Assessment of CYP3A4 Time-Dependent Inhibition in Plated and Suspended Human Hepatocytes

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 K_I and k_inact values from these data, as well as tips and considerations for robust assay outcomes are also provided.