CYP3A4 Inhibition Assay Protocol

Incubations were performed with 0.1 mg/mL human liver microsomes (purchased from BD Gentest as a pooled batch from 150 donors) in a final incubation volume of 0.25 mL. The incubation medium contained 0.05 M phosphate buffer (pH 7.4) containing an NADPH regenerating system (including 1.3 mM NADP, 3.3 mM glucose 6-phosphate,3.3 mM MgCl2, and 1.0 U/mL glucose 6-phosphate dehydrogenase). Probe CYP3A4 substrates (midazolam at 3 μM and testosterone at 50 μM), were incubated for 10 and 30 min, respectively, with increasing concentrations of GM, MGM or CGM (concentration range: 1 - 30 μM). The reaction was stopped by adding acetonitrile to precipitate the proteins. The incubation mixtures were then centrifuged for 5 min at 10,000 × g, and an aliquot of the supernatant was analyzed using high-performance liquid chromatography coupled to mass spectrometry for the assessment of 1’-hydroxymidazolam and 6β-hydroxytestosterone metabolite formation.