Abstract

It has been known that diclofenac is biotransformed into chemically reactive metabolites, which bind covalently to liver microsomal proteins, including cytochrome P450 enzyme(s). We have investigated the ability and selectivity of diclofenac to inactivate P450 enzymes. Preincubation of microsomes of untreated rats with diclofenac in the presence of NADPH resulted in time-dependent loss of testosterone 2α- and 16α-hydroxylation activities. No effect of the preincubation was observed on ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, or testosterone 6β-hydroxylation activity. The time-dependent decreases in testosterone 2α- and 16α-hydroxylation activities followed the pseudo-first order kinetics and were saturable with increasing diclofenac concentrations. Reduced glutathione was not capable of protecting against the decrease in the enzyme activities. These data establish that a mechanism-based inactivation of CYP2C11 occurs during the oxidative metabolism of diclofenac. The diclofenac concentrations required to achieve the half-maximal rate of inactivation (K_I) were 3 to 4 μM, which were close toK_m for the low-K_mcomponents for diclofenac 4′- and 5-hydroxylation activities (7.29 and 4.43 μM, respectively). Anti-CYP2C11 IgG inhibited diclofenac 4′- and 5-hydroxylation activities, indicating that CYP2C11 is a major isozyme responsible for these aromatic oxidations. The preincubation of microsomes with 4′- or 5-hydroxydiclofenac did not cause a decrease in testosterone 2α- or 16α-hydroxylation activity, suggesting that neither of the primary metabolites is a precursor of the metabolite that inactivates CYP2C11. Therefore, a highly reactive intermediate(s) inactivating CYP2C11, probably arene-oxide, appears to be generated during the process of diclofenac 4′- and/or 5-hydroxylation. Diclofenac metabolism in human liver microsomes did not cause inactivation of CYP2C9, a major isozyme involved in diclofenac 4′-hydroxylation. Because the human microsomes have high diclofenac 4′-hydroxylation but not 5-hydroxylation activity, importance of the latter pathway in the inactivation is suggested.