Identification and Quantification of Drugs, Metabolites, Drug Metabolizing Enzymes, and Transporters: Concepts, Methods and Translational Sciences

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IDENTIFICATION AND QUANTIFICATION
OF DRUGS, METABOLITES,
DRUG METABOLIZING ENZYMES,
AND TRANSPORTERS:
Concepts, Methods, and Translational Sciences
Copyright
Contributors
Foreword
	References
Preface
Part I: Techniques for identifying and quantifying drugs and metabolites
Bioanalysis of small and large molecule drugs, metabolites, and biomarkers by LC-MS
	Introduction
	Complexity of contemporary bioanalysis
	Bioanalytical requirements for supporting discovery, nonclinical, and clinical studies
	Current regulatory landscape for bioanalysis
	General considerations for bioanalysis for sample collection
	Diagnosis and mitigation of nonspecific adsorption loss for urine bioanalysis
	Tissue bioanalysis
	Managing unstable metabolites such as acyl glucuronide
	General considerations for bioanalysis for extraction, chromatography, and MS detection
	Selected applications for LC-MS bioanalysis
		LC-MS of large molecules
			LC-MS bioanalysis of mAb
			LC-MS bioanalysis of ADC
			LC-MS bioanalysis of PDC
			LC-MS bioanalysis of protein biomarkers
			LC-MS bioanalysis of drug metabolizing enzymes and transporters
			LC-MS bioanalysis of half-life extended biotherapeutics
		LC-MS bioanalysis using microsampling
			Microsampling sample collection in bioanalysis
			Microsampling in blood and plasma
			Bioanalytical method development for microsampling in blood and plasma
		Biomarkers quantitation
			Leukotriene B4 (authentic matrix and authentic analyte)
			4β-Hydroxylcholesterol and 7α-hydroxy-4-cholesten-3-one (C4) (surrogate matrix and authentic analyte)
			Fatty acid amide hydrolase biomarkers (authentic matrix and surrogate analytes)
		LC-MS analysis of metabolites and MIST
			Metabolite quantitation strategy
			LC-MS bioanalysis of polar metabolites
			Bioanalysis of chiral compounds
			Unexpected assay challenge during analysis of patient samples
	Conclusion and future perspective
	References
Recent advances in mass spectrometric and other analytical techniques for the identification of drug metabolites
	Introduction
	Sample preparation strategies
		Solid-phase extraction
		Supported phase liquid extraction
		Liquid-liquid extraction
		Protein precipitation
	Optical detectors and chromatographic separation techniques
		Ultra-performance liquid chromatography
		Supercritical fluid chromatography
	Different types of Ionization techniques and mass spectrometric scan functions
		Ionization techniques
		Mass analyzers and different acquisition modes
		Inductively coupled mass spectrometry (ICP-MS)
	Wet chemistry techniques combined with MS
		Hydrogen/deuterium (H/D) exchange
		Chemical derivatization
			Increased LC retention and sensitivity in LC-MS for polar metabolites
			Unusual metabolite characterization
			Unstable metabolites
			Isomeric metabolite characterization
			Determination of site of glucuronidation
		Nuclear magnetic resonance spectroscopy
	Conclusion and future trends
	References
High-resolution mass spectrometry-based data acquisition and data-mining technologies for
	Introduction
	HRMS-based data acquisition technologies for metabolite identification
		Data-dependent MS/MS acquisition
			Ion intensity-dependent acquisition
			Accurate mass inclusion list-dependent acquisition
			Mass defect-dependent acquisition
			Isotope pattern-dependent acquisition
			Pseudo-neutral loss-dependent acquisition
			Background-exclusion data-dependent acquisition
		Data-independent acquisition
			MSE
			Sequential windowed acquisition of all theoretical fragment ions (SWATH)
			All ion fragmentation
	HRMS-based data-processing techniques for metabolite identification
		Targeted data-mining technology
			Extracted ion chromatography
			Mass defect filter
			Isotope pattern filter
			Product ion filter and neutral loss filter
		Nontargeted data-processing approaches
			Background subtraction
			Metabolomics approach
		Software-assisted metabolite prediction and identification
			Software for site of metabolism prediction
			Software for metabolite structural identification
	Applications of HRMS technologies in metabolite identification experiments
		Metabolic soft-spot analysis
		Reactive metabolite screening
		In vivo metabolite profiling and identification
	Detection and structural characterization of traditional Chinese medicine components in biological systems
		HRMS-based data acquisition techniques for profiling and characterizing TCM components in biological samples
		Targeted data-mining techniques applied to detection and identification of in vivo TCM components
		Untargeted data-mining techniques for detection and characterization of TCM components
		LC-HRMS-based techniques for determining metabolic pathways of individual TCM components
		A comprehensive analytical strategy for study of exposure, metabolism, and disposition of TCM components
	Conclusion and future perspectives
	Acknowledgments
	References
Methods for metabolite generation and characterization by NMR
	Introduction
	Methods for scaled-up production of drug metabolites
		Chemical synthesis
			Synthesis of phase I oxidized metabolites
				Example: Synthesis of multiple oxidized metabolites for bioactivity testing
				Example: Synthetic routes to active metabolites of clopidogrel
			Synthesis of phase II conjugated metabolites
		Biomimetic chemistry
		Electrochemistry
			Example: Production of CYP1A1 and CYP1B1-mediated amodiaquine metabolite M2 by electrochemistry
		Mammalian tissue fractions
			Example: Species-dependent regioselective hydroxylation of drugs
		Whole-cell microbial biotransformation
			Example: Accessing disproportionate human metabolites for toxicology studies
			Example: Accessing multipathway-derived metabolites
		Recombinant enzymes
			Cytochrome P450 enzymes
			Non-CYP phase I enzymes
			Use of other oxidizing enzymes
			Recombinant UGTs
		Combined synthetic and biosynthetic approach
			Example: Accessing human metabolites of drugs subject to metabolic shunting
	Purification and structure elucidation of metabolites
		Metabolite purification
			Example: Purification of metabolites from excreta for use as analytical standards
		Metabolite structure elucidation using NMR spectroscopy
			1D and 2D NMR experiments for metabolite structure elucidation
			Stepwise determination of metabolite structures
	Conclusions and future direction
	Acknowledgments
	References
Application of SFC for bioanalysis
	Introduction
	Considerations for SFC method development
	Detection in SFC
	Sample preparation
	Examples
		Drugs of abuse, doping control, and toxicological analysis
		Pharmaceuticals and clinical analysis
		Endogenous metabolites, metabolomics, and lipidomics
	Conclusion
	References
AMS in drug development: Exploring the current utility of AMS and future opportunities for absolute bioavailab ...
	Introduction
	Introduction of the AMS technique
		AMS vs. conventional methods
		Application of AMS to drug development
		Sample preparation (``graphitization´´)
		Analysis by AMS
		Data acquisition and calculations
		Calculation of analyte concentration
		Validation of LC+AMS
		Evolution of AMS
	Clinical study design definitions
	First drug development clinical study application of AMS
	Hybrid studies (macrotracer)
	Low tracer dose studies
	Concomitant microtracer: Design and delivery
		Absolute bioavailability assessments using AMS
	Improved efficiencies in drug development
	Conclusions
	Future perspectives
	References
Part II: Drug metabolism enzymes, transporters and drug-drug interaction
Using in vitro methods to determine P450s responsible for metabolism and discrimination from other oxidative p ...
	Introduction
	Recombinant P450 assays
	Human liver microsome-based methods
		Antibodies
		Chemical inhibitors
		Silensomes
		Correlation analysis
	P450 vs FMO/AO metabolism
	Determination of fmCYP3A
		Current methods for fmCYP3A
	Regulatory guidance/risk assessment/examples
	Conclusion and future directions
	References
Evaluation of the clearance mechanism of non-CYP-mediated drug metabolism and DDI as a victim drug
	Introduction
	UDP-glucuronosyltransferase (UGT)
		Reaction phenotyping of UGT enzyme(s)
		Incubation with pooled HLM
		Incubation with recombinant human UGT enzymes
		Inhibition study with chemical inhibitors of UGTs
		Challenges
		Clinical relevance and drug-drug interactions (DDI)
	Flavin monooxygenase (FMO)
		The FMO catalytic cycle
		Determination of in vitro relative contribution of FMO vs CYP
		Experimental design for FMO phenotyping
		Challenges
		Clinical significance and DDI
	Monoamine oxidase (MAO)
		Experimental designs for MAO phenotyping
		Challenges
		Clinical significance
	Aldehyde oxidase
		Experimental designs for AOX1 reaction phenotyping
		Challenges
		Clinical significance and DDI
	Xanthine oxidase (XO)
		Experimental designs for XO phenotyping
		Challenges
		Clinical significance and DDI
	Carboxylesterases (CES)
		Experimental design for CES reaction phenotyping
		Challenges
		Clinical significance: CES and DDI
	Aldo-keto reductase (AKR)
		Experimental design for AKR1C reaction phenotyping
		AKR: Human-animal comparison
		Challenges
		Clinical significance
	Future trends
	Conclusion
	Acknowledgment
	References
In vitro characterization and in vitro to in vivo predictions of drug-drug interactions
	Introduction
	In vitro assessment of metabolism-based drug interaction potential
		Characterization of the substrate
		Characterization of the inhibitor
			Reversible inhibitors
			Time-dependent inhibitors
		Enzyme induction
	Quantitative in vitro to in vivo predictions
		Basic principles of reversible inhibition predictions
		Selection of inhibitor concentration, [I]
		Prediction of irreversible inhibition
		Prediction of drug metabolizing enzyme induction
		Inhibition and induction of first-pass intestinal metabolism and integration of multiple DDI mechanisms
	Clinical drug interaction assessment
	Conclusions
	References
Role of transporters in drug disposition and drug-drug interactions
	Introduction
	Overview of membrane transporters
		The ATP-binding cassette superfamily
			P-glycoprotein
			BCRP
			Multidrug resistance proteins
			Bile salt export protein
		The solute carrier protein superfamily
			Organic anion transporting polypeptides
			Organic anion transporters
			Organic cation transporters
			Multidrug and toxin extrusion transporters
	Clinical significance of transporter-mediated drug disposition and drug-drug interactions
	Tools to assess transporter liabilities in drug discovery and development
		Recombinant transfected cell-based systems
		Membrane vesicle-based assay
		Polarized cell-based systems and bidirectional transporter assays
		Primary cell-based assays
		The use of animal models to assess transporter liabilities in drug development
		In silico modeling of transporter proteins
		Application of biomarkers for in vivo assessment of drug transporter activity
	Regulatory landscape of evaluating transporter-mediated drug interactions
		Determining if the investigational drug is a substrate of transporters
		Determining if the investigational drug is an inhibitor of transporters
	Challenges and perspectives on transporter-mediated drug interactions
	References
Mechanisms and clinical relevance of pharmacokinetic-based clinical drug-drug interactions for drugs r
	Introduction
	Enzyme-mediated DDIs
		NMEs as substrates of enzymes
		NMEs as inhibitors of enzymes
		NMEs as inducers of enzymes
	Transporter-mediated DDIs
		NMEs as substrates of transporters
		NMEs as inhibitors of transporters
	PBPK modeling and simulations in DDI prediction
	PGx studies
	Other mechanisms: Absorption-based DDIs
	Conclusion
	References
Quantifying drug metabolizing enzymes and transporters by LC-MS/MS proteomics
	Introduction
	Basic workflow of DMET quantitative proteomics
		Selection of proteotypic peptides
		Sample procurement, homogenization, and protein extraction
		Protein extraction and digestion methods
		Post-digestion processing
		Peptide separation using liquid chromatography (LC)
		Mass spectrometry (MS) analysis of peptide signal
			Untargeted MS acquisition approaches
		Protein quantification approaches
	Factors affecting DMET protein quantification
	Optimized quantitative analysis approaches
		Use of multiple peptides
		Use of multiple product ions
		Inclusion of a positive control sample
		Calibration curve, surrogate matrix, and LLOQ
		SIL peptide and exogenous protein internal standards
		Optimized practices in targeted quantitative proteomics
	Applications of quantitative DMET proteomics
		Characterization of in vitro and in vivo models
		In vitro to in vivo extrapolation (IVIVE) of drug clearance
		Interspecies differences in protein expression
		Differential tissue and regional protein distribution
		Subcellular localization of proteins
		Interindividual variability and precision medicine
		Drug-drug interaction (DDI) potential (induction/suppression)
		Drug/metabolite-protein interactions
	Conclusion
	References
Protein drug-drug interactions for therapeutic modalities
	Introduction
	DDI mechanisms
		Major mechanisms of TP-DDI
		In vitro effects on CYP enzymes and transporters
	TP-DDI observed in clinical studies
		Cytokine-dependent interactions
			Cytokines and therapeutic proteins targeting cytokines
			Immunomodulatory therapeutic proteins
		Immunogenicity-dependent interactions
		Target-dependent interactions
		Other interactions
			Interactions based on physiology
			Interactions based on binding to proteoglycans
			Interactions of antibody-drug conjugates
	Potential DDI between emerging modalities
		Oligonucleotide and mRNA-based drugs
		Cell-based therapies
		Oncolytic viruses
		Immunocytokines
	Risk assessment and strategies to evaluate potential TP-DDI
		Risk assessment
		Exploratory studies to assess TP-DDI risk
		Dedicated TP-DDI studies
		Design of studies
		Specific study considerations
	Conclusion and future perspectives
	References
Part III: Strategy related to drug metabolism and safety
Metabolites in safety testing (MIST)
	Introduction
		History of guidance on safety testing of drug metabolites
		Summary of the metabolites in safety testing (MIST) guidance
	Technological approaches for MIST assessment
		High-performance liquid chromatography separation and radiometric detection
		High-resolution mass spectrometry
		Nuclear magnetic resonance spectroscopy
		Accelerator mass spectrometry and cavity ring-down spectroscopy
		Post-acquisition software tools for metabolite identification
		Semiquantitative and quantitative assessment of metabolite coverage
		Calibrator approaches
		Mixed-matrix approach for direct comparison of metabolite levels in human and animal plasma samples and AUC pooling
		Bioanalytical methods
	A typical MIST strategy
		Stage 1. Before entering human studies
		Stage 2. During Phase 1 human clinical studies
		Stage 3. Before the start of Phase 3 human clinical studies
	Metabolite safety assessment beyond the MIST guidance documents
	Conclusion and future outlook
	References
The use of stable isotopes in drug metabolism studies
	Introduction
	Use of stable labels for metabolite detection and identification
		Stable labels and their application
			Beyond typical choices of isotopes
			Types of studies in drug metabolism
		Cases studies
			Vismodegib and oxidative pyridine ring cleavage
			Using H218O and D2O to understand tofacitinib metabolism
			Stable-labeled glutathione as a trapping agent for detection of reactive metabolites
			A disconnect between endogenous and [13C]-labeled niacin
	Deuterated drugs
		Deuterium in drug design
		Relevant drug-metabolizing enzymes for utilizing kinetic isotope effects
			Cytochrome P450 metabolism
			Monoamine oxidase metabolism
			Aldehyde oxidase metabolism
		Case studies: Deuterated versions of old drugs
			Lowering clearance
			Improving bioavailability
			Mitigating reactive metabolite formation/drug-drug interaction
			Slowing chiral inversion
		How deuterium should be assessed in drug discovery?
	Conclusions and future perspectives
	References
Assessment of stereoselectivity in pharmacology, toxicology, and drug metabolism
	Introduction
	Regulatory considerations on developing chiral drugs
	Stereoselectivity in pharmacodynamics
		Selective competitive antagonism
		Enantiomers have the opposite effects
		One enantiomer has side effects
		Different biological activities
		Complementarity of enantiomeric effects
		The biological activity of a drug is produced by single enantiomer
		Different action targets present different characteristics
		Same biological activity
	Stereoselectivity in pharmacokinetics and ADME properties
		Stereoselectivity of chiral drug absorption and transport
		Stereoselectivity of chiral drug distribution
			Stereoselectivity of interaction between chiral drugs and plasma proteins
			Stereoselectivity of interaction between chiral drugs and tissues
		Chirality in drug metabolism
		Stereoselectivity of chiral drug excretion
		Methods for studying chiral drug interactions
	Stereoselectivity in toxicity
		Toxicity of chiral drugs
		Enantiomeric biotransformation increases toxicity
		Chiral inversion increases toxicity
		Toxicity or adverse reactions are inseparable from pharmacological activity
		Active enantiomers are potentially toxic
	Chiral inversion mechanisms
		Chiral metabolic inversion of nonsteroidal anti-inflammatory drugs
		Inversion of the opposite metabolic pathway
	Stereoselective analytical methods
		High-performance liquid chromatography
			Chiral stationary phases
			Chiral mobile phase additives
			Chiral derivatization reagents
		Gas chromatography
			Chiral stationary phases
			Chiral derivatization reagents
		Supercritical fluid chromatography
		Capillary electrophoresis
		Immunoassay
	Conclusion
	References
Progress of derisking strategies for drug-induced liver injury (DILI) in the last two decades
	Introduction
	Challenges in predicting DILI
		Bioactivation of drugs to reactive intermediates
			Metabolite identification via trapping studies
			Covalent binding studies in vitro and in vivo
			Reactive phase II conjugates of carboxylic acid-containing molecules
			Glaxo Smith Kline multi-assay approach to assess DILI risk from bioactivation
			Transcriptomics approach
			Immunomics approach
	Mitochondrial impairment
	Transporter inhibition
	Overarching derisking approaches independent of mechanism
		Dose, exposure, and physiochemical properties
		Multiparametric approaches
			European Federation of Pharmaceutical Industries and Associations (EFPIA) three-tiered roadmap
			Roche approach using a combination of assays
			Astra Zeneca integrated in vitro hazard matrix and Bayesian machine learning approach
			Pfizer hepatic risk matrix (HRM) approach
	Summary
	References
Predictive and translational models for renal drug safety evaluation
	Background and introduction
	2D in vitro models for nephrotoxicity screening
	Emerging models for renal safety screening
	Translatable kidney safety biomarkers
	Context of use of in vitro PTEC models-Mechanistic vs. predictive
	Outlook and future perspectives
	References
Immunogenicity: An introduction to its role in the safety and efficacy of biotherapeutics
	Introduction
	Overview of immunogenicity
	Overview of immune response mechanisms
		Humoral immunogenicity
		Cellular immunogenicity
			Presentation via MHC I on the cell surface and binding to CD8+ T cells
			Activation and cellular response of CD8+ T cells
	Humoral immunogenicity: Overall risk assessment and mitigation strategies
		Case studies
			Case study 1: PK assessment in the presence of ADA in animal models
			Case study 2: Immune response to AAV vectors in animal models
			Case study 3: In silico immunogenicity prediction
	Cellular immunogenicity risk assessments
		In silico prediction tool
		ELISpot
		Chromium 51
		Flow cytometry
		In vivo analysis
	Mitigation strategy and case studies for cellular immunogenicity
		Modification of AAV
			Case study: AAV2 YF engineered AAV2 capsid
		Humanization of CAR construct
			Case study: Reducing the immunogenicity of CAR construct
		Lymphodepletion
	Chapter summary
	Acknowledgments
	References
Part IV: Translational sciences
Application of genetically modified rodent models in drug discovery and development for translation of clinical ADME properties
	Introduction
		The rise of novel animal models in drug research
	Knockout animal models
	Xenobiotic receptor KO models
		Advantages
		Disadvantages
	Humanized transgenic animal models
		Utility of humanized CYP mouse models to understand human ADME
	Humanized liver chimeric mouse models
		Advantages
		Disadvantages
	Conclusions and future perspectives
	References
Advances in CRISPR technologies enable novel in vitro tools for ADME studies
	Introduction
	In vitro applications of CRISPR
		Genome editing with CRISPR
		Repression of target genes with CRISPR
		Activation of target genes with CRISPR
		Genetic screens with CRISPR
	Examples of CRISPR application in ADME studies in vitro
		Genetic modification of CYP3A5*3 in Huh-7 cells
		Endogenous Canine P-gp knockout in Madin-Darby canine kidney cells
	Potential opportunities and limitations for CRISPR applications in ADME studies in vitro
		Limitations of CRISPR in ADME studies in vitro
		Potential opportunities for CRISPR applications in ADME studies in vitro
	References
In vitro-in vivo extrapolation of human hepatic and renal clearance
	Introduction
	ECCS framework to identify rate-determining process for CL
	Current IVIVE approaches to predict clearance
		Hepatic clearance
			Metabolic clearance and in vitro methodologies
				IVIVE of metabolic clearance
				In vitro-to-in vivo correlation (IVIVC) of metabolic clearance
			Transporter and metabolism interplay
				In vitro tools and IVIVE of overall hepatic clearance
				IVIVC of overall hepatic clearance
		Renal clearance
			Metabolic clearance and IVIVE
			IVIVE of transporter-mediated CL and tubular reabsorption
	Conclusions
	References
The role of quantitative modeling and simulation in translational science
	Introduction
	Pharmacokinetic modeling
		Pharmacokinetic modeling for small molecule drugs
			Compartmental and population-based PK models
			Physiologically-based pharmacokinetic modeling
				Physiological-based absorption modeling to assess food's and pH's effects on pharmacokinetics
				Modeling the interplay between gut transporters and CYP3A
				Distribution modeling using PBPK
				Prediction of human clearance (hepatic, renal, and biliary excretion)
				Gaps and challenges in PBPK modeling
		Pharmacokinetic modeling for biologics
			Modeling the absorption and distribution of biologics
			Target-mediated drug disposition models and their role in understanding clearance of biologics
			General dynamic of TMDD model
			Constant receptor amount (Rtotal) model
			Quasi-equilibrium model (QE)
			Quasi-steady-state model
			Michaelis-Menten model
	Modeling pharmacodynamic response
		Pharmacodynamic modeling for small molecule drugs
			PKPD modeling to predict efficacy of D2 receptor antagonists for the treatment of schizophrenia
			Using translational PKPD modeling to accelerate EGFR inhibitor development for the treatment of lung cancer
		Special considerations for biologic pharmacodynamic modeling
	Conclusions
	References
PK/PD-driven starting and effective human dose determination for immuno-oncology drugs
	Introduction
	Dose selection for immune-activating agents
	Dose selection for T-cell engaging bispecific molecules
	Dose selection for emerging class of I-O therapies
	Future directions
	References
Index
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