DRUGS AND THE PHARMACEUTICAL SCIENCES

VOLUME 186

Handbook of Drug MetabolismSecond Edition

edited by

Paul G. Pearson Larry C. Wienkers

Handbook of Drug Metabolism

DRUGS AND THE PHARMACEUTICAL SCIENCESA Series of Textbooks and Monographs

Executive Editor James SwarbrickPharmaceuTech, Inc. Pinehurst, North Carolina

Advisory BoardLarry L. AugsburgerUniversity of Maryland Baltimore, Maryland

Harry G. BrittainCenter for Pharmaceutical Physics Milford, New Jersey

Jennifer B. DressmanUniversity of Frankfurt Institute of Pharmaceutical Technology Frankfurt, Germany

Robert GurnyUniversite de Geneve Geneve, Switzerland

Anthony J. HickeyUniversity of North Carolina School of Pharmacy Chapel Hill, North Carolina

Jeffrey A. HughesUniversity of Florida College of Pharmacy Gainesville, Florida

Ajaz HussainSandoz Princeton, New Jersey

Vincent H. L. LeeUS FDA Center for Drug Evaluation and Research Los Angeles, California

Joseph W. PolliGlaxoSmithKline Research Triangle Park North Carolina

Kinam ParkPurdue University West Lafayette, Indiana

Stephen G. SchulmanUniversity of Florida Gainesville, Florida

Jerome P. SkellyAlexandria, Virginia

Elizabeth M. ToppUniversity of Kansas Lawrence, Kansas

Yuichi SugiyamaUniversity of Tokyo, Tokyo, Japan

Geoffrey T. TuckerUniversity of Sheffield Royal Hallamshire Hospital Sheffield, United Kingdom

Peter YorkUniversity of Bradford School of Pharmacy Bradford, United Kingdom

1. Pharmacokinetics, Milo Gibaldi and Donald Perrier 2. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control, Sidney H. Willig, Murray M. Tuckerman, and William S. Hitchings IV 3. Microencapsulation, edited by J. R. Nixon 4. Drug Metabolism: Chemical and Biochemical Aspects, Bernard Testa and Peter Jenner 5. New Drugs: Discovery and Development, edited by Alan A. Rubin 6. Sustained and Controlled Release Drug Delivery Systems, edited by Joseph R. Robinson 7. Modern Pharmaceutics, edited by Gilbert S. Banker and Christopher T. Rhodes 8. Prescription Drugs in Short Supply: Case Histories, Michael A. Schwartz 9. Activated Charcoal: Antidotal and Other Medical Uses, David O. Cooney 10. Concepts in Drug Metabolism (in two parts), edited by Peter Jenner and Bernard Testa 11. Pharmaceutical Analysis: Modern Methods (in two parts), edited by James W. Munson 12. Techniques of Solubilization of Drugs, edited by Samuel H. Yalkowsky 13. Orphan Drugs, edited by Fred E. Karch 14. Novel Drug Delivery Systems: Fundamentals, Developmental Concepts, Biomedical Assessments, Yie W. Chien 15. Pharmacokinetics: Second Edition, Revised and Expanded, Milo Gibaldi and Donald Perrier 16. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control, Second Edition, Revised and Expanded, Sidney H. Willig, Murray M. Tuckerman, and William S. Hitchings IV 17. Formulation of Veterinary Dosage Forms, edited by Jack Blodinger 18. Dermatological Formulations: Percutaneous Absorption, Brian W. Barry 19. The Clinical Research Process in the Pharmaceutical Industry, edited by Gary M. Matoren 20. Microencapsulation and Related Drug Processes, Patrick B. Deasy 21. Drugs and Nutrients: The Interactive Effects, edited by Daphne A. Roe and T. Colin Campbell 22. Biotechnology of Industrial Antibiotics, Erick J. Vandamme 23. Pharmaceutical Process Validation, edited by Bernard T. Loftus and Robert A. Nash 24. Anticancer and Interferon Agents: Synthesis and Properties, edited by Raphael M. Ottenbrite and George B. Butler 25. Pharmaceutical Statistics: Practical and Clinical Applications, Sanford Bolton 26. Drug Dynamics for Analytical, Clinical, and Biological Chemists, Benjamin J. Gudzinowicz, Burrows T. Younkin, Jr., and Michael J. Gudzinowicz 27. Modern Analysis of Antibiotics, edited by Adjoran Aszalos 28. Solubility and Related Properties, Kenneth C. James 29. Controlled Drug Delivery: Fundamentals and Applications, Second Edition, Revised and Expanded, edited by Joseph R. Robinson and Vincent H. Lee 30. New Drug Approval Process: Clinical and Regulatory Management, edited by Richard A. Guarino 31. Transdermal Controlled Systemic Medications, edited by Yie W. Chien

32. Drug Delivery Devices: Fundamentals and Applications, edited by Praveen Tyle 33. Pharmacokinetics: Regulatory . Industrial . Academic Perspectives, edited by Peter G. Welling and Francis L. S. Tse 34. Clinical Drug Trials and Tribulations, edited by Allen E. Cato 35. Transdermal Drug Delivery: Developmental Issues and Research Initiatives, edited by Jonathan Hadgraft and Richard H. Guy 36. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, edited by James W. McGinity 37. Pharmaceutical Pelletization Technology, edited by Isaac Ghebre-Sellassie 38. Good Laboratory Practice Regulations, edited by Allen F. Hirsch 39. Nasal Systemic Drug Delivery, Yie W. Chien, Kenneth S. E. Su, and Shyi-Feu Chang 40. Modern Pharmaceutics: Second Edition, Revised and Expanded, edited by Gilbert S. Banker and Christopher T. Rhodes 41. Specialized Drug Delivery Systems: Manufacturing and Production Technology, edited by Praveen Tyle 42. Topical Drug Delivery Formulations, edited by David W. Osborne and Anton H. Amann 43. Drug Stability: Principles and Practices, Jens T. Carstensen 44. Pharmaceutical Statistics: Practical and Clinical Applications, Second Edition, Revised and Expanded, Sanford Bolton 45. Biodegradable Polymers as Drug Delivery Systems, edited by Mark Chasin and Robert Langer 46. Preclinical Drug Disposition: A Laboratory Handbook, Francis L. S. Tse and James J. Jaffe 47. HPLC in the Pharmaceutical Industry, edited by Godwin W. Fong and Stanley K. Lam 48. Pharmaceutical Bioequivalence, edited by Peter G. Welling, Francis L. S. Tse, and Shrikant V. Dinghe 49. Pharmaceutical Dissolution Testing, Umesh V. Banakar 50. Novel Drug Delivery Systems: Second Edition, Revised and Expanded, Yie W. Chien 51. Managing the Clinical Drug Development Process, David M. Cocchetto and Ronald V. Nardi 52. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control, Third Edition, edited by Sidney H. Willig and James R. Stoker 53. Prodrugs: Topical and Ocular Drug Delivery, edited by Kenneth B. Sloan 54. Pharmaceutical Inhalation Aerosol Technology, edited by Anthony J. Hickey 55. Radiopharmaceuticals: Chemistry and Pharmacology, edited by Adrian D. Nunn 56. New Drug Approval Process: Second Edition, Revised and Expanded, edited by Richard A. Guarino 57. Pharmaceutical Process Validation: Second Edition, Revised and Expanded, edited by Ira R. Berry and Robert A. Nash 58. Ophthalmic Drug Delivery Systems, edited by Ashim K. Mitra 59. Pharmaceutical Skin Penetration Enhancement, edited by Kenneth A. Walters and Jonathan Hadgraft 60. Colonic Drug Absorption and Metabolism, edited by Peter R. Bieck

61. Pharmaceutical Particulate Carriers: Therapeutic Applications, edited by Alain Rolland 62. Drug Permeation Enhancement: Theory and Applications, edited by Dean S. Hsieh 63. Glycopeptide Antibiotics, edited by Ramakrishnan Nagarajan 64. Achieving Sterility in Medical and Pharmaceutical Products, Nigel A. Halls 65. Multiparticulate Oral Drug Delivery, edited by Isaac Ghebre-Sellassie rg 66. Colloidal Drug Delivery Systems, edited by Jo Kreuter 67. Pharmacokinetics: Regulatory . Industrial . Academic Perspectives, Second Edition, edited by Peter G. Welling and Francis L. S. Tse 68. Drug Stability: Principles and Practices, Second Edition, Revised and Expanded, Jens T. Carstensen 69. Good Laboratory Practice Regulations: Second Edition, Revised and Expanded, edited by Sandy Weinberg 70. Physical Characterization of Pharmaceutical Solids, edited by Harry G. Brittain ran 71. Pharmaceutical Powder Compaction Technology, edited by Go m Alderborn and Christer Nystro 72. Modern Pharmaceutics: Third Edition, Revised and Expanded, edited by Gilbert S. Banker and Christopher T. Rhodes 73. Microencapsulation: Methods and Industrial Applications, edited by Simon Benita 74. Oral Mucosal Drug Delivery, edited by Michael J. Rathbone 75. Clinical Research in Pharmaceutical Development, edited by Barry Bleidt and Michael Montagne 76. The Drug Development Process: Increasing Efficiency and Cost Effectiveness, edited by Peter G. Welling, Louis Lasagna, and Umesh V. Banakar 77. Microparticulate Systems for the Delivery of Proteins and Vaccines, edited by Smadar Cohen and Howard Bernstein 78. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control, Fourth Edition, Revised and Expanded, Sidney H. Willig and James R. Stoker 79. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms: Second Edition, Revised and Expanded, edited by James W. McGinity 80. Pharmaceutical Statistics: Practical and Clinical Applications, Third Edition, Sanford Bolton 81. Handbook of Pharmaceutical Granulation Technology, edited by Dilip M. Parikh 82. Biotechnology of Antibiotics: Second Edition, Revised and Expanded, edited by William R. Strohl 83. Mechanisms of Transdermal Drug Delivery, edited by Russell O. Potts and Richard H. Guy 84. Pharmaceutical Enzymes, edited by Albert Lauwers and Simon Scharpe 85. Development of Biopharmaceutical Parenteral Dosage Forms, edited by John A. Bontempo 86. Pharmaceutical Project Management, edited by Tony Kennedy 87. Drug Products for Clinical Trials: An International Guide to Formulation . Production . Quality Control, edited by Donald C. Monkhouse and Christopher T. Rhodes

88. Development and Formulation of Veterinary Dosage Forms: Second Edition, Revised and Expanded, edited by Gregory E. Hardee and J. Desmond Baggot 89. Receptor-Based Drug Design, edited by Paul Leff 90. Automation and Validation of Information in Pharmaceutical Processing, edited by Joseph F. deSpautz 91. Dermal Absorption and Toxicity Assessment, edited by Michael S. Roberts and Kenneth A. Walters 92. Pharmaceutical Experimental Design, Gareth A. Lewis, Didier Mathieu, and Roger Phan-Tan-Luu 93. Preparing for FDA Pre-Approval Inspections, edited by Martin D. Hynes III 94. Pharmaceutical Excipients: Characterization by IR, Raman, and NMR Spectroscopy, David E. Bugay and W. Paul Findlay 95. Polymorphism in Pharmaceutical Solids, edited by Harry G. Brittain 96. Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products, edited by Louis Rey and Joan C. May 97. Percutaneous Absorption: DrugsCosmeticsMechanismsMethodology, Third Edition, Revised and Expanded, edited by Robert L. Bronaugh and Howard I. Maibach 98. Bioadhesive Drug Delivery Systems: Fundamentals, Novel Approaches, and Development, edited by Edith Mathiowitz, Donald E. Chickering III, and Claus-Michael Lehr 99. Protein Formulation and Delivery, edited by Eugene J. McNally 100. New Drug Approval Process: Third Edition, The Global Challenge, edited by Richard A. Guarino 101. Peptide and Protein Drug Analysis, edited by Ronald E. Reid 102. Transport Processes in Pharmaceutical Systems, edited by Gordon L. Amidon, Ping I. Lee, and Elizabeth M. Topp 103. Excipient Toxicity and Safety, edited by Myra L. Weiner and Lois A. Kotkoskie 104. The Clinical Audit in Pharmaceutical Development, edited by Michael R. Hamrell 105. Pharmaceutical Emulsions and Suspensions, edited by Francoise Nielloud and Gilberte Marti-Mestres 106. Oral Drug Absorption: Prediction and Assessment, edited by Jennifer s B. Dressman and Hans Lennerna 107. Drug Stability: Principles and Practices, Third Edition, Revised and Expanded, edited by Jens T. Carstensen and C. T. Rhodes 108. Containment in the Pharmaceutical Industry, edited by James P. Wood 109. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control from Manufacturer to Consumer, Fifth Edition, Revised and Expanded, Sidney H. Willig 110. Advanced Pharmaceutical Solids, Jens T. Carstensen 111. Endotoxins: Pyrogens, LAL Testing, and Depyrogenation, Second Edition, Revised and Expanded, Kevin L. Williams 112. Pharmaceutical Process Engineering, Anthony J. Hickey and David Ganderton 113. Pharmacogenomics, edited by Werner Kalow, Urs A. Meyer and Rachel F. Tyndale

114. Handbook of Drug Screening, edited by Ramakrishna Seethala and Prabhavathi B. Fernandes 115. Drug Targeting Technology: Physical . Chemical . Biological Methods, edited by Hans Schreier 116. DrugDrug Interactions, edited by A. David Rodrigues 117. Handbook of Pharmaceutical Analysis, edited by Lena Ohannesian and Anthony J. Streeter 118. Pharmaceutical Process Scale-Up, edited by Michael Levin 119. Dermatological and Transdermal Formulations, edited by Kenneth A. Walters 120. Clinical Drug Trials and Tribulations: Second Edition, Revised and Expanded, edited by Allen Cato, Lynda Sutton, and Allen Cato III 121. Modern Pharmaceutics: Fourth Edition, Revised and Expanded, edited by Gilbert S. Banker and Christopher T. Rhodes 122. Surfactants and Polymers in Drug Delivery, Martin Malmsten 123. Transdermal Drug Delivery: Second Edition, Revised and Expanded, edited by Richard H. Guy and Jonathan Hadgraft 124. Good Laboratory Practice Regulations: Second Edition, Revised and Expanded, edited by Sandy Weinberg 125. Parenteral Quality Control: Sterility, Pyrogen, Particulate, and Package Integrity Testing: Third Edition, Revised and Expanded, Michael J. Akers, Daniel S. Larrimore, and Dana Morton Guazzo 126. Modified-Release Drug Delivery Technology, edited by Michael J. Rathbone, Jonathan Hadgraft, and Michael S. Roberts 127. Simulation for Designing Clinical Trials: A Pharmacokinetic-Pharmacodynamic Modeling Perspective, edited by Hui C. Kimko and Stephen B. Duffull 128. Affinity Capillary Electrophoresis in Pharmaceutics and Biopharmaceutics, ttinger edited by Reinhard H. H. Neubert and Hans-Hermann Ru 129. Pharmaceutical Process Validation: An International Third Edition, Revised and Expanded, edited by Robert A. Nash and Alfred H. Wachter 130. Ophthalmic Drug Delivery Systems: Second Edition, Revised and Expanded, edited by Ashim K. Mitra 131. Pharmaceutical Gene Delivery Systems, edited by Alain Rolland and Sean M. Sullivan 132. Biomarkers in Clinical Drug Development, edited by John C. Bloom and Robert A. Dean 133. Pharmaceutical Extrusion Technology, edited by Isaac Ghebre-Sellassie and Charles Martin 134. Pharmaceutical Inhalation Aerosol Technology: Second Edition, Revised and Expanded, edited by Anthony J. Hickey 135. Pharmaceutical Statistics: Practical and Clinical Applications, Fourth Edition, Sanford Bolton and Charles Bon 136. Compliance Handbook for Pharmaceuticals, Medical Devices, and Biologics, edited by Carmen Medina 137. Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products: Second Edition, Revised and Expanded, edited by Louis Rey and Joan C. May 138. Supercritical Fluid Technology for Drug Product Development, edited by Peter York, Uday B. Kompella, and Boris Y. Shekunov

139. New Drug Approval Process: Fourth Edition, Accelerating Global Registrations, edited by Richard A. Guarino 140. Microbial Contamination Control in Parenteral Manufacturing, edited by Kevin L. Williams 141. New Drug Development: Regulatory Paradigms for Clinical Pharmacology and Biopharmaceutics, edited by Chandrahas G. Sahajwalla 142. Microbial Contamination Control in the Pharmaceutical Industry, edited by Luis Jimenez 143. Generic Drug Product Development: Solid Oral Dosage Forms, edited by Leon Shargel and Isadore Kanfer 144. Introduction to the Pharmaceutical Regulatory Process, edited by Ira R. Berry 145. Drug Delivery to the Oral Cavity: Molecules to Market, edited by Tapash K. Ghosh and William R. Pfister 146. Good Design Practices for GMP Pharmaceutical Facilities, edited by Andrew Signore and Terry Jacobs 147. Drug Products for Clinical Trials, Second Edition, edited by Donald Monkhouse, Charles Carney, and Jim Clark 148. Polymeric Drug Delivery Systems, edited by Glen S. Kwon 149. Injectable Dispersed Systems: Formulation, Processing, and Performance, edited by Diane J. Burgess 150. Laboratory Auditing for Quality and Regulatory Compliance, Donald Singer, Raluca-Ioana Stefan, and Jacobus van Staden 151. Active Pharmaceutical Ingredients: Development, Manufacturing, and Regulation, edited by Stanley Nusim 152. Preclinical Drug Development, edited by Mark C. Rogge and David R. Taft 153. Pharmaceutical Stress Testing: Predicting Drug Degradation, edited by Steven W. Baertschi 154. Handbook of Pharmaceutical Granulation Technology: Second Edition, edited by Dilip M. Parikh 155. Percutaneous Absorption: DrugsCosmeticsMechanismsMethodology, Fourth Edition, edited by Robert L. Bronaugh and Howard I. Maibach 156. Pharmacogenomics: Second Edition, edited by Werner Kalow, Urs A. Meyer and Rachel F. Tyndale 157. Pharmaceutical Process Scale-Up, Second Edition, edited by Michael Levin 158. Microencapsulation: Methods and Industrial Applications, Second Edition, edited by Simon Benita 159. Nanoparticle Technology for Drug Delivery, edited by Ram B. Gupta and Uday B. Kompella 160. Spectroscopy of Pharmaceutical Solids, edited by Harry G. Brittain 161. Dose Optimization in Drug Development, edited by Rajesh Krishna 162. Herbal Supplements-Drug Interactions: Scientific and Regulatory Perspectives, edited by Y. W. Francis Lam, Shiew-Mei Huang, and Stephen D. Hall 163. Pharmaceutical Photostability and Stabilization Technology, edited by Joseph T. Piechocki and Karl Thoma 164. Environmental Monitoring for Cleanrooms and Controlled Environments, edited by Anne Marie Dixon 165. Pharmaceutical Product Development: In Vitro-In Vivo Correlation, edited by Dakshina Murthy Chilukuri, Gangadhar Sunkara, and David Young

166. Nanoparticulate Drug Delivery Systems, edited by Deepak Thassu, Michel Deleers, and Yashwant Pathak 167. Endotoxins: Pyrogens, LAL Testing and Depyrogenation, Third Edition, edited by Kevin L. Williams 168. Good Laboratory Practice Regulations, Fourth Edition, edited by Anne Sandy Weinberg 169. Good Manufacturing Practices for Pharmaceuticals, Sixth Edition, edited by Joseph D. Nally 170. Oral-Lipid Based Formulations: Enhancing the Bioavailability of Poorly Water-soluble Drugs, edited by David J. Hauss 171. Handbook of Bioequivalence Testing, edited by Sarfaraz K. Niazi 172. Advanced Drug Formulation Design to Optimize Therapeutic Outcomes, edited by Robert O. Williams III, David R. Taft, and Jason T. McConville 173. Clean-in-Place for Biopharmaceutical Processes, edited by Dale A. Seiberling 174. Filtration and Purification in the Biopharmaceutical Industry, Second Edition, edited by Maik W. Jornitz and Theodore H. Meltzer 175. Protein Formulation and Delivery, Second Edition, edited by Eugene J. McNally and Jayne E. Hastedt 176. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, Third Edition, edited by James McGinity and Linda A. Felton 177. Dermal Absorption and Toxicity Assessment, Second Edition, edited by Michael S. Roberts and Kenneth A. Walters 178. Preformulation Solid Dosage Form Development, edited by Moji C. Adeyeye and Harry G. Brittain 179. Drug-Drug Interactions, Second Edition, edited by A. David Rodrigues 180. Generic Drug Product Development: Bioequivalence Issues, edited by Isadore Kanfer and Leon Shargel 181. Pharmaceutical Pre-Approval Inspections: A Guide to Regulatory Success, Second Edition, edited by Martin D. Hynes III 182. Pharmaceutical Project Management, Second Edition, edited by Anthony Kennedy 183. Modified Release Drug Delivery Technology, Second Edition, Volume 1, edited by Michael J. Rathbone, Jonathan Hadgraft, Michael S. Roberts, and Majella E. Lane 184. Modified-Release Drug Delivery Technology, Second Edition, Volume 2, edited by Michael J. Rathbone, Jonathan Hadgraft, Michael S. Roberts, and Majella E. Lane 185. The Pharmaceutical Regulatory Process, Second Edition, edited by Ira R. Berry and Robert P. Martin 186. Handbook of Drug Metabolism, Second Edition, edited by Paul G. Pearson and Larry C. Wienkers

Handbook of Drug MetabolismSecond Editionedited by Pearson Pharma Partners Westlake Village, California, USA Amgen, Inc. Seattle, Washington, USA

Paul G. Pearson

Larry C. Wienkers

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2009 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number 10: 1 4200 7647 7(Hardcover) International Standard Book Number 13: 978 1 4200 7647 9(Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978 750 8400. CCC is a not for profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of drug metabolism. 2nd ed. / edited by Paul G. Pearson, Larry C. Wienkers. p. ; cm. (Drugs and the pharmaceutical sciences; v. 186) Includes bibliographical references and index. ISBN 13: 978 1 4200 7647 9 (hardcover : alk. paper) ISBN 10: 1 4200 7647 7 (hardcover : alk. paper) 1. Drugs Metabolism Handbooks, manuals, etc. I. Pearson, Paul G. (Paul Gerard), 1960 II. Wienkers, Larry C. III. Series. [DNLM: 1. Pharmaceutical Preparations metabolism. W1 DR893B v.186 2008/QV 38 H23655 2008] RM301.55.H36 2008 6150 .7 dc22 2008035272 For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

Preface

It is almost a decade since the first edition of the Handbook of Drug Metabolism was published. The goal of the first edition was to provide a comprehensive text to serve as a graduate course in Drug Metabolism, a useful reference for academic and industrial drug metabolism scientists, but also to serve as an important reference tool for those pursuing a career in drug metabolism. The second edition of the Handbook of Drug Metabolism has been completely updated to capture a decade of advances in our understanding of factors that impact the pharmacokinetics and metabolism of therapeutic agents in humans. Moreover, we have sought to include new chapters that reflect significant advances that have occurred in seven major areas, viz. analytical methodologies, pharmacokinetics, biotransformation reactions, molecular biology, pharmacogenetics, drug interactions, and transgenic animals. The second edition of the Handbook of Drug Metabolism is organized into four parts. The first three parts capture scientific and experimental concepts around drug metabolism. Part I reviews fundamental aspects of drug metabolism, including a history of drug metabolism, a review of oxidative and nonoxidative biotransformation mechanisms, a review of liver structure and function, and pharmacokinetics of drugs metabolites. Part II details factors that impact drug metabolism including pharmacogenetics, drug-drug interactions, and the role of extrahepatic organs in drug biotransformation. Part III provides in depth insights into analytical technologies and methodologies to study drug metabolism at the molecular, subcellular, and cellular levels, and considerations of factors, viz. enzyme inhibition and induction that influence drug metabolism and therapeutic response. Part IV has been expanded substantially from the first edition to illustrate the highly integrated role of drug metabolism in drug discovery and drug development. In this regard, Part IV focuses on clinical and preclinical drug metabolism studies and safety considerations for drug metabolites (chemically reactive and nonreactive metabolites) in the selection and development of promising therapeutic candidates, and highlights the increased focus of regulatory agencies on safety considerations of drug metabolites. This book is dedicated to two groups of exceptional individuals. First, we thank the distinguished academic and industrial leaders (many of whom have contributed to this book) who have trained a generation, or more, of high-quality drug metabolism scientists. Second, we thank the many graduate students, postdoctoral fellows, and industrial colleagues who have challenged us and enriched our lives over the last two decades. We thank all of you for advancing the field of drug metabolism; your efforts have enabled ouriii

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Preface

discipline to advance promising therapeutic agents with increased probability of success in finding medicines to treat serious illness. Lastly, it has been a privilege to interact with this collection of expert authors, and we would like to express our sincere gratitude to them for their contributions to this second edition of the Handbook of Drug Metabolism. Paul G. Pearson, Ph.D. Larry C. Wienkers, Ph.D.

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. PART I: FUNDAMENTAL ASPECTS OF DRUG METABOLISM 1. The Evolution of Drug Metabolism Research Patrick J. Murphy Pharmacokinetics of Drug Metabolites Philip C. Smith ....................

iii ix

1 17 61 85 109 137

2.

....................... .

3.

Hepatic Architecture: Functional and Subcellular Correlates Felix A. de la Iglesia and Jeffrey R. Haskins The Cytochrome P450 Oxidative System Paul R. Ortiz de Montellano

....... .

4.

...................... .

5.

Transformation Enzymes: Oxidative; Non-P450 Carrie M. Mosher and Allan E. Rettie

............... .

6.

UDP-Glucuronosyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rory P. Remmel, Jin Zhou, and Upendra A. Argikar

PART II: FACTORS WHICH AFFECT DRUG METABOLISM 7. Pharmacogenetics Ann K. Daly ...................................... . 179 203 227

8.

Inhibition of Drug Metabolizing Enzymes F. Peter Guengerich

.................... .

9.

The Ontogeny of Drug Metabolizing Enzymes/Pediatric Exclusivity Ronald N. Hines

. .

v

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Contents

10.

Sites of Extra Hepatic Metabolism, Part I: Lung Christopher A. Reilly and Garold S. Yost Sites of Extra Hepatic Metabolism, Part II: Gut Mary F. Paine

.............. .

243 273 299 327

11.

............... .

12.

Sites of Extra Hepatic Metabolism, Part III: Kidney Lawrence H. Lash Sites of Extra Hepatic Metabolism, Part IV: Brain Ying Wang, Jordan C. Bell, and Henry W. Strobel

............ .

13.

............. .

PART III: TECHNOLOGIES TO STUDY DRUG METABOLISM 14. LC-MS Analysis in Drug Metabolism Studies Stacy L. Gelhaus and Ian A. Blair ................. . 355 373

15.

The Practice of NMR Spectroscopy in Drug Metabolism Studies Ian D. Wilson, John C. Lindon, and Jeremy K. Nicholson

... .

16.

Applications of Recombinant and Purified Human Drug-Metabolizing Enzymes: An Industrial Perspective . . . . . . . . . . . . . . . . . . . . . . . . . Ming Yao, Mingshe Zhu, Shu-Ying Chang, Donglu Zhang, and A. David Rodrigues In Vitro Metabolism: Subcellular Fractions .................. . Michael A. Mohutsky, Steven A. Wrighton, and Barbara J. Ring In Vitro Metabolism: Hepatocytes Michael W. Sinz ......................... .

393

17.

445 465

18.

19.

Drug Interaction Studies in the Drug Development Process: Studies In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Scott Obach and Robert L. Walsky Enzyme Induction Studies in Drug Discovery & Development J. Greg Slatter and Leslie J. Dickmann ..... .

493 521

20.

21.

Experimental Characterization of Cytochrome P450 Mechanism Based Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dan Rock, Michael Schrag, and Larry C. Wienkers

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vii

PART IV: APPLICATIONS OF METABOLISM STUDIES IN DRUG DISCOVERY AND DEVELOPMENT 22. Clinical Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kirk R. Henne, George R. Tonn, William F. Pool, and Bradley K. Wong Minimizing Metabolic Activation in Drug Discovery Sanjeev Kumar and Thomas A. Baillie ............ . 571 597

23.

24.

Kinetic Differences between Generated and Preformed Metabolites: A Dilemma in Risk Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomayant Prueksaritanont and Jiunn H. Lin The Use of Transgenic Animals to Study Drug Metabolism Colin J. Henderson, C. Roland Wolf, and Nico Scheer ....... .

619 637

25.

26.

Preclinical Pharmacokinetic Models for Drug Discovery and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kevin L. Salyers ................................................... .

659 675

Index

Contributors

Upendra A. Argikar

Novartis Pharmaceuticals, Cambridge, Massachusetts, U.S.A.

Thomas A. Baillie Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Rahway, New Jersey, and West Point, Pennsylvania, U.S.A. Jordan C. Bell Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas, U.S.A. Ian A. Blair Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Shu-Ying Chang Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey, U.S.A. Ann K. Daly Institute of Cellular Medicine, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K. Felix A. de la Iglesia Department of Pathology, University of Michigan Medical School and Michigan Technology and Research Institute, Ann Arbor, Michigan, U.S.A. Leslie J. Dickmann Washington, U.S.A. Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle,

Stacy L. Gelhaus Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. F. Peter Guengerich Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A. Jeffrey R. Haskins Cellular Imaging & Analysis, Thermo Fisher Scientific, Pittsburgh, Pennsylvania, U.S.A.

ix

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Contributors

Colin J. Henderson Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, U.K. Kirk R. Henne Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California, U.S.A. Ronald N. Hines Departments of Pediatrics and Pharmacology and Toxicology, Medical College of Wisconsin, and Childrens Research Institute, Childrens Hospital and Health Systems, Milwaukee, Wisconsin, U.S.A. Sanjeev Kumar Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Rahway, New Jersey, U.S.A. Lawrence H. Lash Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan, U.S.A. Jiunn H. Lin Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, West Point, Pennsylvania, U.S.A. John C. Lindon Department of Biomolecular Medicine, Imperial College London, South Kensington, London, U.K. Michael A. Mohutsky Indiana, U.S.A. Lilly Research Laboratories, Eli Lilly & Company, Indianapolis,

Carrie M. Mosher Department of Medicinal Chemistry, University of Washington, Seattle, Washington, U.S.A. Patrick J. Murphy College of Pharmacy and Health Sciences, Butler University, Indianapolis, Indiana, U.S.A. Jeremy K. Nicholson Department of Biomolecular Medicine, Imperial College London, South Kensington, London, U.K. R. Scott Obach Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Global Research and Development, Groton, Connecticut, U.S.A. Paul R. Ortiz de Montellano Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California, U.S.A. Mary F. Paine Division of Pharmacotherapy and Experimental Therapeutics, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, U.S.A. William F. Pool Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, San Diego, California, U.S.A. Thomayant Prueksaritanont Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, West Point, Pennsylvania, U.S.A.

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Christopher A. Reilly Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, U.S.A. Rory P. Remmel Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, U.S.A. Allan E. Rettie Department of Medicinal Chemistry, University of Washington, Seattle, Washington, U.S.A. Barbara J. Ring Indiana, U.S.A. Lilly Research Laboratories, Eli Lilly & Company, Indianapolis,

Dan Rock Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, Washington, U.S.A. A. David Rodrigues Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey, U.S.A. Kevin L. Salyers Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California, U.S.A. Nico Scheer TaconicArtemis GmbH, Cologne, Germany

Michael Schrag Department of Drug Metabolism, Array BioPharma Inc., Boulder, Colorado, U.S.A. Michael W. Sinz Bristol-Myers Squibb Co., Wallingford, Connecticut, U.S.A.

J. Greg Slatter Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, Washington, U.S.A. Philip C. Smith School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Henry W. Strobel Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas, U.S.A. George R. Tonn Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California, U.S.A. Robert L. Walsky Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Global Research and Development, Groton, Connecticut, U.S.A. Ying Wang Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas, U.S.A. Larry C. Wienkers Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, Washington, U.S.A.

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Ian D. Wilson Departments of Clinical Pharmacology, Drug Metabolism and Pharmacokinetics, AstraZeneca, Macclesfield, Cheshire, U.K. C. Roland Wolf Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, U.K. Bradley K. Wong Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California, U.S.A. Steven A. Wrighton Indiana, U.S.A. Lilly Research Laboratories, Eli Lilly & Company, Indianapolis,

Ming Yao Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey, U.S.A. Garold S. Yost Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, U.S.A. Donglu Zhang Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey, U.S.A. Jin Zhou Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, U.S.A. Mingshe Zhu Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, New Jersey, U.S.A.

1

The Evolution of Drug Metabolism ResearchPatrick J. MurphyCollege of Pharmacy and Health Sciences, Butler University, Indianapolis, Indiana, U.S.A.

INTRODUCTION Drug metabolism research has grown from a desire to understand the workings of the human body in chemical terms to a major force in the effort to develop drugs tailored to the individual. This essay will trace the beginnings of what are now major branches of drug metabolism to provide some background to the current state of the art represented in the many chapters of this book.

CHEMISTRYMAJOR METABOLIC ROUTES In 1828, the laboratory of Friedrich Woehler was abuzz with the synthesis of urea, the first organic synthetic achievement. Woehler then turned his attention to potential chemical transformations in the body. He had been interested in compounds found in urine since his undergraduate days, and when Liebig identified hippuric acid as a normal urinary product, Woehler suggested that it might be formed from benzoic acid and glycine in the body. His initial experiments in dogs, however, were inconclusive (1). Alexander Ure, a physician seeking a cure for gout, heard about Woehlers idea and reasoned that if it was correct then administration of benzoic acid to man might lead to a diminished excretion of urea due to the use of nitrogen in the glycine conjugate. Ure took benzoic acid and isolated hippuric acid from his urine (2). Woehler had his associate Keller repeat the experiment and confirmed that ingested benzoic acid was indeed excreted in the urine as hippuric acid (3). These studies initiated a period lasting to the end of the century where scientists and their collaborators subjected themselves to interesting molecules to see what would happen. Many of the studies followed logical extensions of the earlier work.

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Erdmann and Marchand administered cinnamic acid to volunteers and isolated a product tentatively identified as hippuric acid (4,5). They proposed that the cinnamic acid was oxidized to benzoic acid and then conjugated with glycine. Woehler and Friederick Frerichs confirmed this transformation in dogs (6). They also showed that benzaldehyde was converted to hippuric acid in dogs and rabbits. The oxidation of benzene to phenol was discovered by the clinician Bernhard Naunyn during the course of experimental treatment of stomach fermentation with benzene. He was surprised to find that phenol was excreted following the administration of benzene. Naunyn then collaborated with the chemist, Schultzen, to study the fate of a number of hydrocarbons, including toluene, xylene, and larger molecules (7). Aromatic hydroxylation, which had proven difficult for the chemists of the day, was readily accomplished in humans. Studies on aromatic hydroxylation led Stadeler to discover conjugated phenols in human and animal urine (8). Munk, after ingesting varying amounts of benzene, monitored the excretion of phenol-forming substance in his urine by hydrolyzing the urine with acid and measuring the released phenol (9). Baumann, using the color of indigo as his guide, purified an indigo-forming substance from urine and showed that upon hydrolysis both indigo and sulfate were released (10). Baumann made many pioneering studies on sulfates formed from a variety of compounds including, catechol, bromobenzene, indole, and aniline. The surprising ability of the body to methylate compounds was discovered by His in 1887 when he was able to isolate and identify N-methyl pyridinium hydroxide from the urine of dogs dosed with pyridine (11). Over 60 years later, MacLagan and Wilkinson discovered the more significant O-methylation pathway using the phenol butyl-4-hydroxy 3,5 diiodobenzoate (12). This is the pathway that led Axelrod to his Nobel Prize related to the methylation of catecholamines. N-Acetylation was first described by Cohn in his studies on the fate of mnitrobenzaldehyde. The oxidized, reduced compound is acetylated and conjugated with glycine to yield the hippurate of N-acetyl m-aminobenzoic as a major metabolite (13). Mercapturic acids were initially isolated in the laboratories of Baumann and Preuss studying the fate of bromobenzene and by Jaffe looking at chloro and iodobenzene (14,15). The actual structure of these acetyl cysteine conjugates was determined by Baumann in 1884 (16). The nature of the cofactors in the conjugation reactions would not be known until the 20th century. A unique, primarily human, conjugation of glutamine with aryl acetic acids was discovered by Thierfelder and Sherwin in 1914 (17). ACTIVE METABOLITES By the early part of the 20th century the major drug-metabolizing reactions had been identified. A unifying theory on the role of metabolism was developed by John Paxson Sherwin. Sherwin was one of the most prominent Americans in the field of metabolism (18). A native of Bristol, Indiana, he was educated in Indiana and Illinois and then spent two years in Tubingen, Germany, before returning to the Midwest. He formulated the chemical defense theory elaborated in his reviews on drug metabolism in 1922, 1933, and 1935 (19 21). The latter two reviews carried the title Detoxication Mechanisms. This same title was used by R.T. Williams in his groundbreaking summaries of drug metabolism in 1947 and 1959 (22). Although Williams was troubled with the general classification of all metabolic reactions as detoxication, he accepted it as the most practical appellation. A mind-set that metabolism led to detoxication was so logical and

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had so many examples that when a compound was actually made more active, it engendered disbelief. But even at this early stage of drug development the examples of activation began to accumulate. The worlds first major drug, arsphenamine (Fig. 1), an arsenical used for the treatment of syphilis, was ineffective in vitro (23). Twelve years after its launch, studies showing that the drug worked through an oxidation product were published by Voegtlin and coworkers (24). This compound, which evolved from the magic bullet concept of Ehrlich, set the stage for future worldwide blockbusters.

Figure 1 Examples of drug activation.

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A more dramatic impact of metabolism occurred with the launch of prontosil (Fig. 1), the first major antibacterial agent. Prontosil was discovered in the early 1930s in the laboratories of I.G. Farben, the worlds largest chemical company. G. Domagk and coworkers used the concept of Ehrlich, wherein compounds that could be shown to bind to tissues may lead to specific antagonists of infectious agents. The early work, therefore, concentrated on derivatives of azo dyes. After numerous failures they came across the compound prontosil, an azo dye containing a sulfonamide moiety (25). This molecule had striking activity and was an instant success. Launched initially in Europe, it quickly stormed the United States when President Roosevelts son was cured by its administration (26). Domagk was awarded the Nobel Prize in 1939 for his work. But, like arsphenamine, prontosil had very low activity in vitro. This puzzled workers in the laboratories of Trefouel in France. They proceeded to test both prontosil and the sulfonamide breakdown product and came to the conclusion that it was the metabolite formed by azo reduction that was the true antibacterial (27). This was confirmed by studies in England that showed the presence of aminobenzenesulfonamide in plasma and urine in patients treated with prontosil (28). Once it became clear that any derivative that would release the active sulfonamide in vivo could represent effective therapy, chemical companies around the world began making variations that would spawn the birth of the modern pharmaceutical industry. Acetanilide (Fig. 1) provides a bridge from active to toxic metabolites. Brodie and Axelrod found that acetanilide was converted to aniline, which explained the methemoglobinemia, which had been observed at high doses and to acetaminophen, a superior analgesic (29). This study launched the illustrious career of Julius Axelrod in the field of metabolism. There are numerous examples of prodrugs, either by fortune or design, that have to be activated for full pharmacological effect. Esters such as enalapril or clofibrate have to be hydrolyzed for activity. Methyl DOPA is decarboxylated and hydroxylated for activation, cyclophosphamide is hydroxylated for activation. There are also many other examples where the metabolites had some or all of the activity designated for the parent. One of the most striking and significant of these is the antihistamine terfenadine (Fig. 1). The parent is oxidized to the active carboxylic acid and other metabolites by cytochrome P450 enzymes. When the P450s are inhibited, parent terfenadine reaches higher than normal levels (30,31). This interaction led to cardiovascular problems in patients taking terfenadine and ketoconazole or erythromycin. Because of this interaction, terfenadine was taken off the market and new regulatory guidelines were put in place by the Food and Drug Administration (FDA) to alert drug developers to the need for interaction studies before approval. TOXIC AND REACTIVE METABOLITES The products of metabolism are determined by reaction mechanism of the enzymes involved and by the chemical structure of the reactant. Whether a metabolic product is more or less active is independent of these two interacting forces. Humans have evolved over the years, whereby we have a certain capacity to handle whatever the environment and our diets present. Certain toxins, which cannot be handled metabolically, we learn to avoid. The time frame of evolution does not permit the type of adaptation necessary to dispose of every new compound in a safe and beneficial manner. It is impossible to estimate what percentage of new molecular entities are converted to active or toxic

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metabolites, but it is clear that it has to be a higher percentage than we see just looking at marketed drugs. The fact is that if a toxic metabolite is produced during drug development, the candidate is usually eliminated from consideration. Therefore the number of compounds actually activated by metabolism is necessarily higher than the overall documented occurrences. With any new compound, there is a significant chance that metabolism will yield pharmacologically active derivatives. REACTIVE INTERMEDIATES The formation of reactive intermediates is of particular concern in the development of new agents. Guroff and coworkers found that during the course of aromatic hydroxylation the hydrogen on the position to be hydroxylated could shift to the adjacent position (32). This was termed the NIH shift and was subsequently explained by the formation of a reactive epoxide intermediate. The formation of green pigments during administration of 2-allyl-2-isopropylacetamide was shown by de Matteis to be due to destruction of P450 (33). Ethylene and other olefins had similar destructive properties (34). The most significant chemical moieties giving rise to reactive molecules and/or P450 inhibition have been reviewed (35). Many of these compounds will react with glutathione in an inactivation step. Excretion of mercapturic acids is often taken as a sign of the formation of the reactive species. In the absence of adequate levels of glutathione or when the kinetics are favorable for protein binding, the formation of chemical-protein conjugates can lead to systemic toxicity. The prototypical reactive intermediate is the quinone-imine formed from metabolism of acetaminophen. In a classic series of papers, Brodie and coworkers revealed the metabolic fate of acetaminophen and its potential tissue-binding metabolite (36 39). While this compound is known to be hepatotoxic and readily binds to protein in vitro, it nonetheless remains a best-selling analgesic. Generally, at recommended doses, acetaminophen is efficiently removed by conjugation or, after oxidation, by glucuronidation and/or reaction with glutathione. At elevated doses or in conjunction with CYP2E1 induction, high levels of quinone-imine can lead to tissue damage (40). BIOANALYTICAL The progress in drug metabolism is paralleled by, indeed dependent on, the advances in bioanalytical techniques. For most of the first century of drug metabolism research, identification of metabolites involved isolation, purification, and chemical manipulation leading to characterization. At the end of World War II, new technology developed during the war came into use in metabolism research. A study on the distribution of radioactivity in the mouse after administration of 14C-dibenzanthracene set new standards for metabolic research (41). The use of high-speed centrifuges to separate cellular components was another legacy of the Manhattan Project. The development of liquid-liquid partition chromatography by Martin and Synge (42) heralded the addition of new separation tools, including paper, thin layer, and gas chromatography. Spectrometry in biological media became routine with the Cary 14 spectrophotometer. Mass spectrometry moved from the hands of specialists to the analytical laboratory with the launch of the LKB 9000 GC/MS. A crucial development for the eventual linking of mass spectrometry and liquid chromatography was the discovery of electrospray

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ionization by Fenn in 1980 (43). LC/MS instruments from Sciex and Finnegan revolutionized the bioanalytical laboratories leading to increasingly more rapid and more efficient delineation of metabolic pathways. Newer analytical techniques permit the analysis of chemical bound to protein and the characterization of the proteins involved. For example, Shin and coworkers recently identified binding of electrophiles to 263 proteins in human microsomal incubations (44). Doss and Baillie (45) have suggested that drug developers use in vitro binding ability as a screen for potential reactive intermediates.

ENZYMOLOGY-MECHANISMS OF METABOLISM Conjugation The discovery of cofactor structures started with acetyl coA. This important cofactor, vital for intermediary metabolism, was identified using the acetylation of sulfanilamide as an assay. Lippman and coworkers painstakingly isolated and identified coenzyme A as the energy-containing component driving acetylation (46). The principle of active cofactors led researchers to solving the structures of 30 -phosphoadenosine-50 -phosphosulfate (PAPS) (47), uridinediphosphoglucuronic acid (UDPGA) (48), and S-adenosylmethionine (SAM) (49). Defining mercapturic acid formation took slightly longer because of the fact that the actual conjugating moiety was altered before elimination. The actual structure of mercapturic acids was solved when Baumann correctly identified the acetyl cysteine moiety (16). Glutathione, originally isolated by M.J. de Rey Pailhade (50), was fully characterized by Hopkins in 1929 (51). But it was not until 1959 when the relationship between glutathione conjugation and the formation of mercapturic acids was elucidated by Barnes and associates (52). In 1961, Booth, Boyland, and Sims published data on the enzymatic formation of glutathione conjugates (53). As a variation in the theme, it became clear that conjugation with amino acids such as glycine or glutamine involved initial activation of the substrate rather than the linking agent. The unique ability of humans and Old World monkeys to conjugate with glutamine was found to be due to the specificity of acyl transferase enzymes found in the mitochondria (54). The structures of most of the human conjugating enzymes have now been elucidated, and in many cases, the enzymes have been cloned. Crystal structures have been slow to emerge for glucuronyl transferases because of the membrane-bound nature of these enzymes. There are 13 human sulfotransferases (55), 16 glucuronyl-transferases (56), multiple N-, O-, and S-methyl transferases, 2 N-acetyl transferases (57), and 24 glutathione transferases (58). The multiplicity of isozymes and overlapping specificities require extensive evaluation to understand which enzymes may be critical for a given drug. Reduction Some of the earliest in vitro experiments in metabolism dealt with enzymatic reduction. The importance of the azo derivatives of sulfanilamide led to initial experiments using neoprontosil as a model substrate. Bernheim reported the reduction of neoprontosil by liver homogenates (59). Mueller and Miller studied the metabolism of the carcinogen dimethylaminoazobenzene (DAB) and found that in rat liver homogenates DAB was hydroxylated and demethylated and the azo linkage was reduced (60). The reducing

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enzyme was shown to require reduced nicotinamide adenine dinucleotide phosphate (NADPH) and to reside in the particulate portion of the fragmented cells (61). Oxidation The incorporation of oxygen into drugs and endogenous molecules was found to occur directly from molecular oxygen by the use of isotopically labeled oxygen (62,63). This led to the definition of a new category of oxidases termed monooxygenases by Hayaishi and mixed-function oxidases by Mason. These oxidases required oxygen and a reductant, usually NADPH. IN VITRO METHODOLOGY/ENZYMOLOGY The unraveling of the secrets of the cell began with the development of the techniques of gently breaking the cell developed by Potter and Elvejham and then fractionating the fragments and components of the cell by differential centrifugation (64). The first drug to be studied using these techniques was amphetamine. Axelrod examined the deamination of amphetamine and showed the activity to be dependent on oxygen and NADPH and to reside in the microsomal fraction of the cell (65). Brodies laboratory quickly examined a number of drug substrates and found them to be metabolized by the same microsomal system (66). Further examination of microsomes by Klingenberg and Garfinkel revealed the presence of a pigment that had some of the properties of a cytochrome and a peak absorbance after reduction in the presence of CO at 450 nm (67,68). The pigment was shown by Omura and Sato to be a cytochrome (69). Estabrook Cooper and Rosenthal used light activation of the CO-inhibited system to prove that cytochrome P450 was the terminal oxidase in the oxidation of many classes of drug substrates (70). The use of microsomes became standard practice in drug metabolism studies. However, the enzymes defied purification because of the fact that they were embedded in the membrane and solubilization inevitably led to denaturation. Lu and Coon solved the problem of releasing the enzyme from the membrane using sodium deoxycholate in the presence of dithiotheitol and glycerol and our knowledge of multiple P450s rapidly expanded (71). Coons laboratory, Wayne Levin and associates, and Fred Guengerich were among the pioneers in separation and purification of P450s (72). Nebert and coworkers developed a unifying nomenclature on the basis of the degree of similarity of the P450s, and the field began to blossom (73). The culmination of the efforts to define human P450s came with the sequencing of the human genome. At that point, it was clear that there were 57 variants of human P450. The major ones involved in drug metabolism have been well characterized, while there are still some isozymes whose function is yet to be defined (72). The physical characteristics of the enzymes are rapidly being defined. The first P450 to be crystallized and the first structure determined were from a pseudomonad, Pseudomonas putida (74). This provided a blueprint for all the structures to come. Human P450s resisted crystallization until they were modified by shortening the amino terminus. It is this portion of the protein that binds the membrane, and by removing the amino terminal segment, it was possible to obtain a soluble, active P450 that could be crystallized and analyzed. The crystal structures of all of the major drug-metabolizing p450s now have been determined (75). The knowledge of the crystal structures has helped in our understanding of the broad specificity of this class of enzymes, helped to determine the

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necessary properties of potential substrates, and led to the development of computer programs to predict whether a compound will be a substrate or not (76). While the P450s have been the stars of drug metabolism, there are many other oxidative enzymes that can play a role, sometimes dominant, in the fate of new molecules. The flavin monooxygenases, which are often involved in the metabolism of heterocyclic amines, sulfur, or phosphorous-containing compounds, have been characterized and five forms identified (77). Aldehyde oxidase, a molybdenum-containing enzyme oxidizes nitrogen heterocycles and aldehydes (78), xanthine oxidase and xanthine dehydrogenase mainly involved in the production of uric acid (79), aldehyde dehydrogenase [3 classes, at least 17 genes (80)], and alcohol dehydrogenase (23 distinct human forms) (81) are among the enzymes most prominent in drug metabolism. GENETIC CHARACTERISTICS OF DRUG-METABOLIZING ENZYMES The drug-metabolizing enzymes showed early indications of genetic polymorphism on the basis of the individual variations in therapeutic effectiveness. The discovery of the utility of isoniazid in the treatment of tuberculosis was quickly followed by the realization that a significant portion of the patient population had elevated levels of isoniazid in the plasma. This was traced to a genetically determined deficiency in the N-acetyl transferase responsible for the inactivation of isoniazid (82). Similarly, genetic variations in serum cholinesterase led to altered susceptibility to the effects of the muscle-relaxant succinyl choline (83). A major breakthrough in the enzymes involved with drug oxidation came with the studies of Smith and coworkers on debrisoquine metabolism (84) and the work of Eichelbaum and coworkers on sparteine metabolism (85). These discoveries led to a broad range of population studies on debrisoquine hydroxylase, later to be identified as CYP450 2D6. The variation in blood levels of these agents could be traced to whether patients had diminished levels of CYP2D6 or, in some cases, enhanced levels of CYP2D6. As we learned more about the role of the isozymes of P450, genetic variations became a major topic of study. Significant polymorphic variations in CYP2C9, CYP 2C19, CYP2A6, and CYP2B6 must be taken into account for substrates of these enzymes (86). Other oxidizing enzymes such as flavin monooxygenase (FMOs) (87) and dihydropyrimidine dehydrogenase (88) also show genetic variation leading to drug toxicity. Conjugating enzymes such as thiopurine methyltransferase, N-acetyl transferase, and glucuronyl transferase have variants that have been shown to be important in altered response to drugs (89). The message for drug development is clear. It is vital to know the enzymes involved in the breakdown of the administered drug. If a specific isozyme is responsible for either the majority of the inactivation or for the activation of an agent, then appropriate studies are required to determine the efficacy and/or toxicity of the drug over a spectrum of the population, including the genetic variants. INDUCTION-CONTROL MECHANISMS One of the most striking features of drug-metabolizing enzymes is their ability to adapt to the substrate load. Early studies showed that ethanol administration to rats increased the ability of the kidney to metabolize ethanol (90), while borneol administration to dogs or

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menthol administration to mice led to increased b-glucuronidase activity in these species (91). Conney et al. discovered enzyme induction by aromatic hydrocarbons (92), while Remmer and Merker reported phenobarbital induction of smooth endoplasmic reticulum in rabbits, rats, and dogs (93). Studies on the induction phenomenon eventually led to discovery of the Ah receptor (94). The mechanism of transcriptional regulation has been elucidated and forms the basis for our understanding of this superfamily of regulators (95). Other receptors integral to the initiation of induction include peroxisome proliferator activated receptor (PPAR) (96), constitutive androstane receptor (CAR), and pregnane X receptor (PXR). Interactions between CAR and PXR have recently been reviewed (97). The crystal structure of the human PXR ligand-binding domain in the presence and absence of ligands has been reported (98,99). INHIBITORS Compounds that had broad specificity as inhibitors of P450 played a major role in the understanding of this class of enzymes. The discovery of SKF525a in the laboratories of SKF and its expanded use by Brodie and coworkers defined the microsomal oxidases before the discovery of P450 (100,101). That one inhibitor could decrease the metabolism of so many diverse compounds argued for an enzyme with broad specificity or multiple enzymes with a common site of inhibition. Other inhibitors such as metyrapone, ketoconazole, and AIA were similarly employed. Attention was drawn to the role of inhibition in drug interactions when cimetidine, a popular proton pump inhibitor, was found to be a weak inhibitor of P450-catalyzed reactions (102). The observation that grapefruit juice had inhibitory properties stimulated the studies of endogenous and environmental inhibitors resulting in adverse drug reactions (103). Once the multiplicity of P450s was clear, specific inhibitors of individual isozymes were used to define activity (35). These inhibitors included antibodies with unique specificities (104). The field of specific inhibitors for targeted therapy is rapidly developing, as the roles of all 57 P450s are unraveled (105,106). TRANSPORTERS The discovery of p-glycoprotein (pgp or mdr1) in 1976 created an enhanced appreciation of the role of transporters in drug disposition (107). In addition to playing an important role in drug penetration through the intestine, pgp plays a significant role in controlling the penetration of many drugs into the brain (108). Umbenhauer and coworkers showed that a genetic deficiency in pgp was correlated with the penetration of avermectin into the brain in mice (109,110). Later studies showed a wide range of compounds controlled in a similar fashion. There are many other transporters that have yet to be characterized with regard to drug disposition. A total of 770 transporter proteins were predicted from analysis of the human genome. The ABC family, which contains pgp, consists of 47 members. The latest information and structural details on transporters can be found in the transporter protein analysis database (111). The first crystal structure of an S. Aureus ABC transporter was reported in 2007 (112). In the drug development process, knowledge as to whether the candidate compounds are substrates for transporters is crucial to predicting bioavailability.

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DRUG METABOLISM RESEARCH IN DRUG DEVELOPMENT The overall progression of drug metabolism from the determination of metabolic pathways to the current position in metabolic profiling is shown in Figure 2. The evolution of drug metabolism research has changed the role of the drug metabolism scientist in a most dramatic fashion. The modern day metabolism scientist must understand and evaluate not just the chemistry of metabolism but also the enzymatic, genetic, environmental, mechanistic, and interactive aspects of any new agent. The metabolism scientist must be able to develop a compound profile detailing all the nuances involved in proposed therapy. This profile, or metafile (Fig. 3), must encompass a breadth of understanding enabling the design of clinical studies that facilitates the tailoring of the new agent to the most appropriate patient population.

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Chemistry starts with Woehler, by 1900 all major pathways, glutathione conjugation, active metabolites, epoxide intermediates, mechanistic studies, electrophiles In vitro starts, Millers define role of liver, Axelrod identifies metabolism in microsomes, Lu and Coon solubilize P450, cloned enzymes, knockout and humanized animals Enzymology starts with co factors, P450, isolation, crystallization, isozymes, human genome details Transporters start with MDR, anion and cation transporters, then human genome reveals > 700 genes, crystallization Genetic polymorphisms start with NAT, accelerate with debrisoquine, then deletions, overexpression, allelic variants Expression and control start with Ah receptor, then CAR, PXR, PPAR Active metabolites start with chloral hydrate, arsphenamine, accelerates with prontosil reaches heightened regulatory awareness with terfenadine Induction starts with b glucuronidase increase with borneol or menthol, accelerates with DMAB, then Phenobarbital, then Ah receptor, nuclear activation Inhibition starts with SK&F 525a, accelerates with P450 discovery, high impact with macrolides, ketoconazole Figure 2 The evolution of drug metabolism research from the earliest days of discovery to the current broad ranging research effort. Abbreviations: Peroxisome proliferator activated receptor, PPAR; constitutive androstane receptor, CAR; pregnane X receptor, PXR.

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Metabolic Profile (Metafile) Transformation products Identification Human in vitro/in vivo Animal in reference to efficacy models and toxicity models Activity Desirable Extraneous, adverse Bioavailability Methods of analysis parent/metabolites Pharmacokinetics Enzymology Enzymes involved in metabolism Location of enzymes Genetics Genotype for enzymes involved in metabolism/disposition Phenotype for enzymes Impact of genetic polymorphisms species comparisons

Interactions Inhibition by or of other drugs Parent, metabolites Induction potentiality Parent, metabolites Transporters Transporters involved in absorption and elimination

Figure 3 Summary of the research required for the creation of a compound profile. Many of these areas have greatly expanded in the last 25 years.

REFERENCES1. Conti A, Bickel MH. History of drug metabolism. Discoveries of the major pathways in the 19th century. Drug Metab Rev 1977; 6:1 50. 2. Ure A. On gouty concretions; with a new method of treatment. Pharm J Transact 1841; 1:24. 3. Keller W. On the conversion of benzoic acid into hippuric acid. Ann Chem Pharm 1842; 43:108. 4. Erdmann OL, Marchand RF. Metabolism of cinnamic acid to hippuric acid in animals. Ann Chem Pharm 1842; 44:344. 5. Erdmann OL, Marchand RF. Umwandlung der Zimmtsaure in Hippursaure im thierishchen Organismus. J Prakt Chem 1842; 26:491 498. 6. Woehler F, Frerichs FT. Concerning the modifications which particular organic materials undergo in their transition to the urine. Ann Chem Pharm 1848; 63:335. 7. Schultzen O, Naunyn B. The behavior of benzene derived hydrocarbons in the animal organism. duBois Reymonds Arch Anat Physiol 1867:349. 8. Stadeler G. Ueber die fluchtigen Sauren des Harns. Ann Chem Liebigs 1851; 77:17 37. 9. Munk I. Zur Kenntniss der phenolbildenden Substanz im Harn. Arch Ges Physiol Pfluegers 1876; 12:142 151. 10. Baumann E. Concerning the occurrence of Brenzcatechin in the urine. Pflugers Arch Physiol 1876; 12:69. 11. His W. On the metabolic products of pyridine. Arch exp Path Pharmak 1887; 22:253. 12. MacLagan NF, Wilkinson JH. The biological action of substances related to thyroxine. 7. The metabolism of butyl 4 hydroxy 3:5 diiodobenzoate. Biochem J 1954; 56:211 215. 13. Cohn R. Concerning the occurrence of acetylated conjugates following the administration of aldehydes. Z Physiol Chem 1893; 17:274. 14. Baumann E, and Preuss C. Concerning Bromophenylmercapturic acid. Ber deut chem Ges 1879; 12:806. 15. Jaffe M. Ueber die nach einfuhrung von brombenzol und chlorbenzol im organismus entstehenden schwefelhaltigen sauren. BerDeut Chem Ges 1879; 12:1092 1098. 16. Baumann E. Ueber cystin und cystein. Z Physiol Chem 1884; 8:299 305.

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Pharmacokinetics of Drug MetabolitesPhilip C. SmithSchool of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

INTRODUCTION Drug metabolites and their disposition in vivo are well recognized by scientists, clinicians, and regulatory agencies to be important when evaluating a new drug entity. In the past several decades, increased attention has been placed on drug metabolism for several reasons. Firstly, the number of drugs with active metabolites, by design (i.e., prodrugs) or by chance, has increased (1 3). This is exemplified by the transition from terfenadine to its active metabolite fexofenadine (3) and interest in the contributions of morphine-6-glucuronide (M6G) toward the analgesic activity of the age-old drug, morphine, and potential development of this active metabolite (4). In addition, with the advent of methods to establish the metabolic genotype and characterize the phenotype of individual patients (5,6) and the identification of specific isoforms of enzymes of metabolism, there is an increased appreciation of how elimination of a drug by metabolism can influence drug bioavailability and clearance, and ultimately affect its efficacy and toxicity. These rapidly evolving methods can be translated to permit costeffective individual optimization of drug therapy on the basis of a subjects metabolic capability (5,6), just as renal creatinine clearance has been used for years to assess renal function and permits individualized dose adjustment for drugs cleared by the kidney (7). Finally, the well accepted, though still poorly understood role of bioactivation in the potential toxicity of drugs and other xenobiotics (8,9) requires that metabolites continue to be evaluated and scrutinized for possible contributions to adverse effects observed in vivo. Though the importance of drug metabolism is seldom questioned, the interpretation and use of pharmacokinetic data on the disposition of metabolites is not well understood or fully implemented by some investigators. The objective of this chapter is to provide a basis for the interpretation and use of metabolite pharmacokinetic data from preclinical and clinical investigations. A number of previous authors have reviewed methods and theory for the analysis of metabolite pharmacokinetics, with literature based upon simple models, as early as 1963 by Cummings and Martin (10). Thorough theoretical analyses and reviews have been published, notably by Houston (11,12), Pang (13), and Weiss (14). The topic of metabolite kinetics is not found in commonly employed textbooks on pharmacokinetics17

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(15), though the topic is usually not presented or taught in a first, introductory course on pharmacokinetics. This chapter is not intended to present all aspects of basic pharmacokinetics that may be necessary for a thorough understanding of metabolite kinetics, and for this reason, motivated readers are recommended to consult other sources (15 19) if an introduction to basic pharmacokinetic principles is needed. This review will also not attempt to present or discuss all possible permutations of metabolite pharmacokinetics, but will make an effort to present and distinguish what can be assessed in humans and animals in vivo given commonly available experimental methods, which in some cases may be augmented by in vitro studies. METABOLITE KINETICS FOLLOWING A SINGLE INTRAVENOUS DOSE OF PARENT DRUG General Considerations in Metabolite Disposition Much of the theory presented here will be based upon primary metabolites, as shown in Scheme 1, which are formed directly from the parent drug or xenobiotic whose initial dose is known. In contrast, secondary or sequential metabolites, as indicated in Scheme 2, are formed from one or more primary metabolites. The theory and resultant equations for the analysis of sequential metabolite kinetics are often more complex (see sect. Sequential Metabolism) (13). Since most metabolites of interest are often primary metabolites, this review will focus on these, unless otherwise noted. Scheme 1 is the simplest model for one metabolite that can be measured in vivo, with other elimination pathways for the parent drug, either by metabolism or excretion (e.g., biliary or renal), represented