Mechanism-based inactivation of CYP2C11 by diclofenac.
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 2alpha- and 16alpha-hydroxylation activities. No effect of the preincubation was observed on ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, or testosterone 6beta-hydroxylation activity. The time-dependent decreases in testosterone 2alpha- and 16alpha-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 microM, which were close to K(m) for the low-K(m) components for diclofenac 4'- and 5-hydroxylation activities (7.29 and 4.43 microM, 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 2alpha- or 16alpha-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.
Masubuchi
Y
Y
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Ose
A
A
Horie
T
T
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D065607:Cytochrome P-450 Enzyme Inhibitors; D000072467:Cytochrome P450 Family 2; D004008:Diclofenac / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors*; D013739:Testosterone / Q000378:metabolism; D014044:Tolbutamide / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004008:Diclofenac; D013739:Testosterone; D014044:Tolbutamide; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; D005978:Glutathione