Metabolic Stability Determination Using Hepatic Microsomes

The metabolic stability of test compounds was determined as described.⁵⁷ (link)^,58 (link) Incubation and experimental conditions with human hepatic microsomal fractions (purchased from BD Gentest as a pooled batch from 6 donors) were as follows: microsomal proteins, 1 mg/mL; bovine serum albumin, 1 mg/mL; substrate, 5 μM; incubation duration, 20 min; cytochrome P-450 monooxygenases (CYPs) and flavin containing monooxygenases (FMOs) cofactor, 1 mM NADPH. Enzyme activity was stopped with 1 volume of acetonitrile. Ketoconazole at a final concentration of 1.5 μM (100-fold above its Ki for CYP3A4) was used for the specific and potent inhibition of enzyme reactions catalyzed by CYP3A4. For each test compound and for each microsomal preparation, three incubations were prepared: absolute reference in buffer (without enzyme material, i.e., microsomes); incubation without NADPH cofactor (with microsomal fractions); and incubation with NADPH (with microsomal fractions). For most compounds, biotransformation, as observed in hepatic microsomal fractions in the presence of the NADPH cofactor, consists of oxidative reactions catalyzed by either CYPs or FMOs. In these conditions, the percentage of total metabolism, which corresponds to oxidative metabolism, was determined as follows: [% total metabolism] ≈ [% oxidative metabolism] = [unchanged compound (UC) peak area − NADPH UC peak area + NADPH] × 100%, where NADPH corresponds to the enzyme cofactor for oxidation reactions catalyzed by either CYP or FMO.