Cytochrome P450: In Vitro Methods and Protocols

ISBN-13: 9781071615447 / Angielski / Miękka / 2022

Yan, Zhengyin

Opis

This collection explores detailed experimental protocols necessary for setting up a variety of in vitro cytochrome P450 (CYP) assays that are vital in selecting drug candidates in a drug discovery pipeline. Major factors affecting drug metabolism include CYP expression levels, kinetic parameters for individual CYP enzymes, CYP inhibition and induction, time-dependent inhibition (TDI), CYP stability, non-CYP stability, UDP-glucuronosyltransferases (UGT) stability, excretion mechanisms, and drug-drug interactions (DDI), all addressed in this volume. Written for the Methods in Pharmacology and Toxicology series, chapters include helpful background information on the in vitro assay, a list of all the materials, reagents, and equipment necessary to carry out the assay, a step-by-step protocol, notes containing common and unexpected experimental problems in the assay, as well as references containing important supplementary reading. 

Authoritative and practical, Cytochrome P450: In Vitro Methods and Protocols serves as a key guide for researchers in the area of discovery and development of new medicines. 

Spis treści

1. Cytochrome P450: In Vitro Methods and Protocols

            Zhengyin Yan and Gary W. Caldwell

 

2. Characteristics of Major Drug Metabolizing Cytochrome P450 Enzymes

            Kevin Johnson, Dian Su, and Donglu Zhang

 

3. Quantitative Determination of Cytochrome P450 Using LC/MS/MS

            Ryan H. Takahashi, Brahim Achour, and Bhagwat Prasad

 

4. Spectral Quantitation of Total Cytochrome P450 in Microsomes Using a Single-Beam Spectrophotometer

            David M. Stresser

 

5. Cytochrome P450 Enzyme Kinetics: Km and Vmax Determinations Using Metabolite Formation and Substrate Depletion Methods

            Jing Wang

 

6. Cytochrome P450 Inhibition Assay Using Human Liver Microsomes

            Jia Jia

 

7. Evaluation of Time-Dependent Cytochrome P450 Inhibition Using the Area Under the Curve Shift Method

            Julie Huang

 

8. Assessing Cytochrome P450 Time Dependent Inhibition (IC50 Shift Assay) Using Both Diluted and Non-Diluted Methods

            Yu Wang, Lili Xie, and Jing Ni

 

9. Cytochrome P450 Gene Regulation: Reporter Assays to Assess Pregnane X Receptor (PXR, NR1I2) Activation

            Marija Pinne and Judy L. Raucy

 

10. Cytochrome P450 Gene Regulation: Reporter Assays to Assess Aryl Hydrocarbon Receptor (HLHE76, AhR) Activation and Antagonism

            Marija Pinne and Judy L. Raucy

 

11. Cytochrome P450 Gene Regulation: Reporter Assays to Assess Direct and Indirect Activation of Constitutive Androstane Receptor (CAR, NR1I3)

            Marija Pinne and Judy L. Raucy

 

12. Determination of In Vitro Cytochrome P450 Induction Potential Using Cryopreserved Human Hepatocytes

            Susan Wong

 

13. Assessing Cytochrome P450 3A (CYP3A) Induction and Suppression Using Primary Human Hepatocyte Spheroids

            Jing Wang and Eugene Chen

 

14. Cytochrome P450-Mediated Metabolic Stability Assay in Liver Microsomes

            Pasquale Carione

 

15. Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension

            Jingwei Cai and Hadil Shalan

 

16. Cytochrome P450 Knock-Out Assay with Non-Selective Inhibitors

            Pasquale Carione and Zhengyin Yan

 

17. Differentiation of Cytochrome P450-Mediated from Non-CYP-Mediated Metabolism: Aldehyde Oxidase and Xanthine Oxidoreductase

            Kirk D. Kozminski and Michael A. Zientek

 

18. In Vitro Reaction Phenotyping of Cytochrome P450 Enzymes

            Susan Wong and Zhengyin Yan

 

19. Cytochrome P450-Mediated Drug Bioactivation Assay: An Untargeted High Resolution Accurate Mass LC/MS Assay

            Kevin M. Johnson

 

20. Simultaneously Assessing Concentration Changes in 20 Biochemical Pathways as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism: An Untargeted Metabolomics GC/MS Assay

            Yanping Lin, Ying Li, and Gary W. Caldwell

 

21. Simultaneously Assessing Concentration Changes in 17 Biochemical Pathways as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism: A Quasi-Untargeted Metabolomics LC/MS Assay

            John A. Masucci, Feng Liang, Kerem Bingol, Vince Windisch, and Gary W. Caldwell

 

22. Assessing Amino Acid Concentration Changes as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism

            Kerem Bingol, John A. Masucci, and Gary W. Caldwell