CYP inhibition screening, direct and time-dependent

In the CYP assay, the test compound is preincubated with human liver microsomes at selected concentrations in the presence and absence of cofactor NADPH. Thereafter, CYP activity is measured by incubating the human liver microsomes with a cocktail of CYP-specific substrates (1A2, 2B6, 2C8, 2C9, 2D6, 2C19 and 3A) in the presence of NADPH. Direct inhibition is observed from the samples pre-incubated in the absence of NADPH as reduced level of CYP-specific metabolite formation in the incubations compared to solvent samples. Time-dependent (TDI) inhibition is detected as changes in inhibition curves after preincubation in the absence and presence of NADPH. One concentration of specific positive control inhibitors for CYP3A are used; for direct and TDI inhibition, respectively (to determine % inhibition and % TDI).