Metabolic Activation of Diclofenac by Human Cytochrome P450 3A4:  Role of 5-Hydroxydiclofenac

Abstract

Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4'-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirus-expressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low.

Increase in covalent binding of 5-hydroxydiclofenac to hepatic tissues in rats co-treated with lipopolysaccharide and diclofenac: involvement in the onset of diclofenac-induced idiosyncratic hepatotoxicity

“…These findings are supported by recent reports (Mohamadin et al, 2011;Zou et al, 2010) and considered attributable to the oxidative stress generated by co-treatment with LPS. Shen et al (1999) have reported that covalent binding of reactive metabolites of 5-OH-DCF to human liver microsomes was inhibited by GSH. Based on this finding, the decreased GSH level in hepatic tissues is considered to imply a reduced detoxication reaction of GSH with 5-OH-DCF reactive metabolites, resulting in increased adducts of the reactive metabolites to hepatic tissues.…”

Drug-induced liver injury in older adults

“…Diclofenac is metabolized by the CYP enzymes 2C9 and 3A4, resulting in reactive metabolite(s) that bind to microsomal proteins [Naisbitt et al 2007;Bort et al 1999;Shen et al 1999]. Diclofenac-induced dose-and concentrationdependant liver injury has been demonstrated in rat hepatocytes [Kretzrommel and Boelsterli, 1993].…”

Diclofenac-Induced Inactivation of CYP3A4 and Its Stimulation by Quinidine

“…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.…”