CYTOCHROME-P450 MEDIATED METABOLISM OF DIAZEPAM IN HUMAN AND RAT - INVOLVEMENT OF HUMAN CYP2C IN N-DEMETHYLATION IN THE SUBSTRATE CONCENTRATION-DEPENDENT MANNER

Metabolism of diazepam (DZP) was studied in vitro to clarify the invol vement of different forms of hepatic cytochrome P450 (CYP) in rats, an d humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by a nti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxyl ation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3 approximat ely 4 times higher than N-demethylation at 0.2 mm; the two activities were essentially the same at a lower substrate concentration (0.02 mm) . Inhibitions of the N-demethylation by anti-CYP2C antibody and S-meph enytoin also depended on the substrate concentration and was detectabl e only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low K(m) and low V(max), and high K(m) and high V(max). The former ac tivity seems to be mediated by a CYP2C P450 form. On the other hand, D ZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consist ent with the observation in vivo that DZP N-demethylation and S-mephen ytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms i n DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.