DRUG METABOLISM IN DRUG DESIGN AND DEVELOPMENT DRUG METABOLISM IN DRUG DESIGN AND DEVELOPMENT Basic Concepts and Practice EDITED BY DONGLU ZHANG MINGSHE ZHU W. GRIFFITH HUMPHREYS WILEY-INTERSCIENCE A JOHN WILEY & SONS, INC., PUBLICATION Copyright # 2008 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Dancers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at . Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at /go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damage, including but not limited to special, incidental, consequential, or other damages. For general information on our other products
CONTENTS
Preface xvii
Contributors xix
PART I BASIC CONCEPTS OF DRUG METABOLISM 1
1 Overview: Drug Metabolism in the Modern Pharmaceutical
Industry 3
Scott J. Grossman
1.1 Introduction 3
1.2 Technology 4
1.3 Breadth of Science 5
1.3.1 Chemistry 5
1.3.2 Enzymology and Molecular Biology 6
1.4 Impact of Drug Metabolism on Efficacy and Safety 7
1.4.1 Efficacy 7
1.4.2 Safety 8
1.5 Regulatory Impact and IP Position 9
1.6 Summary 12
References 12
2 Oxidative, Reductive, and Hydrolytic Metabolism of Drugs 15
F. Peter Guengerich
2.1 Introduction 15
2.2 Nomenclature and Terminology 15
2.3 General Features of the Enzymes 16
2.4 Fractional Contributions of Different Enzymes 17
2.5 Oxidation Enzymes 18
2.5.1 Cytochrome P450 (P450, CYP) 18
2.5.2 Flavin-Containing Monooxygenase (FMO) 20
v
2.5.3 Monoamine Oxidase (MAO) 22
2.5.4 Aldehyde Oxidase and Xanthine Dehydrogenase 23
2.5.5 Peroxidases 24
2.5.6 Alcohol Dehydrogenases (ADH) 25
2.5.7 Aldehyde Dehydrogenases (ALDH) 25
2.6 Reduction 26
2.6.1 P450, ADH 26
2.6.2 NADPH-P450 Reductase 27
2.6.3 Aldo-Keto Reductases (AKR) 28
2.6.4 Quinone Reductase (NQO) 28
2.6.5 Glutathione Peroxidase (GPX) 29
2.7 Hydrolysis 29
2.7.1 Epoxide Hydrolase 29
2.7.2 Esterases and Amidases 30
2.8 Summary 31
References 31
3 Conjugative Metabolism of Drugs 37
Rory Remmel, Swati Nagar and Upendra Argikar
3.1 UDP-Glucuronosyltransferases 37
3.1.1 Location Within the Cell 38
3.1.2 Endogenous Substrates 39
3.1.3 Enzyme Multiplicity 40
3.1.4 Inducibility 42
3.1.5 Pharmacogenetics 56
3.1.6 Experimental Considerations 56
3.1.7 Enzyme Selective Substrates and Inhibitors 59
3.1.8 Drug–Drug Interactions and Glucuronidation 60
3.1.9 Summary 62
3.2 Cytosolic Sulfotransferases 62
3.2.1 Cellular Location and Tissue Expression 64
3.2.2 The SULT Superfamily of Cytosolic Enzymes 64
3.2.3 Inducibility 65
3.2.4 SULT Pharmacogenetics 65
3.2.5 Analytical Detection of Sulfonated Metabolites 67
3.2.6 SULT Inhibitors (Pacifici and Coughtrie, 2005) 68
3.2.7 Drug–Drug Interactions and Sulfonation 68
3.2.8 Summary 72
3.3 Glutathione-S-Transferases 72
3.3.1 General Overview 72
3.3.2 Classification of the GST Enzymes 72
3.3.3 Localization and Expression 73
3.3.4 Reactions Catalyzed by GSTs 73
3.3.5 Regulation of GSTs 73
vi CONTENTS
3.3.6 GST Alpha Class 75
3.3.7 GST Mu Class 77
3.3.8 GST Pi Class 79
3.3.9 GST Theta Class 81
3.3.10 GST Zeta Class 81
3.3.11 Incubation Conditions and Analytical Methods 81
3.3.12 Glutathione Conjugate Metabolism (Mercapturic Acid
Pathway) 83
References 84
4 Enzyme Kinetics 89
Timothy S. Tracy
4.1 Introduction 89
4.2 Enzyme Catalysis 90
4.3 Michaelis–Menten Kinetics 90
4.3.1 Meanings of Km, Vmax and Their Clinical Relevance 91
4.4 Graphical Kinetic Plots 92
4.5 Atypical Kinetics–Allosteric Effects 94
4.5.1 Overview of Atypical Kinetic Phenomena 94
4.5.2 Homotropic Cooperativity 95
4.5.3 Heterotropic Cooperativity 99
4.6 Graphical Analysis of Atypical Kinetic Data 101
4.7 Enzyme Inhibition Kinetics 101
4.7.1 Overview 101
4.7.2 Competitive Inhibition 102
4.7.3 Mixed Inhibition 103
4.7.4 Noncompetitive Inhibition 104
4.7.5 Uncompetitive Inhibition 105
4.7.6 Summary of Effects of Various Inhibition Types of
Kinetic Parameters 106
4.7.7 Meanings of IC50 and Ki Parameters 106
4.8 Inhibition Kinetics Graphical Plots 106
4.9 Mechanism-Based Enzyme Inactivation Kinetics 108
Acknowledgment 110
References 111
5 Metabolism-Mediated Drug–Drug Interactions 113
Hongjian Zhang, Michael W. Sinz, and A. David Rodrigues
5.1 Introduction 113
5.2 Enzyme Inhibition 114
5.2.1 Types of Inhibition 114
5.2.2 In vitro Evaluation of Inhibition 116
5.2.3 Prediction of CYP Inhibition Using In vitro Data 116
5.2.4 Clinical Evaluation of Inhibition 119
CONTENTS vii
5.3 Enzyme Induction 120
5.3.1 Enzyme and Pharmacokinetic Changes 120
5.3.2 Mechanisms of Enzyme Induction 122
5.3.3 Induction Models 125
5.4 Reaction Phenotyping 126
5.4.1 Experimental Considerations 127
5.4.2 Data Interpretation and Integration 128
5.4.3 Clinical Evaluation 129
References 130
6 Drug Transporters in Drug Disposition, Drug Interactions,
and Drug Resistance 137
Cindy Q. Xia, Johnny J. Yang, and Suresh K. Balani
6.1 Introduction 137
6.2 Roles of Transporters in Drug Disposition
and Toxicity 139
6.2.1 Transporters in Drug Absorption 139
6.2.2 Transporters in Drug Distribution 148
6.2.3 Transporters in Drug Metabolism 150
6.2.4 Transporters in Drug Excretion 150
6.2.5 Transporters in Toxicity 153
6.3 Transporters in Drug Resistance 154
6.4 Polymorphism of Transporters and Interindividual Variation 157
6.5 Transporters in Drug–Drug or Drug–Food Interactions 158
6.5.1 Oral Absorption 171
6.5.2 Brain Penetration 172
6.5.3 Renal Excretion and Hepatic Clearance 172
6.5.4 Food Effect 173
6.5.5 Formulation Effect 174
6.5.6 In vitro–In vivo Correlation 175
6.6 Methods to Evaluate Transporter Substrate, Inhibitor,
or Inducer 176
6.6.1 In vitro Models 176
6.6.2 In situ/Ex vivo Models 182
6.6.3 In vivo Models 182
6.7 Conclusions and Perspectives 184
References 185
7 Regulatory Considerations of Drug Metabolism and
Drug Interaction Studies 203
Xiaoxiong Wei and Mingshe Zhu
7.1 Introduction 203
7.2 Regulatory Guidances Relevant to Drug Metabolism 204
7.2.1 Toxicokinetic Studies 206
7.2.2 Use of Radiolabeled Materials 207
viii CONTENTS
7.2.3 Metabolite Safety Assessment 207
7.2.4 Drug–Drug Interaction Studies 208
7.2.5 Analytical Method Validation and Compliance 209
7.2.6 Regulatory Submission Format and Content 210
7.3 Metabolism Studies Relevant to Metabolite Safety Assessment 211
7.3.1 Goals and General Strategies 211
7.3.2 In vitro Metabolite Profiling Studies 212
7.3.3 ADME Studies 213
7.3.4 Analytical Methods for Metabolite Profiling 213
7.3.5 Special Considerations 214
7.4 Drug–Drug Interaction Studies 218
7.4.1 General Strategies 218
7.4.2 In vivo Studies 226
7.4.3 Case Study 230
7.5 Conclusions 231
Acknowledgment 232
References 232
PART II ROLE OF DRUG METABOLISM IN THE
PHARMACEUTICAL INDUSTRY 237
8 Drug Metabolism Research as an Integral Part of the
Drug Discovery Process 239
W. Griffith Humphreys
8.1 Introduction 239
8.2 Metabolic Clearance 240
8.2.1 General 240
8.2.2 Prediction of Human Clearance 241
8.2.3 In vivo Methods to Study Metabolism 242
8.2.4 Screening Strategies 243
8.2.5 In silico Methods to Study Metabolism 244
8.3 Metabolite Profiling 245
8.4 Reaction Phenotyping 246
8.5 Assessment of Potential Toxicology of Metabolites 246
8.5.1 Reactive Metabolite Studies—In vitro 246
8.5.2 Reactive Metabolite Studies—In vivo 248
8.5.3 Toxicology Mediated Through Metabolite Interaction
with Off-Target Receptors 249
8.6 Assessment of Potential for Active Metabolites 249
8.6.1 Detection of Active Metabolites During Drug Discovery 251
8.6.2 Methods for Assessing and Evaluating the Biological
Activity of Metabolites 252
8.6.3 Methods for Generation of Metabolites 253
CONTENTS ix
8.7 Summary 253
References 254
9 Role of Drug Metabolism in Drug Development 261
Ramaswamy Iyer and Donglu Zhang
9.1 Introduction to the Role of Drug Metabolism in Drug Development 261
9.1.1 Drug Metabolism and Clinical Interactions 261
9.1.2 Drug Metabolism and Issue Resolution 262
9.1.3 Drug Metabolism and Regulatory Requirements 263
9.2 Staging and Types of Drug Metabolism Studies in Drug
Development 265
9.3 In vivo ADME Studies 266
9.3.1 Use of Radiolabeled Compound in ADME Studies 267
9.3.2 Tissue Distribution Study to Support the Human
ADME Study 268
9.3.3 Nonclinical and Clinical ADME Studies 268
9.4 Metabolites in Safety Testing (MIST) 273
9.5 In vitro Drug Metabolism Studies in Drug Development 274
9.5.1 In vitro Metabolite Profiling 275
9.5.2 Identification of Drug-Metabolizing Enzyme(s) 275
9.5.3 Evaluation of CYP Inhibition 277
9.5.4 Evaluation of CYP Induction 278
9.6 Examples of Role of Drug Metabolism to Address Safety Issues 278
9.6.1 Rat-Specific Toxicity of Efavirenz Caused by
Species-Specific Bioactivation 279
9.6.2 UGT1A1 Inhibition-Mediated Hyperbilirubinemia by
HIV Protease Inhibitors 279
9.6.3 Mechanism-Based Inactivation of CYP2C8 by
Gemfibrozil Glucuronide 280
9.7 Impact of Metabolism Information on NDA
Filing and Labeling 281
References 281
PART III ANALYTICAL TECHNIQUES IN DRUG
METABOLISM 287
10 Applications of Liquid Radiochromatography Techniques in
Drug Metabolism Studies 289
Mingshe Zhu, Weiping Zhao, and W. Griffith Humphreys
10.1 Introduction 289
10.2 Traditional Radiochromatography Techniques 290
10.2.1 HPLC-RFD 290
10.2.2 HPLC-LSC 291
x CONTENTS
10.3 New Radiochromatography Techniques 293
10.3.1 HPLC-MSC 293
10.3.2 Stop-Flow HPLC-RFD 298
10.3.3 Dynamic Flow HPLC-RFD 300
10.3.4 UPLC-Radiodetection 301
10.3.5 HPLC-AMS 301
10.4 Radiochromatography in Conjunction with Mass Spectrometry 302
10.4.1 LC-RFD-MS 302
10.4.2 Stop-Flow and Dynamic Flow LC–RFD–MS 303
10.4.3 LC-MSC-MS 303
10.4.4 An Integrated Radiochromatography–Mass Spectrometry
Approach 305
10.5 Application of New Radiochromatography Techniques in
Drug Metabolism Studies 306
10.5.1 Profiling of Radiolabeled Metabolites in Plasma 306
10.5.2 Analysis of Metabolites of Nonradiolabeled Drugs
Using Radiolabeled Cofactors or Trapping Agents 308
10.5.3 Determination of Structures and Formation Pathways
of Sequential Metabolites 308
10.5.4 Enzyme Kinetic Studies 310
10.6 Summary 313
References 313
11 Application of Liquid Chromatography/Mass Spectrometry
for Metabolite Identification 319
Shuguang Ma and Swapan K. Chowdhury
11.1 Introduction 319
11.2 LC/MS Instrumentation 320
11.2.1 High Performance Liquid Chromatography (HPLC) 320
11.2.2 Atmospheric Pressure Ionization Methods 321
11.2.3 Mass Analyzers 325
11.2.4 Tandem Mass Spectrometry 329
11.3 Metabolite Identification––Role of LC/MS 329
11.3.1 Metabolite Characterization in Drug Discovery 329
11.3.2 Metabolite Identification in Preclinical and
Clinical Development 337
11.4 Techniques for Improving Metabolite Detection and
Identification 339
11.4.1 Chemical Derivatization 339
11.4.2 Stable Isotope Labeling 340
11.4.3 Hydrogen/deuterium (H/D) Exchange MS 341
11.4.4 Accurate Mass Measurement 342
11.4.5 Nanospray Ionization (NSI) MS for
Metabolite Identification 343
CONTENTS xi
11.5 Software-Assisted Metabolite Identification 345
11.5.1 Data-Dependent Acquisition (DDA) 345
11.5.2 Mass Defect Filter (MDF) 346
11.6 Additional MS-Related Techniques for Metabolite
Identification 348
11.6.1 LC/NMR/MS 348
11.6.2 LC/ICPMS 349
11.7 Characterization of Unstable Metabolites 349
11.7.1 Glucuronides 349
11.7.2 N-Oxides 350
11.7.3 Differentiation of Molecular Ions from In-Source Fragment
Ions by the Presence of Alkali Adducts 352
11.8 Detection and Characterization of Reactive Metabolites and
Intermediates 353
11.8.1 Trapping Reactive Metabolites 353
11.8.2 Screening for Glutathione Conjugates 354
11.9 Conclusions and Future Directions 357
Acknowledgments 359
References 359
12 Introduction to NMR and Its Application in Metabolite
Structure Determination 369
Xiaohua Huang, Robert Powers, Adrienne Tymiak, Robert Espina,
and Vikram Roongta
12.1 Introduction 369
12.2 Theory 370
12.3 NMR Hardware 372
12.4 NMR Observables 373
12.4.1 Chemical Shifts 377
12.4.2 Coupling Constants 377
12.4.3 Integration 379
12.5 Sample Requirements for NMR 380
12.6 Most Commonly Used NMR Experiments and Techniques 381
12.6.1 1D NMR Experiments 381
12.6.2 2D NMR Experiments 382
12.6.3 Solvent Suppression Techniques 385
12.6.4 Hyphenated NMR Methods 387
12.7 General protocol for NMR Analysis of Unknown Compounds
or Metabolites 389
12.8 Examples of Metabolite Structure Determination from
Known Biotransformations 404
References 405
xii CONTENTS
PART IV COMMON EXPERIMENTAL APPROACHES
AND PROTOCOLS 411
13 Determination of Metabolic Rates and Enzyme Kinetics 413
Zhi-Yi Zhang and Laurence S. Kaminsky
13.1 Introduction 413
13.2 Determination of Metabolic Stability 414
13.2.1 Aims 414
13.2.2 Experimental Procedures 415
13.3 Characterization of Enzyme Kinetics 425
13.3.1 Basic Theory 425
13.3.2 Experimental Design 426
13.3.3 Determination of Kinetic Parameters 427
13.4 Quantitative Analytical Methods 432
13.4.1 HPLC/UV/FL 432
13.4.2 LC/MS/MS 432
13.5 Prediction of Human Hepatic Clearance 434
13.5.1 Aims 434
13.5.2 Procedure of In vitro–In vivo Correlation 435
13.5.3 Examples 438
Abbreviations 439
References 441
14 Protocols for Assessment of In vitro and In vivo Bioactivation
Potential of Drug Candidates 447
Zhoupeng Zhang and Jinping Gan
14.1 Glutathione, N-Acetylcysteine, and Potassium Cyanide as
Trapping Agents 450
14.1.1 Introduction 450
14.1.2 Detection of Glutathione/N-Acetylcysteine or Cyano Adducts
Using Ion-Trap Mass Spectrometer 452
14.1.3 Protocol for Detection of Glutathione or Cyano
Adducts Using Constant Neutral Loss Scanning of
Triple Quadrupole Mass Spectrometer 455
14.1.4 Protocol for Qualitative and Quantitative Analysis
of Thiol Adducts Using Dansyl Glutathione (dGSH) 457
14.1.5 Notes 460
14.2 Protocols for In vitro and In vivo Covalent Protein Binding Studies 461
14.2.1 Introduction 461
14.2.2 Protocol for In vitro Covalent Protein Binding in Human
or Rat Liver Microsomes—A Test-Tube Method 462
14.2.3 Protocol for In vitro Covalent Protein Binding in Human
or Rat Hepatocytes 464
CONTENTS xiii
14.2.4 Protocol for In vitro Covalent Protein Binding
in Human or Rat Liver Microsomes—A Semiautomated
Method 464
14.2.5 Protocol For In vivo Covalent Protein Binding in Rats 465
14.2.6 Notes 466
14.3 Protocol for Measurement of Intracellular GSH and GSSG
Concentrations in Hepatocytes 467
14.3.1 Introduction 467
14.3.2 Measurement of Intracellular GSH/GSSG in
Hepatocytes 469
14.4 Perspectives 470
Acknowledgments 472
References 472
15 Reaction Phenotyping 477
Susan Hurst, J. Andrew Williams, and Steven Hansel
15.1 Introduction 477
15.2 Cytochrome P450 Reaction Phenotyping 479
15.3 Noncytochrome P450 Reaction Phenotyping 481
15.3.1 Flavin-Containing Monooxygenases 481
15.3.2 Monoamine Oxidases A and B (MAO-A and
MAO-B) 482
15.3.3 Esterases 483
15.4 Conjugation Phenotyping 484
15.4.1 UGT Reaction Phenotyping 484
15.4.2 N-Acetylation Reaction Phenotyping 487
15.4.3 Sulfation Reaction Phenotyping 488
15.5 Transporter Phenotyping 488
15.6 Nonradiolabeled Reaction Phenotyping 489
15.6.1 Objective 489
15.6.2 Selection of Appropriate Experimental Systems 489
15.6.3 Experimental Approach Considerations 491
15.6.4 Selection of Appropriate Experimental Designs 493
15.6.5 Quantitative Reaction Phenotyping: Expressed or Purified
Enzyme Systems 497
15.7 Radiolabeled Reaction Phenotyping 499
15.7.1 Quantitative In vitro Radiolabeled Reaction
Phenotyping Studies 500
15.7.2 In vivo Quantitative ADME Studies 501
15.7.3 Drug–Drug Interaction Potential 502
15.7.4 Specialized Clinical Studies 504
15.8 Summary and Future Directions 504
Acknowledgments 504
References 505
xiv CONTENTS
Appendix A: Reaction Phenotyping—Expressed cDNA
Enzyme Incubation Method Sheet 511
Appendix B: Reaction Phenotyping—Microsomal
Chemical Inhibition 512
16 Analysis of In vitro Cytochrome P450 Inhibition in Drug
Discovery and Development 513
Magang Shou and Renke Dai
16.1 Introduction 513
16.2 Reversible Inhibition 515
16.2.1 Materials and Reagents 516
16.2.2 Instrument 516
16.2.3 Optimization of Kinetic Reaction 517
16.2.4 LC/MS/MS Analysis 520
16.2.5 Automated Sample Preparation and Incubation 521
16.2.6 Data Analysis 523
16.3 Irreversible Inhibition 526
16.3.1 Kinetic Model for Mechanism-Based Inhibition 528
16.3.2 Measurements of Kinetic Parameters 529
16.3.3 General Incubation Procedure and Sample Preparation 531
16.3.4 Data Analysis 532
16.4 Fluorescent Assay 532
16.5 Prediction of Human Drug–Drug Interactions from In vitro
CYP Inhibition Data 534
16.5.1 Reversible CYP Inhibition 534
16.5.2 Prediction of Human Drug–Drug Interactions
from Mechanism-Based CYP Inhibition 535
16.5.3 Factors Affecting the Prediction of Drug–Drug Interactions 537
16.6 Conclusion 538
Acknowledgment 538
References 538
17 Testing Drug Candidates for CYP3A4 Induction 545
Gang Luo, Liang-Shang Gan, and Thomas M. Guenthner
17.1 Introduction 545
17.2 Assessments 548
17.2.1 Assessment of Induction Potential Using Intact
Animal Models 548
17.2.2 Assessment of Induction Potential Using In vitro Models 552
17.2.3 Direct Assessment of CYP3A4 Induction In vivo
in Humans 562
17.3 Final Comments 565
References 566
CONTENTS xv
18 ADME Studies in Animals and Humans: Experimental Design,
Metabolite Profiling and Identification, and Data Presentation 573
Donglu Zhang and S. Nilgun Comezoglu
18.1 Objectives, Rational, and Regulatory Compliance 573
18.2 Study Designs 575
18.2.1 Choice of Radiolabel 575
18.2.2 Preparation of Animals and Human Subjects 576
18.2.3 Dose Selection, Formulation, and Administration 578
18.2.4 In-Life Studies in Animals and Humans and
Sample Collection/Pooling 579
18.3 Sample Analysis 580
18.3.1 Sample Preparation: Plasma, Urine, Bile, and Feces 580
18.3.2 Radioactivity Determination 581
18.3.3 LC/MS/MS Quantification and Pharmacokinetic Analysis 582
18.3.4 Metabolite Profiling 583
18.3.5 Metabolite Identification 584
18.3.6 Metabolite Isolation from In vivo Samples
and Generation in Bioreactors 585
18.4 Data Presentation Using Metabolism of [14C]Muraglitazar as
an Example 586
18.4.1 Pharmacokinetic Results and Excretion of
Radioactivity 586
18.4.2 Metabolite Profiling in Plasma, Urine, Bile, and Feces 591
18.4.3 Metabolite Identification by LC/MS/MS 594
18.5 Conclusions and Path Forward 597
Acknowledgments 597
Appendix A: Rat Tissue Distribution and Dosimetry Calculation 597
References 602
Index 605
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