Investigation of the inhibition of eight major human cytochrome P450 isozymes by a probe substrate cocktail in vitro with emphasis on CYP2E1.

1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data. 2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes. 3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study. 4. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.