Title Details

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

1.5.1 Plasma Protein Binding 6

1.5.2 Tissue Distribution 6

1.5.3 Lacteal and Placental Distribution Studies 7

1.6 Metabolism 7

1.6.1 In vitro Metabolism Studies 7

1.6.2 Drug–Drug Interaction Studies 8

1.6.3 In vivo Metabolism (ADME) Studies 10

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

2.3.1 Absorption 18

2.3.2 Metabolism 20

2.3.3 PK 22

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

3.2.1 Pgp (MDR1, ABCB1) 31

3.2.2 BCRP (ABCG2) 32

3.2.3 MRP2 (ABCC2) 32

3.3 SLC Transporters 33

3.3.1 OCT1 (SLC22A1) and OCT2 (SLC22A2) 34

3.3.2 MATE1 (SLC47A1) and MATE2K (SLC47A2) 35

3.3.3 OAT1 (SLC22A6) and OAT3 (SLC22A8) 36

3.3.4 OATP1B1 (SLCO1B1, SLC21A6), OATP1B3 (SLCO1B3, SLC21A8), and OATP2B1 (SLCO2B1, SLC21A9) 37

3.4 In vitro Assays in Drug Development 39

3.4.1 Considerations for Assessing Candidate Drugs as Inhibitors 39

3.4.2 Considerations for Assessing Candidate Drugs as Substrates 39

3.4.3 Assay Systems 40

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

4.2.1 Detection of Active Metabolites during Drug Discovery 58

4.2.2 Methods for Assessing and Evaluating the Biological Activity of Metabolite Mixtures 58

4.2.3 Methods for Generation of Metabolites 59

4.3 Assessment of the Potential Toxicology of Metabolites 59

4.3.1 Methods to Study the Formation of Reactive Metabolites 60

4.3.2 Reactive Metabolite Studies: In vitro 61

4.3.3 Reactive Metabolite Studies: In vivo 61

4.3.4 Reactive Metabolite Data Interpretation 61

4.3.5 Metabolite Contribution to Off-Target Toxicities 62

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

6.2.1 PK 80

6.2.2 PD 81

6.2.3 Biomarkers 81

6.3 Model-Based Clinical Drug Development 83

6.3.1 Modeling 83

6.3.2 Simulation 84

6.3.3 Population Modeling 85

6.3.4 Quantitative Pharmacology (QP) and Pharmacometrics 85

6.4 First-in-Human Dose 86

6.4.1 Drug Classification Systems as Tools for Development 86

6.4.2 Interspecies and Allometric Scaling 87

6.4.3 Animal Species, Plasma Protein Binding, and in vivo–in vitro Correlation 88

6.5 Examples 89

6.5.1 First-in-Human Dose 89

6.5.2 Pediatric Dose 90

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

8.5.1 Technical Guidelines for PK Studies in China 113

8.5.2 Studies on New Molecular Entity (NME) Drugs 114

8.5.3 PK Calculation Program 117

8.6 Studies on the PK of Biotechnological Products 117

8.7 Studies on the PK of TCMS 118

8.7.1 The Challenge in PK Research of TCMs 118

8.7.2 New Concept on PK Markers 120

8.7.3 Identification of Nontarget Components from Herbal Preparations 122

8.8 PK and Bioavailability of Nanomaterials 123

8.8.1 Research and Development of Nanopharmaceuticals 123

8.8.2 Biopharmaceutics and Therapeutic Potential of Engineered Nanomaterials 123

8.8.3 Biodistribution and Biodegradation 123

8.8.4 Doxorubicin Polyethylene Glycol-Phosphatidylethnolamine (PEG-PE) Nanoparticles 124

8.8.5 Micelle-Encapsulated Alprostadil (M-Alp) 124

8.8.6 Paclitaxel Magnetoliposomes 125

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

9.2.1 Solubility and Dissolution 131

9.2.2 GI Permeability and Transporters 136

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

9.3.1 Hepatic Metabolism 139

9.3.2 CYPs and Drug Metabolism 140

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

9.4.1 Blood/Plasma Impact on Drug Distribution 142

9.4.2 Plasma Stability 143

9.4.3 PPB 144

9.4.4 Blood/Plasma Partitioning 144

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

9.7.1 In vitro CYP Inhibition 147

9.7.2 Human Liver Microsomes (HLM) + Prototypical Probe Substrates with Quantification by LC-MS 147

9.7.3 Implementation Strategy 149

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

9.8.1 Characteristics of CYP3A TDI 150

9.8.2 In vitro Screening for CYP3A TDI 150

9.8.3 Inactivation Rate (kobs) 150

9.8.4 IC50-Shift 151

9.8.5 Implementation Strategy 152

9.9 CYP Induction 152

9.10 Reactive Metabolites 153

9.10.1 Qualitative in vitro Assays 153

9.10.2 Quantitative in vitro Assay 154

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

10.2.1 PAMPA 162

10.2.2 Cell Models (Caco-2 Cells) 162

10.2.3 P-glycoprotein (Pgp) Models 162

10.3 Transporter Models 163

10.3.1 Intact Cells 164

10.3.2 Transfected Cells 165

10.3.3 Xenopus Oocyte 165

10.3.4 Membrane Vesicles 165

10.3.5 Transgenic Animal Models 166

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

11.3.1 In vivo Brain PK in Combination with in vitro Brain Homogenate Binding Studies 171

11.3.2 Use of CSF Drug Concentration as a Surrogate for Free Drug Concentration in the Brain 171

11.4 Methods for BBB Permeability 172

11.4.1 In situ Brain Perfusion Assay 172

11.4.2 High-throughput PAMPA-BBB 173

11.4.3 Lipophilicity (LogD7.4) 173

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

13.2.1 Clearance Mechanism 190

13.2.2 Selecting the Appropriate in vitro System 191

13.2.3 Substrate Concentration 191

13.2.4 Effect of Incubation Time and Protein Concentration 192

13.2.5 Determination of Kinetic Constant Km and Vmax 192

13.2.6 Development of Analytical Methods 192

13.3 CYP Reaction Phenotyping 193

13.3.1 Specifi c Chemical Inhibitors 194

13.3.2 Inhibitory CYP Antibodies 195

13.3.3 Recombinant CYP Enzymes 196

13.3.4 Correlation Analysis for CYP Reaction Phenotyping 198

13.3.5 CYP Reaction Phenotyping in Drug Discovery versus Development 198

13.4 Non-P450 Reaction Phenotyping 199

13.4.1 FMOs 199

13.4.2 MAOs 200

13.4.3 AO 200

13.5 UGT Conjugation Reaction Phenotyping 201

13.5.1 Initial Considerations in UGT Reaction Phenotyping 202

13.5.2 Experimental Approaches for UGT Reaction Phenotyping 202

13.5.3 Use of Chemical Inhibitors for UGTs 203

13.5.4 Correlation Analysis for UGT Reaction Phenotyping 204

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

14.3.1 Choice of Substrate and Specific Inhibitors 217

14.3.2 Optimization of Incubation Conditions 217

14.3.3 Incubation Procedures 217

14.3.4 LC-MS/MS Analysis 221

14.3.5 Data Calculation 221

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

14.4.1 Choice of Substrate and Specific Inhibitors 222

14.4.2 Optimization of Incubations 223

14.4.3 Incubation Procedures 223

14.4.4 LC-MS/MS Analysis 224

14.4.5 Data Calculation 224

14.5 Time-Dependent CYP Inhibition Assay 226

14.5.1 IC50 Shift Assay 226

14.5.2 KI and Kinact Measurements 227

14.5.3 Data Calculation 228

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

15.3.1 Model-Based Drug Design 234

15.3.2 Computational Models 234

15.4 In vitro Approaches 235

15.4.1 Ligand Binding Assays 235

15.4.2 Reporter Gene Assays 236

15.5 In vitro Hepatocyte and Hepatocyte-Like Models 238

15.5.1 Hepatocyte Cell-Based Assays 238

15.5.2 Hepatocyte-Like Cell-Based Assays 239

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

15.6.1 mRNA Quantification 240

15.6.2 Protein Quantification 241

15.6.3 Assessment of Enzyme Activity 244

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

16.2.1 Section Objectives 253

16.2.2 In vivo Models to Study the Roles of DMEs in Determining Oral Bioavailability 254

16.2.3 In vivo Models to Predict Human Drug Metabolism and Toxicity 257

16.2.4 In vivo Models to Study the Regulation of DMEs 259

16.2.5 In vivo Models to Predict Induction-Based DDIs in Humans 260

16.2.6 In vivo Models to Predict Inhibition-Based DDIs in Humans 261

16.2.7 In vivo Models to Study the Function of DMEs in Physiological Homeostasis and Human Diseases 262

16.2.8 Summary 263

16.3 Animal Models of Drug Transporters 263

16.3.1 Section Objectives 263

16.3.2 In vivo Models to Characterize Transporters in Drug Absorption 264

16.3.3 In vivo Models Used to Study Transporters in Brain Penetration 266

16.3.4 In vivo Models to Assess Hepatic and Renal Transporters 268

16.3.5 Summary 270

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

17.2.1 Passive Diffusion 278

17.2.2 Drug Transporters 279

17.2.3 Metabolism 280

17.3 Study Design 281

17.3.1 Placental Transfer Studies 281

17.3.2 Lacteal Excretion Studies 285

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

18.4.1 Invasive Methods 293

18.4.2 Noninvasive Methods 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

20.2.1 Linear Trap Quadrupole-Orbitrap (LTQ-Orbitrap) Mass Spectrometer 318

20.2.2 Q-tof and Triple Time-of-Flight (TOF) 318

20.2.3 Hybrid Ion Trap Time-of-Flight Mass Spectrometer (IT-tof) 318

20.3 Postacquisition Data Processing 318

20.3.1 MDF 319

20.3.2 Background Subtraction Software 319

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

20.4.1 Fast Metabolite Identification of Metabolically Unstable Compounds 320

20.4.2 Identification of Unusual Metabolites 322

20.4.3 Identification of Trapped Adducts of Reactive Metabolites 325

20.4.4 Analysis of Major Circulating Metabolites of Clinical Samples of Unlabeled Compounds 327

20.4.5 Applications in Metabolomics 328

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

21.2.1 Sample Preparation 332

21.2.2 AMS Instrumentation 332

21.2.3 AMS Analysis 333

21.3 AMS Applications in Mass Balance/Metabolite Profi ling 334

21.4 AMS Applications in Pharmacokinetics 335

21.5 Conclusion 337

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

22.2.1 Conventional Technologies 340

22.2.2 Recent Technologies 341

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

23.2.1 Liver Microsome Incubations of Trazodone 354

23.2.2 HPLC and Metabolite Purification 354

23.2.3 HPLC-MS/MS 355

23.2.4 NMR 355

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

24.3.1 Detectors Used in SFC 365

24.3.2 Mobile Phases Used in SFC 366

24.3.3 Stationary Phases Used in SFC 367

24.3.4 Comparison of SFC with Other Chromatographic Techniques 367

24.3.5 Selectivity in SFC 368

24.4 SFC in Drug Discovery and Development 369

24.4.1 SFC Applications for Pharmaceuticals and Biomolecules 370

24.4.2 SFC Chiral Separations 372

24.4.3 SFC Applications for High-Throughput Analysis 374

24.4.4 Preparative Separations 375

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

25.1.1 A Historical Perspective 381

25.1.2 The Need for Separation in ADME Studies 381

25.1.3 Challenges for Current Chromatographic Techniques in Support of ADME Studies 382

25.2 LC Separation Techniques 383

25.2.1 Basic Practical Principles of LC Separation Relevant to ADME Studies 383

25.2.2 Major Modes of LC Frequently Used for ADME Studies 385

25.2.3 Chiral LC 387

25.3 Sample Preparation Techniques 388

25.3.1 Off-Line Sample Preparation 388

25.3.2 Online Sample Preparation 389

25.3.3 Dried Blood Spots (DBS) 390

25.4 High-Speed LC-MS Analysis 390

25.4.1 UHPLC 390

25.4.2 Monolithic Columns 391

25.4.3 Fused-Core Silica Columns 392

25.4.4 Fast Separation Using HILIC 393

25.5 Orthogonal Separation 394

25.5.1 Orthogonal Sample Preparation and Chromatography 394

25.5.2 2D-LC 395

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

26.1.1 Imaging Techniques for ADMET Studies 401

26.1.2 Mass Spectrometric Imaging (MSI) Background 401

26.2 MSI Instrumentation 403

26.2.1 Microprobe Ionization Sources 403

26.2.2 Mass Analyzers 404

26.3 MSI Workfl ow 406

26.3.1 Postdissection Tissue/Organ Preparation and Storage 406

26.3.2 Tissue Sectioning and Mounting 406

26.3.3 Tissue Section Preparation, MALDI Matrix Selection, and Deposition 407

26.3.4 Spatial Resolution: Relationship between Laser Spot Size and Raster Step Size 407

26.4 Applications of MSI for in situ ADMET Tissue Studies 408

26.4.1 Determination of Drug Distribution and Site of Action 408

26.4.2 Analysis of Whole-Body Tissue Sections Utilizing MSI 409

26.4.3 Increasing Analyte Specificity for Mass Spectrometric Images 411

26.4.4 DESI Applications for MSI 412

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

27.3.1 Choice of Radiolabel 420

27.3.2 Choice of Animals 420

27.3.3 Dose Selection, Formulation, and Administration 420

27.4 QWBA Experimental Procedures 420

27.4.1 Embedding 420

27.4.2 Whole-Body Sectioning 421

27.4.3 Whole-Body Imaging 421

27.4.4 Quantifi cation of Radioactivity Concentration 421

27.5 Applications of QWBA 421

27.5.1 Case Study 1: Drug Delivery to Pharmacology Targets 421

27.5.2 Case Study 2: Tissue Distribution and Metabolite Profi ling 422

27.5.3 Case Study 3: Tissue Distribution and Protein Covalent Binding 424

27.5.4 Case Study 4: Rat Tissue Distribution and Human Dosimetry Calculation 425

27.5.5 Case Study 5: Placenta Transfer and Tissue Distribution in Pregnant Rats 430

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

28.2.1 CYP1A1/CYP1A2 438

28.2.2 CYP2A6/Cyp2a5 438

28.2.3 CYP2C19 439

28.2.4 CYP2D6 439

28.2.5 CYP2E1 440

28.2.6 CYP3A4 440

28.2.7 Cytochrome P450 Reductase (CPR) 441

28.2.8 Glutathione S-Transferase pi (GSTP) 441

28.2.9 Sulfotransferase 1E1 (SULT1E1) 442

28.2.10 Uridine 5′-Diphospho-Glucuronosyltransferase 1 (UGT1) 442

28.3 Drug Transporter Genetically Modifi ed Mouse Models 442

28.3.1 P-Glycoprotein (Pgp/MDR1/ABCB1) 442

28.3.2 Multidrug Resistance-Associated Proteins (MRP/ABCC) 442

28.3.3 Breast Cancer Resistance Protein (BCRP/ABCG2) 444

28.3.4 Bile Salt Export Pump (BSEP/ABCB11) 444

28.3.5 Peptide Transporter 2 (PEPT2/SLC15A2) 444

28.3.6 Organic Cation Transporters (OCT/SLC22A) 445

28.3.7 Multidrug and Toxin Extrusion 1 (MATE1/SLC47A1) 445

28.3.8 Organic Anion Transporters (OAT/SLC22A) 445

28.3.9 Organic Anion Transporting Polypeptides (OATP/SLCO) 445

28.3.10 Organic Solute Transporter α (OSTα) 446

28.4 Xenobiotic Receptor Genetically Modified Mouse Models 446

28.4.1 Aryl Hydrocarbon Receptor (AHR) 446

28.4.2 Pregnane X Receptor (PXR/NR1I2) 446

28.4.3 Constitutive Androstane Receptor (CAR/NR1I3) 446

28.4.4 Peroxisome Proliferator-Activated Receptor α (PPARα/NR1C1) 447

28.4.5 Retinoid X Receptor α (RXRα/NR2B1) 447

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

30.1.1 Food and Drug Administration (FDA) Guidance 461

30.1.2 Third Clinical Study after Single Ascending Dose (SAD) and Multiple Ascending Dose (MAD) Studies 462

30.1.3 Formation of the ADME Team 462

30.1.4 Human Dosimetry Projection 462

30.1.5 cGMP Synthesis Conditions 462

30.1.6 Formation of One Covalent Bond 462

30.2 Radiosynthesis Strategies and Goals 463

30.2.1 Determination of the Most Suitable Radioisotope for the Human ADME Study 463

30.2.2 Synthesize the API with the Radiolabel in the Most Metabolically Stable Position 463

30.2.3 Incorporate the Radiolabel as Late in the Synthesis as Possible 465

30.2.4 Use the Radiolabeled Reagent as the Limiting Reagent 465

30.2.5 Consider Alternative Labeled Reagents and Strategies 466

30.2.6 Develop One-Pot Reactions and Minimize the Number of Purifi cation Steps 467

30.2.7 Safety Considerations 467

30.3 Preparation and Synthesis 467

30.3.1 Designated cGMP-Like Area 467

30.3.2 Cleaning 467

30.3.3 Glassware 468

30.3.4 Equipment and Calibration of Analytical Instruments 468

30.3.5 Reagents and Substrates 468

30.3.6 Practice Reactions 468

30.3.7 Actual Radiolabel Synthesis 468

30.4 Analysis and Product Release 469

30.4.1 Validated HPLC Analysis 469

30.4.2 Orthogonal HPLC Method 469

30.4.3 Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis 469

30.4.4 Proton and Carbon-13 NMR 469

30.4.5 Determination of the SA of the High Specific Activity API 469

30.4.6 Mixing of the High Specifi c Activity API with Unlabeled Clinical-Grade API 470

30.4.7 Determination of the SA of the Low Specific Activity API 470

30.4.8 Other Potential Analyses 470

30.4.9 Establishment of Use Date and Use Date Extensions 470

30.4.10 Analysis and Release of the Radiolabeled Drug Product 471

30.5 Documentation 471

30.5.1 QA Oversight 471

30.5.2 TSE and BSE Assessment 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

31.13.1 Solubility 481

31.13.2 Chemical Stability Assessment 481

31.13.3 Monitoring of the Physical and Chemical Stability 482

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

32.2.1 The Gold Standard Manual Patch Clamp System 486

32.2.2 Semiautomated System 487

32.2.3 Automated System 487

32.2.4 Comparison between Isolated Cardiomyocytes and Stably Transfected Cell Lines 489

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

33.2.1 Advantages and Disadvantages of Technology and Study Designs 494

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

33.3.1 Sample Analysis 496

33.3.2 Comparison and Validation versus Traditional Approaches 497

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

33.4.1 Drug Exposure Measurement 497

33.4.2 Protein Binding and Unbound Concentrations 498

33.4.3 Metabolism and Active Metabolites 500

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

33.5.1 Characterization of the Dopamine D2 PET Tracer Raclopride by LC-MS/MS 502

33.5.2 Discovery of Novel Tracers 503

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

34.2.1 siRNA Design 514

34.2.2 Methods for siRNA Production 515

34.2.3 Controls and Delivery Methods Selection 517

34.2.4 Gene Silencing Effects Detection 520

34.2.5 Challenges in siRNA 524

34.3 Applications of RNAi in Drug Metabolizing Enzymes and Transporters 527

34.3.1 Applications of Silencing Drug Transporters 527

34.3.2 Applications of Silencing Drug Metabolizing Enzymes 534

34.3.3 Applications of Silencing Nuclear Receptors (NRs) 534

34.3.4 Applications in in vivo 535

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.2.1 P450 547

A.2.2 FMOs 548

A.2.3 MAOs 549

A.2.4 Molybdenum Hydroxylases (AO and XO) 549

A.2.5 ADHs 550

A.2.6 ALDHs 550

A.3 Reductive Enzymes 550

A.3.1 AKRs 550

A.3.2 AZRs and NTRs 551

A.3.3 QRs 551

A.3.4 ADH, P450, and NADPH-P450 Reductase 551

A.4 Hydrolytic Enzymes 551

A.4.1 Epoxide Hydrolases (EHs) 551

A.4.2 Esterases and Amidases 552

A.5 Conjugative (Phase II) DMEs 553

A.5.1 UGTs 553

A.5.2 SULTs 553

A.5.3 Methyltransferases (MTs) 553

A.5.4 NATs 554

A.5.5 GSTs 554

A.5.6 Amino Acid Conjugation 555

A.6 Factors Affecting DME Activities 555

A.6.1 Species and Gender 556

A.6.2 Polymorphism of DMEs 556

A.6.3 Comedication and Diet 556

A.7 Biotransformation Reactions 557

A.7.1 Oxidation 557

A.7.2 Reduction 560

A.7.3 Conjugation Reactions 561

A.8 Summary 561

Acknowledgment 562

References 562

Index 567

“This book fills time needs of ADME researchers and provides a fine reference book for scientists engaged in the areas of medicinal chemistry, pharmaceutics, bioanalytical sciences, pharmacology and toxicology in academia and pharmaceutical industry.”  (British Toxicology Society, 1 July 2013)