Drug Metabolism in Drug Design and Development Basic Concepts and Practice

  - 2.5.3 Monoamine Oxidase (MAO)

  - 2.5.4 Aldehyde Oxidase and Xanthine Dehydrogenase

  - 2.5.5 Peroxidases

  - 2.5.6 Alcohol Dehydrogenases (ADH)

  - 2.5.7 Aldehyde Dehydrogenases (ALDH)

• 2.6 Reduction
  
  
  • 2.6.1 P450, ADH
  
  
  • 2.6.2 NADPH-P450 Reductase
  
  
  • 2.6.3 Aldo-Keto Reductases (AKR)
  
  
  • 2.6.4 Quinone Reductase (NQO)
  
  
  • 2.6.5 Glutathione Peroxidase (GPX)
  
  
  
  


• 2.7 Hydrolysis
  
  
  • 2.7.1 Epoxide Hydrolase
  
  
  • 2.7.2 Esterases and Amidases
  
  
  
  


• 2.8 Summary
  
  
  • References
  
  
  
  


• 3 Conjugative Metabolism of Drugs
  
  
  • 3.1 UDP-Glucuronosyltransferases
    
    
    • 3.1.1 Location Within the Cell
    
    
    • 3.1.2 Endogenous Substrates
    
    
    • 3.1.3 Enzyme Multiplicity
    
    
    • 3.1.4 Inducibility
    
    
    • 3.1.5 Pharmacogenetics
    
    
    • 3.1.6 Experimental Considerations
    
    
    • 3.1.7 Enzyme Selective Substrates and Inhibitors
    
    
    • 3.1.8 Drug–Drug Interactions and Glucuronidation
    
    
    • 3.1.9 Summary
    
    
    
    
  
  
  • 3.2 Cytosolic Sulfotransferases
    
    
    • 3.2.1 Cellular Location and Tissue Expression
    
    
    • 3.2.2 The SULT Superfamily of Cytosolic Enzymes
    
    
    • 3.2.3 Inducibility
    
    
    • 3.2.4 SULT Pharmacogenetics
    
    
    • 3.2.5 Analytical Detection of Sulfonated Metabolites
    
    
    • 3.2.6 SULT Inhibitors (Pacifici and Coughtrie, 2005)
    
    
    • 3.2.7 Drug–Drug Interactions and Sulfonation
    
    
    • 3.2.8 Summary
    
    
    
    
  
  
  • 3.3 Glutathione-S-Transferases
    
    
    • 3.3.1 General Overview
    
    
    • 3.3.2 Classification of the GST Enzymes
    
    
    • 3.3.3 Localization and Expression
    
    
    • 3.3.4 Reactions Catalyzed by GSTs
    
    
    • 3.3.5 Regulation of GSTs
    
    
    • 3.3.6 GST Alpha Class
    
    
    • 3.3.7 GST Mu Class
    
    
    • 3.3.8 GST Pi Class
    
    
    • 3.3.9 GST Theta Class
    
    
    • 3.3.10 GST Zeta Class
    
    
    • 3.3.11 Incubation Conditions and Analytical Methods
      
      
      • Pathway) 3.3.12 Glutathione Conjugate Metabolism (Mercapturic Acid
      
      
      
      
    
    
    • References
    
    
    
    
  
  
  
  


• 4 Enzyme Kinetics
  
  
  • 4.1 Introduction Timothy S. Tracy
  
  
  • 4.2 Enzyme Catalysis
  
  
  • 4.3 Michaelis–Menten Kinetics
    
    
    • 4.3.1 Meanings ofKm,Vmaxand Their Clinical Relevance
    
    
    
    
  
  
  • 4.4 Graphical Kinetic Plots
  
  
  • 4.5 Atypical Kinetics–Allosteric Effects
    
    
    • 4.5.1 Overview of Atypical Kinetic Phenomena
    
    
    • 4.5.2 Homotropic Cooperativity
    
    
    • 4.5.3 Heterotropic Cooperativity
    
    
    
    
  
  
  • 4.6 Graphical Analysis of Atypical Kinetic Data
  
  
  • 4.7 Enzyme Inhibition Kinetics
    
    
    • 4.7.1 Overview
    
    
    • 4.7.2 Competitive Inhibition
    
    
    • 4.7.3 Mixed Inhibition
    
    
    • 4.7.4 Noncompetitive Inhibition
    
    
    • 4.7.5 Uncompetitive Inhibition
      
      
      • Kinetic Parameters 4.7.6 Summary of Effects of Various Inhibition Types of
      
      
      
      
    
    
    • 4.7.7 Meanings of IC 50 andKiParameters
    
    
    
    
  
  
  • 4.8 Inhibition Kinetics Graphical Plots
  
  
  • 4.9 Mechanism-Based Enzyme Inactivation Kinetics
    
    
    • Acknowledgment
    
    
    • References
    
    
    
    
  
  
  
  


• 5 Metabolism-Mediated Drug–Drug Interactions
  
  
  • 5.1 Introduction Hongjian Zhang, Michael W. Sinz, and A. David Rodrigues
  
  
  • 5.2 Enzyme Inhibition
    
    
    • 5.2.1 Types of Inhibition
    
    
    • 5.2.2 In vitroEvaluation of Inhibition
    
    
    • 5.2.3 Prediction of CYP Inhibition UsingIn vitroData
    
    
    • 5.2.4 Clinical Evaluation of Inhibition
    
    
    
    
  
  
  
  


• 5.3 Enzyme Induction
  
  
  • 5.3.1 Enzyme and Pharmacokinetic Changes
  
  
  • 5.3.2 Mechanisms of Enzyme Induction
  
  
  • 5.3.3 Induction Models
  
  
  
  


• 5.4 Reaction Phenotyping
  
  
  • 5.4.1 Experimental Considerations
  
  
  • 5.4.2 Data Interpretation and Integration
  
  
  • 5.4.3 Clinical Evaluation
  
  
  • References
  
  
  • and Drug Resistance 6 Drug Transporters in Drug Disposition, Drug Interactions,
  
  
  
  


• 6.1 Introduction Cindy Q. Xia, Johnny J. Yang, and Suresh K. Balani
  
  
  • and Toxicity 6.2 Roles of Transporters in Drug Disposition
  
  
  • 6.2.1 Transporters in Drug Absorption
  
  
  • 6.2.2 Transporters in Drug Distribution
  
  
  • 6.2.3 Transporters in Drug Metabolism
  
  
  • 6.2.4 Transporters in Drug Excretion
  
  
  • 6.2.5 Transporters in Toxicity
  
  
  
  


• 6.3 Transporters in Drug Resistance


• 6.4 Polymorphism of Transporters and Interindividual Variation


• 6.5 Transporters in Drug–Drug or Drug–Food Interactions
  
  
  • 6.5.1 Oral Absorption
  
  
  • 6.5.2 Brain Penetration
  
  
  • 6.5.3 Renal Excretion and Hepatic Clearance
  
  
  • 6.5.4 Food Effect
  
  
  • 6.5.5 Formulation Effect
  
  
  • 6.5.6 In vitro–In vivoCorrelation
  
  
  • or Inducer 6.6 Methods to Evaluate Transporter Substrate, Inhibitor,
  
  
  • 6.6.1 In vitroModels
  
  
  • 6.6.2 In situ/Ex vivoModels
  
  
  • 6.6.3 In vivoModels
  
  
  
  


• 6.7 Conclusions and Perspectives
  
  
  • References
  
  
  • Drug Interaction Studies 7 Regulatory Considerations of Drug Metabolism and
  
  
  
  


• 7.1 Introduction Xiaoxiong Wei and Mingshe Zhu


• 7.2 Regulatory Guidances Relevant to Drug Metabolism
  
  
  • 7.2.1 Toxicokinetic Studies
  
  
  • 7.2.2 Use of Radiolabeled Materials
  
  
  • 7.2.3 Metabolite Safety Assessment
  
  
  • 7.2.4 Drug–Drug Interaction Studies
  
  
  • 7.2.5 Analytical Method Validation and Compliance
  
  
  • 7.2.6 Regulatory Submission Format and Content
  
  
  
  


• 7.3 Metabolism Studies Relevant to Metabolite Safety Assessment
  
  
  • 7.3.1 Goals and General Strategies
  
  
  • 7.3.2 In vitroMetabolite Profiling Studies
  
  
  • 7.3.3 ADME Studies
  
  
  • 7.3.4 Analytical Methods for Metabolite Profiling
  
  
  • 7.3.5 Special Considerations
  
  
  
  


• 7.4 Drug–Drug Interaction Studies
  
  
  • 7.4.1 General Strategies
  
  
  • 7.4.2 In vivoStudies
  
  
  • 7.4.3 Case Study
  
  
  
  


• 7.5 Conclusions
  
  
  • Acknowledgment
  
  
  • References
    
    
    • PHARMACEUTICAL INDUSTRY PART II ROLE OF DRUG METABOLISM IN THE
    
    
    
    
  
  
  • Drug Discovery Process 8 Drug Metabolism Research as an Integral Part of the
  
  
  
  


• 8.1 Introduction W. Griffith Humphreys


• 8.2 Metabolic Clearance
  
  
  • 8.2.1 General
  
  
  • 8.2.2 Prediction of Human Clearance
  
  
  • 8.2.3 In vivoMethods to Study Metabolism
  
  
  • 8.2.4 Screening Strategies
  
  
  • 8.2.5 In silicoMethods to Study Metabolism
  
  
  
  


• 8.3 Metabolite Profiling


• 8.4 Reaction Phenotyping


• 8.5 Assessment of Potential Toxicology of Metabolites
  
  
  • 8.5.1 Reactive Metabolite Studies—In vitro
  
  
  • 8.5.2 Reactive Metabolite Studies—In vivo
    
    
    • with Off-Target Receptors 8.5.3 Toxicology Mediated Through Metabolite Interaction
    
    
    
    
  
  
  
  


• 8.6 Assessment of Potential for Active Metabolites
  
  
  • 8.6.1 Detection of Active Metabolites During Drug Discovery
    
    
    • Activity of Metabolites 8.6.2 Methods for Assessing and Evaluating the Biological
    
    
    
    
  
  
  • 8.6.3 Methods for Generation of Metabolites
  
  
  
  


• 10.3 New Radiochromatography Techniques
  
  
  • 10.3.1 HPLC-MSC
  
  
  • 10.3.2 Stop-Flow HPLC-RFD
  
  
  • 10.3.3 Dynamic Flow HPLC-RFD
  
  
  • 10.3.4 UPLC-Radiodetection
  
  
  • 10.3.5 HPLC-AMS
  
  
  
  


• 10.4 Radiochromatography in Conjunction with Mass Spectrometry
  
  
  • 10.4.1 LC-RFD-MS
  
  
  • 10.4.2 Stop-Flow and Dynamic Flow LC–RFD–MS
  
  
  • 10.4.3 LC-MSC-MS
    
    
    • Approach
    
    
    
    
  
  
  • Drug Metabolism Studies 10.5 Application of New Radiochromatography Techniques in
  
  
  • 10.5.1 Profiling of Radiolabeled Metabolites in Plasma
    
    
    • Using Radiolabeled Cofactors or Trapping Agents 10.5.2 Analysis of Metabolites of Nonradiolabeled Drugs
    
    
    • of Sequential Metabolites 10.5.3 Determination of Structures and Formation Pathways
    
    
    
    
  
  
  • 10.5.4 Enzyme Kinetic Studies
  
  
  
  


• 10.6 Summary
  
  
  • References
  
  
  • for Metabolite Identification 11 Application of Liquid Chromatography/Mass Spectrometry
  
  
  
  


• 11.1 Introduction Shuguang Ma and Swapan K. Chowdhury


• 11.2 LC/MS Instrumentation
  
  
  • 11.2.1 High Performance Liquid Chromatography (HPLC)
  
  
  • 11.2.2 Atmospheric Pressure Ionization Methods
  
  
  • 11.2.3 Mass Analyzers
  
  
  • 11.2.4 Tandem Mass Spectrometry
  
  
  
  


• 11.3 Metabolite Identification––Role of LC/MS
  
  
  • 11.3.1 Metabolite Characterization in Drug Discovery
    
    
    • Clinical Development 11.3.2 Metabolite Identification in Preclinical and
    
    
    
    
  
  
  • Identification 11.4 Techniques for Improving Metabolite Detection and
  
  
  • 11.4.1 Chemical Derivatization
  
  
  • 11.4.2 Stable Isotope Labeling
  
  
  • 11.4.3 Hydrogen/deuterium (H/D) Exchange MS
  
  
  • 11.4.4 Accurate Mass Measurement
    
    
    • Metabolite Identification 11.4.5 Nanospray Ionization (NSI) MS for
    
    
    
    
  
  
  
  


• 11.5 Software-Assisted Metabolite Identification
  
  
  • 11.5.1 Data-Dependent Acquisition (DDA)
  
  
  • 11.5.2 Mass Defect Filter (MDF)
  
  
  • Identification 11.6 Additional MS-Related Techniques for Metabolite
  
  
  • 11.6.1 LC/NMR/MS
  
  
  • 11.6.2 LC/ICPMS
  
  
  
  


• 11.7 Characterization of Unstable Metabolites
  
  
  • 11.7.1 Glucuronides
  
  
  • 11.7.2 N-Oxides
    
    
    • Ions by the Presence of Alkali Adducts 11.7.3 Differentiation of Molecular Ions from In-Source Fragment
    
    
    
    
  
  
  • Intermediates 11.8 Detection and Characterization of Reactive Metabolites and
  
  
  • 11.8.1 Trapping Reactive Metabolites
  
  
  • 11.8.2 Screening for Glutathione Conjugates
  
  
  
  


• 11.9 Conclusions and Future Directions
  
  
  • Acknowledgments
  
  
  • References
  
  
  • Structure Determination 12 Introduction to NMR and Its Application in Metabolite
  
  
  
  


• 12.1 Introduction and Vikram Roongta


• 12.2 Theory


• 12.3 NMR Hardware


• 12.4 NMR Observables
  
  
  • 12.4.1 Chemical Shifts
  
  
  • 12.4.2 Coupling Constants
  
  
  • 12.4.3 Integration
  
  
  
  


• 12.5 Sample Requirements for NMR


• 12.6 Most Commonly Used NMR Experiments and Techniques
  
  
  • 12.6.1 1D NMR Experiments
  
  
  • 12.6.2 2D NMR Experiments
  
  
  • 12.6.3 Solvent Suppression Techniques
  
  
  • 12.6.4 Hyphenated NMR Methods
  
  
  • or Metabolites 12.7 General protocol for NMR Analysis of Unknown Compounds
  
  
  • Known Biotransformations 12.8 Examples of Metabolite Structure Determination from
  
  
  • References
    
    
    • Method in Human or Rat Liver Microsomes—A Semiautomated
    
    
    
    
  
  
  • 14.2.5 Protocol ForIn vivoCovalent Protein Binding in Rats
  
  
  • 14.2.6 Notes
  
  
  • Concentrations in Hepatocytes 14.3 Protocol for Measurement of Intracellular GSH and GSSG
  
  
  • 14.3.1 Introduction
    
    
    • Hepatocytes 14.3.2 Measurement of Intracellular GSH/GSSG in
    
    
    
    
  
  
  • 14.4 Perspectives
    
    
    • Acknowledgments
    
    
    • References
    
    
    
    
  
  
  
  


• 15 Reaction Phenotyping
  
  
  • 15.1 Introduction Susan Hurst, J. Andrew Williams, and Steven Hansel
  
  
  • 15.2 Cytochrome P450 Reaction Phenotyping
  
  
  • 15.3 Noncytochrome P450 Reaction Phenotyping
    
    
    • 15.3.1 Flavin-Containing Monooxygenases
      
      
      • MAO-B) 15.3.2 Monoamine Oxidases A and B (MAO-A and
      
      
      
      
    
    
    • 15.3.3 Esterases
    
    
    
    
  
  
  • 15.4 Conjugation Phenotyping
    
    
    • 15.4.1 UGT Reaction Phenotyping
    
    
    • 15.4.2 N-Acetylation Reaction Phenotyping
    
    
    • 15.4.3 Sulfation Reaction Phenotyping
    
    
    
    
  
  
  • 15.5 Transporter Phenotyping
  
  
  • 15.6 Nonradiolabeled Reaction Phenotyping
    
    
    • 15.6.1 Objective
    
    
    • 15.6.2 Selection of Appropriate Experimental Systems
    
    
    • 15.6.3 Experimental Approach Considerations
    
    
    • 15.6.4 Selection of Appropriate Experimental Designs
      
      
      • Enzyme Systems 15.6.5 Quantitative Reaction Phenotyping: Expressed or Purified
      
      
      
      
    
    
    
    
  
  
  • 15.7 Radiolabeled Reaction Phenotyping
    
    
    • Phenotyping Studies 15.7.1 QuantitativeIn vitroRadiolabeled Reaction
    
    
    • 15.7.2 In vivoQuantitative ADME Studies
    
    
    • 15.7.3 Drug–Drug Interaction Potential
    
    
    • 15.7.4 Specialized Clinical Studies
    
    
    
    
  
  
  • 15.8 Summary and Future Directions
    
    
    • Acknowledgments
    
    
    • References
    
    
    
    
  
  
  • Enzyme Incubation Method Sheet Appendix A: Reaction Phenotyping—Expressed cDNA
  
  
  • Chemical Inhibition Appendix B: Reaction Phenotyping—Microsomal
    
    
    • Discovery and Development 16 Analysis ofIn vitroCytochrome P450 Inhibition in Drug
    
    
    
    
  
  
  • 16.1 Introduction Magang Shou and Renke Dai
  
  
  • 16.2 Reversible Inhibition
    
    
    • 16.2.1 Materials and Reagents
    
    
    • 16.2.2 Instrument
    
    
    • 16.2.3 Optimization of Kinetic Reaction
    
    
    • 16.2.4 LC/MS/MS Analysis
    
    
    • 16.2.5 Automated Sample Preparation and Incubation
    
    
    • 16.2.6 Data Analysis
    
    
    
    
  
  
  • 16.3 Irreversible Inhibition
    
    
    • 16.3.1 Kinetic Model for Mechanism-Based Inhibition
    
    
    • 16.3.2 Measurements of Kinetic Parameters
    
    
    • 16.3.3 General Incubation Procedure and Sample Preparation
    
    
    • 16.3.4 Data Analysis
    
    
    
    
  
  
  • 16.4 Fluorescent Assay
    
    
    • CYP Inhibition Data 16.5 Prediction of Human Drug–Drug Interactions fromIn vitro
    
    
    • 16.5.1 Reversible CYP Inhibition
      
      
      • from Mechanism-Based CYP Inhibition 16.5.2 Prediction of Human Drug–Drug Interactions
      
      
      
      
    
    
    • 16.5.3 Factors Affecting the Prediction of Drug–Drug Interactions
    
    
    
    
  
  
  • 16.6 Conclusion
    
    
    • Acknowledgment
    
    
    • References
    
    
    
    
  
  
  
  


• 17 Testing Drug Candidates for CYP3A4 Induction
  
  
  • 17.1 Introduction Gang Luo, Liang-Shang Gan, and Thomas M. Guenthner
  
  
  • 17.2 Assessments
    
    
    • Animal Models 17.2.1 Assessment of Induction Potential Using Intact
    
    
    • 17.2.2 Assessment of Induction Potential UsingIn vitroModels
      
      
      • in Humans 17.2.3 Direct Assessment of CYP3A4 InductionIn vivo
      
      
      
      
    
    
    
    
  
  
  • 17.3 Final Comments
    
    
    • References
    
    
    • Metabolite Profiling and Identification, and Data Presentation 18 ADME Studies in Animals and Humans: Experimental Design,
    
    
    
    
  
  
  • 18.1 Objectives, Rational, and Regulatory Compliance Donglu Zhang and S. Nilgun Comezoglu
  
  
  • 18.2 Study Designs
    
    
    • 18.2.1 Choice of Radiolabel
    
    
    • 18.2.2 Preparation of Animals and Human Subjects
    
    
    • 18.2.3 Dose Selection, Formulation, and Administration
      
      
      • Sample Collection/Pooling 18.2.4 In-Life Studies in Animals and Humans and
      
      
      
      
    
    
    
    
  
  
  • 18.3 Sample Analysis
    
    
    • 18.3.1 Sample Preparation: Plasma, Urine, Bile, and Feces
    
    
    • 18.3.2 Radioactivity Determination
    
    
    • 18.3.3 LC/MS/MS Quantification and Pharmacokinetic Analysis
    
    
    • 18.3.4 Metabolite Profiling
    
    
    • 18.3.5 Metabolite Identification
      
      
      • and Generation in Bioreactors 18.3.6 Metabolite Isolation fromIn vivoSamples
      
      
      
      
    
    
    • an Example 18.4 Data Presentation Using Metabolism of [^14 C]Muraglitazar as
      
      
      • Radioactivity 18.4.1 Pharmacokinetic Results and Excretion of
      
      
      
      
    
    
    • 18.4.2 Metabolite Profiling in Plasma, Urine, Bile, and Feces
    
    
    • 18.4.3 Metabolite Identification by LC/MS/MS
    
    
    
    
  
  
  • 18.5 Conclusions and Path Forward
    
    
    • Acknowledgments
    
    
    • Appendix A: Rat Tissue Distribution and Dosimetry Calculation
    
    
    • References
    
    
    
    
  
  
  
  


• Index