Assay Method Information

Assay Name:  Inhibition Assay
Description:  Compounds were assessed for inhibition (IC50, n=2) of CYP2C8, CYP2C9 and CYP3A4 in pooled human liver microsomes using selective probe substrates at their previously determined Km values (CYP2C8: paclitaxel, 4 μM; CYP2C9: diclofenac, 5 μM; CYP3A4: midazolam, 0.5 μM). Incubations contained 0.1 mg/mL human liver microsomes, 3 mM MgCl2, probe substrate and various concentrations of inhibitor (12-point IC50 curve) in 100 mM potassium phosphate buffer (pH 7.4). Concentrations of organic solvents were kept to <1% (v/v). All incubations were pre-incubated at 37 C. for 5 minutes prior to addition of 1 mM NADPH (final concentration). Incubations were stopped after 5 (CYP3A4) or 15 minutes (CYP2C8 and CYP2C9) with one volume (v/v) of ice-cold acetonitrile containing 0.1 μM tolbutamide as an internal standard. All samples were vortexed and centrifuged prior to LC-MS/MS analysis.
Affinity data for this assay

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