Bioreactor Systems in Drug Metabolism: Synthesis of Cytochrome P450-Generated Metabolites

In this communication, we report that suspension cultures of Sf 21 insect cells, co-infected with baculovirus containing the cDNA for a single cytochrome P450 and NADPH–cytochrome P450 oxidoreductase, can be employed successfully as “bioreactors” for the synthesis of milligram quantities of cytochrome P450-generated metabolite(s). Three standard or probe substrates for the human P450s were chosen for the initial biosynthetic experiments: testosterone, diazepam, and diclofenac. Testosterone (100 μM, 2.88 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to 6β-hydroxytestosterone (2.3 mg) and 15β-hydroxytestosterone (0.18 mg). Diazepam (100 μM, 2.9 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to temazepam (1.1 mg), N-demethyldiazepam (0.35 mg), and oxazepam (0.15 mg). Diclofenac (100 μM, 3.18 mg/100 ml), added to a 100-ml CYP2C9 bioreactor, was converted to 4′-hydroxydiclofenac (2.6 mg). Since the goal for the development of the bioreactors was to provide a platform for both the production and subsequent purification of milligram quantities of P450-generated metabolite(s), a second 100-ml CYP2C9 bioreactor was used for the large-scale production and subsequent purification of 4′-hydroxydiclofenac. After 55 h of incubation, 7.95 mg of diclofenac was converted to 4.35 mg of 4′-hydroxydiclofenac, while 3.55 mg of unchanged diclofenac remained in the bioreactor. Using a simple preparative HPLC method, approximately 2.2 mg of 4′-hydroxydiclofenac and 1.9 mg of diclofenac were recovered from this experiment (28% yield). These results indicate clearly that suspension cultures of Sf 21 insect cells coexpressing a cytochrome P450 and NADPH–cytochrome P450 oxidoreductase can be used effectively as bioreactors for the production and subsequent purification of milligram quantities of P450-derived metabolite(s).