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.
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Acknowledgements
The authors thank Thuy Ho and Alanna Callendrello for their assistance in performing TDI experiments necessary for the development of these assays.
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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
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DOI: https://doi.org/10.1007/978-1-62703-742-6_15
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