Roles of Human Hepatic Cytochrome P450s 2C9 and 3A4 in the Metabolic Activation of Diclofenac

Abstract

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed 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 incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. 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 inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between 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 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, 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 diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Diclofenac-Induced Inactivation of CYP3A4 and Its Stimulation by Quinidine

“…Because CYP3A4, a major component of diclofenac 5-hydroxylation (Shen et al, 1999;Tang et al, 1999b), was selectively inactivated, it is reasonable to postulate that the 5-hydroxylation pathway of diclofenac is responsible for the inactivation of CYP3A4. Because recent reports (Tang et al, 1999a;Ngui et al, 2000) showed that diclofenac 5-hydroxylation was stimulated by quinidine, the effect of the simulation on the inactivation of CYP3A4 was investigated.…”

“…CYP2C11 was shown to be responsible for major diclofenac metabolisms, 4Ј-hydroxylation and 5-hydroxylation ( Fig. 1), in rat liver microsomes (Masubuchi et al, 2001), whereas distinct P450 isoforms, CYP2C9 and CYP3A4, were involved in these pathways, respectively, in human liver microsomes (Leemann et al, 1993;Shen et al, 1999;Tang et al, 1999b). Because CYP2C9 was not inactivated during diclofenac metabolism (Masubuchi et al, 2001), it was suggested that the inactivation of CYP2C11 was independent of 4Ј-hydroxylation pathway, supposing alternatively that 5-hydroxylation is involved in the inactivation of CYP2C11.…”

“…Thus in humans, it is possible that CYP3A4, a major isoform responsible for diclofenac 5-hydroxylation, is susceptible to inactivation by diclofenac metabolism. It has been demonstrated that benzoquinone imine, a further metabolite of 5-hydroxydiclofenac, is chemically reactive and binds covalently to cellular protein (Shen et al, 1999;Tang et al, 1999b). Although implication of 5-hydroxydiclofenac for diclofenac-induced liver toxicity has been thus suggested, the effect on CYP3A4 catalytic ability has not been investigated.…”

Atypical Kinetic Profiles in Drug Metabolism Reactions

“…In previous studies, Tang et al (1999b) observed that the formation of diclofenac metabolites via CYP3A4 increased more than 4-fold in human liver microsomes in the presence of quinidine. Since similar results were seen in monkey liver microsomes, Tang and coworkers (1999a) followed this work with an in vivo study in monkeys and observed that in the presence of quinidine, diclofenac clearance was enhanced in three male rhesus monkeys by 57, 56, and 56%, respectively.…”

<i>In vitro</i> inhibitory activities of the extract of <i>Hibiscus sabdariffa</i> L. (family malvaceae) on selected cytochrome p450 isoforms

“…These drugs are metabolised by more than one CYP isoform: acetaminophen is metabolised by CYP isoforms 1A2, 2A6, 2D6, 2E1 and 3A4 (Patten et al, 1993); diclofenac by CYPs isoforms 2C9 and 3A4 (Tang et al, 1999) and chloroquine is metabolised by CYP2C8, 2D6 and 3A4/5 (Ducharme and Farinotti 1996; Kim et al, 2003). Drugs metabolised by more than one CYP isoforms or drugs that have other dominant metabolic pathways such as glucoronidation and sulphoxidation may not be significantly affected by inhibitors of only one of the CYP isoform since there is an alternate route of metabolism except if the inhibitor is able to inhibit all the metabolic pathways of the drug concerned.…”