About the Editors xvii
List of Contributors xix
Preface xxiii
Acknowledgments xxvii

Part I Introduction to Drug Metabolism 1

1 Historical Perspective on Drug Metabolism 3
F. Peter Guengerich

1.1 Introduction 3
1.2 Humble Beginnings – The 18th and 19th Centuries 3
1.3 The First Half of the 20th Century 4
1.4 The Age of P450 5
1.5 The 1980s: More P450s, Other Enzymes, Technological Advances 7
1.6 The 1990s – Shifting into High Gear 7
1.7 2000–2020 8
1.8 2020–2025 9

2 Two Lives Dedicated to Biotransformation: Interviews 15
Emre M. Isin

2.1 Introduction 15
2.2 Professor Neal Castagnoli, Jr. 15
2.3 Interview with Professor Neal Castagnoli, Jr. (Conducted on July 17th, 2024) 16
2.4 Professor Thomas A. Baillie 19
2.5 Interview with Professor Thomas A. Baillie (Conducted on 21st of August 2024) 20

3 Species Differences in Drug Metabolizing Enzymes 27
Hiroshi Yamazaki

3.1 Introduction 27
3.2 Case Studies 27
3.3 Liver P450 and FMO Levels and Their Contributions to Drug Metabolism 30
3.4 Conclusions 31

4 Extrahepatic Drug Metabolism 37
Aneesh A. Argikar and Upendra A. Argikar

4.1 Introduction 37
4.2 Brain 38
4.3 Eye 39
4.4 Heart 41
4.5 Intestine 42
4.6 Kidney 44
4.7 Lungs 47
4.8 Mesentery 48
4.9 Microbiome 48
4.10 Placenta 51
4.11 Skin 52
4.12 Extrahepatic Metabolism Considerations for Novel Modalities 53
4.13 Systems to Study Extrahepatic Biotransformation, Mechanistic Models, and Future Directions 53

5 Mitigating Bioactivation Risk in Drug Discovery by Designing Around Structural Alerts 71
Lloyd W. T. Tang and Amit S. Kalgutkar

5.1 Idiosyncratic Adverse Drug Reactions (IADRs) and Drug-Induced Liver Injury (DILI) 71
5.2 Structural Alert Concept 72
5.3 Methodologies to Detect Reactive Metabolite Formation 76
5.4 Designing Around Structural Alerts 80
5.5 Shortcomings of the Structural Alert Avoidance Philosophy 83
5.6 Detoxification of Reactive Metabolites 84
5.7 Total Daily Dose and Reactive Metabolite Body Burden 86
5.8 Concluding Remarks 87

Part II Current, New, and Emerging Tools and Technologies for Biotransformation Studies 95

6 Current and Future Applicability of Complex In Vitro Models to Biotransformation Studies 97
Benoit Cox, Reiner Class, Lloyd King, and Amélie Moreau

6.1 Introduction 98
6.2 Complex In Vitro Models 101
6.3 The Need for Standards, Quality Control, and Harmonization 109
6.4 Recommendation and Future Perspectives 109

7 Analytical Technologies and Software Tools for Biotransformation Studies 115
Daniel J. Weston

7.1 Introduction 115
7.2 Mass Spectrometry 116
7.3 Data Interrogation 120
7.4 Concerted Analytical Techniques – Better Together 127
7.5 Conclusion 134

8 Preclinical In Vivo Models for Drug Metabolism Studies and Strategies for Replacing, Reducing, and Refining the Use of Animals: A Perspective 141
Ian D. Wilson

8.1 Introduction and Background 141
8.2 Preclinical Animal Models 142
8.3 Radiolabeled Studies 143
8.4 Alternatives to the Radiolabeled Study 143
8.5 Advanced Animal Models for In Vivo Studies 145
8.6 Genetically Modified Rodents in DMPK Studies 146
8.7 Human-Relevant Animal Models: Genetically Modified Humanized and Chimeric Mice 147
8.8 Chimeric Liver Humanized Mouse Models 148
8.9 Humanized versus Chimeric Animal Models 149
8.10 Future Perspectives 150

9 Application of Isotope Labeling Approaches to the Study of Drug Metabolism 157
Scott G. Summerfield, Tim Smith, and Grant Johnston

9.1 Introduction 157
9.2 Importance of Isotopes in the Study of Drug Metabolism and Disposition 157
9.3 The Regulatory Case for Isotope Labeling 159
9.4 A Brief History of Isotopic Markers in the Life Sciences 159
9.5 Radioactive Properties of Isotopic Labels 161
9.6 Measuring Radioactivity 161
9.7 Points to Consider for Utilizing 14C 162
9.8 Points to Consider for Utilizing Tritium (3H) 164
9.9 Mass Properties of Isotopic Labels 165
9.10 Spin Properties of Isotopic Labels 167
9.11 Spectroscopic Isotopic Properties 168
9.12 Summary 168

10 Imaging in Drug Discovery and Development 173
Andreas Dannhorn

10.1 Introduction 173
10.2 Commonly Used Imaging Modalities in Drug Discovery and Development 173
10.3 Choosing the Right Imaging Strategy for Every Development Stage 179
10.4 Concluding Remarks 186

11 Approaches for Metabolite Synthesis (Biocatalytic, Electrochemical, and Chemical) 191
Valerie M. Kramlinger, Marta Wenzler, David Cantillo, and Elizabeth M. J. Gillam

11.1 Introduction 191
11.2 Chemo-Catalysis for Metabolite Synthesis 194
11.3 Electrochemical Synthesis of Drug Metabolites 199
11.4 Biocatalysis 203
11.5 Integrated Chemo-Enzymatic Approaches for Metabolite Synthesis 208
11.6 Conclusions and Perspectives for the Future 209

Part III Role of Biotransformation in Drug Discovery and Development 221

12 Metabolites and Drug–Drug Interactions 223
Sandeepraj Pusalkar and Yannick Parmentier

12.1 Introduction 223
12.2 Mechanism of Drug–Drug Interactions 223
12.3 Metabolites and Drug–Drug Interaction 224
12.4 Discussion 231

13 Metabolite Profiling and Identification in Early Drug Development Without Radiolabel 237
Alexander D. James and Christian Lanshoeft

13.1 Introduction 237
13.2 Regulatory Guidelines Relevant to Drug Metabolism Investigations 237
13.3 Metabolite Screening in First-in-Human (FIH) Studies 238
13.4 Assessing Metabolite Coverage Across Species 244

14 Current Approaches to hADME Studies and Future Perspectives 253
Graeme C. Young

14.1 Introduction 253
14.2 Traditional Human ADME Study Design 254
14.3 Characteristics of the Modern Human ADME Study Design 256
14.4 Extracting More Information from the Data 257
14.5 The Missing Piece of the ADME Jigsaw: Duodenal Bile Collection 259
14.6 Advances in the Detection of 14C (Radiocarbon) 262
14.7 Future Perspectives and the Paradigm Shift 265
14.8 Conclusion 266

15 Drug Metabolism in Patients and Special Populations 271
Anne Smits, Isabel Spriet, Karel Allegaert, and Pieter Annaert

15.1 Introduction 271
15.2 Inter-Patient Variability in Drug Metabolizing Enzymes 273
15.3 Special Populations and Unique Challenges 280
15.4 Advances in Studying Drug Metabolism in Patient Populations 287
15.5 Regulatory Considerations 289
15.6 Future Directions 290
15.7 Conclusion 292

Part IV "New" Modalities 301

16 Biotransformation Aspects of Targeted Covalent Inhibitors 303
Holger Scheible, Hanno Schieferstein, and Rowan Stringer

16.1 Introduction 303
16.2 Mode of Binding of TCIs 304
16.3 Considerations on Radiolabeling Position 305
16.4 Relevant Pathways and Enzymes for TCIs 311
16.5 Warhead Metabolism 315
16.6 Considerations for hADME Studies and Metabolite Profiling Methods for TCIs 318
16.7 Summary and Outlook 320

17 Biotransformation of Peptides and Proteins 331
Jesper K. Christensen and Jørgen Olsen

17.1 Peptides and Proteins 331
17.2 Metabolism Reactions 331
17.3 Proteolytic Enzymes 332
17.4 In Vitro and In Vivo Metabolism 332
17.5 Analytical Tools to Investigate Metabolism 333
17.6 Metabolism Case Stories 334
17.7 Summary 340

18 Biotransformation of Antibody-Drug Conjugates (ADCs): Mechanisms and Bioanalytical Strategies 345
Israel Rodríguez and Jintang He

18.1 Introduction 345
18.2 Fundamentals of Biotransformation in Therapeutic Proteins 345
18.3 Antibody-Drug Conjugates: Structure, Function, and Mechanism of Action 346
18.4 Overview of ADC Biotransformation: Processes and Factors Affecting ADC Biotransformation 350
18.5 Analytical Techniques for Studying ADC Biotransformation 350Contents xv
18.6 Influence of Biotransformation on ADC Pharmacokinetics, Pharmacodynamics, Efficacy, and Safety 353
18.7 Case Study: Understanding the Biotransformation of Kadcyla (T-DM1) 353
18.8 Future Directions for Studying ADC Biotransformation 357
18.9 Conclusion 357

19 Biotransformation of Oligonucleotide (ON) Therapeutics 363
Marie Ahlqvist, Bo Lindmark, Xue-Qing Li, and Lars Weidolf

19.1 Introduction and Scope: Antisense Oligonucleotides, siRNA, and Oligonucleotide Conjugates 364
19.2 Number of ONs in Development, Therapy Areas Beyond Rare Diseases 365
19.3 General Information on Biotransformation of ONs 366
19.4 Regulatory Guidance 366
19.5 Quantitative and Qualitative Bioanalytical Guide 367
19.6 Analysis of Raw Data 370
19.7 Example of Oligonucleotide Data Analysis 370
19.8 Biotransformation of Oligonucleotide Conjugates 370
19.9 Study Types and Test Systems 371
19.10 Radiolabeling on Oligonucleotides and Oligonucleotide Conjugates 374
19.11 Possible On- and Off-Target Effects Due to Formed Metabolite(s) 377
19.12 Proposed Strategy for Biotransformation of Oligonucleotides and Oligonucleotide Conjugates 378
19.13 Concluding Remarks 379

References 379
Index 383