CYTOCHROME-P450TB (CYP2C) - A MAJOR MONOOXYGENASE CATALYZING DICLOFENAC 4'-HYDROXYLATION IN HUMAN LIVER

The nature of the enzyme(s) catalyzing the major metabolic pathway of diclofenac, 4'-hydroxylation, was investigated in human liver microsomes. Inhibition studies were performed with tolbutamide and sulfaphenazole (respectively the prototype substrate and a selective inhibitor of cytochrome P450TB - CYP2C subfamily), and with phenytoin and (+/-)-warfarin, other proposed substrates of P450TB. Diclofenac 4'-hydroxylation displayed single enzyme Michaelis-Menten kinetics and was similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6), with a K(m) of 5.6 +/- 1.5 muM (mean +/- sd) and a V(max) of 60.6 +/- 23.5 nmol/mgP/h. Inhibition by tolbutamide, sulfaphenazole, phenytoin and (+/-)-warfarin was comparable in all livers, with values predicted from their K(m) or K(i) for cytochrome P450TB determined in separate studies and a competitive inhibition model. Sulfaphenazole competitively inhibited diclofenac 4'-hydroxylation (K(i) = 0.11 +/- 0.08 muM, n = 3). Diclofenac 4'-hydroxylation is predominantly catalyzed by a cytochrome P450 isozyme of the CYP2C subfamily, most likely CYP2C9. This particular isozyme therefore appears to be responsible for the oxidation of polar acidic substances such as non-steroidal anti-inflammatory drugs from different chemical classes. It also constitutes a common site for drug interactions involving these compounds, as well as tolbutamide, phenytoin and warfarin.