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ADME-Enabling Technologies in Drug Design and Development

 

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Word Power Books

ADME-Enabling Technologies in Drug Design and Development


Sekhar Surapaneni (Editor)
Donglu Zhang (Editor)

 

Hardback

ISBN: 9780470542781

 

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This book comprehensively covers the state-of-the-art and cutting-edge technologies in an integrated fashion for applications in ADME studies of small molecular drugs. Each chapter provides descriptions of the technologies, application scope and limitations, optimal conditions for intended results, protocols, case studies, and future developments.


This book comprehensively covers the state-of-the-art and cutting-edge technologies in an integrated fashion for applications in ADME studies of small molecular drugs. Each chapter provides descriptions of the technologies, application scope and limitations, optimal conditions for intended results, protocols, case studies, and future developments. By concisely describing these technologies, the book provides a useful tool for drug metabolism scientists and as a reference for scientists in the fields of pharmacology, medicinal chemistry, pharmaceutics, toxicology, bioanalytical science in academia and industry.


 

ISBN 470542780
ISBN13 9780470542781
Publisher Wiley-Blackwell (an imprint of John Wiley & Sons Ltd)
Format Hardback
Publication date 25/05/2012
Pages 622
Weight (grammes) 1616
Published in United Kingdom
Height (mm) 284
Width (mm) 218

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 alpha (OSTalpha) 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 alpha (PPARalpha/NR1C1) 447
28.4.5 Retinoid X Receptor alpha (RXRalpha/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