In vitro enzymic synthesis of mammalian liver xenobiotic metabolites catalysed by ovine liver microsomal cytochrome P450

Ovine microsomal Cytochrome P450 was used preparatively in vitro to make milligram quantities of Nordiazepam, a known metabolite of the anxiolytic drug Diazepam. The product was purified by reversed phase chromatography and preparative HPLC. This synthetic approach should be of use in the preparation of purified metabolites for analytical reference purposes or research. Using demethylation of p-nitroanisole as a model reaction, the conditions required for optimum activity of ovine liver microsomal cytochrome P450 in vitro have been determined. Variation of both enzyme and substrate concentrations revealed saturation behavior in both cases. Above the optimal concentrations, no increase in yield of product was obtained. End-product inhibition was shown to occur and this accounts for the lower conversion rate at higher substrate concentrations. The enzyme showed reasonable resistance to denaturation by organic cosolvents (essential for substrate solubilisation). Up to 3% v/v of acetonitrile, methanol or DMSO had no apparent effect on the enzyme, reaction yields being unaffected. Another xenobiotic metabolizing enzyme, Glucuronyl transferase is also present in microsomes. Attempted simultaneous hydroxylation and glucuronidation, in the presence of UDPGA, the cofactor for Glucuronyl transferase was not successful, due to the effects of contaminating β-glucuronidase in the microsomes. The glucuronidation/de-glucuronidation did, however, increase the yield of hydroxylation product. Conditions which favour glucuronidation allowed some glucuronide to accumulate but the total yield of hydroxylation products (glucuronidated and free) was significantly reduced.