Liver microsomal metabolic stability

UNC10201652 (1 μM) was incubated with human, mouse and rat liver microsomes at a final protein concentration of 0.5 mg/mL in 0.1 M phosphate buffer pH 7.4. The final DMSO concentration in the incubation was 0.25%. Samples were pre-incubated at 37 °C with NADPH (at a final concentration of 1 mM) added to initiate the reaction. A control incubation, with NADPH replaced with 0.1 M (pH 7.4) phosphate buffer, was also performed. Two positive control compounds were incubated in addition to UNC10201652 for each species (human, dextromethorphan and verapamil; mouse and rat, diazepam and diphenhydramine). All incubations were performed on the individual test compounds.

UNC10201652 was incubated with NADPH for 0, 5, 15, 30 and 45 min (the control incubation without NADPH was 45 min only) with incubations terminated by transferring one volume of incubate into three volumes of acetonitrile (1:3 v/v). The precipitated proteins were removed by centrifugation at 1530g for 30 min at 4 °C.

For determination of the intrinsic clearance (CLint) of UNC10201652, an aliquot (50 μL) of the supernatant was diluted with 50 μL of deionised water containing 0.55 μM metoprolol which was employed as an internal standard. For the metabolite identification studies (see below), 80 μL of the supernatant was diluted with 80 lL of deionised water.