ADME-Enabling Technologies in Drug Design and Development

Donglu Zhang, PhD, is a Principal Scientist in Pharmaceutical Candidate Optimization at Bristol-Myers Squibb in Princeton, New Jersey. He has published seventy peer-reviewed articles, codiscovered the Mass Defect Filtering technique, and coedited two books. Sekhar Surapaneni, PhD, is Director, DMPK, at Celgene Corporation in New Jersey. He has published extensively in peer-reviewed journals and is a member of ISSX and ACS.

FOREWORD xxi
Lisa A. Shipley

PREFACE xxv
Donglu Zhang and Sekhar Surapaneni

CONTRIBUTORS xxvii

PART A ADME: OVERVIEW AND CURRENT TOPICS 1

1 Regulatory Drug Disposition and NDA Package Including MIST 3
Sekhar Surapaneni

1.1 Introduction 3

1.2 Nonclinical Overview 5

1.3 PK 5

1.4 Absorption 5

1.5 Distribution 6

1.6 Metabolism 7

1.7 Excretion 11

1.8 Impact of Metabolism Information on Labeling 11

1.9 Conclusions 12

References 12

2 Optimal ADME Properties for Clinical Candidate and Investigational New Drug (IND) Package 15
Rajinder Bhardwaj and Gamini Chandrasena

2.1 Introduction 15

2.2 NCE and Investigational New Drug (IND) Package 16

2.3 ADME Optimization 17

2.4 ADME Optimization for CNS Drugs 23

2.5 Summary 24

References 25

3 Drug Transporters in Drug Interactions and Disposition 29
Imad Hanna and Ryan M. Pelis

3.1 Introduction 29

3.2 ABC Transporters 31

3.3 SLC Transporters 33

3.4 In vitro Assays in Drug Development 39

3.5 Conclusions and Perspectives 45

References 46

4 Pharmacological and Toxicological Activity of Drug Metabolites 55
W. Griffith Humphreys

4.1 Introduction 55

4.2 Assessment of Potential for Active Metabolites 56

4.3 Assessment of the Potential Toxicology of Metabolites 59

4.4 Safety Testing of Drug Metabolites 62

4.5 Summary 63

References 63

5 Improving the Pharmaceutical Properties of Biologics in Drug Discovery: Unique Challenges and Enabling Solutions 67
Jiwen Chen and Ashok Dongre

5.1 Introduction 67

5.2 Pharmacokinetics 68

5.3 Metabolism and Disposition 70

5.4 Immunogenicity 71

5.5 Toxicity and Preclinical Assessment 74

5.6 Comparability 74

5.7 Conclusions 75

References 75

6 Clinical Dose Estimation Using Pharmacokinetic/Pharmacodynamic Modeling and Simulation 79
Lingling Guan

6.1 Introduction 79

6.2 Biomarkers in PK and PD 80

6.3 Model-Based Clinical Drug Development 83

6.4 First-in-Human Dose 86

6.5 Examples 89

6.6 Discussion and Conclusion 90

References 93

7 Pharmacogenomics and Individualized Medicine 95
Anthony Y.H. Lu and Qiang Ma

7.1 Introduction 95

7.2 Individual Variability in Drug Therapy 95

7.3 We Are All Human Variants 96

7.4 Origins of Individual Variability in Drug Therapy 96

7.5 Genetic Polymorphism of Drug Targets 97

7.6 Genetic Polymorphism of Cytochrome P450s 98

7.7 Genetic Polymorphism of Other Drug Metabolizing Enzymes 100

7.8 Genetic Polymorphism of Transporters 100

7.9 Pharmacogenomics and Drug Safety 101

7.10 Warfarin Pharmacogenomics: A Model for Individualized Medicine 102

7.11 Can Individualized Drug Therapy Be Achieved? 104

7.12 Conclusions 104

Disclaimer 105

Contact Information 105

References 105

8 Overview of Drug Metabolism and Pharmacokinetics with Applications in Drug Discovery and Development in China 109
Chang-Xiao Liu

8.1 Introduction 109

8.2 PK–PD Translation Research in New Drug Research and Development 109

8.3 Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME/T) Studies in Drug Discovery and Early Stage of Development 110

8.4 Drug Transporters in New Drug Research and Development 111

8.5 Drug Metabolism and PK Studies for New Drug Research and Development 113

8.6 Studies on the PK of Biotechnological Products 117

8.7 Studies on the PK of TCMS 118

8.8 PK and Bioavailability of Nanomaterials 123

References 125

PART B ADME SYSTEMS AND METHODS 129

9 Technical Challenges and Recent Advances of Implementing Comprehensive ADMET Tools in Drug Discovery 131
Jianling Wang and Leslie Bell

9.1 Introduction 131

9.2 “A” Is the First Physiological Barrier That a Drug Faces 131

9.3 “M” Is Frequently Considered Prior to Distribution Due to the “First-Pass” Effect 139

9.4 “D” Is Critical for Correctly Interpreting PK Data 142

9.5 “E”: The Elimination of Drugs Should Not Be Ignored 145

9.6 Metabolism- or Transporter-Related Safety Concerns 146

9.7 Reversible CYP Inhibition 147

9.8 Mechanism-Based (Time-Dependent) CYP Inhibition 149

9.9 CYP Induction 152

9.10 Reactive Metabolites 153

9.11 Conclusion and Outlook 154

Acknowledgments 155

References 155

10 Permeability and Transporter Models in Drug Discovery and Development 161
Praveen V. Balimane, Yong-Hae Han, and Saeho Chong

10.1 Introduction 161

10.2 Permeability Models 162

10.3 Transporter Models 163

10.4 Integrated Permeability–Transporter Screening Strategy 166

References 167

11 Methods for Assessing Blood–Brain Barrier Penetration in Drug Discovery 169
Li Di and Edward H. Kerns

11.1 Introduction 169

11.2 Common Methods for Assessing BBB Penetration 170

11.3 Methods for Determination of Free Drug Concentration in the Brain 170

11.4 Methods for BBB Permeability 172

11.5 Methods for Pgp Efflux Transport 173

11.6 Conclusions 174

References 174

12 Techniques for Determining Protein Binding in Drug Discovery and Development 177
Tom Lloyd

12.1 Introduction 177

12.2 Overview 178

12.3 Equilibrium Dialysis 179

12.4 Ultracentrifugation 180

12.5 Ultrafiltration 181

12.6 Microdialysis 182

12.7 Spectroscopy 182

12.8 Chromatographic Methods 183

12.9 Summary Discussion 183

Acknowledgment 185

References 185

13 Reaction Phenotyping 189
Chun Li and Nataraj Kalyanaraman

13.1 Introduction 189

13.2 Initial Considerations 190

13.3 CYP Reaction Phenotyping 193

13.4 Non-P450 Reaction Phenotyping 199

13.5 UGT Conjugation Reaction Phenotyping 201

13.6 Reaction Phenotyping for Other Conjugation Reactions 204

13.7 Integration of Reaction Phenotyping and Prediction of DDI 205

13.8 Conclusion 205

References 206

14 Fast and Reliable CYP Inhibition Assays 213
Ming Yao, Hong Cai, and Mingshe Zhu

14.1 Introduction 213

14.2 CYP Inhibition Assays in Drug Discovery and Development 215

14.3 HLM Reversible CYP Inhibition Assay Using Individual Substrates 217

14.4 HLM RI Assay Using Multiple Substrates (Cocktail Assays) 222

14.5 Time-Dependent CYP Inhibition Assay 226

14.6 Summary and Future Directions 228

References 230

15 Tools and Strategies for the Assessment of Enzyme Induction in Drug Discovery and Development 233
Adrian J. Fretland, Anshul Gupta, Peijuan Zhu, and Catherine L. Booth-Genthe

15.1 Introduction 233

15.2 Understanding Induction at the Gene Regulation Level 233

15.3 In silico Approaches 234

15.4 In vitro Approaches 235

15.5 In vitro Hepatocyte and Hepatocyte-Like Models 238

15.6 Experimental Techniques for the Assessment of Induction in Cell-Based Assays 239

15.7 Modeling and Simulation and Assessment of Risk 244

15.8 Analysis of Induction in Preclinical Species 245

15.9 Additional Considerations 245

15.10 Conclusion 246

References 246

16 Animal Models for Studying Drug Metabolizing Enzymes and Transporters 253
Kevin L. Salyers and Yang Xu


16.1 Introduction 253

16.2 Animal Models of DMEs 253

16.3 Animal Models of Drug Transporters 263

16.4 Conclusions and the Path Forward 270

Acknowledgments 271

References 271

17 Milk Excretion and Placental Transfer Studies 277
Matthew Hoffmann and Adam Shilling

17.1 Introduction 277

17.2 Compound Characteristics That Affect Placental Transfer and Lacteal Excretion 277

17.3 Study Design 281

17.4 Conclusions 289

References 289

18 Human Bile Collection for ADME Studies 291
Suresh K. Balani, Lisa J. Christopher, and Donglu Zhang

18.1 Introduction 291

18.2 Physiology 291

18.3 Utility of the Biliary Data 292

18.4 Bile Collection Techniques 293

18.5 Future Scope 297

Acknowledgment 297

References 297

PART C ANALYTICAL TECHNOLOGIES 299

19 Current Technology and Limitation of LC-MS 301
Cornelis E.C.A. Hop

19.1 Introduction 301

19.2 Sample Preparation 302

19.3 Chromatography Separation 302

19.4 Mass Spectrometric Analysis 304

19.5 Ionization 304

19.6 MS Mode versus MS/MS or MSn Mode 305

19.7 Mass Spectrometers: Single and Triple Quadrupole Mass Spectrometers 306

19.8 Mass Spectrometers: Three-Dimensional and Linear Ion Traps 308

19.9 Mass Spectrometers: Time-of-Flight Mass Spectrometers 308

19.10 Mass Spectrometers: Fourier Transform and Orbitrap Mass Spectrometers 309

19.11 Role of LC-MS in Quantitative in vitro ADME Studies 309

19.12 Quantitative in vivo ADME Studies 311

19.13 Metabolite Identification 312

19.14 Tissue Imaging by MS 313

19.15 Conclusions and Future Directions 313

References 314

20 Application of Accurate Mass Spectrometry for Metabolite Identification 317
Zhoupeng Zhang and Kaushik Mitra

20.1 Introduction 317

20.2 High-Resolution/Accurate Mass Spectrometers 317

20.3 Postacquisition Data Processing 318

20.4 Utilities of High-Resolution/Accurate Mass Spectrometry (HRMS) in Metabolite Identification 320

20.5 Conclusion 328

References 329

21 Applications of Accelerator Mass Spectrometry (AMS) 331
Xiaomin Wang, Voon Ong, and Mark Seymour

21.1 Introduction 331

21.2 Bioanalytical Methodology 332

References 337

22 Radioactivity Profiling 339
Wing Wah Lam, Jose Silva, and Heng-Keang Lim

22.1 Introduction 339

22.2 Radioactivity Detection Methods 340

22.3 AMS 346

22.4 Intracavity Optogalvanic Spectroscopy 349

22.5 Summary 349

Acknowledgments 349

References 349

23 A Robust Methodology for Rapid Structure Determination of Microgram-Level Drug Metabolites by NMR Spectroscopy 353
Kim A. Johnson, Stella Huang, and Yue-Zhong Shu

23.1 Introduction 353

23.2 Methods 354

23.3 Trazodone and Its Metabolism 355

23.4 Trazodone Metabolite Generation and NMR Sample Preparation 356

23.5 Metabolite Characterization 356

23.6 Comparison with Flow Probe and LC-NMR Methods 361

23.7 Metabolite Quantification by NMR 361

23.8 Conclusion 361

References 362

24 Supercritical Fluid Chromatography 363
Jun Dai, Yingru Zhang, David B. Wang-Iverson, and Adrienne A. Tymiak

24.1 Introduction 363

24.2 Background 363

24.3 SFC Instrumentation and General Considerations 364

24.4 SFC in Drug Discovery and Development 369

24.5 Future Perspective 375

References 376

25 Chromatographic Separation Methods 381
Wenying Jian, Richard W. Edom, Zhongping (John) Lin, and Naidong Weng

25.1 Introduction 381

25.2 LC Separation Techniques 383

25.3 Sample Preparation Techniques 388

25.4 High-Speed LC-MS Analysis 390

25.5 Orthogonal Separation 394

25.6 Conclusions and Perspectives 395

References 396

26 Mass Spectrometric Imaging for Drug Distribution in Tissues 401
Daniel P. Magparangalan, Timothy J. Garrett, Dieter M. Drexler, and Richard A. Yost

26.1 Introduction 401

26.2 MSI Instrumentation 403

26.3 MSI Workfl ow 406

26.4 Applications of MSI for in situ ADMET Tissue Studies 408

26.5 Conclusions 413

References 414

27 Applications of Quantitative Whole-Body Autoradiography (QWBA) in Drug Discovery and Development 419
Lifei Wang, Haizheng Hong, and Donglu Zhang

27.1 Introduction 419

27.2 Equipment and Materials 419

27.3 Study Designs 420

27.4 QWBA Experimental Procedures 420

27.5 Applications of QWBA 421

27.6 Limitations of QWBA 432

References 433

PART D NEW AND RELATED TECHNOLOGIES 435

28 Genetically Modified Mouse Models in ADME Studies 437
Xi-Ling Jiang and Ai-Ming Yu

28.1 Introduction 437

28.2 Drug Metabolizing Enzyme Genetically Modified Mouse Models 438

28.3 Drug Transporter Genetically Modifi ed Mouse Models 442

28.4 Xenobiotic Receptor Genetically Modified Mouse Models 446

28.5 Conclusions 448

References 448

29 Pluripotent Stem Cell Models in Human Drug Development 455
David C. Hay

29.1 Introduction 455

29.2 Human Drug Metabolism and Compound Attrition 455

29.3 Human Hepatocyte Supply 456

29.4 hESCS 456

29.5 hESC HLC Differentiation 456

29.6 iPSCS 456

29.7 CYP P450 Expression in Stem Cell-Derived HLCs 457

29.8 Tissue Culture Microenvironment 457

29.9 Culture Defi nition for Deriving HLCS from Stem Cells 457

29.10 Conclusion 457

References 458

30 Radiosynthesis for ADME Studies 461
Brad D. Maxwell and Charles S. Elmore

30.1 Background and General Requirements 461

30.2 Radiosynthesis Strategies and Goals 463

30.3 Preparation and Synthesis 467

30.4 Analysis and Product Release 469

30.5 Documentation 471

30.6 Summary 471

References 471

31 Formulation Development for Preclinical in vivo Studies 473
Yuan-Hon Kiang, Darren L. Reid, and Janan Jona

31.1 Introduction 473

31.2 Formulation Consideration for the Intravenous Route 473

31.3 Formulation Consideration for the Oral, Subcutaneous, and Intraperitoneal Routes 474

31.4 Special Consideration for the Intraperitoneal Route 475

31.5 Solubility Enhancement 475

31.6 pH Manipulation 476

31.7 Cosolvents Utilization 477

31.8 Complexation 479

31.9 Amorphous Form Approach 479

31.10 Improving the Dissolution Rate 479

31.11 Formulation for Toxicology Studies 479

31.12 Timing and Assessment of Physicochemical Properties 480

31.13 Critical Issues with Solubility and Stability 481

31.14 General and Quick Approach for Formulation Identification at the Early Discovery Stages 482

References 482

32 In vitro Testing of Proarrhythmic Toxicity 485
Haoyu Zeng and Jiesheng Kang

32.1 Objectives, Rationale, and Regulatory Compliance 485

32.2 Study System and Design 486

32.3 Good Laboratory Practice (GLP)-hERG Study 489

32.4 Medium-Throughput Assays Using PatchXpress as a Case Study 490

32.5 Nonfunctional and Functional Assays for hERG Traffi cking 491

32.6 Conclusions and the Path Forward 491

References 492

33 Target Engagement for PK/PD Modeling and Translational Imaging Biomarkers 493
Vanessa N. Barth, Elizabeth M. Joshi, and Matthew D. Silva

33.1 Introduction 493

33.2 Application of LC-MS/MS to Assess Target Engagement 494

33.3 LC-MS/MS-Based RO Study Designs and Their Calculations 494

33.4 Leveraging Target Engagement Data for Drug Discovery from an Absorption, Distribution, Metabolism, and Excretion (ADME) Perspective 497

33.5 Application of LC-MS/MS to Discovery Novel Tracers 502

33.6 Noninvasive Translational Imaging 503

33.7 Conclusions and the Path Forward 507

References 508

34 Applications of iRNA Technologies in Drug Transporters and Drug Metabolizing Enzymes 513
Mingxiang Liao and Cindy Q. Xia

34.1 Introduction 513

34.2 Experimental Designs 514

34.3 Applications of RNAi in Drug Metabolizing Enzymes and Transporters 527

34.4 Conclusions 538

Acknowledgment 539

References 539

Appendix Drug Metabolizing Enzymes and Biotransformation Reactions 545
Natalia Penner, Caroline Woodward, and Chandra Prakash

A.1 Introduction 545

A.2 Oxidative Enzymes 547

A.3 Reductive Enzymes 550

A.4 Hydrolytic Enzymes 551

A.5 Conjugative (Phase II) DMEs 553

A.6 Factors Affecting DME Activities 555

A.7 Biotransformation Reactions 557

A.8 Summary 561

Acknowledgment 562

References 562

Index 567