Abstract
This chapter provides a step-by-step description of methodology used to assess time-dependent inhibition/inactivation (TDI) potential of cytochrome P450 3A4 (CYP3A4) by a test compound using human hepatocytes in a 96-well plate format. Human hepatocytes in suspension or plated cultures are pre-incubated with the test compound for different time periods and at different concentrations, prior to incubation with midazolam, a CYP3A4 probe substrate. The metabolite 1′-hydroxymidazolam is then quantified by LC/MS/MS. The TDI potential of the test compound may then be evaluated by determining the enzyme activity remaining after each condition. Methods to determine KI and kinact values from these data, as well as tips and considerations for robust assay outcomes are also provided.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Grimm SW, Einolf HJ, Hall SD, He K, Lim HK, Ling KH, Lu C, Nomeir AA, Seibert E, Skordos KW et al (2009) The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America. Drug Metab Dispos 37:1355–1370
Lin JH, Lu AY (1998) Inhibition and induction of cytochrome P450 and the clinical implications. Clin Pharmacokinet 35:361–390
Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102:1016–1023
FDA guidance (draft) (2012) Drug interaction studies—study design, data analysis, implications for dosing, and labeling recommendations http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm292362.pdf
European Medicines Agency (2012) Guideline on the investigation of drug interactions (final) http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf
Xu L, Chen Y, Pan Y, Skiles GL, Shou M (2009) Prediction of human drug-drug interactions from time-dependent inactivation of CYP3A4 in primary hepatocytes using a population-based simulator. Drug Metab Dispos 37:2330–2339
Zhao P, Kunze KL, Lee CA (2005) Evaluation of time-dependent inactivation of CYP3A in cryopreserved human hepatocytes. Drug Metab Dispos 33:853–861
Chen Y, Liu L, Monshouwer M, Fretland AJ (2011) Determination of time-dependent inactivation of CYP3A4 in cryopreserved human hepatocytes and assessment of human drug-drug interactions. Drug Metab Dispos 39:2085–2092
Van LM, Swales J, Hammond C, Wilson C, Hargreaves JA, Rostami-Hodjegan A (2007) Kinetics of the time-dependent inactivation of CYP2D6 in cryopreserved human hepatocytes by methylenedioxymethamphetamine (MDMA). Eur J Pharm Sci 31:53–61
McGinnity DF, Berry AJ, Kenny JR, Grime K, Riley RJ (2006) Evaluation of time dependent cytochrome P450 inhibition using cultured human hepatocytes. Drug Metab Dispos 34:1291–1300
Albaugh DR, Fullenwider CL, Fisher MB, Hutzler JM (2012) Time-dependent inhibition and estimation of CYP3A clinical pharmacokinetic drug-drug interactions using plated human cell systems. Drug Metab Dispos 40:1336–1344
Li AP, Doshi U (2011) Higher throughput human hepatocyte assays for the evaluation of time-dependent inhibition of CYP3A4. Drug Metab Lett 5:183–191
Mao J, Mohutsky MA, Harrelson JP, Wrighton SA, Hall SD (2011) Prediction of CYP3A mediated drug-drug interactions using human hepatocytes suspended in human plasma. Drug Metab Dispos 39:591–602
Mao J, Mohutsky MA, Harrelson JP, Wrighton SA, Hall SD (2012) Predictions of cytochrome P450-mediated drug-drug interactions using cryopreserved human hepatocytes: comparison of plasma and protein-free media incubation conditions. Drug Metab Dispos 40:706–716
Parkinson A, Kazmi F, Buckley DB, Yerino P, Ogilvie BW, Paris BL (2010) System-dependent outcomes during the evaluation of drug candidates as inhibitors of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) enzymes: human hepatocytes versus liver microsomes versus recombinant enzymes. Drug Metab Pharmacokinet 25:16–27
Aasa J, Hu Y, Eklund G, Lindgren A, Baranczewski P, Malmquist J, Turek D, Bueters T (2013) Effect of solvents on the time-dependent inhibition of CYP3A4 and the biotransformation of AZD3839 in human liver microsomes and hepatocytes. Drug Metab Dispos 41:159–169
Acknowledgements
The authors thank Thuy Ho and Alanna Callendrello for their assistance in performing TDI experiments necessary for the development of these assays.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zhang, J.G., Stresser, D.M. (2014). Assessment of CYP3A4 Time-Dependent Inhibition in Plated and Suspended Human Hepatocytes. In: Caldwell, G., Yan, Z. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-742-6_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-742-6_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-741-9
Online ISBN: 978-1-62703-742-6
eBook Packages: Springer Protocols