Preface
Contents
Contributors
Part I: Small Molecules
Chapter 1: Measurement of Transcellular Transport Rates and Intracellular Drug Sequestration in the Presence of an Extracellul...
1 Introduction
1.1 Cell Membranes as Functional Barriers Determining the Drug Transport Rates Between Body Compartments
1.2 Transcellular Permeability as a Key Component Determining Transport and Distribution of Drugs in the Body
2 In Vitro Cell Models to Measure Transcellular Drug Transport in the Presence of an Extracellular Concentration Gradient
2.1 Intestinal Cell Models
2.2 Airway Cell Models
2.3 Ocular Cell Models
3 Theoretical Aspects Related to the Measurement of Passive Drug Transport in the Presence of an Extracellular Concentration G...
4 Materials and Methods to Measure Transcellular Drug Transport Across Epithelial Cell Monolayers Differentiated on Porous Mem...
4.1 Preparation of Reagents
4.2 Experimental Equipment
4.3 Protocol for Measuring Drug Transport Rates and Intracellular Accumulation
5 Factors Affecting Reproducibility and Reliability of Experimental Measurements
6 The Mass Balance Problem Defined: Measuring the Rates and Absolute Extent of Intracellular Drug Sequestration in Relation to...
7 Future Prospects
References
Chapter 2: Kinetic Design for Establishing Long-Term Stationary Cytosol Concentrations During Drug Transport across P-gp Expre...
1 Introduction
2 Transcellular Transport Across Confluent Cell Monolayers
3 Drug Partitioning into Membrane Monolayers
4 Passive Permeability with Drug Loss
5 Stationary Cytosolic Concentrations During Active Transport by P-gp
6 Kinetic Design to achieve Stationary Cytosol Concentrations
6.1 Case 1: Ketoconazole
6.2 Case 2: Digoxin
7 Simulation to Experiment Extrapolation
References
Chapter 3: In Vitro Methodologies to Assess Potential for Transporter-Mediated Drug-Drug Interactions
Abbreviations
1 Introduction
1.1 Drug Transporters
2 Transporter Substrate
2.1 Efflux Transporters (P-gp and BCRP)
2.1.1 Experimental Procedures
2.1.2 Data Analysis
2.1.3 Typical Results for Efflux Transporter Probe Substrates
2.1.4 Results Interpretation
2.2 Uptake Transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2K)
2.2.1 Experimental Procedures
2.2.2 Data Analysis
2.2.3 Typical Results of Uptake Transporter Probe Substrates
2.2.4 Results Interpretation
3 Transporter Inhibition
3.1 Efflux Transporters (P-gp and BCRP)
3.1.1 Experimental Procedures
3.1.2 Data Analysis
3.1.3 Typical Results of Efflux Transporter Inhibitors
P-gp Inhibitors
BCRP Inhibitors
3.1.4 Results Interpretation
3.2 Uptake Transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2K) (See Table 6)
3.2.1 Experimental Procedures
3.2.2 Data Analysis
3.2.3 Typical Results of Uptake Transporter Inhibition
3.2.4 Results Interpretation
4 Conclusions
References
Chapter 4: Determination of Fraction Unbound and Unbound Partition Coefficient to Estimate Intracellular Free Drug Concentrati...
1 Introduction
2 Measurement of Total Drug Concentration
3 Measurement of fu
3.1 Equilibrium Dialysis Method
3.1.1 Preparation of Tissue and Cell Homogenate
Tissue Homogenate Preparation
Cell Homogenate Preparation
3.1.2 Preparation of Equilibrium Dialysis Device
3.1.3 Equilibrium Dialysis Experiment
Standard Method
Pre-Saturation Method
Equilibrium Dialysis for Unstable Compounds
Calculations of fu from Equilibrium Dialysis Experiments
3.2 Partition Coefficient Method with Cells at 4 C
3.2.1 Protocol for fu,cell Determination Using Partition Coefficient Method with Cells at 4 C
Partition Coefficient Measurement in Suspension Cells
Partition Coefficient Measurement in Plated Cells
3.2.2 Preparation of Standard Curves
3.2.3 Calculations of fu from Partition Coefficient Method with Cells at 4 C
4 Measurement of In Vitro Kpuu Between Cells and Media
5 Bioanalysis Using LC-MS/MS
References
Chapter 5: Quantitative Analysis of Intracellular Drug Concentrations in Hepatocytes
1 Introduction
1.1 Importance of Measuring Hepatic Intracellular Drug Concentrations
1.2 Introduction to the Isolated Perfused Liver (IPL)
1.2.1 Background
1.2.2 Technical Considerations
1.2.3 Applications
1.3 Introduction to Sandwich-Cultured Hepatocytes (SCH)
1.3.1 Background
1.3.2 Technical Considerations
1.3.3 Applications
2 Materials
2.1 Buffers, Media, and Equipment Required to Perform a Basic Rat IPL Experiment
2.2 Buffers, Media, and Equipment Required for Rat SCH, Differential Centrifugation, Protein Binding, and Sample Analysis
3 Methods
3.1 Perfusion of the Isolated Liver, Collection of Samples, and Liver Tissue Homogenization
3.2 Use of Pharmacokinetic Modeling to Simulate Hepatocellular Concentrations from IPL Data
3.3 Measuring Hepatic Unbound Intracellular Concentrations in SCH
3.3.1 Sample and Data Analysis
3.4 Use of Imaging Methods to Estimate Hepatic Concentrations in the IPL
4 Notes
5 Emerging Tools and Technologies
References
Chapter 6: Quantification of Intracellular Drug Aggregates and Precipitates
1 Introduction
2 Drug Administration to Animals
3 Cellular Isolation Techniques
3.1 Peritoneal Macrophage Isolation and Culture
3.2 Alveolar Macrophage Isolation and Culture
3.3 Kupffer Cell Isolation and Culture
3.4 Bone Marrow Monocyte Isolation and Culture
4 Cellular Drug Quantification
5 Multiparameter Imaging and Determination of Sequestering Vs. Non-sequestering Cell Populations
5.1 Calibration of LC-PolScope and Imaging
5.2 Imaging of Xenobiotic-Sequestering Cells
5.3 Image Analysis and Quantification of Xenobiotic-Sequestering Cell Populations
6 Cellular Drug Measurements from Tissue Cryosections
6.1 Isolation of Insoluble CFZ and Quantification
6.2 Total Macrophage or Other Drug-Sequestering Cell Population Determination and Measurement of Percentage of Drug-Sequesteri...
7 Conclusions and Future Applications
References
Chapter 7: Quantitative Phenotypic Analysis of Drug Sequestering Macrophage Subpopulations
1 Introduction
2 Materials and Methods
2.1 Mice Clofazimine Treatment (8 Weeks)
2.2 Alveolar Macrophage Isolation
2.3 Immunocytochemistry Analysis
2.4 Fluorescence Microscopy
2.5 Image Analysis and Statistics
2.6 Physical and Biological Markers of Macrophage Differentiation into Xenobiotic Sequestering Cells
2.6.1 Cell Area/Size
2.6.2 TLR2 and TLR4
2.6.3 TFEB
2.6.4 NF-kB (p65)
2.6.5 V-ATPase and CLC7
3 Conclusion
References
Chapter 8: Using an Integrated QSAR Model to Check Whether Small-Molecule Xenobiotics Will Accumulate in Biomembranes, with Pa...
1 What Types of Membranes Are Considered?
2 What Types of Xenobiotic Are Considered?
3 What Types of Physicochemical Processes Are Involved?
4 Additional Factors Influencing Membrane Uptake and Accumulation
5 QSAR Modelling of Uptake in Various Membranes
6 The Integrated Model: Comments and Extensions
7 Assessment of Validity and Demonstrations of Applicability
8 Conclusions, and the Range of Applicability of the Model
References
Chapter 9: Diversity-Oriented Fluorescence Library Approach (DOFLA) for Discovery of Cell-Permeable Probes for Applications in...
1 Introduction
2 Cell Based High-Throughput Screening: Case Study
2.1 Embryonic Stem Cell (ESC) Probes
2.2 Alpha and Beta-Cells in Pancreatic Islets Probes
2.3 Tumor Initiating Cells (TICs)
2.4 Brain-Related Cells
2.5 Taming Probes
3 Summary
References
Part II: Macromolecules, Biologics, and Nanoparticles
Chapter 10: Overcoming Cellular and Systemic Barriers to Design the Next Wave of Peptide Therapeutics
1 Introduction
2 Evolution of Peptide Therapeutics: From Natural Products to Targeted Design of Diverse Peptide Therapeutics
3 Peptide Permeability and Exposure: Mechanistic Concepts and In Vitro Models
4 Peptide Permeability Mechanisms
4.1 Chameleon-Like Passive Partitioning
4.2 Cationic Partitioning and Endosomal Escape
4.3 Lipophilic Partitioning
5 Intracellular Metabolism
6 In Vivo Systemic Exposure: Absorption, Distribution, Metabolism, Elimination
6.1 Absorption
6.2 Distribution
6.3 Metabolism and Elimination
7 Hierarchical Strategies for Peptide Drug Discovery
References
Chapter 11: Intracellular Targeting of Cyclotides for Therapeutic Applications
1 Introduction
2 Materials
2.1 Labeling Peptides
2.2 Detecting Cells with Internalized Peptide Using Flow Cytometry
2.2.1 Cell Culture
2.2.2 Internalization Assay
2.2.3 Flow Cytometry
2.3 Examining Peptide Intracellular Location Using Confocal Microscopy of Live Cells
3 Methods
3.1 Labeling Peptides
3.1.1 Incorporating a Fluorescent Label at a Lys Residue (as per Manufacturer Recommendation)
3.1.2 Incorporating a Fluorescent Label at an Azidoalanine (Method Developed from Ref.)
3.2 Detecting Cells with Internalized Peptide Using Flow Cytometry
3.2.1 Cell Culture
3.2.2 Internalization Assay
3.2.3 Characterizing Endocytosis
3.2.4 Flow Cytometry
3.3 Examining Peptide Intracellular Location Using Confocal Microscopy of Live Cells
4 Notes
References
Chapter 12: Cellular Trafficking of Monoclonal and Bispecific Antibodies
1 Introduction
1.1 Factors Influencing Antibody Binding, Trafficking, and Disposition
1.2 Considerations for Fluorescent Labeling of Proteins
1.3 Quantitative Assessment of Cellular Internalization
2 Quantitative Assessment of Antibody Internalization and Intracellular Trafficking
2.1 Protocol for Determining Fluorescence Quenching Efficiency
2.2 Protocol for Assessing Cellular Internalization of Antibodies
2.3 Quantitative Assessment of Intracellular Trafficking of Antibodies
2.4 Methods for Imaging the Intracellular Trafficking of mAbs
3 Binding and Internalization of Antibodies and Bispecific Antibodies by Fluorescence Microscopy
3.1 High Content Microscopy Method
3.2 Quantitative Image Analysis
4 Conclusions
5 Notes
References
Chapter 13: Quantitative Drug Target Imaging Using Paired-Agent Principles
1 Introduction
2 Materials
2.1 Targeted Imaging Agent(s)
2.1.1 Targeting Moiety (Vector)
2.1.2 Labeling the Targeting Vector with a Reporter
2.2 Control Imaging-Agent
2.3 Kinetic Modeling
2.3.1 Complex Model Example
2.3.2 Simplified Model Example
3 Methods
3.1 Imaging-Agent Preparation
3.2 Imaging-Agent Dosing
3.3 Imaging-Agent Administration
3.4 Data Collection
3.5 Data Preprocessing
3.5.1 Motion Correction
3.5.2 Background Subtraction
3.5.3 Targeted and Control Agent Signal Normalization
3.5.4 Input Function Correction
3.6 Data Analysis
3.7 Simulations to Identify Adequate Paired-Agent Imaging Protocol for a Specific Application
4 Notes
References
Chapter 14: Quantitative Determination of Intracellular Bond Cleavage
1 Introduction: Components of Stimuli-Responsive Delivery Systems
1.1 Nanoparticle- and Conjugate-Based Carriers
1.2 Receptor-Mediated and Nonspecific Internalization Pathways
1.3 Types of Cleavable Bonds
1.4 Phenomenological Models of Intracellular Processing
2 Materials
2.1 Design of Antibody Conjugate to Probe Intracellular Bond Cleavage
2.1.1 Production and Purification of Recombinant Trastuzumab
2.1.2 Site-Specific Modification of Trastuzumab with Dibenzocyclooctyne Functional Handles
2.1.3 Copper-Free Click´´ Chemistry Attachment of Fluorescence Probe to Trastuzumab Carrier Protein
2.2 Confocal Microscopy-Based Visualization of Bond Cleavage
2.2.1 Confocal Analysis of Kinetic Bond Cleavage
2.2.2 Confocal Analysis of FRET Probe Colocalization
2.3 Flow Cytometry-Based Quantification of Bond Cleavage
3 Methods
3.1 Design of Fluorescent Probe to Detect Bond Cleavage
3.2 Design of Antibody Conjugate to Probe Intracellular Bond Cleavage
3.2.1 Production and Purification of Recombinant Trastuzumab
3.2.2 Site-Specific Modification of Trastuzumab with Dibenzocyclooctyne Functional Handles
3.2.3 Copper-FreeClick´´ Chemistry Attachment of Fluorescence Probe to Trastuzumab Carrier Protein
3.3 Confocal Microscopy-Based Visualization of Bond Cleavage
3.3.1 Confocal Analysis of Kinetic Bond Cleavage
3.3.2 Confocal Analysis of FRET Probe Colocalization
3.4 Phenomenological Model of Intracellular Bond Cleavage
3.5 Flow Cytometry-Based Quantification of Bond Cleavage
3.6 Extraction of Bond Degradation Rate from Flow Cytometry Data
4 Future Outlook
5 Notes
References
Chapter 15: Development and Application of a Single Cell-Level PK-PD Model for ADCs
1 Introduction
2 Mathematical Modeling Framework
3 Case Studies
3.1 Case Study 1: Development of a Cellular PK Model to Understand Parameters Responsible for the Cellular Disposition of ADCs
3.2 Case Study 2: Application of the Cellular PK-PD Model to Understand the Bystander Effect of an ADC
3.2.1 In Vitro Bystander Effect
3.2.2 In Vivo Bystander Effect
3.3 Case Study 3: Application of the Cell-Level Model to Support Preclinical-to-Clinical Translation of ADC
4 Summary and Future Outlook
References
Chapter 16: Contribution of Nontarget Cells to the Disposition, Antitumor Activity, and Antigen-Independent Toxicity of Antibo...
1 Introduction
2 Role of Nontarget Cells in ADC Pharmacokinetics
2.1 Components of ADC Clearance
2.2 Consequences of Extratumoral ADC Clearance
2.3 Predicting Conjugation-Induced Clearance from In Vitro Experiments
2.4 Influence of Immune Cell Composition on the Pharmacokinetics of ADCs
3 Mechanisms of ADC Uptake in Tumors
3.1 Role of Antigen
3.2 Contribution from Tumor-Associated Macrophages
4 Conclusions
References
Chapter 17: Tracking siRNA-Nanocarrier Assembly and Disassembly Using FRET
Abbreviations
1 Introduction
2 Materials
2.1 Polymer Synthesis
2.2 Particle Formation
2.3 Polyplex Assembly
2.4 Fluorescence Spectroscopy
2.5 Heparin Competition Assay
3 Methods
3.1 Particle Formation-Solvent Displacement
3.2 Polyplex Assembly
3.3 Fluorescence Spectroscopy-Proof of FRET Capability
3.4 Fluorescence Spectroscopy- Assessing Polyplex Stability
3.5 Heparin Competition Assay
3.6 Translation of FRET Approach and the Difficulties of Quantitative Analysis
3.7 Analyzing the Data
4 Notes
References
Chapter 18: Subcellular Drug Depots as Reservoirs for Small-Molecule Drugs
1 Introduction
2 Materials
2.1 Fluorescent Drug and Polymer Conjugates
2.2 TNP Synthesis and Characterization
2.2.1 Nanoprecipitation
2.2.2 TNP Characterization
2.3 Image-Based Reporters of Drug Action and Subcellular Distribution at a Single-Cell Level
2.4 Cell Lines and Animal Models
2.5 Microscopy Platforms
3 Methods: In Vitro Experiments
3.1 Building a Standard Curve for Correlating Pt Fluorescent Intensity and Concentration
3.1.1 Equipment, Experimental Setup, Protocol
3.1.2 Quantitative Data Analysis
3.2 Quantifying Subcellular Localization of TNP
3.2.1 Equipment, Experimental Setup, Protocol
Imaging the Subcellular Localization of TNP and its Payloads
Single-Fluorophore Control Experiment to Evaluate the Degree of Fluorescence Bleed-through
3.2.2 Quantitative Data Analysis
4 Methods: In Vivo Experiments
4.1 Building an In Vivo Standard Curve that Correlates TNP Fluorescent Intensity with Concentration
4.1.1 Equipment, Experimental Setup, and Protocol
4.1.2 Quantitative Data Analysis
4.2 IVM Quantification of In Vivo PK of TNP Vehicle and Payload
4.2.1 Equipment, Experimental Setup, and Protocol
Establishment of HT1080-53BP1-mApple Tumors in the Dorsal Window Chamber
Evaluating the Pharmacokinetics of TNP Via IVM
4.2.2 Quantitative Data Analysis
4.3 IVM Quantification of In Vivo Distribution of TNP Vehicles and Payloads
4.3.1 Equipment, Experimental Setup, and Protocol
4.3.2 Quantitative Data Analysis
4.4 IVM Quantification of In Vivo Payload Redistribution
4.4.1 Equipment, Experimental Setup, and Protocol
4.4.2 Quantitative Data Analysis
Evaluate the Gradient of Pt Payload and TNP Vehicle around TAM
Evaluate the Gradient of DNA Damage around TAM
5 Notes
6 Future Directions
References
Index