An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes – application to establishing CYP2C8 inhibitor selectivity

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity CYP2C8 inhibitors gemfibrozil 1-O-?-glucuronide and clopidogrel acyl-?-D-glucuronide. In incubations selective probe substrates pooled human liver microsomes were conducted 96-well plates automated liquid handler techniques metabolite concentrations measured quantitative UHPLC-MS/MS analysis. After determination inter-substrate interactions Km values each reaction, divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 midazolam/CYP3A4/5) II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 astemizole/CYP2J2). Time-dependent (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, 1-O-?-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 ritonavir/CYP3A) direct inhibitor (terfenadine/CYP2J2) showed similar inhibition single substrate methods. Established caused IC50 fold shifts ranging from 2.2 30 method. Under conditions, a strong (>90% inhibition) (<< 20% other CYPs) at 60 300 ?M, while acyl-?-D-glucuronide limited above its IC80 CYP2C8. these glucuronides 8.1 38 µM, respectively. conclusion, reliable including most important enzymes developed, validated detecting Moreover, established as use diagnostic studies.