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TECHNOLOGIES IN DRUG DESIGN AND DEVELOPMENT
EDITED BY
DONGLU ZHANG SEKHAR SURAPANENI
WILEY A JOHN WILEY & SONS, INC., PUBLICATION
CONTENTS
FOREWORD xxi Lisa A. Shipley
PREFACE xxv Donglu Zhang and Sekhar Surapaneni
CONTRIBUTORS xxvii
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 Chandrasena
2.1 Introduction 15 2.2 NCE and Investigational New Drug (IND) Package 16
vi CONTENTS
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 Hanna and Ryan M.
3.1 Introduction 29 3.2 ABC Transporters 31
3.2.1 Pgp(MDRl,ABCBl) 31 3.2.2 BCRP (ABCG2) 32 3.2.3 MRP2 (ABCC2) 32
3.3 SLC Transporters 33 3.3.1 (SLC22A1) and (SLC22A2) 34 3.3.2 MATE1 (SLC47A1) and MATE2K (SLC47A2) 35 3.3.3 (SLC22A6) and OAT3 (SLC22A8) 36 3.3.4 SLC21A6), (SLC01B3,
SLC21A8), and OATP2B1 (SLC02B1, 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 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 in Drug Discovery: Unique Challenges and Enabling Solutions 67
Chen and Ashok Dongre
5.1 Introduction 67 5.2 Pharmacokinetics 68 5.3 Metabolism and Disposition 70
CONTENTS
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 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 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 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
viii CONTENTS
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 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 the TCMs 118
8.7.1 The Challenge in Research 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
(PEG-PE) Nanoparticles 124
8.8.5 Micelle-Encapsulated Alprostadil 124 8.8.6 Paclitaxel Magnetoliposomes 125
References 125
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 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
CONTENTS
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 150 9.8.4 151 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 (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
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
In situ Brain Perfusion Assay 172 11.4.2 High-throughput PAMPA-BBB 173 11.4.3 Lipophilicity 173
11.5 Methods for Pgp Efflux Transport 173 11.6 Conclusions 174 References
x CONTENTS
12 Techniques for Determining Protein Binding in Drug Discovery and Development 177
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 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 and 192 13.2.6 Development of Analytical Methods 192
13.3 CYP Reaction Phenotyping 193 13.3.1 Specific 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 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 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
CONTENTS xi
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 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 Shift Assay 226 14.5.2 and 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 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
xii CONTENTS
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
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 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
ANALYTICAL TECHNOLOGIES 299
19 Current Technology and Limitation of LC-MS 301 Cornells Hop
19.1 Introduction 301
CONTENTS
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 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 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 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 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 Profiling 334
xiv CONTENTS
21.4 AMS Applications in Pharmacokinetics 335 21.5 Conclusion 337 References 337
22 Radioactivity Profiling 339 Wing Wah Lam, Jose and Heng-Keang
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 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, Zhang, David B. and Adrienne A.
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
CONTENTS xv
25 Chromatographic Separation Methods 381 Wenying Jian, Richard W. 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 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 Workflow 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, 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 Applications for MSI 412
26.5 Conclusions 413 References 414
xvi CONTENTS
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 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 Quantification 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
Profiling 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 Modified Mouse Models 442 28.3.1 442 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 2 444 28.3.6 Organic Cation Transporters 445
CONTENTS xvii
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 a 446
28.4 Xenobiotic Receptor Genetically Modified Mouse Models 446 28.4.1 Hydrocarbon Receptor (AHR) 446 28.4.2 Pregnane X Receptor 446 28.4.3 Constitutive Androstane Receptor 446 28.4.4 Peroxisome Proliferator-Activated Receptor a
447 28.4.5 Retinoid X Receptor a (RXRa/NR2Bl) 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 Definition 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
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 Purification 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
xviii CONTENTS
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 Spectrometry (LC-MS)
Analysis 469 30.4.4 Proton and NMR 469 30.4.5 Determination of the SA of the High Specific
Activity API 469 30.4.6 Mixing of the High Specific 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 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 Utilization 477 31.8 479 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
CONTENTS xix
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 Trafficking 491 32.6 Conclusions and the Path Forward 491 References 492
33 Target Engagement for Modeling and Translational Imaging Biomarkers 493 Vanessa N. Barth, Elizabeth M. Joshi, and Matthew D.
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 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
XX CONTENTS
34.4 Conclusions 538 Acknowledgment 539 References 539
Appendix Drug Metabolizing Enzymes and Biotransformation Reactions 545 Natalia Penner, Caroline Woodward, and Chandra Prakash
Introduction 545 A.2 Oxidative Enzymes 547
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 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 Epoxide Hydrolases (EHs) 551
A.4.2 Esterases and Amidases 552 A.5 Conjugative (Phase II) DMEs 553
UGTs 553 A.5.2 SULTs 553
Methyltransferases (MTs) 553 A.5.4 NATs 554 A.5.5 GSTs 554
Amino Acid Conjugation 555 A.6 Factors Affecting DME Activities 555
Species and Gender 556 A.6.2 Polymorphism of DMEs 556 A.6.3 Comedication and Diet 556
Biotransformation Reactions 557 Oxidation 557
A.7.2 Reduction 560 A.7.3 Conjugation Reactions 561
A.8 Summary 561 Acknowledgment 562 References 562
INDEX 567
