Bioreactor systems in drug metabolism: synthesis of cytochrome P450-generated metabolites.
In this communication, we report that suspension cultures of Sf21 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 microM, 2.88 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to 6beta-hydroxytestosterone (2.3 mg) and 15beta-hydroxytestosterone (0.18 mg). Diazepam (100 microM, 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 microM, 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 Sf21 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).
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. tom_rushmore@merck.com
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D000818:Animals; D016367:Baculoviridae; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism
D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase