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ADME-Enabling Technologies in Drug Design and Development

 

You are here: Sciences > Chemistry > Analytical Chemistry > Qualitative Analytical Ch... > Chemical Spectroscopy, Sp... 

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) 1668
Published in United Kingdom
Height (mm) 285
Width (mm) 226

<
p>
FOREWORD xxi Lisa A. Shipley <
p>
PREFACE xxv Donglu Zhang and Sekhar Surapaneni <
p>
CONTRIBUTORS xxvii <
p>
PART A ADME: OVERVIEW AND CURRENT TOPICS 1 <
p>
1 Regulatory Drug Disposition and NDA Package Including MIST 3 Sekhar Surapaneni <
p>
1.1 Introduction 3 <
p>
1.2 Nonclinical Overview 5 <
p>
1.3 PK 5 <
p>
1.4 Absorption 5 <
p>
1.5 Distribution 6 <
p>
1.5.1 Plasma Protein Binding 6 <
p>
1.5.2 Tissue Distribution 6 <
p>
1.5.3 Lacteal and Placental Distribution Studies 7 <
p>
1.6 Metabolism 7 <
p>
1.6.1 In vitro Metabolism Studies 7 <
p>
1.6.2 Drug
Drug Interaction Studies 8 <
p>
1.6.3 In vivo Metabolism (ADME) Studies 10 <
p>
1.7 Excretion 11 <
p>
1.8 Impact of Metabolism Information on Labeling 11 <
p>
1.9 Conclusions 12 <
p>
References 12 <
p>
2 Optimal ADME Properties for Clinical Candidate and Investigational New Drug (IND) Package 15 Rajinder Bhardwaj and Gamini Chandrasena <
p>
2.1 Introduction 15 <
p>
2.2 NCE and Investigational New Drug (IND) Package 16 <
p>
2.3 ADME Optimization 17 <
p>
2.3.1 Absorption 18 <
p>
2.3.2 Metabolism 20 <
p>
2.3.3 PK 22 <
p>
2.4 ADME Optimization for CNS Drugs 23 <
p>
2.5 Summary 24 <
p>
References 25 <
p>
3 Drug Transporters in Drug Interactions and Disposition 29 Imad Hanna and Ryan M. Pelis <
p>
3.1 Introduction 29 <
p>
3.2 ABC Transporters 31 <
p>
3.2.1 Pgp (MDR1, ABCB1) 31 <
p>
3.2.2 BCRP (ABCG2) 32 <
p>
3.2.3 MRP2 (ABCC2) 32 <
p>
3.3 SLC Transporters 33 <
p>
3.3.1 OCT1 (SLC22A1) and OCT2 (SLC22A2) 34 <
p>
3.3.2 MATE1 (SLC47A1) and MATE2K (SLC47A2) 35 <
p>
3.3.3 OAT1 (SLC22A6) and OAT3 (SLC22A8) 36 <
p>
3.3.4 OATP1B1 (SLCO1B1, SLC21A6), OATP1B3 (SLCO1B3, SLC21A8), and OATP2B1 (SLCO2B1, SLC21A9) 37 <
p>
3.4 In vitro Assays in Drug Development 39 <
p>
3.4.1 Considerations for Assessing Candidate Drugs as Inhibitors 39 <
p>
3.4.2 Considerations for Assessing Candidate Drugs as Substrates 39 <
p>
3.4.3 Assay Systems 40 <
p>
3.5 Conclusions and Perspectives 45 <
p>
References 46 <
p>
4 Pharmacological and Toxicological Activity of Drug Metabolites 55 W. Griffith Humphreys <
p>
4.1 Introduction 55 <
p>
4.2 Assessment of Potential for Active Metabolites 56 <
p>
4.2.1 Detection of Active Metabolites during Drug Discovery 58 <
p>
4.2.2 Methods for Assessing and Evaluating the Biological Activity of Metabolite Mixtures 58 <
p>
4.2.3 Methods for Generation of Metabolites 59 <
p>
4.3 Assessment of the Potential Toxicology of Metabolites 59 <
p>
4.3.1 Methods to Study the Formation of Reactive Metabolites 60 <
p>
4.3.2 Reactive Metabolite Studies: In vitro 61 <
p>
4.3.3 Reactive Metabolite Studies: In vivo 61 <
p>
4.3.4 Reactive Metabolite Data Interpretation 61 <
p>
4.3.5 Metabolite Contribution to Off-Target Toxicities 62 <
p>
4.4 Safety Testing of Drug Metabolites 62 <
p>
4.5 Summary 63 <
p>
References 63 <
p>
5 Improving the Pharmaceutical Properties of Biologics in Drug Discovery: Unique Challenges and Enabling Solutions 67 Jiwen Chen and Ashok Dongre <
p>
5.1 Introduction 67 <
p>
5.2 Pharmacokinetics 68 <
p>
5.3 Metabolism and Disposition 70 <
p>
5.4 Immunogenicity 71 <
p>
5.5 Toxicity and Preclinical Assessment 74 <
p>
5.6 Comparability 74 <
p>
5.7 Conclusions 75 <
p>
References 75 <
p>
6 Clinical Dose Estimation Using Pharmacokinetic/Pharmacodynamic Modeling and Simulation 79 Lingling Guan <
p>
6.1 Introduction 79 <
p>
6.2 Biomarkers in PK and PD 80 <
p>
6.2.1 PK 80 <
p>
6.2.2 PD 81 <
p>
6.2.3 Biomarkers 81 <
p>
6.3 Model-Based Clinical Drug Development 83 <
p>
6.3.1 Modeling 83 <
p>
6.3.2 Simulation 84 <
p>
6.3.3 Population Modeling 85 <
p>
6.3.4 Quantitative Pharmacology (QP) and Pharmacometrics 85 <
p>
6.4 First-in-Human Dose 86 <
p>
6.4.1 Drug Classification Systems as Tools for Development 86 <
p>
6.4.2 Interspecies and Allometric Scaling 87 <
p>
6.4.3 Animal Species, Plasma Protein Binding, and in vivo
in vitro Correlation 88 <
p>
6.5 Examples 89 <
p>
6.5.1 First-in-Human Dose 89 <
p>
6.5.2 Pediatric Dose 90 <
p>
6.6 Discussion and Conclusion 90 <
p>
References 93 <
p>
7 Pharmacogenomics and Individualized Medicine 95 Anthony Y.H. Lu and Qiang Ma <
p>
7.1 Introduction 95 <
p>
7.2 Individual Variability in Drug Therapy 95 <
p>
7.3 We Are All Human Variants 96 <
p>
7.4 Origins of Individual Variability in Drug Therapy 96 <
p>
7.5 Genetic Polymorphism of Drug Targets 97 <
p>
7.6 Genetic Polymorphism of Cytochrome P450s 98 <
p>
7.7 Genetic Polymorphism of Other Drug Metabolizing Enzymes 100 <
p>
7.8 Genetic Polymorphism of Transporters 100 <
p>
7.9 Pharmacogenomics and Drug Safety 101 <
p>
7.10 Warfarin Pharmacogenomics: A Model for Individualized Medicine 102 <
p>
7.11 Can Individualized Drug Therapy Be Achieved? 104 <
p>
7.12 Conclusions 104 <
p>
Disclaimer 105 <
p>
Contact Information 105 <
p>
References 105 <
p>
8 Overview of Drug Metabolism and Pharmacokinetics with Applications in Drug Discovery and Development in China 109 Chang-Xiao Liu <
p>
8.1 Introduction 109 <
p>
8.2 PK
PD Translation Research in New Drug Research and Development 109 <
p>
8.3 Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME/T) Studies in Drug Discovery and Early Stage of Development 110 <
p>
8.4 Drug Transporters in New Drug Research and Development 111 <
p>
8.5 Drug Metabolism and PK Studies for New Drug Research and Development 113 <
p>
8.5.1 Technical Guidelines for PK Studies in China 113 <
p>
8.5.2 Studies on New Molecular Entity (NME) Drugs 114 <
p>
8.5.3 PK Calculation Program 117 <
p>
8.6 Studies on the PK of Biotechnological Products 117 <
p>
8.7 Studies on the PK of TCMS 118 <
p>
8.7.1 The Challenge in PK Research of TCMs 118 <
p>
8.7.2 New Concept on PK Markers 120 <
p>
8.7.3 Identification of Nontarget Components from Herbal Preparations 122 <
p>
8.8 PK and Bioavailability of Nanomaterials 123 <
p>
8.8.1 Research and Development of Nanopharmaceuticals 123 <
p>
8.8.2 Biopharmaceutics and Therapeutic Potential of Engineered Nanomaterials 123 <
p>
8.8.3 Biodistribution and Biodegradation 123 <
p>
8.8.4 Doxorubicin Polyethylene Glycol-Phosphatidylethnolamine (PEG-PE) Nanoparticles 124 <
p>
8.8.5 Micelle-Encapsulated Alprostadil (M-Alp) 124 <
p>
8.8.6 Paclitaxel Magnetoliposomes 125 <
p>
References 125 <
p>
PART B ADME SYSTEMS AND METHODS 129 <
p>
9 Technical Challenges and Recent Advances of Implementing Comprehensive ADMET Tools in Drug Discovery 131 Jianling Wang and Leslie Bell <
p>
9.1 Introduction 131 <
p>
9.2

A

Is the First Physiological Barrier That a Drug Faces 131 <
p>
9.2.1 Solubility and Dissolution 131 <
p>
9.2.2 GI Permeability and Transporters 136 <
p>
9.3

M

Is Frequently Considered Prior to Distribution Due to the

First-Pass

Effect 139 <
p>
9.3.1 Hepatic Metabolism 139 <
p>
9.3.2 CYPs and Drug Metabolism 140 <
p>
9.4

D

Is Critical for Correctly Interpreting PK Data 142 <
p>
9.4.1 Blood/Plasma Impact on Drug Distribution 142 <
p>
9.4.2 Plasma Stability 143 <
p>
9.4.3 PPB 144 <
p>
9.4.4 Blood/Plasma Partitioning 144 <
p>
9.5

E
: The Elimination of Drugs Should Not Be Ignored 145 <
p>
9.6 Metabolism- or Transporter-Related Safety Concerns 146 <
p>
9.7 Reversible CYP Inhibition 147 <
p>
9.7.1 In vitro CYP Inhibition 147 <
p>
9.7.2 Human Liver Microsomes (HLM) + Prototypical Probe Substrates with Quantification by LC-MS 147 <
p>
9.7.3 Implementation Strategy 149 <
p>
9.8 Mechanism-Based (Time-Dependent) CYP Inhibition 149 <
p>
9.8.1 Characteristics of CYP3A TDI 150 <
p>
9.8.2 In vitro Screening for CYP3A TDI 150 <
p>
9.8.3 Inactivation Rate (kobs) 150 <
p>
9.8.4 IC50-Shift 151 <
p>
9.8.5 Implementation Strategy 152 <
p>
9.9 CYP Induction 152 <
p>
9.10 Reactive Metabolites 153 <
p>
9.10.1 Qualitative in vitro Assays 153 <
p>
9.10.2 Quantitative in vitro Assay 154 <
p>
9.11 Conclusion and Outlook 154 <
p>
Acknowledgments 155 <
p>
References 155 <
p>
10 Permeability and Transporter Models in Drug Discovery and Development 161 Praveen V. Balimane, Yong-Hae Han, and Saeho Chong <
p>
10.1 Introduction 161 <
p>
10.2 Permeability Models 162 <
p>
10.2.1 PAMPA 162 <
p>
10.2.2 Cell Models (Caco-2 Cells) 162 <
p>
10.2.3 P-glycoprotein (Pgp) Models 162 <
p>
10.3 Transporter Models 163 <
p>
10.3.1 Intact Cells 164 <
p>
10.3.2 Transfected Cells 165 <
p>
10.3.3 Xenopus Oocyte 165 <
p>
10.3.4 Membrane Vesicles 165 <
p>
10.3.5 Transgenic Animal Models 166 <
p>
10.4 Integrated Permeability
Transporter Screening Strategy 166 <
p>
References 167 <
p>
11 Methods for Assessing Blood
Brain Barrier Penetration in Drug Discovery 169 Li Di and Edward H. Kerns <
p>
11.1 Introduction 169 <
p>
11.2 Common Methods for Assessing BBB Penetration 170 <
p>
11.3 Methods for Determination of Free Drug Concentration in the Brain 170 <
p>
11.3.1 In vivo Brain PK in Combination with in vitro Brain Homogenate Binding Studies 171 <
p>
11.3.2 Use of CSF Drug Concentration as a Surrogate for Free Drug Concentration in the Brain 171 <
p>
11.4 Methods for BBB Permeability 172 <
p>
11.4.1 In situ Brain Perfusion Assay 172 <
p>
11.4.2 High-throughput PAMPA-BBB 173 <
p>
11.4.3 Lipophilicity (LogD7.4) 173 <
p>
11.5 Methods for Pgp Efflux Transport 173 <
p>
11.6 Conclusions 174 <
p>
References 174 <
p>
12 Techniques for Determining Protein Binding in Drug Discovery and Development 177 Tom Lloyd <
p>
12.1 Introduction 177 <
p>
12.2 Overview 178 <
p>
12.3 Equilibrium Dialysis 179 <
p>
12.4 Ultracentrifugation 180 <
p>
12.5 Ultrafiltration 181 <
p>
12.6 Microdialysis 182 <
p>
12.7 Spectroscopy 182 <
p>
12.8 Chromatographic Methods 183 <
p>
12.9 Summary Discussion 183 <
p>
Acknowledgment 185 <
p>
References 185 <
p>
13 Reaction Phenotyping 189 Chun Li and Nataraj Kalyanaraman <
p>
13.1 Introduction 189 <
p>
13.2 Initial Considerations 190 <
p>
13.2.1 Clearance Mechanism 190 <
p>
13.2.2 Selecting the Appropriate in vitro System 191 <
p>
13.2.3 Substrate Concentration 191 <
p>
13.2.4 Effect of Incubation Time and Protein Concentration 192 <
p>
13.2.5 Determination of Kinetic Constant Km and Vmax 192 <
p>
13.2.6 Development of Analytical Methods 192 <
p>
13.3 CYP Reaction Phenotyping 193 <
p>
13.3.1 Specifi c Chemical Inhibitors 194 <
p>
13.3.2 Inhibitory CYP Antibodies 195 <
p>
13.3.3 Recombinant CYP Enzymes 196 <
p>
13.3.4 Correlation Analysis for CYP Reaction Phenotyping 198 <
p>
13.3.5 CYP Reaction Phenotyping in Drug Discovery versus Development 198 <
p>
13.4 Non-P450 Reaction Phenotyping 199 <
p>
13.4.1 FMOs 199 <
p>
13.4.2 MAOs 200 <
p>
13.4.3 AO 200 <
p>
13.5 UGT Conjugation Reaction Phenotyping 201 <
p>
13.5.1 Initial Considerations in UGT Reaction Phenotyping 202 <
p>
13.5.2 Experimental Approaches for UGT Reaction Phenotyping 202 <
p>
13.5.3 Use of Chemical Inhibitors for UGTs 203 <
p>
13.5.4 Correlation Analysis for UGT Reaction Phenotyping 204 <
p>
13.6 Reaction Phenotyping for Other Conjugation Reactions 204 <
p>
13.7 Integration of Reaction Phenotyping and Prediction of DDI 205 <
p>
13.8 Conclusion 205 <
p>
References 206 <
p>
14 Fast and Reliable CYP Inhibition Assays 213 Ming Yao, Hong Cai, and Mingshe Zhu <
p>
14.1 Introduction 213 <
p>
14.2 CYP Inhibition Assays in Drug Discovery and Development 215 <
p>
14.3 HLM Reversible CYP Inhibition Assay Using Individual Substrates 217 <
p>
14.3.1 Choice of Substrate and Specific Inhibitors 217 <
p>
14.3.2 Optimization of Incubation Conditions 217 <
p>
14.3.3 Incubation Procedures 217 <
p>
14.3.4 LC-MS/MS Analysis 221 <
p>
14.3.5 Data Calculation 221 <
p>
14.4 HLM RI Assay Using Multiple Substrates (Cocktail Assays) 222 <
p>
14.4.1 Choice of Substrate and Specific Inhibitors 222 <
p>
14.4.2 Optimization of Incubations 223 <
p>
14.4.3 Incubation Procedures 223 <
p>
14.4.4 LC-MS/MS Analysis 224 <
p>
14.4.5 Data Calculation 224 <
p>
14.5 Time-Dependent CYP Inhibition Assay 226 <
p>
14.5.1 IC50 Shift Assay 226 <
p>
14.5.2 KI and Kinact Measurements 227 <
p>
14.5.3 Data Calculation 228 <
p>
14.6 Summary and Future Directions 228 <
p>
References 230 <
p>
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 <
p>
15.1 Introduction 233 <
p>
15.2 Understanding Induction at the Gene Regulation Level 233 <
p>
15.3 In silico Approaches 234 <
p>
15.3.1 Model-Based Drug Design 234 <
p>
15.3.2 Computational Models 234 <
p>
15.4 In vitro Approaches 235 <
p>
15.4.1 Ligand Binding Assays 235 <
p>
15.4.2 Reporter Gene Assays 236 <
p>
15.5 In vitro Hepatocyte and Hepatocyte-Like Models 238 <
p>
15.5.1 Hepatocyte Cell-Based Assays 238 <
p>
15.5.2 Hepatocyte-Like Cell-Based Assays 239 <
p>
15.6 Experimental Techniques for the Assessment of Induction in Cell-Based Assays 239 <
p>
15.6.1 mRNA Quantification 240 <
p>
15.6.2 Protein Quantification 241 <
p>
15.6.3 Assessment of Enzyme Activity 244 <
p>
15.7 Modeling and Simulation and Assessment of Risk 244 <
p>
15.8 Analysis of Induction in Preclinical Species 245 <
p>
15.9 Additional Considerations 245 <
p>
15.10 Conclusion 246 <
p>
References 246 <
p>
16 Animal Models for Studying Drug Metabolizing Enzymes and Transporters 253 Kevin L. Salyers and Yang Xu
<
p>
16.1 Introduction 253 <
p>
16.2 Animal Models of DMEs 253 <
p>
16.2.1 Section Objectives 253 <
p>
16.2.2 In vivo Models to Study the Roles of DMEs in Determining Oral Bioavailability 254 <
p>
16.2.3 In vivo Models to Predict Human Drug Metabolism and Toxicity 257 <
p>
16.2.4 In vivo Models to Study the Regulation of DMEs 259 <
p>
16.2.5 In vivo Models to Predict Induction-Based DDIs in Humans 260 <
p>
16.2.6 In vivo Models to Predict Inhibition-Based DDIs in Humans 261 <
p>
16.2.7 In vivo Models to Study the Function of DMEs in Physiological Homeostasis and Human Diseases 262 <
p>
16.2.8 Summary 263 <
p>
16.3 Animal Models of Drug Transporters 263 <
p>
16.3.1 Section Objectives 263 <
p>
16.3.2 In vivo Models to Characterize Transporters in Drug Absorption 264 <
p>
16.3.3 In vivo Models Used to Study Transporters in Brain Penetration 266 <
p>
16.3.4 In vivo Models to Assess Hepatic and Renal Transporters 268 <
p>
16.3.5 Summary 270 <
p>
16.4 Conclusions and the Path Forward 270 <
p>
Acknowledgments 271 <
p>
References 271 <
p>
17 Milk Excretion and Placental Transfer Studies 277 Matthew Hoffmann and Adam Shilling <
p>
17.1 Introduction 277 <
p>
17.2 Compound Characteristics That Affect Placental Transfer and Lacteal Excretion 277 <
p>
17.2.1 Passive Diffusion 278 <
p>
17.2.2 Drug Transporters 279 <
p>
17.2.3 Metabolism 280 <
p>
17.3 Study Design 281 <
p>
17.3.1 Placental Transfer Studies 281 <
p>
17.3.2 Lacteal Excretion Studies 285 <
p>
17.4 Conclusions 289 <
p>
References 289 <
p>
18 Human Bile Collection for ADME Studies 291 Suresh K. Balani, Lisa J. Christopher, and Donglu Zhang <
p>
18.1 Introduction 291 <
p>
18.2 Physiology 291 <
p>
18.3 Utility of the Biliary Data 292 <
p>
18.4 Bile Collection Techniques 293 <
p>
18.4.1 Invasive Methods 293 <
p>
18.4.2 Noninvasive Methods 293 <
p>
18.5 Future Scope 297 <
p>
Acknowledgment 297 <
p>
References 297 <
p>
PART C ANALYTICAL TECHNOLOGIES 299 <
p>
19 Current Technology and Limitation of LC-MS 301 Cornelis E.C.A. Hop <
p>
19.1 Introduction 301 <
p>
19.2 Sample Preparation 302 <
p>
19.3 Chromatography Separation 302 <
p>
19.4 Mass Spectrometric Analysis 304 <
p>
19.5 Ionization 304 <
p>
19.6 MS Mode versus MS/MS or MSn Mode 305 <
p>
19.7 Mass Spectrometers: Single and Triple Quadrupole Mass Spectrometers 306 <
p>
19.8 Mass Spectrometers: Three-Dimensional and Linear Ion Traps 308 <
p>
19.9 Mass Spectrometers: Time-of-Flight Mass Spectrometers 308 <
p>
19.10 Mass Spectrometers: Fourier Transform and Orbitrap Mass Spectrometers 309 <
p>
19.11 Role of LC-MS in Quantitative in vitro ADME Studies 309 <
p>
19.12 Quantitative in vivo ADME Studies 311 <
p>
19.13 Metabolite Identification 312 <
p>
19.14 Tissue Imaging by MS 313 <
p>
19.15 Conclusions and Future Directions 313 <
p>
References 314 <
p>
20 Application of Accurate Mass Spectrometry for Metabolite Identification 317 Zhoupeng Zhang and Kaushik Mitra <
p>
20.1 Introduction 317 <
p>
20.2 High-Resolution/Accurate Mass Spectrometers 317 <
p>
20.2.1 Linear Trap Quadrupole-Orbitrap (LTQ-Orbitrap) Mass Spectrometer 318 <
p>
20.2.2 Q-tof and Triple Time-of-Flight (TOF) 318 <
p>
20.2.3 Hybrid Ion Trap Time-of-Flight Mass Spectrometer (IT-tof) 318 <
p>
20.3 Postacquisition Data Processing 318 <
p>
20.3.1 MDF 319 <
p>
20.3.2 Background Subtraction Software 319 <
p>
20.4 Utilities of High-Resolution/Accurate Mass Spectrometry (HRMS) in Metabolite Identification 320 <
p>
20.4.1 Fast Metabolite Identification of Metabolically Unstable Compounds 320 <
p>
20.4.2 Identification of Unusual Metabolites 322 <
p>
20.4.3 Identification of Trapped Adducts of Reactive Metabolites 325 <
p>
20.4.4 Analysis of Major Circulating Metabolites of Clinical Samples of Unlabeled Compounds 327 <
p>
20.4.5 Applications in Metabolomics 328 <
p>
20.5 Conclusion 328 <
p>
References 329 <
p>
21 Applications of Accelerator Mass Spectrometry (AMS) 331 Xiaomin Wang, Voon Ong, and Mark Seymour <
p>
21.1 Introduction 331 <
p>
21.2 Bioanalytical Methodology 332 <
p>
21.2.1 Sample Preparation 332 <
p>
21.2.2 AMS Instrumentation 332 <
p>
21.2.3 AMS Analysis 333 <
p>
21.3 AMS Applications in Mass Balance/Metabolite Profi ling 334 <
p>
21.4 AMS Applications in Pharmacokinetics 335 <
p>
21.5 Conclusion 337 <
p>
References 337 <
p>
22 Radioactivity Profiling 339 Wing Wah Lam, Jose Silva, and Heng-Keang Lim <
p>
22.1 Introduction 339 <
p>
22.2 Radioactivity Detection Methods 340 <
p>
22.2.1 Conventional Technologies 340 <
p>
22.2.2 Recent Technologies 341 <
p>
22.3 AMS 346 <
p>
22.4 Intracavity Optogalvanic Spectroscopy 349 <
p>
22.5 Summary 349 <
p>
Acknowledgments 349 <
p>
References 349 <
p>
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 <
p>
23.1 Introduction 353 <
p>
23.2 Methods 354 <
p>
23.2.1 Liver Microsome Incubations of Trazodone 354 <
p>
23.2.2 HPLC and Metabolite Purification 354 <
p>
23.2.3 HPLC-MS/MS 355 <
p>
23.2.4 NMR 355 <
p>
23.3 Trazodone and Its Metabolism 355 <
p>
23.4 Trazodone Metabolite Generation and NMR Sample Preparation 356 <
p>
23.5 Metabolite Characterization 356 <
p>
23.6 Comparison with Flow Probe and LC-NMR Methods 361 <
p>
23.7 Metabolite Quantification by NMR 361 <
p>
23.8 Conclusion 361 <
p>
References 362 <
p>
24 Supercritical Fluid Chromatography 363 Jun Dai, Yingru Zhang, David B. Wang-Iverson, and Adrienne A. Tymiak <
p>
24.1 Introduction 363 <
p>
24.2 Background 363 <
p>
24.3 SFC Instrumentation and General Considerations 364 <
p>
24.3.1 Detectors Used in SFC 365 <
p>
24.3.2 Mobile Phases Used in SFC 366 <
p>
24.3.3 Stationary Phases Used in SFC 367 <
p>
24.3.4 Comparison of SFC with Other Chromatographic Techniques 367 <
p>
24.3.5 Selectivity in SFC 368 <
p>
24.4 SFC in Drug Discovery and Development 369 <
p>
24.4.1 SFC Applications for Pharmaceuticals and Biomolecules 370 <
p>
24.4.2 SFC Chiral Separations 372 <
p>
24.4.3 SFC Applications for High-Throughput Analysis 374 <
p>
24.4.4 Preparative Separations 375 <
p>
24.5 Future Perspective 375 <
p>
References 376 <
p>
25 Chromatographic Separation Methods 381 Wenying Jian, Richard W. Edom, Zhongping (John) Lin, and Naidong Weng <
p>
25.1 Introduction 381 <
p>
25.1.1 A Historical Perspective 381 <
p>
25.1.2 The Need for Separation in ADME Studies 381 <
p>
25.1.3 Challenges for Current Chromatographic Techniques in Support of ADME Studies 382 <
p>
25.2 LC Separation Techniques 383 <
p>
25.2.1 Basic Practical Principles of LC Separation Relevant to ADME Studies 383 <
p>
25.2.2 Major Modes of LC Frequently Used for ADME Studies 385 <
p>
25.2.3 Chiral LC 387 <
p>
25.3 Sample Preparation Techniques 388 <
p>
25.3.1 Off-Line Sample Preparation 388 <
p>
25.3.2 Online Sample Preparation 389 <
p>
25.3.3 Dried Blood Spots (DBS) 390 <
p>
25.4 High-Speed LC-MS Analysis 390 <
p>
25.4.1 UHPLC 390 <
p>
25.4.2 Monolithic Columns 391 <
p>
25.4.3 Fused-Core Silica Columns 392 <
p>
25.4.4 Fast Separation Using HILIC 393 <
p>
25.5 Orthogonal Separation 394 <
p>
25.5.1 Orthogonal Sample Preparation and Chromatography 394 <
p>
25.5.2 2D-LC 395 <
p>
25.6 Conclusions and Perspectives 395 <
p>
References 396 <
p>
26 Mass Spectrometric Imaging for Drug Distribution in Tissues 401 Daniel P. Magparangalan, Timothy J. Garrett, Dieter M. Drexler, and Richard A. Yost <
p>
26.1 Introduction 401 <
p>
26.1.1 Imaging Techniques for ADMET Studies 401 <
p>
26.1.2 Mass Spectrometric Imaging (MSI) Background 401 <
p>
26.2 MSI Instrumentation 403 <
p>
26.2.1 Microprobe Ionization Sources 403 <
p>
26.2.2 Mass Analyzers 404 <
p>
26.3 MSI Workfl ow 406 <
p>
26.3.1 Postdissection Tissue/Organ Preparation and Storage 406 <
p>
26.3.2 Tissue Sectioning and Mounting 406 <
p>
26.3.3 Tissue Section Preparation, MALDI Matrix Selection, and Deposition 407 <
p>
26.3.4 Spatial Resolution: Relationship between Laser Spot Size and Raster Step Size 407 <
p>
26.4 Applications of MSI for in situ ADMET Tissue Studies 408 <
p>
26.4.1 Determination of Drug Distribution and Site of Action 408 <
p>
26.4.2 Analysis of Whole-Body Tissue Sections Utilizing MSI 409 <
p>
26.4.3 Increasing Analyte Specificity for Mass Spectrometric Images 411 <
p>
26.4.4 DESI Applications for MSI 412 <
p>
26.5 Conclusions 413 <
p>
References 414 <
p>
27 Applications of Quantitative Whole-Body Autoradiography (QWBA) in Drug Discovery and Development 419 Lifei Wang, Haizheng Hong, and Donglu Zhang <
p>
27.1 Introduction 419 <
p>
27.2 Equipment and Materials 419 <
p>
27.3 Study Designs 420 <
p>
27.3.1 Choice of Radiolabel 420 <
p>
27.3.2 Choice of Animals 420 <
p>
27.3.3 Dose Selection, Formulation, and Administration 420 <
p>
27.4 QWBA Experimental Procedures 420 <
p>
27.4.1 Embedding 420 <
p>
27.4.2 Whole-Body Sectioning 421 <
p>
27.4.3 Whole-Body Imaging 421 <
p>
27.4.4 Quantifi cation of Radioactivity Concentration 421 <
p>
27.5 Applications of QWBA 421 <
p>
27.5.1 Case Study 1: Drug Delivery to Pharmacology Targets 421 <
p>
27.5.2 Case Study 2: Tissue Distribution and Metabolite Profi ling 422 <
p>
27.5.3 Case Study 3: Tissue Distribution and Protein Covalent Binding 424 <
p>
27.5.4 Case Study 4: Rat Tissue Distribution and Human Dosimetry Calculation 425 <
p>
27.5.5 Case Study 5: Placenta Transfer and Tissue Distribution in Pregnant Rats 430 <
p>
27.6 Limitations of QWBA 432 <
p>
References 433 <
p>
PART D NEW AND RELATED TECHNOLOGIES 435 <
p>
28 Genetically Modified Mouse Models in ADME Studies 437 Xi-Ling Jiang and Ai-Ming Yu <
p>
28.1 Introduction 437 <
p>
28.2 Drug Metabolizing Enzyme Genetically Modified Mouse Models 438 <
p>
28.2.1 CYP1A1/CYP1A2 438 <
p>
28.2.2 CYP2A6/Cyp2a5 438 <
p>
28.2.3 CYP2C19 439 <
p>
28.2.4 CYP2D6 439 <
p>
28.2.5 CYP2E1 440 <
p>
28.2.6 CYP3A4 440 <
p>
28.2.7 Cytochrome P450 Reductase (CPR) 441 <
p>
28.2.8 Glutathione S-Transferase pi (GSTP) 441 <
p>
28.2.9 Sulfotransferase 1E1 (SULT1E1) 442 <
p>
28.2.10 Uridine 5
-Diphospho-Glucuronosyltransferase 1 (UGT1) 442 <
p>
28.3 Drug Transporter Genetically Modifi ed Mouse Models 442 <
p>
28.3.1 P-Glycoprotein (Pgp/MDR1/ABCB1) 442 <
p>
28.3.2 Multidrug Resistance-Associated Proteins (MRP/ABCC) 442 <
p>
28.3.3 Breast Cancer Resistance Protein (BCRP/ABCG2) 444 <
p>
28.3.4 Bile Salt Export Pump (BSEP/ABCB11) 444 <
p>
28.3.5 Peptide Transporter 2 (PEPT2/SLC15A2) 444 <
p>
28.3.6 Organic Cation Transporters (OCT/SLC22A) 445 <
p>
28.3.7 Multidrug and Toxin Extrusion 1 (MATE1/SLC47A1) 445 <
p>
28.3.8 Organic Anion Transporters (OAT/SLC22A) 445 <
p>
28.3.9 Organic Anion Transporting Polypeptides (OATP/SLCO) 445 <
p>
28.3.10 Organic Solute Transporter

(OST
) 446 <
p>
28.4 Xenobiotic Receptor Genetically Modified Mouse Models 446 <
p>
28.4.1 Aryl Hydrocarbon Receptor (AHR) 446 <
p>
28.4.2 Pregnane X Receptor (PXR/NR1I2) 446 <
p>
28.4.3 Constitutive Androstane Receptor (CAR/NR1I3) 446 <
p>
28.4.4 Peroxisome Proliferator-Activated Receptor

(PPAR
/NR1C1) 447 <
p>
28.4.5 Retinoid X Receptor

(RXR
/NR2B1) 447 <
p>
28.5 Conclusions 448 <
p>
References 448 <
p>
29 Pluripotent Stem Cell Models in Human Drug Development 455 David C. Hay <
p>
29.1 Introduction 455 <
p>
29.2 Human Drug Metabolism and Compound Attrition 455 <
p>
29.3 Human Hepatocyte Supply 456 <
p>
29.4 hESCS 456 <
p>
29.5 hESC HLC Differentiation 456 <
p>
29.6 iPSCS 456 <
p>
29.7 CYP P450 Expression in Stem Cell-Derived HLCs 457 <
p>
29.8 Tissue Culture Microenvironment 457 <
p>
29.9 Culture Defi nition for Deriving HLCS from Stem Cells 457 <
p>
29.10 Conclusion 457 <
p>
References 458 <
p>
30 Radiosynthesis for ADME Studies 461 Brad D. Maxwell and Charles S. Elmore <
p>
30.1 Background and General Requirements 461 <
p>
30.1.1 Food and Drug Administration (FDA) Guidance 461 <
p>
30.1.2 Third Clinical Study after Single Ascending Dose (SAD) and Multiple Ascending Dose (MAD) Studies 462 <
p>
30.1.3 Formation of the ADME Team 462 <
p>
30.1.4 Human Dosimetry Projection 462 <
p>
30.1.5 cGMP Synthesis Conditions 462 <
p>
30.1.6 Formation of One Covalent Bond 462 <
p>
30.2 Radiosynthesis Strategies and Goals 463 <
p>
30.2.1 Determination of the Most Suitable Radioisotope for the Human ADME Study 463 <
p>
30.2.2 Synthesize the API with the Radiolabel in the Most Metabolically Stable Position 463 <
p>
30.2.3 Incorporate the Radiolabel as Late in the Synthesis as Possible 465 <
p>
30.2.4 Use the Radiolabeled Reagent as the Limiting Reagent 465 <
p>
30.2.5 Consider Alternative Labeled Reagents and Strategies 466 <
p>
30.2.6 Develop One-Pot Reactions and Minimize the Number of Purifi cation Steps 467 <
p>
30.2.7 Safety Considerations 467 <
p>
30.3 Preparation and Synthesis 467 <
p>
30.3.1 Designated cGMP-Like Area 467 <
p>
30.3.2 Cleaning 467 <
p>
30.3.3 Glassware 468 <
p>
30.3.4 Equipment and Calibration of Analytical Instruments 468 <
p>
30.3.5 Reagents and Substrates 468 <
p>
30.3.6 Practice Reactions 468 <
p>
30.3.7 Actual Radiolabel Synthesis 468 <
p>
30.4 Analysis and Product Release 469 <
p>
30.4.1 Validated HPLC Analysis 469 <
p>
30.4.2 Orthogonal HPLC Method 469 <
p>
30.4.3 Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis 469 <
p>
30.4.4 Proton and Carbon-13 NMR 469 <
p>
30.4.5 Determination of the SA of the High Specific Activity API 469 <
p>
30.4.6 Mixing of the High Specifi c Activity API with Unlabeled Clinical-Grade API 470 <
p>
30.4.7 Determination of the SA of the Low Specific Activity API 470 <
p>
30.4.8 Other Potential Analyses 470 <
p>
30.4.9 Establishment of Use Date and Use Date Extensions 470 <
p>
30.4.10 Analysis and Release of the Radiolabeled Drug Product 471 <
p>
30.5 Documentation 471 <
p>
30.5.1 QA Oversight 471 <
p>
30.5.2 TSE and BSE Assessment 471 <
p>
30.6 Summary 471 <
p>
References 471 <
p>
31 Formulation Development for Preclinical in vivo Studies 473 Yuan-Hon Kiang, Darren L. Reid, and Janan Jona <
p>
31.1 Introduction 473 <
p>
31.2 Formulation Consideration for the Intravenous Route 473 <
p>
31.3 Formulation Consideration for the Oral, Subcutaneous, and Intraperitoneal Routes 474 <
p>
31.4 Special Consideration for the Intraperitoneal Route 475 <
p>
31.5 Solubility Enhancement 475 <
p>
31.6 pH Manipulation 476 <
p>
31.7 Cosolvents Utilization 477 <
p>
31.8 Complexation 479 <
p>
31.9 Amorphous Form Approach 479 <
p>
31.10 Improving the Dissolution Rate 479 <
p>
31.11 Formulation for Toxicology Studies 479 <
p>
31.12 Timing and Assessment of Physicochemical Properties 480 <
p>
31.13 Critical Issues with Solubility and Stability 481 <
p>
31.13.1 Solubility 481 <
p>
31.13.2 Chemical Stability Assessment 481 <
p>
31.13.3 Monitoring of the Physical and Chemical Stability 482 <
p>
31.14 General and Quick Approach for Formulation Identification at the Early Discovery Stages 482 <
p>
References 482 <
p>
32 In vitro Testing of Proarrhythmic Toxicity 485 Haoyu Zeng and Jiesheng Kang <
p>
32.1 Objectives, Rationale, and Regulatory Compliance 485 <
p>
32.2 Study System and Design 486 <
p>
32.2.1 The Gold Standard Manual Patch Clamp System 486 <
p>
32.2.2 Semiautomated System 487 <
p>
32.2.3 Automated System 487 <
p>
32.2.4 Comparison between Isolated Cardiomyocytes and Stably Transfected Cell Lines 489 <
p>
32.3 Good Laboratory Practice (GLP)-hERG Study 489 <
p>
32.4 Medium-Throughput Assays Using PatchXpress as a Case Study 490 <
p>
32.5 Nonfunctional and Functional Assays for hERG Traffi cking 491 <
p>
32.6 Conclusions and the Path Forward 491 <
p>
References 492 <
p>
33 Target Engagement for PK/PD Modeling and Translational Imaging Biomarkers 493 Vanessa N. Barth, Elizabeth M. Joshi, and Matthew D. Silva <
p>
33.1 Introduction 493 <
p>
33.2 Application of LC-MS/MS to Assess Target Engagement 494 <
p>
33.2.1 Advantages and Disadvantages of Technology and Study Designs 494 <
p>
33.3 LC-MS/MS-Based RO Study Designs and Their Calculations 494 <
p>
33.3.1 Sample Analysis 496 <
p>
33.3.2 Comparison and Validation versus Traditional Approaches 497 <
p>
33.4 Leveraging Target Engagement Data for Drug Discovery from an Absorption, Distribution, Metabolism, and Excretion (ADME) Perspective 497 <
p>
33.4.1 Drug Exposure Measurement 497 <
p>
33.4.2 Protein Binding and Unbound Concentrations 498 <
p>
33.4.3 Metabolism and Active Metabolites 500 <
p>
33.5 Application of LC-MS/MS to Discovery Novel Tracers 502 <
p>
33.5.1 Characterization of the Dopamine D2 PET Tracer Raclopride by LC-MS/MS 502 <
p>
33.5.2 Discovery of Novel Tracers 503 <
p>
33.6 Noninvasive Translational Imaging 503 <
p>
33.7 Conclusions and the Path Forward 507 <
p>
References 508 <
p>
34 Applications of iRNA Technologies in Drug Transporters and Drug Metabolizing Enzymes 513 Mingxiang Liao and Cindy Q. Xia <
p>
34.1 Introduction 513 <
p>
34.2 Experimental Designs 514 <
p>
34.2.1 siRNA Design 514 <
p>
34.2.2 Methods for siRNA Production 515 <
p>
34.2.3 Controls and Delivery Methods Selection 517 <
p>
34.2.4 Gene Silencing Effects Detection 520 <
p>
34.2.5 Challenges in siRNA 524 <
p>
34.3 Applications of RNAi in Drug Metabolizing Enzymes and Transporters 527 <
p>
34.3.1 Applications of Silencing Drug Transporters 527 <
p>
34.3.2 Applications of Silencing Drug Metabolizing Enzymes 534 <
p>
34.3.3 Applications of Silencing Nuclear Receptors (NRs) 534 <
p>
34.3.4 Applications in in vivo 535 <
p>
34.4 Conclusions 538 <
p>
Acknowledgment 539 <
p>
References 539 <
p>
Appendix Drug Metabolizing Enzymes and Biotransformation Reactions 545 Natalia Penner, Caroline Woodward, and Chandra Prakash <
p>
A.1 Introduction 545 <
p>
A.2 Oxidative Enzymes 547 <
p>
A.2.1 P450 547 <
p>
A.2.2 FMOs 548 <
p>
A.2.3 MAOs 549 <
p>
A.2.4 Molybdenum Hydroxylases (AO and XO) 549 <
p>
A.2.5 ADHs 550 <
p>
A.2.6 ALDHs 550 <
p>
A.3 Reductive Enzymes 550 <
p>
A.3.1 AKRs 550 <
p>
A.3.2 AZRs and NTRs 551 <
p>
A.3.3 QRs 551 <
p>
A.3.4 ADH, P450, and NADPH-P450 Reductase 551 <
p>
A.4 Hydrolytic Enzymes 551 <
p>
A.4.1 Epoxide Hydrolases (EHs) 551 <
p>
A.4.2 Esterases and Amidases 552 <
p>
A.5 Conjugative (Phase II) DMEs 553 <
p>
A.5.1 UGTs 553 <
p>
A.5.2 SULTs 553 <
p>
A.5.3 Methyltransferases (MTs) 553 <
p>
A.5.4 NATs 554 <
p>
A.5.5 GSTs 554 <
p>
A.5.6 Amino Acid Conjugation 555 <
p>
A.6 Factors Affecting DME Activities 555 <
p>
A.6.1 Species and Gender 556 <
p>
A.6.2 Polymorphism of DMEs 556 <
p>
A.6.3 Comedication and Diet 556 <
p>
A.7 Biotransformation Reactions 557 <
p>
A.7.1 Oxidation 557 <
p>
A.7.2 Reduction 560 <
p>
A.7.3 Conjugation Reactions 561 <
p>
A.8 Summary 561 <
p>
Acknowledgment 562 <
p>
References 562 <
p>
Index 567