Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metaboli tes also were detected in human hepatocyte cultures in the form of glutathi one (GSH) adducts. This report describes the results of further studies aim ed at characterizing the human hepatic P450-mediated bioactivation of diclo fenac. The reactive metabolites formed in vitro were trapped by GSH and ana lyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion -S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubation s of diclofenac with human liver microsomes in the presence of NADPH and GS H. The formation of the adducts was taken to reflect the intermediacy of th e corresponding putative benzoquinone imines. While M2 was the dominant met abolite over a substrate concentration range of 10-50 mu M, M1 and M3 becam e equally important products at greater than or equal to 100 mu M diclofena c. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibite d by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M 2 was generated only by P450 2C9catalyzed reaction, while M1 and M3 were pr oduced by P450 3A4-catalyzed reaction. Good correlations were established b etween the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activiti es (r. = 0.98, n = 10) in human liver microsomal incubations. Taken togethe r, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of dic lofenac, P450 2C9 is capable of producing the benzoquinone imine intermedia te at lower drug concentrations which may be more clinically relevant.