, 53, 635 Grepa oxacin, 75 GREs. See Glucocorticoid response elements Griseo ulvin, 664f, 665, 670t GRKs. See GPCR-specif c protein kinases Growth actor receptor antagonists, 611, 757–761, 765t–767t, 778t Growth actor receptors, cancer biochemistry and, 750–751, 751f, 752t, 753f, 756–757, 756t, 757f Growth actors cancer biochemistry and, 750–751, 751f, 752t, 753f, 756–757, 756t, 757f hematopoietic, 830–836, 831t, 832f, 834f, 835t, 836f Growth raction, 609–610 Growth hormone (GH), 500, 500t, 959t def ciency o , 502–503, 502f excess o , 502f, 503–504, 510t in hypothalamic-pituitary-growth hormone axis, 501–502, 502f replacement o , 510t Growth hormone-releasing hormone (GHRH), 123, 501–503, 502f, 975, 976t Guanabenz, 158, 163t, 476 Guanadrel, 157, 162t Guanethidine, 157, 162t, 476 Guan acine, 158, 163t Guanidine, 60t Guanine, 622, 624f synthesis o , 724–725, 725f, 726f, 726t, 727f Guanylyl cyclase, 363 GVHD. See Gra t-versus-host disease Gynecomastia, 536

H

H1-antihistamines, 823–826, 824f, 828t–829t H1 receptor, 821, 821t H2 receptor, 821, 821t, 864 H2 receptor antagonists, 826, 826f, 829t, 865, 869–871, 869f, 870f, 875t H3 receptor, 821, 821t H3 receptor antagonists, 826 H4 receptor, 821t, 822 H4 receptor antagonists, 826 HAART. See Highly active antiretroviral therapy Hageman actor, 408, 409f Hal -li e, 38–39, 38t Halichondrin, 922 Hallucinations, schizophrenia, 216, 216b Halo antrine, 678f Haloperidol, 51t, 72–73, 90, 217, 218f, 219, 219f, 225t Halothane, 52t, 89t, 267, 279, 284t Meyer-Overton rule and, 270f properties o , 268t recovery rom, 279f in uptake model, 273, 273t, 274f, 276f Haptens, 76 Harm ul immune responses, 76–79, 78t Hashimoto’s thyroiditis, 518, 518f Hatch-Waxman Act, 944 HbA1c. See Glycohemoglobin HbF. See Fetal hemoglobin HBsAg. See Hepatitis B sur ace antigen HBV. See Hepatitis B virus hCG. See Human chorionic gonadotropin HCl. See Hydrochloric acid HCV. See Hepatitis C virus HD. See Hodgkin’s disease HDL. See High-density lipoprotein

Headache, 137, 236. See also Migraine headaches Health Canada, 943 Healthy user e ect, 952 Heart, 433 electrical dys unction o , 438–440, 439f, 440f electrical physiology o cardiac action potentials, 433–437, 435f, 436f, 436t, 437b, 437f f ring rate determination, 437–438 pacemaker and nonpacemaker cells, 433 Heart ailure (HF), 454 a terload reduction or, 494–495 case study or, 488 clinical management o , 492–495, 493t combination therapy or, 495 diastolic, 459, 489 doxorubicin causing, 740 Frank-Starling relationship in, 490–491, 491f inotropic agents or, 495 pathophysiology o , 459–461, 460f, 488–492, 489f, 489t, 490f, 491f, 492f preload reduction or, 493–494 sodium retention and, 370–371, 371f systolic, 459, 489 Heart rate, 454, 490 Helicobacter pylori, in peptic ulcer disease, 865, 867–868, 867f, 870, 874 Helminths case study, 685 drugs targeting, 686–687 physiology o , 685–686, 686f Helper T cells (TH cells), 786t, 787–789, 788f in asthma, 879, 880f, 881–882, 883f Hemagglutinin, viral, 698–699, 700f Hematocrit, 833 Hematopoiesis case study, 831 drug classes targeting, 836–839 drug summary table, 841t–843t uture directions, 840 physiology o , 830–836, 831t, 832f, 834f, 835t, 836f Hematopoietic growth actors, 830–833 Heme metabolism, o malarial plasmodia, 676, 676f inhibitors o , 677–679, 678f, 679f, 689t Heme protein monooxygenases, 47 Hemicholinium-3, 128, 137, 145t Hemoglobin, 676, 676f, 833. See also Fetal hemoglobin Hemoglobinopathies, 833 Hemolysis, primaquine causing, 680 Hemophilia A, 413b, 958 Hemophilia B, 958 Hemorrhagic disorders, 412–414, 413b, 413t Hemostasis, 403 physiology o , 403–404, 405f primary, 403, 405–407, 406f, 407f, 408f regulation o , 410–411, 411f secondary, 403–404, 407–410, 409f vasoconstriction, 404 Heparan sul ate proteoglycans, 342 Heparin, 23, 88, 925t or acute coronary syndromes, 485f, 486–487 clinical uses o , 422 mechanism o action o , 420–422, 421f structure o , 420 Heparin-induced thrombocytopenia (HIT), 412, 413t, 422 Hepatic lipase, 342 Hepatitis B immune globulin, 971t Hepatitis B sur ace antigen (HBsAg), 974, 974t Hepatitis B virus (HBV), 697, 716, 958 nucleoside and nucleotide analogues targeting, 703f, 707–708, 719t vaccine or, 974 Hepatitis C antigens, 976t Hepatitis C virus (HCV), 695–696, 716–717, 958 nonnucleoside RNA polymerase inhibitors targeting, 709f, 710, 720t NS5A inhibitors targeting, 709f, 710, 720t nucleoside and nucleotide analogues targeting, 703f, 708, 720t protease inhibitors targeting, 696f, 700–702, 701f, 701f, 718t

Index 1005 Hepatorenal re ex, 371, 371f Hepatotoxicity, drug-induced, 79–80, 80f Hereditary nonpolyposis colon cancer, 727 Heroin, 24, 123, 309, 310t, 320, 328 Herpesviruses, 697, 708, 709f, 720t nucleoside and nucleotide analogues targeting, 702–706, 703f, 704f, 718t–719t Hesperidin, 60t, 65t, 66 Heterologous desensitization, 15, 16t Heteroreceptors, 134 Heterozygous amilial hypercholesterolemia, 337 HETEs. See Hydroxyeicosatetraenoic acids Hexahydrosiladi enidol, 131t Hexamethonium, 132, 135f, 151, 476 Hexose transporter, 123 HF. See Heart ailure; Hydro uoric acid HGPRT. See Hypoxanthine-guanine phosphoribosyltrans erase HIF-1 . See Hypoxia-inducible actor-1 High-a f nity IgE receptors (Fc RI), 882, 883f, 889 High-density lipoprotein (HDL), 336, 337t, 338 delivery o , to liver, 344, 345f, 346 intravascular maturation o , 344, 345f metabolism o , 342–346, 345f disorders o , 348 High innate tolerance, 318 Highly active antiretroviral therapy (HAART), 707b High-threshold mechanical nociceptors, 169 High-throughput screening, 924 High-voltage-activated (HVA) calcium channel, inhibition o , 258 Himbacine, 131t Hippocampus, 115, 115f dopamine receptors in, 209 Hirudin, 423 Hirudin-related agents, 430t His–Purkinje conduction system, 462 Histamine, 89t, 790, 790t, 882, 883f case study, 820 drug classes targeting, 823–826, 823t, 824f, 826f drug summary table, 828t–829t uture directions, 827 in gastric acid secretion, 864–865, 866f as neurotransmitter, 120, 120t, 121f pathophysiology o , 822–823, 822f physiology o actions, 819–821, 821t receptors, 821–822, 821t synthesis, storage, and release, 819, 820f receptors o , TCAs at, 236 single-source divergent neuronal systems and, 118t, 119 synthesis o , 122–123 vascular tone and, 388f, 389f, 390 Histidine decarboxylase, 819, 820f Histrelin, 512t, 556t, 965t HIT. See Heparin-induced thrombocytopenia Hit, drug discovery and, 921 Hit-to-lead development, 924 HIV. See Human immunodef ciency virus HIV antigens, 976t HMG-CoA reductase, 22, 342 inhibitors o , 350–352, 351f or chronic CAD, 484 Hodgkin’s disease (HD), combination chemotherapy or, 779–780, 780t HoFH. See Homozygous amilial hypercholesterolemia Homatropine, 143t Homologous desensitization, 15, 16t Homologous recombination, 727, 729f, 730 Homovanillic acid (HVA), 209, 209f Homozygous amilial hypercholesterolemia (HoFH), 352 Horizontal transmission, 612 Hormone modulators, or cancer, 777t Hormone replacement therapy (HRT), 554–555, 554t, 560t, 590–591, 599t HPA axis. See Hypothalamic-pituitary-adrenal axis HPETEs. See Hydroperoxyeicosatetraenoic acids

HPV. See Human papillomavirus HRT. See Hormone replacement therapy 5-HT. See Serotonin Hu f ng, 326 Human albumin, 962t Human chorionic gonadotropin (hCG), 546, 958, 963t Human growth hormone, 925t Human immunodef ciency virus (HIV), 694–695 CCR5 receptor and, 606, 698, 698b, 699f drugs targeting. See Anti-HIV drugs drug therapy preventing, 604 li e cycle o , 695–697, 697f postexposure prophylaxis or, 615 Humanized therapeutic antibodies, 853 Human papillomavirus (HPV), 716–717 vaccine or, 974, 974t Human solute carrier (SLC), 28 Humoral immunity, 786t, 787–788, 788f Humoral regulators, 388f, 389f, 390 Huntington’s disease, 198 HVA. See Homovanillic acid HVA calcium channel. See High-voltage-activated calcium channel Hyaluronidase, 967t Hydralazine, 53, 78, 88, 89t, 392, 399t, 473t, 476, 495 Hydrochloric acid (HCl), 909 Hydrochlorothiazide, 376, 383t, 474–475, 474t, 493, 509 Hydrocodone, 298–299, 304t Hydrocortisone, 81, 525, 526, 531t, 539t Hydro umethiazide, 383t Hydro uoric acid (HF), 909 Hydrogen bonds, 3, 5t Hydromorphone, 298–299 Hydroperoxyeicosatetraenoic acids (HPETEs), 799, 801f Hydrophilic protein segments, 3 Hydrophobic e ect, 4 Hydrophobicity, 4 o antidepressants, 235 benzodiazepines and, 190 o local anesthetics, 171–172, 172f Hydrophobic protein segments, 3 Hydrostatic pressure, 359, 360f Hydroxocobalamin, 907 Hydroxyapatite, 580–581 Hydroxydaunorubicin. See Doxorubicin Hydroxyeicosatetraenoic acids (HETEs), 799, 801f 11 -Hydroxysteroid dehydrogenase, 526, 527f 5-Hydroxytryptamine. See Serotonin 5-Hydroxytryptophan, 228, 229f Hydroxyurea, 725f, 727f, 734, 744t, 777t, 837–838, 841t Hydroxyzine, 143t, 824, 825, 829t Hyoscine hydrobromide, 141 Hyoscyamine products, 143b Hyperacute rejection, 844–845, 845t Hyperaldosteronism, 535–536 Hyperalgesia, 199–200, 294–295 Hypercalcemia, 376, 593b associated with malignancy, 592 Hypercholesterolemia, 347, 349t Hypercoagulability, 412–414, 413t Hyperglycemia, 506 Hyperinsulinemia, 573–574 Hyperkalemia, 373, 376–377 Hyperlipidemia, 347–349 secondary, 348–349, 349t Hyperparathyroidism, 531 secondary, 587, 588t, 589, 596 Hyperpolarized membrane, 99 Hyperreactivity, in asthma, 881, 881f Hyperresponsiveness, in asthma, 881, 881f Hypersalivation syndrome, 142 Hypersensitivity, 73f, 293 Hypersensitivity reactions. See also Allergic reactions in asthma, 881–882, 881f, 883f, 884f penicillin, 652, 652f Hypersensitivity responses, 76–78, 78t Hypertension action potentials and, 105 cardiac unction in, 472

case study or, 470 clinical management o , 473–479, 473f, 473t essential, 472 neuroendocrine unction in, 473 pathophysiology o , 470–472, 471f renal unction in, 472–473 secondary, 472 treatment o , 471f vascular unction in, 472 Hypertensive crisis, 478–479 with catecholamines, 235 Hypertensive emergency, 478–479 Hypertensive urgency, 479 Hyperthyroidism, 505, 515, 518, 518f treatment o , 519–521 Hypertriglyceridemia, 347–348, 349t Hypertrophy, 491 Hyperuricemia, 375 Hyperventilation, anesthetics and, 275–276 Hypnozoites, 676, 676f Hypoaldosteronism, 535 Hypocalcemia, 593b Hypoglycemia, 569 insulin-induced, 503 Hypogonadism, 548 Hypokalemia, 462 Hypomanic episode, 232 Hypopituitarism, 503 Hypotension, 156–157, 373 Hypothalamic-pituitary-adrenal (HPA) axis, 505–506, 506f, 529 Hypothalamic-pituitary-gonadal axis, 506–507, 506f Hypothalamic-pituitary-growth hormone axis, 501–504, 502f Hypothalamic-pituitary portal vascular system, 498, 500f Hypothalamic-pituitary-prolactin axis, 504–505, 504f Hypothalamic-pituitary-reproduction axis, 544–545, 544f, 545f disruption o , 547 Hypothalamic-pituitary-thyroid axis, 504f, 505, 517, 518f Hypothalamus, 115 dopamine receptors in, 209 eedback inhibition o , 500t, 501, 501f pituitary gland relationship with, 115, 498–501, 500f, 500t Hypothyroidism, 240, 515, 518, 518f treatment o , 519 Hypoventilation, anesthetics and, 275, 276f Hypoxanthine-guanine phosphoribosyltrans erase (HGPRT), 734, 895–897, 896f Hypoxia-inducible actor-1 (HIF-1 ), 757, 757f, 834, 834f

I

IB. See Investigator’s Brochure Ibandronate, 592–594, 592f, 599t Ibritumomab tiuxetan, 763, 972, 973t Ibrutinib, 79, 760, 766t IBS. See Irritable bowel syndrome Ibupro en, 52t, 78, 89t, 168, 301, 306t, 309, 795, 809f, 810, 815t, 896, 902t, 951 Ibutilide, 446, 451t Icatibant, 971t Idelalisib, 13 Idiopathic hypereosinophilic syndrome, 759 Idiosyncratic toxicity, 75 IDL. See Intermediate-density lipoprotein Idoxuridine, 719t Idursul ase, 961t IECs. See Independent Ethics Committees IFN -2a. See Inter eron alpha-2a IFN -2b. See Inter eron alpha-2b IFN -n3. See Inter eron alpha-n3 IFN- . See Inter eron gamma-1b I os amide, 737, 746t IgE, in asthma, 881–882, 883f IgE-mediated type I hypersensitivity reaction. See Allergic reactions IGF-1. See Insulin-like growth actor 1 IHD. See Ischemic heart disease I B. See Inhibitor o NF B

1006 Index IL-1. See Interleukin-1 IL-1 antagonists, 901 IL-1 cytokine and IL-1 receptor inhibitors, 854 IL-2. See Interleukin-2 IL-3. See Interleukin-3 IL-5. See Interleukin-5 IL-6. See Interleukin-6 IL-6 receptor inhibitor, 854 IL-12/IL-23p40 cytokine inhibitors, 854, 860t IL-17 cytokine and IL-17 receptor inhibitors, 854 Iloperidone, 220, 226t Iloprost, 396, 401t, 817t ILs. See Interleukins Imatinib, 60t, 63t, 65, 65t, 605, 753f, 759–760, 764, 766t, 934, 940 binding interactions o , 4–5, 5f, 12–13 Imatinib mesylate, 750 Imciromab pentetate, 976t IMiD. See Immunomodulatory drug Imidapril, 380t Imidazoles, 662, 663f, 664f, 665–667, 671t, 775 Imiglucerase, 940 Imipenem, 75, 654, 659t Imipramine, 51t, 52t, 90, 143t, 158, 163t, 233, 236, 243t, 301, 306t Imiquimod, 716–717, 722t, 786 Immediate hypersensitivity, 76, 78t Immune cell depletion, 848f, 857, 861t–862t Immune checkpoints, 789, 857 inhibition o , 857, 863t Immune complex-mediated hypersensitivity, 76, 78, 78t Immune diversity, 787 Immune responses, 76–79, 78t Immune system adaptive immunity, 786–790, 786t, 787f, 788f, 789f airway and, 879, 880f cell development, 784, 785f, 835–836 innate immunity, 784–786, 786t Immune system modulators, antiviral actions o , 716–717, 722t Immunization. See Vaccination Immunoglobulin, 787 Immunomodulatory drug (IMiD), 763 or cancer, 777t, 839, 843t Immunosuppressants, 51t, 294 Immunosuppression case study, 845 drugs classes targeting, 846–857, 848f, 849f, 850f, 851f, 852f, 853f drug summary table, 858t–863t uture directions, 857 pathophysiology autoimmunity, 846, 847t transplantation, 844–846, 845t Immunotoxicity, 79 IMP. See Inosine monophosphate Impulse conduction de ects, 439–440, 439f, 440f Impulse ormation de ects, 438–439, 439f Inactivated state, o sodium channel, 250, 251f Inactivation, 8–9, 15, 15f, 16t, 104 Inactive state, 20 Inamrinone, 459f, 464–465, 468t, 493t, 495 Increased intracranial pressure, 375 Incretins, 567, 569t, 572, 576t IND. See Investigational New Drug Indacaterol, 885, 892t Indapamide, 383t, 474t Independent Ethics Committees (IECs), 936 Indinavir, 51t, 63t, 712, 712f, 721t Indirect pathway, 211, 212f Indole acetic acid derivatives, 301 Indomethacin, 63t, 301, 306t, 809f, 810, 815t, 898, 902t Induced f t, 5–6 Inducible nitric oxide synthase (iNOS), 461 Induction therapy, 855 In ectious disease. See Antimicrobial drugs In erior cervical ganglion, 111 In erior mesenteric ganglion, 111, 113f Inf ltration anesthesia, 177, 177b In ammation, 294, 783 in asthma, 881–882, 883f, 884f case study, 784

chemical mediators o , 790–791, 790t chronic, 792 eicosanoids in, 802, 806, 806f, 806t uture directions, 792–793 histamine role in, 821–823, 822f immune system and, 784–790, 785f, 786t, 787f, 788f, 789f In ammatory bowel disease, 966 eicosanoid pathophysiology in, 807 In ammatory response, 791–792, 792f In iximab, 14, 812, 816t, 853, 853f, 860t, 890, 969t In uenza viruses, drugs targeting, 608, 609f, 696f, 698–700, 700f, 715, 715f, 718t, 721t In ormed consent, 936 In ranodal conduction system, 462 INH. See Isoniazid Inhalants, 310t, 326 Inhaled anesthetics agents or, 284t pharmacodynamics o , 265–266, 267f Meyer-Overton rule, 267–268, 269b minimum alveolar concentration, 266, 267b, 268t therapeutic and analgesic indices, 266–267, 268f pharmacokinetics o , 269 applications o uptake model, 275–277, 276f, 277f, 277t, 278f recovery rom, 277–278, 279f respiratory physiology concepts, 269–270 uptake model, 269–274, 271f, 272f, 273t, 274f, 275f pharmacology o , 278–279 structures o , 281f Inhaled glucocorticoids, 532, 533f Inhaled nitric oxide gas, 394, 400t Inhalers, 889 Inhibins, 507, 545 Inhibitor o NF B (I B), 756, 756f Inhibitory G protein (G i), 461 Inhibitory neurotransmitters, 184–185, 185f Inhibitory postsynaptic currents (IPSCs), 187 Inhibitory postsynaptic potentials (IPSPs), 106 in autonomic ganglia, 133, 135f o GABA, 187 Initiation, bacterial translation, 626, 627f Initiation actors, bacterial, 626 Initiators, 82 Innate immunity, 784–786, 786t Innate tolerance, 311, 318 Inner medullary collecting duct, 369 Inorganic phosphate, 597, 601t iNOS. See Inducible nitric oxide synthase Inosine monophosphate (IMP), 724, 725f, 726f, 727f Inositol triphosphate (IP3), 154 Inotropic agents, 495 INR. See International normalized ratio Insomnia, 191 antihistamines or, 825 Inspired partial pressure (PI), 266, 267b alveolar partial pressure and, 271–272, 272f Institutional Review Boards (IRBs), 936 Insulin, 52t, 569t, 922, 924b, 925t, 958, 959t biochemistry o , 563, 565f as neuropeptides, 123 release o , 561 secretion o , 564–566, 565f target tissue action o , 566, 566f Insulin analogues, 5 Insulin aspart, 570, 570t, 575t Insulin detemir, 570t, 571, 575t, 959t Insulin glargine, 570t, 571, 575t, 959t Insulin glulisine, 570, 570t, 575t Insulin-induced hypoglycemia, 503 Insulin-like growth actor 1 (IGF-1), 501–504, 502f, 510t Insulin lispro, 570, 570t, 575t Insulin receptors, 11f, 12, 566 Insulin receptor substrate (IRS) proteins, 11f, 12 Insulin resistance, 568 Insulin sensitizers, 575t, 577t Insulin theory, 547 Integrase, 705 Integrase inhibitors, 609f, 696f, 710, 710f–711f, 721t

Integration, viral, 696–697, 697f Integrins, 14 Intelligent drug delivery, 983–984 Intercalated cells, 369 Intercellular adhesion molecules (ICAMs), 791, 792f Intercellular communication, 98 Intercellular signal transduction, cancer biochemistry and, 750–757, 751f, 752t, 753f, 754f, 755f, 756f, 756t, 757f Intercostal nerve blocks, 177 Inter eron, 958 Inter eron-alpha, 701–702, 716, 722t, 777t Inter eron alpha-2a (IFN -2a), 963t Inter eron alpha-2b (IFN -2b), 963t, 982 Inter eron alpha-n3 (IFN -n3), 963t Inter eron-beta, 716 Inter eron beta-1a, 963t Inter eron beta-1b, 963t Inter eron-gamma, 716, 879, 880f Inter eron gamma-1b (IFN- ), 963t Inter erons, 836 antineoplastic applications o , 839, 843t Interictal spikes, on EEG, 252 Interleukin-1 (IL-1), 854, 860t Interleukin-2 (IL-2), 777t, 835–836, 879, 880f, 964t antineoplastic applications o , 839, 843t Interleukin-3 (IL-3), 832–833 Interleukin-5 (IL-5), 835, 879, 880f Interleukin-6 (IL-6), 854, 879, 880f Interleukin-7 (IL-7), 835–836 Interleukin-11 (IL-11), 962t recombinant human, 839, 842t Interleukin-17 (IL-17), 854 Interleukins (ILs), 790t, 791, 833, 835–836 in asthma, 879, 880f, 881–882, 883f, 887 Intermediate-density lipoprotein (IDL), 337t, 342 Intermediolateral columns, 111 International Con erence on Harmonization, 936 International normalized ratio (INR), 420 Interscalene block, 177 Intra-articular administration, 533–534 Intracellular bacteria, 651 Intracellular calcium, 386–387, 387f Intracellular communication, 98 Intracellular drug concentration, reduced, 613, 613t Intracellular enzymes, 12–13 Intracellular potential, 99 Intracellular receptors, 12–13, 13f Intracellular signal transduction, cancer biochemistry and, 750–757, 751f, 752t, 753f, 754f, 755f, 756f, 756t, 757f Intraocular pressure, 138 Intrathecal anesthetic, 177 Intravascular volume determinants o , 358–359, 360f reduction o , 474–475, 474t Intravenous general anesthetics, 284t–285t pharmacology o , 280, 280f structures o , 281f Intravenous regional and systemic anesthesia, 177–178 Intrinsic genes, or antibiotic resistance, 651 Intrinsic pathways, 408, 409f Inverse agonists, 7, 20, 24, 26t, 823, 824f Inverse tolerance, 309 Investigational New Drug (IND), 936–937, 937f Investigator’s Brochure (IB), 937 Investigator Sponsored INDs, 937 Inward current, 99 Iodide uptake, 511t inhibitors o , 519–520, 519f, 523t Iodine, 514 thyroid hormone and, 519f, 520, 523t Iodoquinol, 683 Iodothyronine 5 -deiodinase, 515f, 516 Ion channel blockers, 609f, 696f, 698–700, 700f Ion channels, 7–9, 8f, 8t in cellular communication, 98 electrical circuit model o , 100, 100f general anesthetic e ect on, 282–283, 282f inactivation period o , 8–9, 104 pharmacology o , 105 selectivity o , 100–102, 100f, 101f, 101t, 102f state-dependent block o , 440–441 voltage dependence o , 103, 104f

Index 1007 Ionic current, 103 Ionic interactions, 3–4, 5t Ionization state (pKa), 4 o lidocaine, 179 o local anesthetics, 172 Ionotropic GABA receptors, 186–188, 187f Ionotropic glutamate receptors, 193f, 196–197, 197t Ionotropic receptors, 106, 107–108 Ion pumps, 101 Ions, Nernst potentials or, 101, 101t, 130 Iontophoresis, 981 IP3. See Inositol triphosphate Ipilimumab, 857, 863t, 969t Ipodate, 521, 523t Ipratropium, 112, 141–142, 148t, 878, 886, 893t Iproniazid, 158, 163t, 233, 234, 242t IPSCs. See Inhibitory postsynaptic currents IPSPs. See Inhibitory postsynaptic potentials Irbesartan, 52t, 381t IRBs. See Institutional Review Boards Irinotecan, 63t, 66t, 89t, 740, 747t, 978 Irreversible antagonists, 21, 21f Irritable bowel syndrome (IBS), 238 IRS proteins. See Insulin receptor substrate proteins Isavuconazole, 666–667, 671t Ischemic heart disease (IHD) case study or, 479 classif cation o , 479, 480f clinical management o , 482 pathophysiology o , 479–482, 480f, 481f, 482f Ischemic stroke, 199, 199f Islets o Langerhans, 561 Isocarboxazid, 234, 242t Isoetharine, 884, 892t Iso urane, 52t, 266, 279, 284t dose–response curves o , 267, 268f Meyer-Overton rule and, 270f properties o , 268t in uptake model, 273, 273t, 274f Iso urophate, 138f Isolated systolic hypertension, 470 Isoniazid (INH), 52, 52t, 78, 88, 89t, 189t, 629, 648f, 654–655, 660t, 771, 773–774 targets o , 607f Isoprostanes, 796f, 802 Isoproterenol, 159, 164t, 464, 467t, 884, 892t Isosorbide dinitrate, 53, 393–394, 394f, 399t Isosorbide 5-mononitrate, 393–394, 399t Isotretinoin, 82 Isradipine, 398t Itching, antihistamines or, 825 Itraconazole, 51t, 607, 666, 671t Ivabradine, 447–448, 453t Ivaca tor, 978 Ivermectin, 685–687, 693t Ixekizumab, 854

J

Jacksonian march, 252 JAK2 inhibitors, 753f, 760, 767t JAK-STAT pathway, 752, 753f JGA. See Juxtaglomerular apparatus Jimson weed, 909 Juxtaglomerular apparatus (JGA), 360

K

K . See Potassium Kainate receptors, 193f, 196–197, 197t Kanamycin, 630, 638t, 773–774 Kaposi’s sarcoma, 958 KCTD subunits. See Potassium channel tetramerization domain subunits Ke auver-Harris Amendments, 935 Keratinocyte growth actor (KGF), 965t Kernicterus, 47, 617 Ketamine, 60t, 237, 280, 285t, 295–296, 302, 307t Ketanserin, 238, 247t Ketoconazole, 50, 51t, 61t, 63t, 75, 505, 534, 534t, 540t, 666, 671t Ketolides, 626f, 627f, 628, 630t, 633–635, 634f, 635f, 639t targets o , 607f Ketone NSAIDs, 809f, 810, 815t Ketopro en, 810, 815t

Ketorolac, 301, 306t, 810, 815t KGF. See Keratinocyte growth actor Kidney ailure. See Renal ailure Kininase II, 362 Kinins, 291 KIT ligand. See Stem cell actor Kolliphor®, 78

L

LABAs. See Long-acting beta-agonists Labetalol, 160t, 161, 165t, 445, 451t, 475 Lacosamide, 256t, 257–258, 261t Lactase, 961t Lactic acidosis, 571 Lactotrophs, 209, 504 LAL. See Lysosomal acid lipase LAL-D. See Lysosomal acid lipase def ciency Lamellae, 582 Lamivudine (3TC), 60t, 63t, 615, 695, 703f, 707–708, 707b, 712, 719t Lamotrigine, 199, 205t, 239, 247t, 256t, 257, 261t, 307t Lanreotide, 503, 510t, 965t Lansoprazole, 52t, 870–873, 871f, 872b, 875t Lapatinib, 758–759, 765t, 779 Laplace’s law, 491 Laronidase, 961t LAs. See Local anesthetics L-asparaginase, resistance to, 613t Latanoprost, 817t Late, slow EPSP, in autonomic ganglia, 133, 135f Late-acting growth actors. See Lineage-specif c growth actors Latency, viral, 702–704 Latent pacemakers, 438 Laterodorsal tegmental area, 323 LCAT. See Lecithin:cholesterol acyltrans erase LDL. See Low-density lipoprotein LDL receptor, 342 LDL receptor-related protein (LRP), 342 L-DOPA. See Levodopa Lead drug discovery and, 921 poisoning with, 907–908, 908f Lead optimization, 926 Learned tolerance, 311 Lebrikizumab, 890, 891f Lecithin:cholesterol acyltrans erase (LCAT), 344, 345f Ledipasvir, 609f, 708, 710, 720t Le unomide, 848–850, 850f, 858t Legionella pneumophila, 623 Leishmaniasis, 685 Lenalidomide, 763, 769t Lennox-Gastaut syndrome, 259–260 Lepirudin, 421f, 423, 430t, 966, 968t Leptin, 562 Lesch classif cation, 319 Lesch-Nyhan syndrome, 897 Lesinurad, 901 Letrozole, 547, 548f, 549, 556t Leucovorin, 618, 733 Leucovorin rescue, 779 Leukocytes production o , 835–836 agents stimulating, 838, 842t recruitment and activation o , suppressors o , 898–899, 899f, 902t Leukotriene B4 (LTB4), 879b Leukotriene inhibitors, 812–813, 813f, 888–889, 890t, 894t Leukotriene receptor antagonists, 813, 813f, 818t, 878 Leukotrienes, 795, 796f, 799, 800f, 802, 806–808, 806f, 806t in asthma, 882, 883f, 884f Leukotriene synthesis inhibitors, 813 Leuprolide, 507, 512t, 549, 556t, 777t, 965t Levalbuterol, 884, 892t Levamisole, 839, 843t Levetiracetam, 256t, 260, 264t Levobunolol, 161, 165t Levobupivacaine, 179 Levocabastine, 829t

Levocetirizine, 825, 829t Levodopa (L-DOPA), 89t, 922 blood–brain barrier and, 123–124, 124f in catecholamine synthesis, 121, 122f, 206, 208f dopamine precursors and, 222t NMDA receptor and, 199 or Parkinson’s disease, 213–214, 214f protein and, 111 striatum and, 114 Levo oxacin, 60t, 75, 628, 628f, 637t, 774, 874 Levonorgestrel, 552, 554, 559t, 981–982, 982f Levosimendan, 465, 468t Levothyroxine, 519, 523t Leydig cells, 544, 545f LFA-3, 855 LH. See Luteinizing hormone LH hypothesis, 547 LH surge, 545 Library o compounds, 924 Lidocaine, 89t, 168, 171f, 173, 177, 179, 181t as antiarrhythmic, 178, 443–444, 450t case study o , 266 in dentistry, 177 dosing o , 41 inf ltration anesthesia, 177 intravenous, 178 metabolism o , 43 on-target adverse e ect with, 72 or pain management, 302 peripheral vasculature and, 178 Li e cycle o Entamoeba histolytica, 682, 683f o helminths, 686, 686f o malarial plasmodia, 674–676, 676f viral, 695–697, 696f, 697f Ligand-binding domain, 8, 8f Ligand-gated conductance, direct, 130, 132f Ligand-gated ion channels, 8, 8f, 8t general anesthetic e ect on, 282–283, 282f nicotinic cholinergic receptors, 127 serotonin, 230, 230t Ligand-gated ionotropic receptors, 107 Limbic system, 115, 115f dopamine receptors in, 209 Linaclotide, 965t Linagliptin, 572, 577t Lincosamides, 626f, 627f, 628, 630t, 634f, 635, 635f, 639t targets o , 607f Lineage-specif c growth actors, hematopoietic, 832–833 Linear bone growth, 532 Linezolid, 612, 634f, 636, 640t, 774 Linoleic acid, 795, 796f Liothyronine, 523t Lipid II, 644f, 645 Lipid-lowering agents, or chronic coronary artery disease, 484 Lipids, 336 deposits o , 337 f brates and, 353, 353f niacin and, 354, 354f Lipid solubility hypothesis, 281 Lipocortins, 795, 808 Lipopeptides, 612 Lipopolysaccharides (LPS), 642f, 643 Lipoprotein lipase (LPL), 341 def ciency o , 348 Lipoproteins, 337t metabolism o , 338 assembly o , 338–340, 339f intravascular, 340–341, 340f receptor-mediated clearance o , 341–342, 341f structure o , 338f Liposomal doxorubicin (Doxil), 740, 931 Liposome-based drug delivery systems, 984 Liposomes, 931, 979, 984 Lipoteichoic acids, 642f, 643 Lipotropin, 505 Lipoxins, 795, 796f, 801f, 802, 806, 806f, 806t, 813 Lipoxin-stable analogues, 813 5-Lipoxygenase, 92 5-Lipoxygenase-activating protein (FLAP), 799, 884f, 888

1008 Index 5-Lipoxygenase-activating protein (FLAP) inhibitors, 800f, 813, 888 Lipoxygenase inhibitors, 812–813, 813f, 818t Lipoxygenase pathway, 795, 796f, 799–802, 799t, 800f, 801f, 803f–805f Liraglutide, 572, 576t, 964t Lisdexam etamine, 234, 242t Lisinopril, 92t, 373, 380t, 402t, 476, 488 Lithium, 227–228, 239–240, 248t, 377, 507, 521 LMW heparin. See Low-molecular-weight heparin Loading dose, 40–41 Local anesthetics (LAs), 170–171 administration o , 176 amide-linked, 171, 171f, 179–180, 181t–182t cocaine as, 158 distribution o , 176 ester-linked, 171, 171f, 178–179, 181t excretion o , 176 ion channel conductance and, 9, 105 major toxicities o , 178 mechanism o action o anatomic actors, 172–173, 173f receptors or, 175–176 voltage-gated sodium channel in, 173–175, 174f, 175f, 175t metabolism o , 176 modulated receptor hypothesis o , 173–174, 173f, 174t molecular determinants o action o , 171, 171f amine group, 172 aromatic group, 171–172, 172f pharmacokinetics o , 176 pharmacologic classes and agents o , 171 systemic absorption o , 176 Local circuit neurons, 117, 117f Local equilibration, 269–270 Local inhibitory regulation, in spinal cord, 292–293, 292f Localized vasoconstriction, 403, 405f Locus ceruleus, 116 cocaine and amphetamine at, 324 norepinephrine release rom, 228 single-source divergent neuronal systems and, 118, 118f, 118t Lo exidine, 327 Log cell kill model, 611–612, 612f Lomitapide, 352, 356t, 940 Lona arnib, 760 Long-acting beta-agonists (LABAs), 885 Long-tract neuronal organization, 117, 117f Loop diuretics, 359, 375–376, 383t, 473t, 474t, 475, 493 Loperamide, 63t, 65t, 66t, 143t, 309 Lopinavir, 50, 59, 60t, 65t, 712, 712f, 721t Loratadine, 51t, 143t, 820, 823t, 824–826, 829t Lorazepam, 202t, 263t abuse o , 310t clinical uses o , 192t GABAA receptor and, 259 with general anesthetic, 280, 285t Losartan, 50, 51t, 52t, 60t, 66, 373–374, 381t, 402t, 476, 900, 903t Lovastatin, 51t, 89t, 351, 356t Lovaza®, 355 Low-density lipoprotein (LDL), 336, 337t, 338 atherosclerosis and, 342, 344f ormation and clearance o , 342, 343f pharmacologic classes and agents or, 349–350, 349t, 350t plasma decrease o , 350–351 Low-molecular-weight (LMW) heparin, 71, 420, 421f, 429t Low-threshold mechanoreceptors, 169 Loxapine, 143t, 225t LP50, 267 LPL. See Lipoprotein lipase LPS. See Lipopolysaccharides LRP. See LDL receptor-related protein LSD. See Lysergic acid diethylamide LTB4. See Leukotriene B4 Luliconazole, 666, 671t Lume antrine, 679 Luminal protozoa, 682–683, 683f, 684f Lumiracoxib, 812

Lurasidone, 220, 226t Luteal phase, 545, 546f Luteinizing hormone (LH), 500, 500t, 506–507, 506f Lutropin al a, 963t Lyme disease, 974 Lymphocytes, 830 development o , 784, 785f, 835–836 Lymphocyte-signaling inhibitors, 848f, 850–851, 851f, 852f, 858t–859t Lymphocyte-stimulating actors, 835–836 Lymphoid stem cells, 784, 785f, 835–836 Lymphopoiesis, 835–836 Lyophilized powder, 931 Lysergic acid diethylamide (LSD), 238, 310t Lysine analogues, 424, 432t Lysosomal acid lipase (LAL), 342 Lysosomal acid lipase def ciency (LAL-D), 348

M

M2 channel, 699, 700f MAC. See Minimum alveolar concentration mAChR. See Muscarinic cholinergic receptors Macitentan, 396, 401t Macrolide antibiotics, 51t, 623, 626f, 627f, 628, 630t, 633–635, 634f, 635f, 639t resistance to, 633 selective targeting by, 605 targets o , 607f Macrolides, 89t Macromolecular biologics and therapeutics, 924b Macrophages, 786, 786t, 830 Macula densa, 360 Magnesium chloride, 593b, 876t Magnesium hydroxide, 873 Magnesium sul ate, 887, 893t Ma-Huang, 884 Maintenance dose, 41, 41f Major basic protein (MBP), 882, 883f Major depressive disorder (MDD), 227, 231–233, 231b Major histocompatibility complex (MHC), 786–788, 787f, 788f Malaria, 607–608 drugs treating. See Antimalarial agents plasmodial physiology and, 674–677, 676f, 677f Malathion, 909–911, 910f Male contraception, 554 Malignant hyperthermia, 279 Manic episode, 231–232 Mannitol, 16, 375, 382t Mannoproteins, 662 MAO. See Monoamine oxidase MAO-A. See Monoamine oxidase A MAO-B. See Monoamine oxidase B MAOIs. See Monoamine oxidase inhibitors MAP kinase inhibitors, 760–761, 767t Maprotiline, 236 Maraviroc, 606, 609f, 696f, 698, 698b, 699f, 718t Maresins, 802, 803f–805f, 813 Margin o sa ety, 70 Maribavir, resistance to, 613t Marijuana, 309, 324f, 325 Mast cells, 784–785, 786t in asthma, 882, 883f, 884f Mast cell stabilizers, 823, 823t, 890t, 894t MATEs. See Multidrug and toxic compound extrusion proteins Matrix metalloproteinases, 879, 882 Maturation, viral, 696, 696f, 697f inhibitors o , 696f, 712–714, 712f, 713f, 721t MBC. See Minimum bactericidal concentration MBP. See Major basic protein mCPP. See Meta-chlorophenylpiperazine M-CSF. See Monocyte colony-stimulating actor MDA. See Methylenedioxyamphetamine MDD. See Major depressive disorder MDMA. See Methylenedioxymethamphetamine MDR. See Multidrug resistance MDR1. See Multidrug resistance protein 1; P-glycoprotein MDR-TB. See Multidrug-resistant tuberculosis MDR transporters. See Multiple drug resistance transporters

Mebendazole, 687, 693t Mecamylamine, 144, 149t, 151 Mecasermin, 503, 510t, 959t Mechanoreceptors, 169 Mechlorethamine, 736, 737f, 746t Mechlorethamine, vincristine, procarbazine, and prednisone (MOPP), 780 Meclizine, 143t, 825, 829t Meclo enamate, 810, 815t MEDI-528, 891f Medial orebrain bundle, 313, 315 Medical review, 937, 937f, 942, 942f Medication Guides, 943 Medium-threshold mechanoreceptors, 169 Medroxyprogesterone acetate, 552f, 554, 559t Medulla, 114, 114f, 116 Me enamate, 809f, 810, 815t Me oquine, 608, 615, 677–679, 678f, 681, 689t Meglitinides, 569t, 576t Meglumine antimonate, 685, 692t MEK inhibitors, 761 Melanocyte-stimulating hormone (MSH), 505 Melarsoprol, 684–685, 692t Meloxicam, 811–812, 811f Melphalan, 82, 736–737, 746t Memantine, 60t, 199, 205t Membrane attack complex, 790, 856–857 Membrane di usion, 28–29, 29f Membrane excitability, local anesthetics and, 105 Membrane hyperpolarization, 185, 185f Membrane potential, 99 inhibitory and excitatory neurotransmitters and, 185, 185f Membrane resistance, 185, 185f Memory, cholinergic link to, 134 Menopause, 548 Menstrual cycle, 545–546, 546f Meperidine, 158, 299, 305t as general anesthetic, 285t Parkinson’s disease and, 213 Mepivacaine, in dentistry, 177 Mepolizumab, 890, 891f MEPP. See Miniature end-plate potential Mercaptopurine, 89t 6-Mercaptopurine, 63t, 90, 725f, 727f, 733–734, 733f, 743t, 777t, 778t, 899–900, 900f or immunosuppression, 848, 848f Meropenem, 654, 659t Merozoites, 675, 676f Mesenteric ganglion, 111, 113f Mesna, 737, 738f, 850 Mesocortical system, 217 Mesolimbic dopamine pathway, 315, 315f, 316f endogenous cannabinoid neurotransmission in, 323, 324f nicotine and, 323 Mesolimbic hyperactivity, 217 Mesolimbic system, 217 Mesoridazine, 224t Mestranol, 552, 552f, 559t Metabolic acidosis, 377 Metabolic blood–brain barrier, 124 Metabolic modulators, or chronic coronary artery disease, 484–485 Metabolic syndrome, 348 Metabolism, 27, 34 Metabotropic GABA receptors, 188, 188f Metabotropic glutamate receptors (mGluR), 197–198, 197f, 198t Metabotropic receptors, 106–108 Meta-chlorophenylpiperazine (mCPP), 238 Metal–ligand complexes, 908, 908f Metals, occupational exposure to, 915–916 Metaproterenol, 159, 164t, 884, 892t Metastasis, 610 Metered-dose inhalers, 889 Met ormin, 59, 60t, 63t, 64–65, 65t, 66t, 67, 569t, 571, 575t, 929t Met ormin-ER, 575t Methacholine, 133, 137, 139–140, 140f, 140t, 146t Methadone, 299, 304t, 309, 327, 328, 332t Methamphetamine, 158 Methanol, 54 Methemoglobin, 393–394

Index 1009 Methemoglobinemia, 617 Methicillin, 651–652 Methicillin-resistant Staphylococcus aureus (MRSA), 612, 642, 646, 649, 651–652, 654 Methimazole, 515, 519f, 520, 523t Methohexital, 194, 203t clinical uses o , 194t Methotrexate (MTX), 60t, 63, 63t, 66t, 81, 616f, 617–618, 617t, 725f, 727f, 777t, 778t, 779, 966 or immunosuppression, 849, 858t resistance to, 613, 613t Methoxamine, 158, 163t Methoxy urane, 52t, 270f Methoxy polyethylene glycol-epoetin beta, 962t Methscopolamine, 141, 147t 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 213 1-Methyl-4-phenyl-pyridinium (MPP ), 213 Methyldopa, 78, 89t, 159, 163t, 476 Methylenedioxyamphetamine (MDA), 310t Methylenedioxymethamphetamine (MDMA), 234, 310t, 325 Methylmorphine, 298–299 Methylnaltrexone, 300, 306t Methylphenidate, 158, 162t, 983 or atypical depression, 232, 234 Methylprednisolone, 530, 530f, 531t, 539t, 816t, 858t, 878, 902t Methylxanthines, 325, 883, 885f, 886–887, 890t, 893t Methysergide, 238 MET inhibitors, 758, 766t Metolazone, 383t, 474t Metoprolol, 51t, 89–90, 111, 160t, 161, 166t, 401t, 445, 451t, 475, 488 Metreleptin, 959t Metronidazole, 420t, 682–683, 684f, 691t, 775, 874 Metyrapone, 505, 534, 534t, 540t Mevalonate pathway, 592 Mexiletine, 51t, 307t, 443–444, 450t Meyer-Overton rule, 267–268, 269b, 287 MG. See Muscle group mGluR. See Metabotropic glutamate receptors MHC. See Major histocompatibility complex Mibe radil, 949t MIC. See Minimum inhibitory concentration Mica ungin, 607, 668, 673t Michaelis-Menten kinetics, 37–38, 38f Miconazole, 52t, 607, 666, 671t Microbiology review, 942, 942f Microneedles, 981 Micronized glyburide, 576t Microparticles, 410, 414 Microsatellite instability, 727 Microsomal triglyceride-trans er protein (MTP), 338 Microspheres, 979–980 Microtubule inhibitors, 610f, 611, 611f, 741–742, 741f, 748t–749t Microtubules, cancer biochemistry and, 731–732, 731f, 732f Midazolam, 51t, 89t, 191, 202t, 263t case study o , 266 clinical uses o , 192t GABAA receptor and, 259 with general anesthetic, 280, 285t Midbrain, 114, 114f Middle cervical ganglion, 111 Mi epristone (RU-486), 51t, 505–506, 534, 539t, 548f, 552, 558t Miglitol, 578t Migraine headaches, 236, 238, 296–297 therapy or, 302 Milnacipran, 237, 245t Milrinone, 464–465, 468t, 493t, 495 Milte osine, 685, 692t Mineral balance, 581 Mineralocorticoid receptor, 526–527, 535 agonists, 536, 540t antagonists, 536, 540t Mineralocorticoids, 524 pathophysiology o , 535–536 pharmacologic classes and agents or, 535 physiology o , 534–535

Miniature end-plate potential (MEPP), 132, 135f Minimum alveolar concentration (MAC), 266, 267b, 268t Minimum bactericidal concentration (MBC), 770–772, 771t, 772f Minimum inhibitory concentration (MIC), antimicrobials, 770–772, 771t, 772f Ministry o Health and Wel are, 943 Minocycline, 632–633, 638t Minoxidil, 392, 399t, 473t, 476 Mipomersen, 13, 352, 356t, 940, 978 Mirtazapine, 237, 245t Mismatch repair (MMR), 725, 727, 728f Misoprostol, 552, 618, 817t, 873–874, 876t MIT. See Monoiodotyrosine Mitomycin, 736, 746t Mitomycin C, 50 Mitosis cancer biochemistry and, 731–732, 731f, 732f ungal, inhibitor o , 665, 670t Mitotane, 505, 534, 534t, 540t Mitotoxicity hypothesis, 611 Mitoxantrone, 63t Mivacurium, 144, 148t, 285t Mixed hyperlipidemia, 348 Mixed venous partial pressure, 287 Mizolastine, 829t MK571, 63t MLCK. See Myosin light chain kinase MMF. See Mycophenolate mo etil MMR. See Mismatch repair Moclobemide, 158, 163t, 235, 243t Modaf nil, 242t or atypical depression, 234 Moderation management, 328 Modulated receptor hypothesis, 173–174, 173f, 174t Moexipril, 380t Molds, 661 Molecular lesions, 614 Molecular mimicry, 846 Molindone, 225t Momelotinib, 760 Mometasone, 532, 533f, 887, 894t Monoamine oxidase (MAO), 47, 153, 153f, 156 in dopamine degradation, 207, 208f in serotonin degradation, 230, 234–235 Monoamine oxidase A (MAO-A), 207–208, 230 inhibition o , 157–158, 235 reversible inhibitors o , 235 Monoamine oxidase B (MAO-B), 207–208, 230 inhibition o , 158, 234–235 or Parkinson’s disease, 214f, 215 Monoamine oxidase inhibitors (MAOIs) or atypical depression, 232 catecholamine metabolism inhibition with, 158, 163t norepinephrine and, 122 serotonin degradation and, 234–235, 235f SSRIs or SNRIs with, 158 Monoamine theory o depression, 233 ketamine and, 237 limitations o , 233, 234f Monobactams, 644f, 650f, 651, 654, 659t targets o , 607f Monoclonal antibodies, 924b or immunosuppression, 855 tumor-specif c, 763, 769t Monocyte colony-stimulating actor (M-CSF), 835 Monoiodotyrosine (MIT), 515 Montelukast, 800f, 813, 813f, 818t, 878, 884f, 888, 894t Mood stabilizers, 233, 238–240, 247t Moperone, 218f MOPP. See Mechlorethamine, vincristine, procarbazine, and prednisone Moricizine, 444, 450t Morphine, 24, 89t, 123, 304t, 923t abuse o , 309, 310t, 320, 321f as analgesia, 293, 296–299 case study o , 266, 289 discovery o , 922 in dorsal horn, 293 with general anesthetic, 280, 285t

glutamate transporters and, 200 methadone and, 328 neuroadaptation to, 312f–313f Motion sickness, 137, 141 antihistamines or, 825 Motor nervous system anatomy o , 112–114, 112f cellular organization o , 116–117, 117f neurotransmitters o , 119, 119f Moxi oxacin, 74, 628, 637t, 774 MPA. See Mycophenolic acid MPP . See 1-Methyl-4-phenyl-pyridinium MPTP. See 1-Methyl-4-phenyl-1,2,3, 6-tetrahydropyridine MraY, 644f, 645, 645b MRP/CFTR amily. See Multidrug resistanceassociated protein amily MRSA. See Methicillin-resistant Staphylococcus aureus MSH. See Melanocyte-stimulating hormone mTOR inhibitors, 12, 753f, 761, 767t, 851, 852f MTP. See Microsomal triglyceride-trans er protein MTX. See Methotrexate Mucous membrane drug administration, 30t, 31 Mucus, 866 agents promoting, 873–874 Multidrug and toxic compound extrusion proteins (MATEs), 63t, 64 Multidrug resistance (MDR), mechanisms o , 612–614, 613t Multidrug resistance-associated protein (MRP/ CFTR) amily, 63, 63t Multidrug resistance protein 1 (MDR1), 49, 56 Multidrug-resistant tuberculosis (MDR-TB), 612, 649, 771, 773–774 Multilineage growth actors, hematopoietic, 832–833 Multiple drug resistance (MDR) transporters, 37, 56, 124 Multiple sclerosis, 114 MurA, 643, 644f, 645b MurB, 643, 644f, 645b MurC, 644f, 645, 645b MurD, 644f, 645, 645b MurE, 644f, 645, 645b Murein. See Peptidoglycan Murein monomers polymerization o , 644f, 645–646 inhibitors o , 649–650, 656t synthesis o , 643–645, 644f, 645b inhibitors o , 648–649, 649f, 656t MurF, 644f, 645, 645b MurG, 644f, 645, 645b Muromonab-CD3, 971t Muscarine, 140, 140f Muscarinic acetylcholine receptors (mAChR), 127, 130, 131t, 878 acetylcholine e ects on, 133–134, 136t agonists o , 139–140, 140f, 140t, 142b, 146t antagonists o , 141–143, 143b, 147t–148t in CNS, 134 TCAs at, 236 Muscarinic toxicity, 142b Muscimol, 189t, 190, 202t Muscle contraction, ACh and, 132, 135f Muscle group (MG), 270–271, 271f Mutual support programs, 327–328 Myasthenia gravis, 108, 128, 137–138 Mycobacteria cell wall o , 642f, 646–647, 648f drugs targeting. See Antimycobacterial drugs Mycolic acid, 646–647, 648f Mycophenolate mo etil (MMF), 66t, 845, 848–849, 849f, 858t Mycophenolic acid (MPA), 849, 849f, 858t Mycoses. See Fungal in ections Mydriasis, 137 Myelin, 169 Myeloid stem cells, 784, 785f, 835–836 Myelopoiesis, 835–836 Myeloproli erative disorders, 760 Myelosuppression, 838 vinblastine causing, 741 Myoclonic seizure, 252t

1010 Index Myocytes anatomy o , 455, 456f, 457t contraction o , 455–456, 457f regulation o contractility in, 456–459 Myosin light chain kinase (MLCK), 387, 387f Myotomal distribution, 112 Myotonia, 104 Myxedema, 517

N

NA. See Narcotics Anonymous Na . See Sodium Nabumetone, 809f, 810, 815t NAc. See Nucleus accumbens N-acetylcysteine (NAC), 79, 80f, 84 N-acetyl-p-benzoquinoneimine (NAPQI), 79, 80f nAChR. See Nicotinic cholinergic receptors nACh receptors. See Nicotinic cholinergic receptors NaCl co-transporter (NCC), 367, 367f Nadolol, 160, 160t, 165t Na arelin, 512t, 549, 556t, 965t Na cillin, 651–652, 657t Na tif ne, 665, 670t NAG-arabinogalactan, 646–647 Na /K ATPase, 535 Nalbuphine, 300, 305t Nalidixic acid, 628, 628f, 637t Naloxone, 84, 300, 306t, 309, 321f, 332t Naltrexone, 280, 300, 306t, 328, 332t NAPQI. See N-acetyl-p-benzoquinoneimine Naproxen, 89t, 301, 306t, 809, 810, 815t Naratriptan, 302, 307t Narcolepsy, 157 Narcotics Anonymous (NA), 327 Naringin, 60t, 65t, 66 Natalizumab, 14, 79, 856, 863t, 970t Natamycin, 667–668, 672t Nateglinide, 576t National Cholesterol Education Program Adult Treatment Panel III (ATPIII) guidelines, 349, 349t Native pacemaker, 438 Natriuresis, 360, 364 Natriuretic peptide receptor-A (NPR-A), 363, 363f Natriuretic peptide receptor-B (NPR-B), 363, 363f Natriuretic peptide receptor-C (NPR-C), 363, 363f Natriuretic peptides, 363–364, 363f Natural products, 922 Nausea, antihistamines or, 825 NBCe1, 365 NBM. See Nucleus basalis o Meynert NCC. See NaCl co-transporter NE. See Norepinephrine Nebivolol, 161, 166t, 445, 451t, 475 Nebulizers, 889 Necrosis, 76 Nedocromil, 823, 823t, 888, 894t Ne azodone, 51t Negative GREs, 527 Negative rein orcement, 317 Negative symptoms, schizophrenia, 216, 216b Nelf navir, 51t, 63t, 712, 712f, 721t Nematodes, 685 Neomycin, 630, 638t Neostigmine, 114, 137, 138, 138f, 145t, 280 Nephrogenic diabetes insipidus, 240, 377, 508–509, 508f Nephrolithiasis, 376 Nephron, 364–365, 364f Nephrotic syndrome, 372 Nephrotoxicity, cisplatin causing, 738–739 NER. See Nucleotide excision repair Nernst equation, 100–102, 101t Nernst potential, 100–102, 101t, 130, 433–434 Nervous system, 110, 124–125 neuroanatomy o , 110 cellular organization, 116–119, 117f, 118f, 118t central, 114–116, 114f, 115f, 116f peripheral, 110–114, 112f, 113f neurophysiology o blood–brain barrier, 123–124, 124f neurotransmitters, 119–123, 119f, 120t, 121f, 122f Nesacaine, 178–179

Nesiritide, 12, 374, 381t, 396, 400t, 965t NESP. See Darbepoetin al a Nestorone®, 554 NET. See Norepinephrine transporter Netilmicin, 630, 638t Neural networks, 250 Neuraminidase inhibitors, 609f, 696f, 714–715, 715f, 721t Neuraxial blockade, 177 Neuroactive peptides, 107, 119, 120t, 123 Neuroanatomy, 110 cellular organization, 116–119, 117f, 118f, 118t o central nervous system, 114–116, 114f, 115f, 116f o peripheral nervous system, 110–114, 112f, 113f Neurodegenerative diseases, 198–199 Neurogenesis, antidepressants and, 234 Neurogenic diabetes insipidus, 508, 508f Neurohormonal control, o gastric acid secretion, 864–865, 866f Neurohumoral activation, 491–492, 492f Neurohypophysis, 498 Neurokinin A, 878 Neuroleptic malignant syndrome (NMS), 218 Neuroleptics, 217 Neuromuscular activity, o helminths, 686 inhibitors o , 686–687 Neuromuscular blockade, 137 aminoglycosides causing, 632 Neuromuscular blockers, 285t Neuromuscular junction (NMJ) cholinergic neurotransmission and, 132, 134f, 135f cholinergic pathways in, 127 nicotine and, 323 nondepolarizing blockers o , 143–144 Neuronal apoptosis, 178 Neuronal death, 120 Neuronal hyperactivity, 185 Neurons, 188 action potential regulation in, 102–103 nociceptor, 290 o striatum, 211 Neuropathic pain, 104, 296, 297f Neuropeptides, 107, 119, 120t, 123 local anesthetics and, 175–176 in pain transmission, 291, 292f Neuropeptide Y, 878 Neurophysiology blood–brain barrier, 123–124, 124f neurotransmitters, 119–123, 119f, 120t, 121f, 122f Neuroprotective therapies, 213 Neurorestorative therapies, 213 Neurosteroids, 187f, 188, 195 Neurotoxicity, drug-induced, 81 Neurotransmitters, 105, 106f amino acid, 119–121, 120t, 121f biogenic amine, 119, 120t, 121–123, 121f, 122f categories o , 119, 121f divergent neurons and, 118–119, 118f, 118t excitatory, 184–185, 185f inhibitory, 184–185, 185f local anesthetics and, 175–176 metabolism and reuptake o , 108–109 neuropeptides, 107, 119, 120t, 123 neurophysiology o , 119–123, 119f, 120t, 121f, 122f release o , 106, 106f, 108f removal o , 106, 106f synthesis o , 105 Neutropenia, 831, 838 Neutrophils, 785, 786t Nevirapine, 51t, 63t, 708, 709f, 720t NF B pathway. See Nuclear actor-kappa B pathway NHEJ. See Nonhomologous end-joining NHE3 Na /H exchanger, 365 Niacin, 351–352, 354, 354f, 357t Nicardipine, 398t Nicorandil, 392, 399t Nicotine, 60t, 89t, 142b, 310t, 316f, 321f, 323, 327–329 Nicotine replacement therapy, 328 Nicotinic acid. See Niacin

Nicotinic acetylcholine receptors (nAChR), 127, 130–131, 131t agonists o , 140–141, 141t, 142b, 147t partial, 334t antagonists o , 143–144, 148t–149t bupropion and, 237 in CNS, 134 kinetics o , 130, 133f nicotine and, 323 structure o , 8, 8f, 132f Nicotinic toxicity, 142b Niemann-Pick C1-like 1 protein (NPC1L1), 339 Ni edipine, 51t, 390, 398t, 437, 447, 476, 483 Ni urtimox, 685, 692t Nigrostriatal system dopamine in, 209–210 physiology o , 211–212, 212f Nigrostriatal tract, 114, 118, 118f, 118t Nilotinib, 753f, 759–760, 766t Nilutamide, 547, 548f, 551, 558t Nimodipine, 398t Nisoldipine, 398t Nitazoxanide, 684, 691t Nitrates, 392, 399t–400t or acute coronary syndromes, 485f, 486 beta-antagonists with, 483 chemical classes o , 393–394, 393f, 394f or chronic coronary artery disease, 484 or heart ailure, 493t, 494 hydralazine with, 495 mechanism o action, 392–393, 393f pharmacokinetics o , 394–395, 394f pharmacologic tolerance to, 395 toxicities and contraindications o , 395 Nitric oxide (NO), 878. See also Nitrates inhaled gas, 394, 400t nebivolol and, 161 as neurotransmitter, 120, 121f, 123 vascular tone and, 388f, 389, 389f Nitrogen, Meyer-Overton rule and, 270f Nitrogen mustards, 82, 723, 735–737 Nitroglycerin (NTG), 75, 386, 392–394, 394f, 399t, 404, 486, 494 Nitrosoureas, 82, 736, 736f Nitrous oxide, 266, 279, 284t abuse o , 310t Meyer-Overton rule and, 270f properties o , 268t recovery rom, 279f in uptake model, 273, 273t, 274f, 276f Nivolumab, 857, 863t Nizatidine, 829t, 869–870, 870f, 875t NKCC2, 366, 366f NMDA receptors, 193f, 197, 197t alcohol abuse and, 323 amantadine and, 215 antagonists, 205t, 302, 307t epilepsy and, 199 glutamate at, 291 hyperalgesia and, 199–200 ketamine and, 237 neurodegenerative diseases and, 198–199 PCP at, 217 seizure activity and, 259 stroke and, 199 NMJ. See Neuromuscular junction NMR. See Nuclear magnetic resonance NMS. See Neuroleptic malignant syndrome NN-Ach. See Non-neuronal acetylcholine NNCS. See Non-neuronal cholinergic system NNRTIs. See Nonnucleoside reverse transcriptase inhibitors NO. See Nitric oxide Nociception analgesia and anesthesia, 170–171 circuit o , 288–289, 289f pain perception, 170, 170f pain sensation transmission, 169, 169t, 170f physiology o , 167–169, 168f Nociceptive pain, 293 clinical, 293–294 Nociceptor neurons, 290–291, 290t Nociceptors, 167, 168f, 289 types o , 169, 169t

Index 1011 Nodes o Ranvier, 169 No etumomab, 976t Nonblinded trials, 939 Noncompetitive antagonists, 7, 22–23, 26t Nondepolarizing neuromuscular blockade, 143–144 Nonhomologous end-joining (NHEJ), 727, 729f, 730 Non-incretin insulin secretagogues, 571–572, 576t Non-neuronal acetylcholine (NN-Ach), 136 Non-neuronal cholinergic system (NNCS), 127, 136 Nonnucleoside DNA polymerase inhibitors, 708, 709f, 720t Nonnucleoside inhibitors, 702 Nonnucleoside reverse transcriptase inhibitors (NNRTIs), 608, 698b, 707b, 708, 709f, 720t Nonnucleoside RNA polymerase inhibitors, anti-HCV, 709f, 710, 720t Nonpacemaker cells, 433 Nonprescription drugs, 944–945 Non-rapid eye movement (NREM) sleep, benzodiazepines and, 191 Nonreceptor antagonists, 21, 23 Nonreceptor-mediated mechanisms, 16 Nonreceptor tyrosine kinases, 12 Nonspecif c cholinesterase, 131–132 Non-ST elevation myocardial in arction (NSTEMI), 481 Nonsteroidal analgesics, 306t Nonsteroidal anti-in ammatory drugs (NSAIDs), 60t, 89t, 294, 300–301, 300f, 794–795, 796t, 797f, 808–811, 809f, 815t ginkgo biloba and, 76 or gout, 896, 897t, 898–899, 902t or migraine therapy, 302 in peptic ulcer disease, 865, 868, 868f, 870, 873 Noradrenaline. See Norepinephrine Noradrenergic neurons, 118, 118f, 118t Norelgestromin, 553 Norepinephrine (NE), 10, 206. See also Catecholamines adrenoceptors or, 153–155, 154t or cardiac contractility, 464, 467t in dorsal horn, 292–293, 292f locus ceruleus and, 118, 118f, 118t neurotransmission with, 228, 230f as neurotransmitter, 120–121, 120t, 121f in peripheral nervous system, 119, 119f physiologic and pharmacologic e ects o , 156 regulation o , 228–230, 230f reuptake inhibitors o , 235f, 235–237 synthesis o , 121–122, 122f, 150–151, 152f, 228–230, 229f TCAs and, 236 tyramine and, 157 Norepinephrine-selective reuptake inhibitors (NRIs), 237, 245t Norepinephrine transporter (NET), 152f, 153, 156, 230, 324 Norethindrone, 52t, 552–553, 552f, 559t Nor oxacin, 51t, 52t, 628, 637t Norgestimate, 553, 553f, 559t Norgestrel, 552, 559t Normeperidine, 299 Nortriptyline, 51t, 143t, 236, 243t, 301, 306t, 951 Novobiocin, 60t, 63t NPC1L1. See Niemann-Pick C1-like 1 protein NPH insulin, 571 NPR-A. See Natriuretic peptide receptor-A NPR-B. See Natriuretic peptide receptor-B NPR-C. See Natriuretic peptide receptor-C NREM sleep. See Non-rapid eye movement sleep NRIs. See Norepinephrine-selective reuptake inhibitors NS5A inhibitors, 709f, 710, 720t NSAID-induced gastropathy, 808–809, 811, 865, 868, 868f, 870, 873 NSAIDs. See Nonsteroidal anti-in ammatory drugs NSTEMI. See Non-ST elevation myocardial in arction NTG. See Nitroglycerin N-type voltage-gated calcium channels, 291 Nuclear actor-kappa B (NF B) pathway, 755–756, 755f–756f Nuclear magnetic resonance (NMR), 925

Nucleic acids, 13 Nucleic acid synthesis cancer biochemistry and, 725 ungal, inhibitors o , 663–665, 664f, 670t Nucleoside and nucleotide analogues, 63t, 608, 702 anti-HBV, 703f, 707–708, 719t anti-HCV, 703f, 708, 720t antiherpesvirus, 702–705, 703f, 704f, 718t–719t anti-HIV, 703f, 705–707, 706t, 707b, 719t Nucleosides, 724 Nucleotide excision repair (NER), 725, 728, 728f Nucleotides, 724 synthesis o , 724–725, 725f, 726f, 726t, 727f Nucleus accumbens (NAc), 115 in dependence, 313, 315 dopamine receptors in, 209, 210f Nucleus basalis o Meynert (NBM), 118f, 118t, 119, 134 Nystatin, 607, 667–668, 672t

O

OAT. See Organic anion transporter OATP. See Organic anion transporting polypeptide OATP1. See Organic anion transporting polypeptide 1 Obinutuzumab, 855, 861t, 969t Observer bias, 938 Obsessive-compulsive disorder, 235–236 Occipital lobes, 114, 115f Occupational exposures, 915–916 Ocriplasmin, 965t OCT. See Organic cation transporter Octopamine, 157 Octreotide, 499–500, 503, 510t, 573–574, 579t, 965t Oculomotor nerve, 112, 113f Odds ratio, 950 O atumumab, 763, 769t, 855, 861t, 969t “O -label” use, 944 O -target adverse e ects, 71, 72f, 73–75 O oxacin, 52t, 628, 637t Ohm’s law, 99, 99f Oil/gas partition coe f cient, 267–268, 268t, 269b Oil/water partition coe f cient, 123 OKT3, 855, 861t Olanzapine, 143t, 218f, 220, 226t Olaparib, 742 Oleamide, 188 Ol actory tubercle, dopamine receptors in, 209, 210f Oligodendrocytes, 169 Olmesartan, 60t, 63t, 381t Olodaterol, 885, 892t Olopatadine, 825, 829t Omalizumab, 878, 883f, 889, 891f, 894t, 971t Ombitasvir, 702, 710, 720t Omega-3 atty acids, 354–355, 357t generation o , 795, 796f Omega-conotoxin, 291 Omeprazole, 52t, 63t, 74, 82, 89t, 865, 870–873, 871f, 872b, 875t Onchocerca volvulus, 685–686, 686f Oncogenic osteomalacia, 586 Oncotic pressure, 359, 360f Ondansetron, 238, 247t One-pot synthesis, 931 On-target adverse e ects, 70–73, 72f Open state, o sodium channel, 250, 251f OPG. See Osteoprotegerin Opioid hyperalgesia, 319 Opioid receptors, 123 agonists, 297, 304t–305t long-acting, 332t mechanisms o action, 297–298, 298f morphine, codeine, and derivatives, 298–299 partial and mixed, 300, 333t synthetic agonists, 299 antagonists, 300, 306t, 332t Opioids, 51t abuse o , 310t, 316f, 319–320, 320f, 321f, 328 as analgesics, 171, 293 benzodiazepines with, 191 in dorsal horn, 292–293, 292f as general anesthetic, 285t in mesolimbic dopamine pathway, 316f

or migraine therapy, 302 naltrexone and, 328 as neuropeptides, 123 spinal cord and, 116 tolerance to, 200 Oprelvekin, 839, 842t, 962t Opsonins, 791–792 Optic tract, 117 Oral drug delivery, 979–980 Oral hydrocortisone, 531 Oral phosphate binders, 591f, 593b, 596, 600t Organic anion transporter (OAT) amily, 49, 59, 60t–61t Organic anion transporting polypeptide (OATP), 49, 59, 60t–61t Organic anion transporting polypeptide 1 (OATP1), 75 Organic cation transporter (OCT) amily, 49, 59, 60t–61t Organif cation, 515 inhibitors o , 519f, 520–521, 523t Organogenesis, 82 Organophosphate inhibition, 137–138 Organophosphate insecticides, 909–911, 910f Organophosphate pesticides, 142b Organ transplantation, 844–846, 845t Oritavancin, 649–650, 656t Orlistat, 929t Ornithine decarboxylase inhibitors, 685 Oropharyngeal candidiasis, 532 Orphan diseases, 940 Orphan Drug Act, 940 Orphenadrine, 143t Oseltamivir, 608, 609f, 696f, 715, 715f, 721t Osmosis, 982f, 983 Osmotic diuretics, 375, 382t OspA, 974, 974t Ospemi ene, 551, 557t Osteitis f brosa cystica, 589 Osteoblasts, 581, 583f Osteoclasts, 581, 583f Osteomalacia, 589 Osteopenia, 587 Osteoporosis, 376, 531, 587–589, 588t, 589f, 590b Osteoprotegerin (OPG), 581 OTC drugs. See Over-the-counter drugs Ototoxicity, 376 aminoglycosides causing, 632 Ouabain, 60t, 461 Outer medullary collecting duct, 369 Outward current, 99 Ovarian hyperstimulation syndrome, 507 Ovarian theory, 547 Overactive bladder syndrome, 142 Overdrive suppression, 438 Over ow model, 371, 371f Overpressure, or anesthesia induction, 277, 278f Over-the-counter (OTC) drugs, 944–945 Oxacillin, 651–652, 657t Oxaliplatin, 60t, 63t, 739, 747t Oxazolidinones, 612, 626f, 627f, 628, 630t, 634f, 636, 640t targets o , 607f Oxcarbazepine, 51t, 256–257, 256t, 261t, 296, 302, 307t Oxicam derivatives, 301 Oxicam NSAIDs, 809f, 810, 815t Oxiconazole, 666, 671t Oxidation/reduction reactions, 34, 45t, 47, 48f Oxotremorine, 131t Oxybutynin, 142, 143t, 148t Oxycodone, 298–299, 304t, 310t, 320, 922, 923t Oxymetazoline, 158, 163t Oxypurinol, 899, 899f, 900f, 902t Oxytocin, 509

P

P2X receptors, 290–291, 290f, 290t P2Y1 receptors, 406, 408f P2Y(ADP) receptors, 406, 408f P2Y receptors, 290–291, 290f, 290t p53, 730–731, 730f p170. See P-glycoprotein PABA. See Para-aminobenzoic acid

1012 Index Pacemaker cells, 433 Package insert, 943 Paclitaxel, 63t, 78, 81, 89t, 487, 741–742, 749t, 777t, 831, 922, 923t, 984 PAI. See Plasminogen activator inhibitor Pain, 288 categories o , 288 conduction rom periphery to spinal cord, 291 dorsal horn transmission o , 291–292, 292f modulation o acetylcholine in, 136 TCAs or, 236 neuropathic, 104, 296, 297f pathophysiology o , 293 central sensitization, 295–296, 295f clinical nociceptive pain, 293–294 peripheral sensitization, 294–295, 294f perception o , 170, 170f pharmacologic classes and agents or, 297, 303, 303f adrenergic agonists, 302 antidepressants, 301 antiepileptic drugs and antiarrhythmics, 301–302 migraine therapy, 302 NMDA receptor antagonists, 302 NSAIDs, 300–301 opioid receptor agonists, 297–300, 298f physiology o , 288–289 spontaneous, 293 transduction o , 289–291, 290f, 290t Pain sensation action potentials and, 169 f rst pain and second pain, 169, 170f transmission o , 169, 169t Pain syndromes, 137 TCAs or, 236 Pali ermin, 965t Paliperidone, 226t Palivizumab, 716, 966, 971t Palv. See Alveolar partial pressure PAM. See Pralidoxime Pamidronate, 592–594, 592f, 599t Pancreas, anatomy o , 561 Pancreatic enzymes, 955, 961t Pancuronium, 60t, 114, 144, 148t, 280, 285t Pancytopenia, 833 Panic reactions, 325 Panitumumab, 758–759, 765t Pantoprazole, 52t, 870–873, 871f, 872b, 875t Papain, 958, 967t PAR-1. See Protease-activated receptor 1 PAR-4. See Protease-activated receptor 4 Para-aminobenzoic acid (PABA), 178–179, 615–617, 616f Para-aminophenols, 301 Paracetamol. See Acetaminophen Para ollicular C cells, 514 Parallel synthesis, 925, 925f Parasitic in ections case studies, 675, 682, 685 drugs targeting. See Antiparasitic drugs uture directions, 687–688 helminths. See Helminths protozoal, 682–685, 683f, 684f. See also Malaria Parasympathetic nervous system anatomy o , 112, 113f cellular organization o , 116–117, 117f neurotransmitters o , 119, 119f Parathion, 909–911, 910f Parathyroid hormone (PTH), 583–584, 584t, 585f, 595–596, 925t Paraventricular nuclei, dopamine in, 211 Parecoxib, 812 Parenteral drug administration, 30t, 31, 31t Parenteral ormulation, 931, 932t Paricalcitol, 596, 601t Parietal cells, gastric acid secretion by, 864–867, 866f Parietal lobe, 114, 115f Paritaprevir, 701–702, 701f, 710, 718t Parkinson’s disease, 206 anatomy o , 114 antimuscarinics or, 142

blood–brain barrier and, 123 catecholamine metabolism inhibition and, 158 central dopamine pathways and, 210 dopamine neurotransmission and, 211 nigrostriatal pathways in, 211–212, 212f nigrostriatal tract in, 118 NMDA receptor and, 199 pathophysiology o , 212–213, 212f pharmacologic classes and agents or, 213 dopamine metabolism inhibitors, 214f, 215, 222t–223t dopamine precursors, 213–214, 214f, 222t dopamine receptor agonists, 214–215, 222t nondopaminergic, 215, 224t protein intake and, 111 treatment or, 215–216 Parkinson’s disease with dementia (PDD), 136, 139 Park nucleotide, 644f, 645 Paromomycin, 630–631, 638t, 682, 683 Paroxetine, 51t, 143t, 236, 244t, 301 Paroxysmal depolarizing shi t (PDS), 251, 255–256 Paroxysmal supraventricular tachycardia (PSVT), 441b PARP1. See Poly(ADP-ribose) polymerase 1 PARP1 inhibitors, 742 PARs. See Protease-activated receptors Pars compacta, 209, 212, 212f Part. See Arterial partial pressure Partial agonists, 7, 20, 23–24, 23f, 26t in addiction treatment, 320, 321f buprenorphine, 328 varenicline, 329 Partial pressure, 267b Pasireotide, 503, 506, 512t Passive di usion, 58, 58f Passive immunization, 716 Passive targeting, 984 Passive transport, 58, 58f Pathogens, drug classes targeting, 606–608, 607f, 609f Patient-controlled analgesia, 299 Pattern recognition, o immune system, 786 Pazopanib, 762, 762t, 768t PBPs. See Penicillin-binding proteins PCNS. See CNS partial pressure PCOS. See Polycystic ovarian syndrome PCP. See Phencyclidine; Pneumocystis jiroveci (P. carinii) pneumonia PCSK9. See Proprotein convertase subtilisin-like kexin type 9 PD1, 857 PDD. See Parkinson’s disease with dementia PDE5 inhibitors. See Phosphodiesterase type V inhibitors PDE inhibitors. See Phosphodiesterase inhibitors PDGF. See Platelet-derived growth actor PDGFR inhibitors, 759–760, 766t, 778t PDS. See Paroxysmal depolarizing shi t Pediatric studies, 940 Pedunculopontine nucleus, 118f, 118t, 119 Peg-asparaginase, 967t PEG-epoetin beta, 837, 841t PEG-f lgrastim, 831, 838, 842t, 962t Peg-G-CSF, 962t Peginesatide acetate, 962t PEG-inter eron, 975 Peginter eron al a-2a, 963t Peginter eron al a-2b, 963t Peginter eron beta-1b, 963t Pegloticase, 901, 903t, 968t PEG-rHuMGDF. See Pegylated recombinant human megakaryocyte growth and development actor Pegvisomant, 499–500, 503–504, 510t, 972t Pegylated recombinant human megakaryocyte growth and development actor (PEG-rHuMGDF), 839 Pembrolizumab, 857, 863t Pemetrexed, 733, 743t Penbutolol, 51t, 161, 165t Penciclovir, 703f, 705, 719t Penicillamine, 908, 908f Penicillin benzathine, 657t Penicillin-binding proteins (PBPs), 644f, 646, 647f

Penicillin G, 651–652, 657t Penicillins, 650–654, 650f, 652f, 657t–658t, 900 bacterial cell wall synthesis and, 641, 644f, 646, 647f combination chemotherapy using, 773–775 discovery o , 922 GABAA receptor and, 187f, 250 resistance to, 613 targets o , 605–606, 607f toxicity with, 75–76 Penicillin V, 652, 657t Pentamidine, 684, 691t Pentobarbital, 203t abuse o , 310t clinical uses o , 194t phenobarbital comparison with, 194t Pentostatin, 734, 734f, 744t PEP. See Phosphoenolpyruvate PEPT amily. See Peptide transporter amily Peptic ulcer disease case studies, 865 drug classes treating, 869–874, 869f, 870f, 871f, 872b drug summary table, 875t–876t uture directions, 874 pathophysiology o , 867–869, 867f, 868f physiology o gastric acid secretion, 864–867, 866f Peptides, 924b. See also Neuropeptides natriuretic, 363–364, 363f neuroactive, 107, 119, 120t, 123 Peptide transporter (PEPT) amily, 60t–61t, 61 Peptidoglycan, 641, 642f monomer synthesis, 643–645, 644f, 645b inhibitors o , 648–649, 649f, 656t polymer cross-linking, 644f, 646, 647f inhibitors o , 650–654, 650f, 652f, 657t–659t polymerization, 644f, 645–646 inhibitors o , 649–650, 656t Peptidoglycan glycosyltrans erases (PGTs), 644f, 645–646 Peptidomimetics, 924b Peptidyl (P) site, 626–627, 626f, 627f Peptidyl trans erase center, 627 Perampanel, 256t, 260, 264t Perchlorate, 519, 519f, 523t Per orins, 787 Per usion-limited anesthetics, 274 ventilation changes and, 275–276, 276f Pergolide, 214 Periaqueductal gray, 116 Perimenopause, 587 Perindopril, 380t Perineurium, 172, 173f Peripheral nerve blockade, 177 Peripheral nerve f bers anatomy o , 172–173, 173f types o , 169, 169t Peripheral nervous system acetylcholine in, 123 cellular organization o , 116–117, 117f neuroanatomy o , 110–114, 112f, 113f neurotransmitters o , 119, 119f pain conduction to spinal cord rom, 291 Peripheral neuropathy paclitaxel causing, 742 vincristine causing, 741 Peripheral sensitization, 294–295, 294f Peripheral tolerance, 846 Peripheral transduction, 289–291, 290f, 290t Peripheral vasculature, local anesthetics and, 178 Permanent plug, 404, 405f Permethrin, 911 Peroxisome proli erator-activated receptor(PPAR ), 563 Perphenazine, 51t, 143t, 224t Pertechnetate, 519, 519f, 523t Pertuzumab, 969t Pesticides, 909–911, 910f Petit mal seizure, 252t, 253, 255f PFA. See Phosphono ormic acid PFOR. See Pyruvate- erredoxin oxidoreductase PGG2. See Prostaglandin G2 PGI2. See Prostacyclin

Index 1013 P-glycoprotein (P-gp, p170, MDR1), 49, 62, 75, 613, 613t, 740 PGTs. See Peptidoglycan glycosyltrans erases Phagocytosis, in inf ammatory response, 791–792 Phalloidin, 60t Phallotoxins, 909 Pharmacodynamic assessments, 939 Pharmacodynamic drug–drug interactions, 75–76 Pharmacodynamics, 7, 17–26 pharmacogenomics, 91–92, 92t Pharmacodynamic tolerance, 311, 312f–313f, 314f Pharmacoepidemiology, 948, 948t, 949t data sources or, 949–950 study design and interpretation, 950–952 study strategies or, 950, 950f, 951f Pharmacogenomics, 42, 50, 52–53, 66–67, 87 case study or, 88 epigenetics, 93–94, 93f modern, 94 pathway-based, 92–93, 93f pharmacodynamics, 91–92, 92t pharmacokinetics, 88–91, 89t, 90f, 91f pharmacology or, 88 physiology o , 87 regulatory science and, 94–95 Pharmacokinetic drug–drug interactions, 75 Pharmacokinetics, 27 clinical applications o , 37–41, 38f, 38t, 39f, 40f, 41f pharmacogenomics, 88–91, 89t, 90f, 91f physiologic barriers, 27–29, 29f Pharmacokinetic tolerance, 311 Pharmacology review, 942, 942f Pharmacology/toxicology review, 937, 937f Phase I reactions, 43 Phase II reactions, 43 Phase III reactions, 43 Phasic inhibition, with local anesthetics, 174–175, 175f Phencyclidine (PCP), 60t, 217, 310t, 316f, 325 Phenelzine, 158, 163t, 234, 242t Phenobarbital, 51t, 185, 204t, 256t, 263t abuse o , 310t clinical uses o , 194t GABAA receptor and, 259 or hyperbilirubinemia, 47, 49 pentobarbital comparison with, 194t or withdrawal symptoms, 327, 333t Phenothiazines, 217, 219f, 224t, 504 Phenotype switching, 663 Phenoxybenzamine, 60t, 159, 164t, 475–476 Phentolamine, 159–160, 164t, 475–476 Phenylacetic acid derivatives, 301 Phenylalanine, 206 Phenylalkylamines, 390, 398t, 437 Phenylbutazone, 52t, 809 Phenylephrine, 158, 163t, 386 Phenylethanolamine N-methyltrans erase (PNMT), 151, 207, 208f Phenylethylamines, 310t Phenylpropanolamine, 157–158, 949t Phenyltrimethylammonium, 131t Phenytoin, 38, 51t, 78, 89t, 125, 254–255, 256t, 261t, 419t, 443–444, 450t, 519 antibiotic interactions with, 635 Philadelphia chromosome, 759 Phosphate, hormonal control o , 583–587, 584t, 585f Phosphatidylinositol 3-kinase (PI3-kinase), 566 Phosphatidylinositol-3-kinase (PI3K)-AKT pathway, 751, 753f inhibitors o , 761 Phosphatonins, 584t, 586 Phosphodiesterase (PDE) inhibitors, 414–415, 416f, 426t, 461, 464–465, 468t, 495 or asthma, 886–887, 893t Phosphodiesterase type V (PDE5) inhibitors, 395–396 Phosphoenolpyruvate (PEP), 643, 644f Phospholamban, 458 Phospholipase A2, 795, 796f Phospholipase C (PLC) activation o , 9–10, 10f -adrenoceptors and, 154 muscarinic receptors and, 130

Phospholipase inhibitors, 808 Phospholipid-based protein–protein complex, 407, 409f Phospholipid trans er protein (PLTP), 344 Phosphomycin. See Fos omycin Phosphono ormic acid (PFA), 708, 709f, 720t Phosphoribosyl pyrophosphate (PRPP), 895–897, 896f pH trapping, 29, 29f, 37 Physical dependence, 298, 309–310 Physiologic antagonists, 21, 23 Physiologic barriers, 27–29, 29f Physostigmine, 128, 137–138, 138f, 142b, 145t Phytophototoxic, 909 PI. See Inspired partial pressure PI3K-AKT pathway. See Phosphatidylinositol-3kinase-AKT pathway PI3-kinase. See Phosphatidylinositol 3-kinase PI3K inhibitors, 753f Picrotoxin, 202t GABAA receptor and, 187f, 189t, 190 Pilocarpine, 123, 140, 140f, 146t Pilsicainide, 60t, 65t Pimozide, 143t, 218f, 225t Pindolol, 60t, 65t, 160t, 161, 166t, 445, 451t Pioglitazone, 573, 577t Piperacillin, 651, 653, 658t Piperazine, 679, 687, 693t Piperine, 53 Pirbuterol, 884, 892t Pirenzepine, 131t, 137, 141, 147t Pirmagrel, 812, 818t Piroxicam, 301, 306t, 809, 809f, 810, 815t Pitavastatin, 60t, 65t, 351, 356t Pitolisant, 826 Pitrakinra, 890, 891f Pituitary gland eedback inhibition o , 500t, 501, 501f hypothalamus relationship with, 115, 498–501, 500f, 500t Pivotal trials, 938 pKa. See Ionization state PKA. See Protein kinase A PKC. See Protein kinase C Placebo control, 938 Plants, toxic, 909 Plant sterols, 352–353 Plasma cells, in asthma, 882, 883f Plasma lipoproteins, 337t Plasma protein binding, 33, 33f Plasmids, in drug resistance, 613, 651 Plasmin, 410–411, 412f Plasminogen, 410–411, 412f, 487 Plasminogen activator inhibitor (PAI), 411, 412f Plasmodia, 607–608 drugs targeting. See Antimalarial agents electron transport chain o , 676–677, 677f heme metabolism o , 676, 676f li e cycle o , 674–676, 676f Platelet-derived growth actor (PDGF), 965t Platelet granule release reaction, 404–406, 406f Platelets, 404, 830 activation o , 403, 405f adhesion o , 404, 406f aggregation and consolidation o , 406–407, 407f, 408f production o , 836, 836f agents stimulating, 838–839, 842t Platinum compounds, 610f, 611f, 738–739, 738f, 739f, 746t–747t, 777t PLC. See Phospholipase C Pleasure centers, 313 Pleiotropic e ects, 350 Pleiotropic growth actors. See Multilineage growth actors Pleuromutilins, 612, 626f, 627f, 628, 630t, 634f, 636, 640t targets o , 607f PLTP. See Phospholipid trans er protein Pluripotent hematopoietic stem cell, 830, 832f PMEA, 63t Pneumocystis jiroveci (P. carinii) pneumonia (PCP), 684 PNMT. See Phenylethanolamine N-methyltrans erase

Podagra, 897 Poiseuille’s law, 385–386 Poly(ADP-ribose) polymerase 1 (PARP1), 727, 729f, 742 Polyclonal antibodies, or immunosuppression, 855 Polycystic ovarian syndrome (PCOS), 536, 542, 547 Polyenes, 607, 661, 664f, 667–668, 672t Polygenic hypercholesterolemia, 347 Polymerase inhibitors, 608, 609f, 696f anti-HBV nucleoside and nucleotide analogues, 703f, 707–708, 719t anti-HCV nonnucleoside RNA polymerase inhibitors, 709f, 710, 720t anti-HCV nucleoside and nucleotide analogues, 703f, 708, 720t antiherpesvirus nucleoside and nucleotide analogues, 702–705, 703f, 704f, 718t–719t anti-HIV nucleoside and nucleotide analogues, 703f, 705–707, 706t, 707b, 719t NNRTIs. See Nonnucleoside reverse transcriptase inhibitors nonnucleoside DNA polymerase inhibitors, 708, 709f, 720t Polymer-based drug delivery, 981–983, 982f, 983f Polymer cross-linking, in bacterial cell wall synthesis, 644f, 646, 647f inhibitors o , 650–654, 650f, 652f, 657t–659t Polymerization, glycan, 644f, 645–646 inhibitors o , 649–650, 656t Polymicrobial in ections, 775 Polymodal nociceptors, 169 Polymyxin, 643 Polythiazide, 383t Polyvalent cations, chelators o , 668, 673t POMC. See Proopiomelanocortin Ponatinib, 79, 760, 766t Pons, 114, 114f, 116 Pooled immunoglobulins, 961t Popliteal block, 177 Porins, 642f, 643 Posaconazole, 666, 671t Positive chronotropic e ect, 458 Positive inotropes, 454 Positive inotropic e ect, 458 Positive lusitropic e ect, 458 Positive rein orcement, 317 Positive symptoms, schizophrenia, 216, 216b Posterior horn, 112f, 113 Posterior pituitary gland, 498, 507 antidiuretic hormone, 507–509, 508f oxytocin, 509 Postexposure prophylaxis, or HIV, 615 Postganglionic neuron, 111, 113f Postglomerular nephron, 365, 365f Postictal period, o seizure, 253, 254f Postmyocardial in arction, management o , 487–488 Postsynaptic ganglionic response, 133 Postsynaptic potentials, 106 Potassium (K ) blood concentration o , 524 GABAB and, 188, 188f lithium and, 239–240 loading o , 535 Nernst equilibrium potential or, 101t resting membrane potential and, 100 contribution o , 102, 102f at synaptic cle t, 107 Potassium channel blockers, action potential and, 105 Potassium channels action potential at, 250, 251f inhibition o , 258 openers o , 392, 399t, 476 potentiators o , 262t Potassium channel tetramerization domain (KCTD) subunits, 188 Potassium hydroxide, 909 Potassium phosphate, 601t Potassium-sparing diuretics, 384t, 473t, 474t, 475 Potency o drug (EC50), 19, 19f PPAR . See Peroxisome proli erator-activated receptor-

1014 Index PPD test. See Purif ed protein derivative test PPIs. See Proton pump inhibitors Pralidoxime (PAM), 138, 911 or cholinergic toxicity, 142b Pramipexole, 63t, 215, 222t Pramlintide, 573, 577t, 959t Prandial bolus insulins, 570, 570t, 575t Prasugrel, 406, 415–417, 427t, 486 Pravastatin, 60t, 62, 63t, 65t, 66, 92t, 351, 356t Praziquantel, 687, 693t Prazosin, 60t, 160, 164t, 397, 401t, 475 pRB. See Retinoblastoma protein Pre-beta-HDL, 343 Preclinical research and development, 936 Prednisolone, 81, 529–530, 530f, 531t, 539t, 808, 816t, 858t Prednisone, 38, 52t, 525, 529, 530f, 539t, 777t, 795, 808, 816t or gout, 896, 902t or immunosuppression, 845, 858t Pregabalin, 256t, 258–259, 262t, 291, 296, 301–302, 307t Preganglionic neuron, 111–112, 113f Pregnancy, glucocorticoids and, 534 Preload, 454, 490, 490f reduction o , 493–494 Pressure-volume loop, 489, 489f Presynaptic terminal, 106 Preventers, in asthma, 882 Prevertebral ganglia, 111, 113f Prilocaine, 179, 181t Primaquine, 677, 680, 690t Primary active transport, 28 Primary (genetic) disorders, 412, 413t Primary hemostasis, 403 platelet adhesion, 404, 406f platelet aggregation and consolidation, 406–407, 407f, 408f platelet granule release reaction, 404–406, 406f Primary hemostatic plug, 403, 407 Primary hyperaldosteronism, 535–536 Primary percutaneous intervention, 487 Primary prevention, 350 Primary structure, o proteins, 2–3, 4f Primase, 624 Principal cells, 369 PR interval, 437b Priority review, 940, 942 Probenecid, 59, 60t, 63t, 65, 65t, 75, 705, 896f, 897t, 900, 903t Procainamide, 60t, 63t, 78, 443, 449t Procaine, 167, 176, 178–179, 181t inf ltration anesthesia, 177–178 Procarbazine, 736, 746t Prochlorperazine, 143t, 218f Procyclidine, 142 Prodrug, 34, 50 Proenzymes, 407 Progesterone, 60t, 188, 541 Progestin-only contraceptives, 552f, 553–554, 559t Progestins replacement o , 554–555, 554t structure o , 552f, 553f synthesis o , 541–542, 543f Proguanil, 677, 680, 691t Prolactin, 500, 500t, 504–505, 504f lowering o , 511t schizophrenia and, 219 Prolactinomas, 504, 547 Proli eration, carcinogenesis and, 608–610 Proli erative phase, 545, 546f Promethazine, 143t, 309, 824, 824f, 825, 828t Promoters, 82 Proneuropeptides, 107 Prontosil, 604, 616 Proopiomelanocortin (POMC), 505, 528 Propa enone, 51t, 444, 450t Propantheline, 142, 143b, 148t Prophylactic chemotherapeutics, 615 Propionic acid derivatives, 301 Propionic acid NSAIDs, 809f, 810, 815t Propo ol, 195, 204t, 280, 284t Propranolol, 51t, 52t, 60t, 61t, 89t, 160, 160t, 165t, 401t, 445, 451t, 475

Proprioception, 112 Proprotein convertase subtilisin-like kexin type 9 (PCSK9), 342 inhibitors o , 355, 357t Propylthiouracil (PTU), 519f, 520, 523t Prorenin, 361 Prorenin receptor (PRR), 361 Prostacyclin (PGI2), 389, 410, 414, 415f, 795, 796f, 797f, 798–799, 798f, 806, 806f, 806t analogues, 396, 401t Prostaglandin G2 (PGG2), 414, 415f Prostaglandins, 795, 796f, 797f, 798, 798f, 798t, 806–808, 806f, 806t in asthma, 882 in gastric acid secretion, 865–866, 866f, 873–874, 876t Prostaglandin synthesis, 414, 415f Prostanoid, 798, 798f Prostanoid receptor mimetics, 812, 817t Prostate cancer, 547, 974–975 Protamine, 23, 424, 431t, 968t Protease, HIV, 705–706 Protease-activated receptor 1 (PAR-1), 405–406, 409–410 antagonists, 417–418, 428t Protease-activated receptor 4 (PAR-4), 405–406, 409–410 Protease-activated receptors (PARs), 409–410 Protease inhibitors HCV, 696f, 700–702, 701f, 701f, 718t HIV, 608, 609f, 696f, 707b, 712–714, 712f, 713f, 714b, 721t resistance to, 614 Proteasome, structure and unction o , cancer biochemistry and, 752–756, 754f, 755f–756f Proteasome inhibitors, 611, 754f, 761–762, 768t, 778t Protectins, 802, 803f–805f, 813 Protection, gastric, 866–867 Protein C, 410, 960t Protein kinase A (PKA) -adrenoceptors and, 154–155 cAMP and, 458 G protein-coupled receptors and, 155 Protein kinase C (PKC) ethanol tolerance and, 196 G protein-coupled receptors and, 155 Proteins drugs and, 111 structural, 13 structure o , 2–3, 4f Protein S, 410 Protein synthesis bacterial, 626–627, 626f, 627f inhibitors o , 630–636, 630t, 631f, 632f, 634f, 635f, 638t–640t plasmodial, inhibitors o , 680 Protein therapeutics, 955 case study or, 956 challenges or, 975–977 classif cation o , 957–958, 957b enzymes and regulatory proteins, 958, 959t–962t, 962t–965t, 966 uture directions or, 977–978 protein diagnostics, 975, 976t protein vaccines, 973–975, 974t targeted proteins, 966, 967t–968t, 968t–972t, 972–973, 973t Protein vaccines, 973–975, 974t Proteolytic cleavage, viral, 695 Prothrombin complex concentrate, 960t Prothrombin G20210A mutation, 412, 413t Prothrombin time (PT), 420 Protirelin, 511t Proton pump inhibitors (PPIs), 52t, 865, 869f, 870–874, 871f, 872b, 875t Protozoal in ections, 682–685, 683f, 684f. See also Malaria Protracted withdrawal syndrome, 313 Protriptyline, 143t, 243t Proximal tubule (PT), 365–366, 365f Proxy an, 826 PRPP. See Phosphoribosyl pyrophosphate

PRR. See Prorenin receptor Prucalopride, 238, 247t P-selectin glycoprotein ligand-1 (PSGL-1), 410 Pseudocholinesterase, 131–132, 176 Pseudoephedrine, 157–158, 162t Pseudomembranous colitis, antibiotics causing, 635 PSGL-1. See P-selectin glycoprotein ligand-1 Psilocybin, 310t P site. See Peptidyl site Psoralen isomers, 909 Psoriasis, 966 PSVT. See Paroxysmal supraventricular tachycardia Psychological dependence, 310 Psychostimulants, 310t, 324 Psychotic depression, 231 PT. See Prothrombin time; Proximal tubule PTH. See Parathyroid hormone Ptissue. See Tissue partial pressure PTU. See Propylthiouracil Pulmonary drug delivery, 980–981 Pulmonary embolism, 553 Pulmonary f brosis, bleomycin causing, 740 Pulmonary toxicity, drug-induced, 82 Pump-based hypertension, 472 Pure Food and Drugs Act, 935 Purif ed protein derivative (PPD) test, 975, 976t Purine analogues, 613, 733f, 734f, 735, 735f, 744t–745t, 777t Purine metabolism, 895–897, 896f Purine metabolism inhibitors, 733–734, 733f, 734f, 743t–744t Purine ribonucleotides, synthesis o , 724–725, 725f, 726f, 726t, 727f Putamen, 114, 115f P wave, 437b Pyrantel pamoate, 687, 693t Pyrazinamide, 607f, 648f, 654–655, 660t, 771, 773 Pyrethroid insecticides, 911 Pyridostigmine, 138, 142b, 145t Pyridoxal phosphate, 186 Pyrilamine, 825, 828t Pyrimethamine, 65, 65t, 616f, 617–618, 617t, 675, 677, 678f, 681, 690t, 774–775 Pyrimidine analogues, 613, 733f, 734f, 735, 735f, 744t–745t, 777t Pyrimidine ribonucleotides, synthesis o , 724–725, 725f, 726f, 726t, 727f Pyrimidines, targets o , 607 Pyruvate- erredoxin oxidoreductase (PFOR), 683, 684f

Q

QRS complex, 437b QT interval, 437b Quality control, 944 Quantal dose–response relationships, 19–20, 20f Quaternary structure, o proteins, 2–3, 4f Quazepam, 191, 203t clinical uses o , 192t Quercetin, 60t, 63t, 65t, 66 Quetiapine, 220, 226t, 825 Quinagolide, 504–505 Quinapril, 380t Quinidine, 51t, 60t, 63t, 64, 65t, 76, 89t, 442–443, 449t, 678, 689t Quinine, 60t, 677–678, 689t Quinolines, 676–679, 676f, 678f Quinolone antibiotics, 607f, 625f, 627–628, 628–629, 628f, 637t Quinolones, 52t Quinone, 680 Quinupristin, 612, 634f, 635–636, 640t

R

RAAS. See Renin-angiotensin–aldosterone system Rabeprazole, 870–873, 871f, 872b, 875t Rabies vaccine, 716 Race, drug metabolism and, 53 Raclopride, 218f Radioactive iodine, 515, 523t Raloxi ene, 24, 82, 549t, 550–551, 557t, 591, 592f, 599t, 929t RAL signaling, 752, 753f

Index 1015 Raltegravir, 609 , 615, 696 , 710, 710 –711 , 721t Ramipril, 373, 380t Ramucirumab, 762, 768t, 968t Randomization o subjects, 938 Ranibizumab, 972t Ranitidine, 60t, 826, 826 , 829t, 865, 869–870, 870 , 875t RANK, 581, 583 RANK ligand (RANKL), 581, 583 antagonists, 594–595, 599t Ranolazine, 65t, 447–448, 453t, 484–485 Rapamycin, 761, 767t, 851, 852 r-APC. See Recombinant activated protein C Raphe nucleus, 116 serotonin release rom, 228 single-source divergent neuronal systems and, 118–119, 118 , 118t Rapid eye movement (REM) sleep, benzodiazepines and, 191 Rasagiline, 158, 163t, 215, 222t–223t Rasburicase, 900, 903t, 967t RAS-MAP kinase pathway, 751, 753 inhibitors o , 760–761, 767t Rational drug design, 6, 925–926 Ravuconazole, 607, 666–667 Raxibacumab, 971t Receptor binding assays, 928 Receptor guanylyl cyclases, 11 , 12 Receptors. See also specif c receptors binding site o , 3–4, 5t con ormation and chemistry o , 2–4, 4 drug binding impact on, 5–6 drug interactions with. See Drug–receptor interactions or local anesthetics, 175–176 types o , 6–7, 7 , 7t Receptor serine/threonine kinases, 11 , 12 Receptor tyrosine kinases, 11–12, 11 . See also Tyrosine kinase inhibitors cancer biochemistry and, 750–751, 751 , 752t, 753 Receptor tyrosine phosphatases, 11 , 12 Recombinant activated protein C (r-APC), 423, 430t Recombinant human bone morphogenic protein 2 (rhBMP-2), 965t Recombinant human bone morphogenic protein 7 (rhBMP-7), 965t Recombinant human erythropoietin (rhEPO), 837, 841t Recombinant human growth hormone, 503 Recombinant human IL-11 (rhIL-11), 839, 842t Recombinant human thrombopoietin (rhTPO), 839, 842t Recombinant IGF-1, 503 Recombinant insulin, 956 Recombinant tissue plasminogen activator (t-PA), 424, 431t, 487 Recovery, rom inhaled anesthetics, 277–278, 279 Recurrent disease, combination chemotherapy or, 780–781 Red blood cells. See Erythrocytes Redistribution o anesthetic, 278 Red man syndrome, 78, 649 5 -Reductase, 542, 548 inhibitors, 548 , 549 Re-entry, 439–440, 439 Re ractory disease, combination chemotherapy or, 780–781 Re ractory period, 8–9, 15, 16t, 436 Re ractory state, 15, 104 Regora enib, 763 Regulatory T cells (Treg), 790 Rejection, transplant, 844–846, 845t Relapse, 315 Relative risk, 950 Release, viral, 696, 696 inhibitors o , 696 , 714–715, 715 , 721t Release actors, bacterial, 627 Relievers, asthma, 882 Remi entanil, 285t, 289, 293–294, 299, 305t Remnant particles, 341–342, 341 Remodeling, 490, 581

Remoxipride, 218 REM sleep. See Rapid eye movement sleep Renal excretion, 36–37, 36 Renal ailure, aminoglycosides causing, 632 Renal glomerulus, 364–365, 364 Renal parenchymal disease, 472 Renal phospholipidosis, 80 Renal sympathetic nerves, 364 Renal toxicity, drug-induced, 80–81 Renal tubular dysgenesis (RTD), 368t Renin, 360 inhibitors o , 372, 373 , 380t, 402t, 473t, 476 release o , 360–361, 362 Renin-angiotensin–aldosterone system (RAAS), 360–363, 361 , 362 , 535 inhibitors o , 372–374, 373 , 397, 473t modulation o , 476–477 Renovascular disease, 473 Repaglinide, 60t, 576t Repeat-dose toxicity studies, 929 Replication bacterial, 623–624, 625 inhibitors o . See Topoisomerase inhibitors viral, 694–697, 696 , 697 inhibitors o , 696 , 702–710, 703 , 704 , 706t, 707b, 709 , 710 –711 , 718t–721t Repolarization inhibitors, 445–447, 445 , 446t Reproduction, pathophysiology o , 546–548, 546t Reproductive hormones action and metabolism o , 542–544, 544 synthesis o , 541–542, 543 Reserpine, 63t, 156, 162t, 230, 233, 476 Reserve pool, 130 Resistance, 99, 99 drug antimalarial agents, 678, 678 , 681 antimycobacterial agents, 655 antineoplastic drugs, 612–614, 613t, 724, 778, 778t antiviral drugs, 613t, 614, 697–698 beta-lactams, 613, 613t, 651 mechanisms o , 612–614, 613t, 646, 650 practices promoting, 614 Reslizumab, 891 Resolution, in in ammatory response, 792 Resolvins, 802, 803 –805 , 813 Resting membrane potential cause o , 100 electrochemical basis o , 100, 101 Goldman-Hodgkin-Katz equation in, 102 ion pumps and, 101 Nernst equation and, 100–101 potassium and sodium contribution to, 102, 102 o ventricular myocyte, 434 Retapamulin, 612, 634 , 636, 640t Reteplase, 424, 431t, 487, 958, 964t Reticular activating system, 116, 134 Reticulocyte, 834 Retigabine, 258, 262t Retinoblastoma protein (pRB), 752, 754 Retinoic acid, 82, 89t Retinoid X receptor (RXR), 516–517 Retrosynthetic analysis, 930–931, 930 Retroviruses. See Human immunodef ciency virus (HIV) Rett syndrome, 136 Reversal potential, 107 Reverse cholesterol transport, 342–346, 345 Reverse transcriptase (RT), 697 , 705 inhibitors o , 703 , 705–707, 706t, 707b, 719t. See also Nonnucleoside reverse transcriptase inhibitors Reverse triiodothyronine (rT3), 514, 515 Reversible antagonists, 21, 21 Reversible inhibitors o MAO-A (RIMAs), 235 Reward system, 310 Reye’s syndrome, 810 rhBMP-2. See Recombinant human bone morphogenic protein 2 rhBMP-7. See Recombinant human bone morphogenic protein 7 rhEPO. See Recombinant human erythropoietin Rheumatoid arthritis, 294, 795, 852–853, 852 , 966 eicosanoid pathophysiology in, 807

rhIL-11. See Recombinant human IL-11 Rhodanese, 907 rhTPO. See Recombinant human thrombopoietin Ribavirin, 701–702, 703 , 708, 716, 722t Ribonucleotide reductase, 724–725, 727 Ribonucleotide reductase inhibitors, 734, 744t Ribonucleotides, synthesis o , 724–725, 725 , 726 , 726t, 727 Ribosomes, bacterial, 626–627, 626 , 627 inhibition o , 630–636, 630t, 631 , 632 , 634 , 635 , 638t–640t Ridogrel, 812, 818t Ri abutin, 51t, 626 , 629, 629 , 638t Ri ampin, 38, 50, 51t, 52t, 54, 60t, 63t, 65t, 66, 66t, 71, 78, 419t, 519, 607 , 626 , 628, 629–630, 629 , 638t combination chemotherapy using, 771, 773–774 Ri amycins, 51t, 60t, 61t, 628, 629–630, 629 , 637t–638t Ri apentine, 51t Rilonacept, 854, 860t, 901, 970t Rilpivirine, 708, 709 , 720t Riluzole, 198, 205t Rimantadine, 608, 609 , 696 , 698–700, 700 , 718t RIMAs. See Reversible inhibitors o MAO-A Rimonabant, 325, 331, 929t Riociguat, 396, 400t Risedronate, 592–594, 592 , 599t Risk evaluation, 950, 951 Risperidone, 51t, 220, 226t Ritonavir, 50, 51t, 52t, 59, 60t, 61t, 63t, 64, 65t, 66, 609 , 695, 696 , 712, 712 , 713 , 714, 714b, 721t, 924, 925 Rituximab, 79, 736, 763, 769t, 855, 861t, 966, 969t Rivaroxaban, 421 , 422, 430t Rivastigmine, 139, 139t, 146t River blindness, 685–686, 686 Rizatriptan, 238, 247t, 302, 307t RNA inter erence (RNAi) therapeutics, 13 RNA polymerases bacterial, 624, 626 cancer biochemistry and, 725 HCV, inhibitors o , 709 , 710, 720t Rocuronium, 144, 148t Ro ecoxib, 70, 295, 301, 811–812, 811 , 947–948, 949t, 951 Ro umilast, 886, 893t Romiplostim, 839, 842t, 958, 963t, 977 ROMK, 366, 366 Ropinirole, 215, 222t Ropivacaine, 179 ROS1 inhibitors, 759, 766t Rosiglitazone, 573, 577t, 947 Rosuvastatin, 60t, 61, 61t, 63t, 65t, 66, 351, 356t Rotigotine, 215, 222t RT. See Reverse transcriptase rT3. See Reverse triiodothyronine RTD. See Renal tubular dysgenesis RU-486. See Mi epristone Ruf namide, 256t, 260, 264t Ruxolitinib, 753 , 760, 767t R-War arin, 52t RXR. See Retinoid X receptor Ryanodine receptor, 279, 457–458, 458

S

Salbutamol. See Albuterol Salicylates, 301, 809–810, 809 Saline diuresis, 593b Salmeterol, 159, 164t, 878, 885, 889, 892t Salmon calcitonin, 595, 600t, 964t Sample size, 938 Saquinavir, 51t, 53, 60t, 61t, 63t, 65t, 712, 712 , 721t Sarcoendoplasmic reticulum calcium ATPase (SERCA), 457–458, 458 Sargramostim, 838, 842t Sarin nerve gas, 142b Satumomab pendetide, 976t Saturation kinetics, 37, 38 , 41 Saxagliptin, 572, 577t Saxitoxins, 908–909 Scavenger receptor class B, type I (SR-BI), 343

1016 Index Scavenger receptors, 342, 343f Schizophrenia, 206 central dopamine pathways and, 211 D2 dopamine receptors in, 209 dopamine neurotransmission and, 211 dopamine receptor antagonists or, 121 lithium and, 239 pathophysiology o , 216–217 pharmacologic classes and agents or, 217 atypical antipsychotic agents, 218f, 220 typical antipsychotic agents, 217–220, 218f, 219f Schwann cells, 169 Sclerostin, 582–583 Scopolamine, 123, 141, 143b, 143t, 147t Secobarbital, 50, 52t, 203t clinical uses o , 194t Secondary active transport, 28 Secondary (acquired) disorders, 412, 413t Secondary hemostasis, 403–404, 407–410, 409f Secondary hemostatic plug, 407 Secondary hyperparathyroidism, 587, 588t, 589, 596 Secondary hypertension, 472 Secondary osteoporosis, 589 Secondary prevention, 350 Secondary prevention trials, 483 Secondary structure, o proteins, 2–3, 4f Second-generation H1-antihistamines, 823–826, 829t Second messenger-regulated ion channels, 8, 8t Second messengers, 9–10, 10f Second pain, 169, 170f Secretins, 975, 976t as neuropeptides, 123 Secretory phase, 545, 546f Secukinumab, 854, 891f Sedation benzodiazepines and, 191 reticular activating system and, 116 Segregation, o bacterial DNA, 623–624, 625f Seizures, 249. See also Antiepileptic drugs; Focal seizure; Generalized seizure classif cation o , 252t pathophysiology o , 250–251 ocal, 251–252, 253f primary generalized, 253, 253f, 255f secondary generalized, 252–253, 253f, 254f pharmacologic classes and agents or, 253–254, 256t physiology o , 249–250 propagation o , 251, 253f Selection bias, 951 Selective estrogen receptor modulators (SERMs), 24, 548f, 549–551, 549t, 550f, 551f, 557t, 591–592, 592f, 599t, 750, 929t Selective actor Xa inhibitors, 421f, 422, 429t–430t Selective inhibition, o antimicrobial and antineoplastic drugs, 604t, 605 Selective progesterone receptor modulators (SPRM), 552, 558t Selective serotonin reuptake inhibitors (SSRIs), 236–237, 244t adverse e ects o , 237 alcohol use disorder and, 319, 329 MAOIs with, 158 neurotransmitters and, 122 in pain management, 301 St. John’s wort and, 76 or substance use disorders, 334t TCAs compared with, 236–237 Selective toxicity, 911 o antimicrobial and antineoplastic drugs, 604–606, 604t olate metabolism inhibitors, 615–618, 616f, 617t Selectivity, o drugs, molecular and cellular determinants o , 6 Selegiline, 158, 163t, 215, 222t–223t, 235, 243t Sel -help programs, 327–328 Seliciclib, 63t Selumetinib, 761 Semisynthetic product, 922 Sensitization, 309

Sensitizing agents, 290 Sensitizing chemical mediators, 295 Sensory nervous system anatomy o , 112–114, 112f cellular organization o , 116–117, 117f neurotransmitters o , 119, 119f nociception and, 167 Sensory transduction, 289–291, 290f, 290t Sequential blockade, 618 SERCA. See Sarcoendoplasmic reticulum calcium ATPase Serine-protease inhibitor, 424, 431t Sermorelin, 503, 510t SERMs. See Selective estrogen receptor modulators Serotonergic neurons, 118, 118f, 118t Serotonin (5-HT, 5-Hydroxytryptamine) degradation inhibitors o , 234–235, 235f, 242t–243t in dorsal horn, 292–293, 292f neurotransmission with, 228, 229f pharmacologic classes and agents or, 233–240 as neurotransmitter, 120, 120t, 121f, 227 raphe nuclei and, 118, 118f, 118t receptors o , 230, 230t agonists o , 238, 246t–247t, 307t antagonists o , 238–239, 247t, 302 regulation o , 228–230, 229f reuptake inhibitors o , 51t, 235f, 236–237 storage inhibitors o , 234, 242t synthesis o , 122, 122f, 228–230, 229f Serotonin-norepinephrine reuptake inhibitors (SNRIs), 237, 244t–245t or analgesia, 306t catecholamine reuptake inhibition with, 158 MAOIs with, 158 neurotransmitters and, 121–122 in pain management, 301 Serotonin syndrome, 76, 158, 236, 299 Serotonin transporter (SERT), 230 Sertaconazole, 666, 671t Sertoli cells, 544, 545f Sertraline, 52t, 236, 244t Serum sickness, 78 SeSAME syndrome, 367, 368t Sevelamer, 596, 600t Seven-transmembrane helix receptors, 153 70S ribosome, 626–627, 626f, 627f Sevo urane, 52t, 279, 284t properties o , 268t in uptake model, 273, 273t SFUs. See Sul onylureas SGLT-2. See Sodium-glucose co-transporter-2 SGLT amily. See Sodium glucose transport protein amily Shared brain mechanisms, 330 Shock, 156 Shotgun approach, 925 Shunting, 187 SIADH. See Syndrome o inappropriate ADH Sibutramine, 929t Sickle cell anemia, 833, 837–838 Side e ects, 70 Signal transduction, cancer biochemistry and, 750–757, 751f, 752t, 753f, 754f, 755f, 756f, 756t, 757f Signal transduction antagonists, 611, 757–761, 765t–767t, 778t Signal transduction molecules, 12–13 Signal transduction pathways, in vascular tone, 387–388, 387f Sildenaf l, 51t, 75, 386, 395, 400t Silent nociceptor f bers, 291 Siltuximab, 970t Simeprevir, 701–702, 701f, 708, 718t Simple di usion, 58, 58f Simvastatin, 66t, 94, 351, 356t Sinemet, 111, 114 Single-blind trials, 939 Single nucleotide polymorphisms (SNPs), 87 Single-source divergent circuit organization, 117f, 118–119, 118f, 118t

Sinoatrial nodal cells (SA nodal cells) action potentials o , 434, 435f f ring cycle phases o , 434, 435f, 436t f ring rate and, 437–438 impulse ormation de ects in, 438–439, 439f Sinus tachycardia, 441b Sipuleucel-T, 974, 974t Sirolimus, 487, 761, 767t, 851, 852f, 859t, 984 Sitagliptin, 572, 577t 16S rRNA, 626–627, 626f, 627f Sjögren’s syndrome, 140 Skeletal muscle relaxants benzodiazepines or, 192 elderly and, 143t Skeletal muscle toxicity, drug-induced, 81 Skin rashes, 78 SLC. See Human solute carrier; Solute carrier SLC amily. See Solute carrier amily Sleep ACh in, 134 benzodiazepines and, 191 relay neurons in, 253, 255f Sleeping nociceptor f bers, 291 Slow acetylator, 52 Slow EPSP, in autonomic ganglia, 133, 135f Slow-reacting substance o anaphylaxis (SRS-A), 882 Slow-wave sleep (SWS) absence seizure and, 253 ACh in, 134 benzodiazepines and, 191 SN-38, 740 SNAP-25, 107, 108f, 137 SNARE/SM protein complex, 107, 108f sNDA/sBLA. See Supplemental NDA/BLA SNPs. See Single nucleotide polymorphisms SNRIs. See Serotonin-norepinephrine reuptake inhibitors Sodium (Na ) Nernst equilibrium potential or, 101t renal control o , excretion o , 364–370, 364f, 365f, 366f, 367f, 367t, 368t, 369f resting membrane potential contribution o , 102, 102f at synaptic cle t, 107 Sodium bicarbonate, 185, 873, 876t Sodium–calcium exchanger, 456, 458f Sodium channel blockers, 254 action potential and, 105 as antiarrhythmic agents, 441–442, 442f, 443f, 450t state-dependent, 440–441 or substance use disorders, 333t Sodium channels action potential at, 250, 251f inhibition o , 254–258, 257f, 261t Sodium-glucose co-transporter-2 (SGLT-2), inhibitors, 572, 577t Sodium glucose transport protein (SGLT) amily, 60t–61t, 62 Sodium nitrite, 907 Sodium nitroprusside, 393–394, 393f, 394f, 400t, 473t Sodium polystyrene sul onate, 519 Sodium pump, 456, 458f Sodium reabsorption, pharmacologic classes and agents or, 374–378, 382t–384t Sodium retention cirrhosis and, 371–372, 371f heart ailure and, 370–371, 371f Sodium stibogluconate, 685, 692t Sodium thiosul ate, 907 So osbuvir, 608, 609f, 702, 703f, 708–710, 720t Solid organ rejection, 844–846, 845t Soli enacin, 142, 143t, 148t Solute carrier (SLC), 56 Solute carrier (SLC) amily, 63t, 64 Solvent activation, 982f, 983 Solvent/gas partition coe f cient, 267b, 268t, 269b Soman, 142b Somatic gene recombination, o immune system, 787 Somatic nervous system anatomy o , 112–114, 112f cellular organization o , 116–117, 117f neurotransmitters o , 119, 119f

Index 1017 Somatostatin, 123, 499, 561, 566 Somatostatin analogues, 579t Somatostatin receptor ligands (SRLs), 503 Somatrem, 510t, 959t Somatropin, 503, 510t, 959t Sonophoresis, 981 Sora enib, 13, 753f, 760, 762–763, 762t, 767t Sort1, 340 Sortases, 643 Sortilin, 340 Sotalol, 60t, 444, 446, 451t Space o Disse, 342 Spare receptors, 19, 24–25, 25f Sparteine, 89 Specif city, 6 Spectinomycin, 607f, 628, 630t, 631f, 632, 638t Spectrum o action, 651 Spinal anesthesia, 175–176 Spinal cord, 114, 114f, 116, 116f descending and local inhibitory regulation in, 292–293, 292f dorsal horn pain transmission in, 291–292, 292f in nociception, 167, 168f pain conduction to, 291 Spinal nerves, 112, 112f Spironolactone, 359, 377, 384t, 397, 455, 474t, 475, 493t, 494, 536, 540t, 548f, 551–552, 558t Spiroperidol, 218f Spontaneous pain, 293 Sporozoites, 674–675, 676f SPRM. See Selective progesterone receptor modulators Squalene epoxidase inhibitors, 662, 663f, 665, 670t SR-BI. See Scavenger receptor class B, type I SRC–ABL inhibitors, 760 SREBP2. See Sterol regulatory element binding protein 2 SRLs. See Somatostatin receptor ligands SRS-A. See Slow-reacting substance o anaphylaxis SSA. See Succinic semialdehyde SSRIs. See Selective serotonin reuptake inhibitors Stabilizers, 931 Stable angina, 480, 480f, 484 Stable exertional angina, 484 Stanols, 352–353 St. Anthony’s Fire, 238 Staphylococcus aureus. See also Methicillin-resistant Staphylococcus aureus; Vancomycinresistant Staphylococcus aureus penicillins targeting, 652 Starvation, 563 Stasis, 412 State-dependent binding, 9 State-dependent ion channel block, 440–441 Statin benef t groups, 349, 350t Statins, 51t, 60t–61t, 81, 92t, 337, 350–352, 351f, 929t or acute coronary syndromes, 485f or chronic CAD, 484 on-target adverse e ect with, 73 pharmacogenomics, 66–67 transport o , 59, 63 Statistical review, 942, 942f Statistical signif cance, 952 STATs, 752, 753f Stavudine (d4T), 703f, 719t Steel actor. See Stem cell actor ST elevation myocardial in arction (STEMI), 481–482 clinical management o , 486–487 Stem cell actor, 832–833 Stem cells cancer, 724 hematopoietic, 830, 832f o immune system, 784, 785f, 835–836 STEMI. See ST elevation myocardial in arction Stents, sirolimus-, everolimus-, and zotarolimuseluting, 851 Stereochemistry, 4 Steroid 21-hydroxylase, 536, 537f Steroid production, 512t–513t Steroids, 89t 14 -Sterol demethylase inhibitors, 662, 663f, 665–667, 671t

Sterol regulatory element binding protein 2 (SREBP2), 350 Sterols, 352–353 Stevens-Johnson syndrome, 78, 301 St. John’s wort, 51t, 53, 65t, 66, 76 Strabismus, 137 Stramonium, 885–886 Stratum corneum, 176 Streptogramin B, resistance to, 633 Streptogramins, 612, 626f, 627f, 628, 630t, 634f, 635–636, 640t targets o , 607f Streptokinase, 76, 423–424, 431t, 487, 922, 925t, 966, 968t Streptomycin, 626f, 627f, 630, 631f, 638t selective targeting by, 605 Striatum, 114, 115f central dopamine pathways, 209–210 dopamine receptors in, 209, 210f neurons o , 211 Stroke, 199, 199f Structural proteins, 13 Structure-based drug design, 925–926 ST segment, 437b Subject bias, 938 Suboxone®, 328 Substance P, 175–176, 291, 878 Substance use disorders, 308 criteria or, 308, 311b depression with, 329 uture directions in, 329–331, 330f learning and development o , 315–317, 317f mechanisms o , 315–319 medical complications o , 326 personality characteristics and co-occurring disorders in, 319 treatments or, 326–327 detoxif cation, 327 pharmacologic treatment, 328–329 sel -help and mutual support programs, 327–328 variables a ecting development o , 317–319, 318f Substantia nigra, 114 central dopamine pathways, 209–210 single-source divergent neuronal systems and, 118, 118f, 118t Substantia nigra pars compacta, 212, 212f Subthalamic nucleus, 211 Subtherapeutic dosing, 39f Succimer, 908, 908f Succinic semialdehyde (SSA), 186 Succinylcholine, 52, 88, 140–141, 147t, 266, 280, 285t Sucral ate, 873, 876t Sudden cardiac death, 482 Su entanil, 299, 305t Sugammadex, 144, 149t Suicide substrate inhibition, 650 Suicide substrates, 4 Sulbactam, 650f, 651, 653, 657t, 775 Sulconazole, 666, 671t Sul adiazine, 615–616, 616f, 618 Sul adoxine, 675 Sul adoxine-pyrimethamine, 608, 677, 678f, 681, 690t, 775 Sul a drugs, 615–618, 616f Sul alene-pyrimethamine, 681, 690t Sul amethoxazole, 89t, 420t, 615–616, 616f, 618, 775 Sul anilamide, 616, 616f Sul asalazine, 63t Sulf npyrazone, 63t, 896f, 897t, 900, 903t Sul obromophthalein, 60t Sul onamides, 78, 615–618, 616f, 774 derivatives o , 376 procaine and, 179 resistance to, 613t targets o , 607f Sul ones, 616–617 Sul onylureas (SFUs), 569t, 571–572, 576t Sul uric acid, 909 Sulindac, 810, 815t Sulpiride, 218f Sumatriptan, 238, 247t, 302, 307t

Sunitinib, 79, 521, 759, 762–763, 762t, 768t Supercoils, DNA, 623–624, 625f Superior cervical ganglion, 111 Superior mesenteric ganglion, 111, 113f Superoxide anions, 685 Supplemental NDA/BLA (sNDA/sBLA), 944 Supplements, 944–945 Supro en, 52t Suramin, 684, 691t Surrogate endpoints, 939 Surround inhibition, 250, 251f SWS. See Slow-wave sleep Sympathetic nervous system activation o , 151–152 anatomy o , 111, 113f antagonists, 397 catecholamines in, 150 cellular organization o , 116–117, 117f neurotransmitters o , 119, 119f renal, 364 Sympathetic tone down-regulation, 475–476 Sympatholytics, 156, 157f or hypertension, 473t, 476 Sympathomimetics, 156, 157f or heart ailure, 495 receptor selectivity o , 463t Symptomatic treatments, 213 Synapses, 105, 106f Synapsins, 107 Synaptic cle t, 106f, 107 ACh release into, 129 Synaptic neuromodulators, 291 Synaptic transmission, 105–106, 106f Synaptic vesicles, 105, 106f ACh storage in, 128 regulation o , 107 Synaptobrevin/VAMP, 107, 108f Synaptotagmins, 107 Syndrome o inappropriate ADH (SIADH), 374, 507 Synergistic drugs antimicrobial examples o , 606, 631–632, 772–775, 773f olate metabolism inhibitors as, 615–618, 616f, 617t Syntaxin-1, 107, 108f Synthetic compounds, 922 Systemic absorption, o local anesthetics, 176 Systemic blood pressure, 472, 472f Systemic mastocytosis, 759 Systolic heart ailure, 459, 489

T

T3. See 3,5,3 -Triiodothyronine T4. See Thyroxine T-20. See En uvirtide TAAR1. See Trace amine-associated receptor 1 Tabun, 142b TAC. See Tetracaine, adrenaline, and cocaine Tachycardia autonomic ganglia and, 133 neurohumoral activation and, 491–492 Tachykinins, 878 as neuropeptides, 123 Tachyphylaxis, 15, 16t, 325 Tacrine, 52t, 139 Tacrolimus (FK506), 51t, 63t, 66t, 89t, 845, 850–851, 851f, 859t, 899f Tactile mechanoreceptors, 169 Tadalaf l, 51t, 395, 400t TAL. See Thick ascending limb Taliglucerase al a, 960t, 977 Talinolol, 60t, 63t, 65t, 66 Tamoxi en, 24, 50, 52t, 82, 89t, 90, 91f, 547, 549t, 550, 557t, 750, 777t Tamsulosin, 160, 164t TAP block. See Transversus abdominis plane block Tapentadol, 293 Tardive dyskinesia, 218 Target-centered drug design, 922–926, 924b, 925t, 925f Target product prof le (TPP), 937 Tariquidar, 63t Taxanes, 610f, 611, 741–742, 741f, 777t

1018 Index Tazobactam, 651, 653, 658t, 775 TBG. See Thyroid-binding globulin Tbo-Filgrastim, 962t TCAs. See Tricyclic antidepressants TC cells. See Cytotoxic T cells T-cell receptors (TCRs), 787–788, 788f T cells, 786t, 787–790, 788f, 789f in asthma, 879, 880f, 881–882, 883f development o , 784, 785f, 835–836 specif c depletion o , 848f, 857, 861t–862t TCRs. See T-cell receptors TDF. See Teno ovir T-DM1. See Trastuzumab emtansine Tebipenem, 60t Technetium anolesomab, 976t Tedizolid phosphate, 636, 640t Teduglutide, 965t Tegaserod, 238, 247t Telaprevir, 701–702, 701f, 718t Telavancin, 649–650, 656t Telbivudine, 703f, 707–708, 719t Telithromycin, 63t, 634–635, 639t Telmisartan, 60t, 381t Telomerase, 727, 731 Telomerase inhibitors, 742 Telomeres, 727, 730–731, 730f Tema oxacin, 74–75 Temazepam, 191, 202t clinical uses o , 192t Temocaprilat, 60t Temozolomide, 736, 746t Temporal lobes, 114, 115f Temsirolimus, 753f, 761, 767t Tenecteplase, 424, 431t, 487, 956, 958, 964t Teniposide (VM-26), 63t, 740, 748t Teno ovir (TDF), 60t, 63t, 66t, 604, 615, 695, 703f, 706, 707b, 710, 719t Teratogen, 82, 83b Teratogenesis, due to drug therapy, 82, 83b Terazosin, 160, 164t, 397, 401t, 475 Terbinaf ne, 665, 670t Terbutaline, 159, 164t, 884, 892t Terconazole, 666–667, 671t Ter enadine, 72, 74, 826 Teriparatide, 595, 600t, 929t, 964t Terlipressin, 374 Termination, bacterial translation, 627 Terodiline, 142, 148t Tertiary active transport, 28 Tertiary structure, o proteins, 2–3, 4f Tesamorelin, 503, 510t, 965t Testicular cancer, combination chemotherapy or, 780, 780f Testosterone, 60t, 420t, 541 DHT conversion o , 542, 544f inhibitors o , 556t transdermal gel or patch, 555, 560t Testosterone cypionate, 555, 560t Testosterone enanthate, 554–555, 560t Testosterone undecanoate, 554 Tetanic ade, 132, 135f Tetanus toxin GABAergic transmission and, 189t SNAREs and, 107 Tetracaine, 179, 181t Tetracaine, adrenaline, and cocaine (TAC), 176 Tetracyclic antidepressants, 236 Tetracyclines, 623, 626f, 627f, 628, 630, 630t, 631f, 632–633, 638t, 677, 680, 690t, 874 combination chemotherapy using, 773, 775 resistance to, 633 targets o , 606, 607f Tetraethylammonium, 64 9 -Tetrahydrocannabinol (THC), 325 Tetrahydro olate (THF), 724, 727f Tetrahydrozoline, 158, 163t 3,5,3 ,5 -Tetraiodothyronine (T4), 514 Tetrodotoxin, 99, 105 TFPI. See Tissue actor pathway inhibitor TGF- super amily. See Trans orming growth actor super amily TH. See Tyrosine hydroxylase Thalamocortical projections, 252 Thalamus, 115

Thalidomide, 74, 763, 769t THC. See 9-Tetrahydrocannabinol TH cells. See Helper T cells Thecal cells, 544, 545f Theobromine, 325 Theophylline, 325, 464–465, 468t, 879b, 885f, 886, 890t, 893t Therapeutic dosing and requency, 39–41, 39f, 40f, 41f Therapeutic e ects, 2 Therapeutic index (TI), 25–26, 605 Therapeutics, concepts in, 25–26 Therapeutic window, 25 Thermal nociceptors, 169 THF. See Tetrahydro olate Thiabendazole, 687, 693t Thiazide diuretics, 376–377, 383t, 473t, 474–475, 474t, 493 Thiazolidinediones (TZDs), 569t, 572–573, 577t, 929t Thick ascending limb (TAL) o loop o Henle, 365–367, 366f, 367t Thiocyanate, 519, 519f, 523t Thioguanine, 89t, 725f, 727f, 733f, 735, 744t Thiomethoxy urane, 270f Thionamides, 519f, 520 Thiopental, 203t, 280 case study o , 266 clinical uses o , 194t as general anesthetic, 284t Thioperamide, 826 Thiopurine S-methyltrans erase (TPMT), 89–91, 90f, 91f Thioridazine, 143t, 218f, 224t Thiotepa, 736, 746t Thiothixene, 218f, 224t Thioxanthenes, 217, 219f 30S ribosomal subunit, 626–627, 626f, 627f antimicrobial drugs targeting, 630–633, 630t, 631f, 632f, 638t–639t Thoracolumbar system, 111 3TC. See Lamivudine Threshold potential, 104 Threshold voltage, 102, 103f Thrombin, 405, 408, 409f, 964t Thrombocytopenia, 838–839 Thrombolytic agents, 423–424, 431t, 485f, 486–487 Thrombomodulin, 410 Thrombopoiesis, 836, 836f agents stimulating, 838–839, 842t Thrombopoietin (TPO), 833, 836 recombinant human, 839, 842t Thrombopoietin receptor agonists, 839, 842t Thrombosis, 403 pathogenesis o , 411–414, 412f, 413b, 413t pharmacologic classes and agents or anticoagulants, 418–423, 419f, 419t, 420t, 421f anticoagulation and f brinolysis inhibitors, 425 antiplatelet agents, 414–418, 415f, 416f uture directions or, 424–425 thrombolytic agents, 423–424 Thromboxane A2 (TxA2), 404 Thromboxane antagonists, 797f, 812, 818t Thromboxanes, 795, 796f, 797f, 798–799, 798f, 806, 806f, 806t, 808 Thrombus, 411 Thymidine kinase (TK), viral, 702, 704f, 706, 706t Thymidylate synthase, 725, 727f Thymidylate synthase inhibitors, 616f, 664, 664f, 733, 733f, 743t Thymine, 622, 624f synthesis o , 724–725, 725f, 726f, 726t, 727f Thyroglobulin, 515 Thyroid-binding globulin (TBG), 516 Thyroid gland case study or, 515 pathophysiology o , 518, 518f pharmacologic classes and agents or, 519–521, 519f physiology o , 514–517, 515f, 516f, 517f, 518f Thyroid hormone receptors (TRs), 516–517, 517f Thyroid hormones calcium and phosphate and, 586 uture directions or, 521–522

metabolism o , 515f, 516 inhibitors o , 521 release inhibition o , 519f, 520–521, 523t synthesis and secretion o , 514–516, 515f, 516f target tissue e ects o , 516–517, 517f Thyroid peroxidase, 515, 516f Thyroid-stimulating hormone (TSH), 500, 500t, 975, 976t lithium and, 240 TRH and, 505, 517, 518f Thyroid-stimulating immunoglobulin (TSIg), 518 Thyrotropin, 505, 511t, 976t Thyrotropin-releasing hormone (TRH), 123, 499, 500t, 504–505, 504f, 517, 518f Thyroxine (T4), 514–515, 515f, 519 TI. See Therapeutic index Tiagabine, 189, 189t, 202t, 256t, 260, 264t Tianeptine, 237, 246t Ticagrelor, 406, 417, 427t, 486 Ticarcillin, 651, 653, 658t Ticlopidine, 406, 415–417, 426t, 486 Tigecycline, 612, 631f, 633, 639t Tiludronate, 592f Time constant, 270 Time-dependent bactericidal agents, 772, 772f Timolol, 51t, 160, 160t, 165t Tinidazole, 684, 691t Tinzaparin, 422, 429t Tioconazole, 666, 671t Tiotropium, 141–142, 148t, 879b, 886, 893t Tipi arnib, 760 Tipranavir, 65t, 712, 712f, 721t Tirof ban, 417, 428t Tissue/blood partition coe f cients, 273, 273t Tissue actor, 403–405, 405f, 408, 409f Tissue actor pathway inhibitor (TFPI), 409f, 410 Tissue partial pressure (Ptissue) alveolar partial pressure and, 272–273, 273t, 274f arterial partial pressure and, 273, 273t, 274f Tissue plasminogen activator (tPA), 964t Tissue schizonts, 675 Tizanidine, 143t TK. See Thymidine kinase TKIs. See Tyrosine kinase inhibitors TLRs. See Toll-like receptors TNF. See Tumor necrosis actor TNF- inhibitors, 294, 795, 812, 852–854, 852f, 853f, 859t–860t, 890 Tobacco, 310t, 323, 879b, 912–913 peptic ulcer disease and, 874 Tobramycin, 630, 638t Tocilizumab, 854, 861t, 970t Tolazamide, 576t Tolbutamide, 92t, 576t Tolcapone, 215, 223t Tolerance, 54, 298, 309–311, 311–313, 312f–313f, 314f immune, 788–790, 789f, 846 opioid use disorder and, 320 Toll-like receptors (TLRs), 786 Tolterodine, 142, 143t, 148t Toluene, 310t Tolvaptan, 374, 381t, 494, 507, 513t Tonic–clonic seizure, 252–253, 252t, 254f Tonic inhibition, with local anesthetics, 174–175, 175f Tonic phase, o seizure, 253, 254f Tophi, 897 Topical anesthesia, 176–177 Topiramate, 256t, 260, 264t, 329, 333t, 375 Topoisomerase inhibitors, 610f, 611f, 628–629, 628f, 637t, 728f, 739f, 740–741, 747t–748t Topoisomerases, 623–624, 625f, 628, 628f, 740 Topotecan, 63t, 64, 65t, 740, 747t Toremi ene, 551 Torsades de pointes, 438, 441b Torsemide, 376, 383t, 474t Torticollis, 137 Tositumomab, 763, 769t, 777t, 973t Toxic dosing, 39f Toxic e ects, 70 Toxic epidermal necrolysis, 78 Toxic metabolites, 50

Index 1019 Toxic mushrooms, 142b Toxicology, 929–930 t-PA. See Recombinant tissue plasminogen activator tPA. See Tissue plasminogen activator TPH. See Tryptophan hydroxylase TPMT. See Thiopurine S-methyltrans erase TPO. See Thrombopoietin TPs. See Transpeptidases Trace amine-associated receptor 1 (TAAR1), 157 Tralokinumab, 891f Tramadol, 158, 293, 304t Trametinib, 753f, 761, 767t Trandolapril, 380t Tranexamic acid, 424, 432t Transcellular biosynthesis, o eicosanoids, 806, 806f Transcortin, 525–526 Transcription bacterial, 624, 626f inhibitors o , 612, 629–630, 629f, 637t–638t viral, 695, 697f Transcription actors, 6, 13, 13f Transdermal drug administration, 30t, 31 Transdermal drug delivery, 981 Transduction, 613 Trans ormation in carcinogenesis, 608 in drug resistance, 613 Trans orming growth actor (TGF- ) super amily, 12 Transient receptor potential vanilloid receptor 1 (TRPV1), 290, 290f Translation bacterial, 626–627, 626f, 627f inhibitors o , 630–636, 630t, 631f, 632f, 634f, 635f, 638t–640t plasmodial, inhibitors o , 680, 690t viral, 695, 697f Translocation, 627 Transmembrane electrochemical gradient, 100, 101f Transmembrane G protein-coupled receptors, 9–11, 9f, 10f Transmembrane ion channels, 7–9, 8f, 8t Transmembrane receptors with linked enzymatic domains, 11–12, 11f Transmigration, 791 Transmission, o drug resistance, 612 Transmission mode, 253, 255f Transpeptidases (TPs), 644f, 646, 647f Transplantation, organ, 844–846, 845t Transporters dopamine, 207, 230 GABA, 186 glutamate, 198 hexose, 123 multiple drug resistance, 124 norepinephrine, 152f, 153, 156, 230 serotonin, 230 Transudative edema, 370 Transversus abdominis plane (TAP) block, 177 Tranylcypromine, 158, 163t Trastuzumab (Anti-ErbB2), 753f, 758–759, 765t, 778t, 925t, 966, 969t Trastuzumab emtansine (T-DM1), 759, 765t, 972t, 973t Trauma, 199, 199f Traumatic brain injury, 139 Travoprost, 817t Trazodone, 237–238, 246t, 825 Treg. See Regulatory T cells Trematodes, 685 Tremor syndrome, 911 Treprostinil, 396, 401t, 817t Tretinoin, 777t antineoplastic applications o , 839, 843t TRH. See Thyrotropin-releasing hormone Triamcinolone, 532, 533f, 539t, 887, 894t Triamterene, 377, 384t, 474t, 475 Triazolam, 51t, 191, 202t abuse o , 310t clinical uses o , 192t Triazoles, 662, 663f, 664f, 665–667, 671t, 775

Tricyclic antidepressants (TCAs), 236, 243t adverse e ects o , 236 or analgesia, 306t catecholamine reuptake inhibition with, 158 elderly and, 143b neurotransmitters and, 121–122 in pain management, 301 SSRIs compared with, 236–237 or substance use disorders, 334t Trientine, 908 Tri uoperazine, 143t, 218f, 224t Tri uperidol, 218f Tri uridine, 719t Triggered activity, 438–439, 439f Triglycerides, 338 absorption o , 339, 339f Trihexyphenidyl, 142, 143t, 215, 224t 3,5,3 -Triiodothyronine (T3), 514, 515f, 519 Trilostane, 534, 534t, 540t Trimethaphan, 131t, 144, 149t, 476 Trimethoprim, 616f, 617–618, 617t, 774–775 targets o , 607f Trimipramine, 143t, 243t Tripelennamine, 824f, 828t Triptans, 238, 302 Triptorelin, 549, 556t Troglitazone, 66t, 70, 80, 949t Troleandomycin, 51t Trophozoites, 682, 683f Trospium, 142, 143t, 148t Trova oxacin, 75 TRPV1. See Transient receptor potential vanilloid receptor 1 TRs. See Thyroid hormone receptors Trypanosoma brucei gambiense, 684–685 Trypanosoma brucei rhodesiense, 684–685 Trypanosoma cruzi, 685 Trypsin, 965t Tryptase, 882 Tryptophan hydroxylase (TPH), 228–229, 229f TSH. See Thyroid-stimulating hormone TSIg. See Thyroid-stimulating immunoglobulin T-type calcium channel, 253, 255f inhibition o , 258 Tuberculosis antibiotics or, 629–630, 649, 649f, 654–655, 660t combination chemotherapy or, 771, 773–774 MDR, 612, 649, 771, 773–774 mycobacterial cell wall and, 642f, 646–647, 648f Tuberoin undibular pathway, 211 Tuberomammillary nucleus, 118t, 119 Tubocurare, 128, 131t, 138, 143–144 Tubocurarine, 280, 285t D-Tubocurarine, 60t, 144, 148t Tubulins, 731–732, 731f, 732f, 741–742, 741f Tubuloglomerular eedback, 360 Tumoral calcinosis, 586 Tumor lysis syndrome, 734, 779, 900 Tumor necrosis actor (TNF), 790t, 791, 852–854, 852f, 859t–860t, 879, 880f Tumors drugs targeting. See Antineoplastic drugs genome synthesis, stability, and maintenance biochemistry in, 724–732, 725f, 726f, 726t, 727f, 728f, 729f, 730f, 731f, 732f initiation and promotion o , 912, 913f Tumor-specif c monoclonal antibodies, 611, 763, 769t Tumor suppressor protein p53, 730–731, 730f Turbulence, 412 T wave, 437b 23S rRNA, 626–627, 626f, 627f TxA2. See Thromboxane A2 Type-1 cannabinoid receptors (CB1), 325 Type 1 diabetes mellitus, 567–568, 567t Type 2 diabetes mellitus, 567t, 568 Type I 5 -deiodinase, 516 Type I hypersensitivity, in asthma, 881–882 Type I inter erons, 716 Type II 5 -deiodinase, 516 Type II inter erons, 716 Type III 5-deiodinase, 516

Type IV hypersensitivity, in asthma, 881–882 Typical antipsychotic agents, or schizophrenia, 217–220, 218f, 219f Tyramine, 157–158, 208, 235 Tyramine toxicity, 235 Tyrosine, in catecholamine synthesis, 121, 150, 152f, 206–207, 208f Tyrosine hydroxylase (TH), 150–151, 152f, 206–207, 208f, 229 Tyrosine kinase-associated receptors, 11f, 12. See also Receptor tyrosine kinases Tyrosine kinase inhibitors (TKIs), 79, 521, 751, 753f, 758–763, 762t, 765t–767t TZDs. See Thiazolidinediones

U

UA. See Unstable angina UA/NSTEMI. See Unstable angina/non-ST elevation myocardial in arction Ubenimex, 60t Ubiquinone, 677, 677f, 680 Ubiquitin–proteasome pathway, 752–756, 754f, 755f–756f U f bers, 252 UGN. See Uroguanylin Ulcers. See Peptic ulcer disease Ulipristal, 548f, 552, 554, 558t Umeclidinium, 886, 893t UMP. See Uridylate Uncoating, viral, 695, 696f, 697f Uncoating inhibitors, 609f, 696f, 698–700, 700f, 718t Uncompetitive antagonists, 7 Underf ll model, 371, 371f Un ractionated heparin, 404, 421f, 422, 429t Unique drug targets, o antimicrobial and antineoplastic drugs, 604t, 605 Unitary hypothesis, 281, 281f Unoprostone, 60t Unstable angina (UA), 481 Unstable angina/non-ST elevation myocardial in arction (UA/NSTEMI), 481 clinical management o , 485–486, 485f Unstable plaques, 481, 482f Uptake 1, 152f, 153 Uptake model, 269–274, 271f, 272f, 273t, 274f, 275f applications o , 275–277, 276f, 277f, 277t, 278f Uptake proteins, 56 Uptake transporters, 58–62, 58f, 60t–61t Uracil, synthesis o , 724–725, 725f, 726f, 726t, 727f Urease, 867 Ureidopenicillins, 653 Uric acid, 895, 897. See also Gout agents decreasing synthesis o , 899–900, 899f, 900f, 902t agents enhancing metabolism o , 900–901, 903t agents increasing excretion o , 900, 903t Uricase, 896f, 900–901 Uricosuric agents, 900 Uridylate (UMP), 725, 726f, 727f Urinary incontinence, 137, 142 Uro ollitropin, 507, 513t Uroguanylin (UGN), 363 Urokinase, 964t US Food and Drug Administration. See FDA Use-dependent block, 104 Ustekinumab, 854, 860t, 970t

V

Vaccination, 603–604 or malaria, 688 or viral in ections, 716 Vaccines or cocaine, 330, 330f or hepatitis B, 974 protein, 973–975, 974t Vacuolar H ATPase (vH ATPase), 365 Vagomimetic, 140 Vagus nerve, 112, 113f Valacyclovir, 60t, 695, 703f, 704, 705, 718t

1020 Index Valdecoxib, 78, 295, 301, 811–812, 811f, 949t Valganciclovir, 67, 703f, 719t Valproic acid, 239, 247t, 254, 256t, 258, 262t, 302 Valsartan, 60t, 63t, 373–374, 381t, 402t, 476 Valspodar, 63t Valv. See Alveolar ventilation Vancomycin, 36, 71, 78, 607f, 629, 642, 643, 644f, 649–650, 656t combination chemotherapy using, 772–773 resistance to, 613, 613t Vancomycin-resistant enterococci (VRE), 612–613, 635, 650, 654 Vancomycin-resistant Staphylococcus aureus (VRSA), 650 van der Waals orces, 3, 5t Vandetanib, 762, 762t, 768t Vardenaf l, 395, 400t Varenicline, 60t, 65t, 321f, 329, 334t Variant angina, 483 Varicella zoster virus (VZV), nucleoside and nucleotide analogues targeting, 702–705, 703f, 704f, 718t–719t Vascular cell adhesion molecules (VCAMs), 791 Vascular endothelial growth actors (VEGFs), 756–757, 756t, 757f receptor inhibitors, 762–763, 762t Vascular endothelium, 388–389 Vascular-ischemic dementia, 139 Vascular resistance-based hypertension, 472 Vascular smooth muscle contraction and relaxation o , 386–387, 387f intracellular signaling in, 387–388, 388f modulation o , 476 Vascular tone, 385 uture directions or, 397 pharmacologic classes and agents or calcium channel blockers, 390–392, 391f cGMP potentiators, 395–396 endothelin receptor antagonists, 396 hydralazine, 392 nitric oxide donors, 392–395, 393f, 394f potassium channel openers, 392 prostacyclin analogues, 396 RAAS blockers, 397 sympathetic nervous system antagonists, 397 physiology o , 385–390, 386f, 387f regulation o , 387–389, 388f, 389f, 390f Vasoactive intestinal polypeptide (VIP), 878 Vasoconstriction, 387–388, 404 localized, 403, 405f neurohumoral activation and, 491–492 Vasoconstrictors, 176 with local anesthetics, 176 Vasodilation, 360, 388 Vasodilators or heart ailure, 495 or hypertension, 473t Vasopressin, 115, 363f, 364, 507 analogue, 513t Vasopressin receptor 2 (V2) agonists, 374 antagonists, 374, 381t, 513t Vasospastic angina, 483–484 Vd. See Volume o distribution Vecuronium, 144, 148t, 285t Vedolizumab, 14, 970t VEGFR inhibitors, 762–763, 762t VEGFs. See Vascular endothelial growth actors Velaglucerase al a, 961t Vemura enib, 13, 605, 753f, 760, 767t resistance to, 613t, 614 Venla axine, 51t, 232, 237, 244t, 301, 306t Venodilators, 494 Ventilation-limited anesthetics, 273–274 ventilation changes and, 275, 276f Ventilation/per usion (V/Q) mismatch, 277 Ventral horns, 112 Ventral roots, 112, 112f Ventral tegmental area (VTA), 210–211, 313, 315, 315f, 316f Ventricular f brillation, 441b

Ventricular myocyte, action potential phases o , 434, 435f, 436f, 436t Ventricular tachycardia (VT), 441b Verapamil, 51t, 60t, 63t, 390, 398t, 437, 447, 452t, 462, 476, 483, 899, 899f Vertical transmission, 612 Very-low-density lipoprotein (VLDL), 337t, 338, 340 inhibitors o secretion o , 352, 356t remnants, 341–342, 341f Vesamicol, 128, 137 Vesicular monoamine transporter (VMAT), 151, 153, 156, 186, 207, 229 Vesnarinone, 468t Vessel dilation, in in ammatory response, 791 Vessel-poor group (VPG), 270–271, 271f Vessel-rich group (VRG), 270–271, 271f VGAT, 186 vH ATPase. See Vacuolar H ATPase VHL protein. See von Hippel-Lindau protein Vidarabine, 719t Vigabatrin, 189–190, 189t, 202t, 256t, 259, 263t Vilanterol, 885, 892t Vilazodone, 238, 246t Vinblastine, 63t, 81, 741, 748t Vinca alkaloids, 610f, 611, 741, 741f, 777t, 778t Vincristine, 63t, 81, 741, 748t, 777t VIP. See Vasoactive intestinal polypeptide Viral in ections case study, 695 drugs targeting. See Antiviral drugs uture directions, 717 replication o , 694–697, 696f, 697f Virchow’s triad, 411, 412f Virions, 695 Virotoxins, 909 Vitamin B3. See Niacin Vitamin B6, 186 Vitamin D, 601t analogues o , 591f, 597, 601t calcium and phosphate and, 584–586, 584t, 585f Vitamin K, 418, 419t Vitamin K epoxide reductase complex 1 (VKORC1), 53, 92–93, 93f VLDL. See Very-low-density lipoprotein VMAT. See Vesicular monoamine transporter Voltage, 99, 99f Voltage-clamping, 103 Voltage-dependent unctions, 98 Voltage di erence, 99 Voltage-gated calcium channels, N-type, 291 Voltage-gated ion channels, 8, 8t action potential and, 102–103 delayed rectif er potassium channels, 104, 105f voltage dependence o , 103, 104f Voltage-gated potassium channels, 102–103, 104f delayed rectif er, 104, 105f time course o , 104, 105f Voltage-gated sodium channels, 102–103, 104f inactivation o , 104 local anesthetics and, 173, 174f modulated receptor hypothesis, 173–174, 173f, 174t tonic and phasic inhibition, 174–175, 175f in pain conduction, 291 time course o , 104, 105f Volume-based hypertension, 472–473 Volume capacity, 270, 271f, 287 Volume-contraction alkalosis, 376 Volume o distribution (Vd), 32–33, 33f, 176 Volume regulation, 358 edema ormation, 370–372 uture directions or, 378–379 pharmacologic classes and agents or renal sodium reabsorption, 374–378 volume regulator modif cation, 372–374, 375f physiology o intravascular volume determinants, 358–359, 360f regulators or, 360–364, 361f, 362f, 363f renal control o sodium excretion, 364–370, 364f, 365f, 366f, 367f, 367t, 368t, 369f sensors or, 360

Volume regulators, 360–364, 361f, 362f, 363f modif cation o , 372–374, 375f Volume sensors, 360 Volume transmission, 107 von Hippel-Lindau (VHL) protein, 757, 757f von Willebrand actor (vWF), 404, 406f, 960t Vorapaxar, 410, 417–418, 428t Voriconazole, 607, 663f, 666–667, 671t Vortioxetine, 238, 246t VP-16. See Etoposide VPG. See Vessel-poor group V/Q mismatch. See Ventilation/per usion mismatch VRE. See Vancomycin-resistant enterococci VRG. See Vessel-rich group VRSA. See Vancomycin-resistant Staphylococcus aureus VT. See Ventricular tachycardia VTA. See Ventral tegmental area Vulnerable plaques, 481, 482f vWF. See von Willebrand actor VZV. See Varicella zoster virus

W

Wake ulness, ACh in, 134 Wall teichoic acids, 642f, 643 War arin, 12, 52t, 71, 88, 89t, 92t, 418, 428t antibiotic interactions with, 635 clinical uses o , 418–420, 419t, 420t mechanism o action o , 418, 419f metabolism o , 53 pharmacogenomics and, 92–93, 93f stereochemistry o , 4 Weak positive allosteric agonists, 191, 191f Wheal-and- are reaction, 76 Wheal-and- are response, histamine causing, 820 White blood cells. See Leukocytes White matter, 114, 115f Withdrawal, 313, 315, 315f, 322f Withdrawal symptoms, 322f, 327 WNK protein kinases, 367–368, 367f, 368t WNT signaling pathway, 755–756, 755f–756f Wol -Chaiko e ect, 520 Workplace exposures, 915–916 Worms. See Helminths

X

Xanthine oxidase, 896, 896f, 899, 899f, 900f, 902t Xanthomas, 337 Xenobiotics, 43 Xenon, 270f Xeroderma pigmentosa, 728 Xerostomia, 137, 140 X-linked hypophosphatemic rickets (XLH), 586 X-ray crystallography, 925

Y

Yeasts, 661 Yohimbine, 160, 165t

Z

Zaf rlukast, 800f, 813, 813f, 818t, 884f, 888, 894t Zalcitabine (ddC), 60t, 703f Zaleplon, 191 Zanamivir, 608, 609f, 696f, 715, 715f, 721t Z-drugs, 191 Zero-order kinetics, 37, 38f Ziconotide, 153, 291 Zidovudine (AZT), 60t, 61, 81, 608, 609f, 615, 695, 696f, 703f, 706, 706t, 707b, 719t Zileuton, 92, 92t, 800f, 812–813, 813f, 818t, 884f, 888, 894t Ziprasidone, 220, 226t Ziv-a ibercept, 968t Zo enopril, 60t, 380t Zoledronate, 592–594, 592f, 599t Zollinger-Ellison syndrome, 868 Zolmitriptan, 238, 247t, 302, 307t Zolpidem, 191, 202t Zonisamide, 256t, 260, 264t Zopiclone, 191 Zotarolimus, 487, 851, 852f, 859t, 984