Human Cytochrome P450 Biocatalysts for the Facile Synthesis of Metabolites

Characterizing the human P450 metabolism of potential drug candidates requires considerable effort, due to the instability and poor activity levels of commercially available human cytochrome enzymes. The investigator can choose from many sources: pooled human liver microsomes, recombinant microsomes, or purified recombinant P450s. These forms have their limitations ‐‐ poor stability, troublesome reconstitution, and high cost. Using these formulations to generate milligram levels of P450 metabolites would be inefficient and prohibitively expensive. To address these limitations, we have developed Human Cytochrome Biocatalysts that can produce preparatively useful levels of metabolite. Each freeze‐dried biocatalyst product incorporates both the P450 and human P450 reductase into a fully functional catalytic system. The corresponding dry Reaction Buffer Mix contains NADPH cofactor, a cofactor recycling system, and stabilizers that improve both the activity and stability of the cytochrome during the reaction. To run a reaction, simply reconstitute with water and add the drug to be metabolized. In most cases, metabolite yields approaching 100% are possible. Using testosterone hydroxylation as a test case, Human Cytochrome Biocatalyst 3A4 converted greater than 70% of the 100 mg testosterone starting material into 40 mg 6β‐hydroxytestosterone (34 mg isolated yield following extraction and flash chromatography) and 40 mg other hydroxylated metabolites. A proprietary drug candidate with unknown P450 metabolism was successfully screened against the Human Cytochrome Biocatalysts. Three metabolites were identified, and the reactions were scaled up to deliver at least 100 μg of each metabolite, purified by preparative HPLC, for structure determination by NMR.