Received for publication March 3, 2008.
Revised June 3, 2008.
Accepted for publication June 4, 2008.

A Novel Bioactivation Pathway for Diclofenac Initiated by P450 Mediated Oxidative Decarboxylation

Abstract

Diclofenac, a nonsteroidal antiinflammatory drug, undergoes bioactivation by cytochrome P450 oxidation to chemically-reactive metabolites that are capable of reacting with endogenous nucleophiles such as glutathione (GSH) and proteins and which may play a role in the idiosyncratic hepatotoxicity associated with the drug. Here, we investigated the ability of diclofenac to be metabolized to 2-(2,6-dichlorophenylamino)-benzyl-S-thioether glutathione (DPAB-SG) in incubations with rat (RLM) and human (HLM) liver microsomes fortified with NADPH and GSH. Thus, after incubation of diclofenac (50 µM) with liver microsomes (1 mg protein/mL), the presence of DPAB-SG was detected in both RLM and HLM incubation extracts by LC-MS/MS techniques. The formation of DPAB-SG was NADPH-, concentration- and time-dependent. Coincubation of diclofenac (10 µM) with ketoconazole (1 µM), an inhibitor of P450 3A4, with HLM led to a 75% decrease in DPAB-SG formation. However, by contrast, coincubation with the P450 2C9 inhibitor sulfaphenazole (10 µM) or the P450 2D6 inhibitor quinidine (40 µM) led to a 1.9 and 1.6 fold increase in DPAB-SG production, respectively. From these data, we propose that P450 3A4 mediates the oxidative decarboxylation of diclofenac resulting in the formation of a transient benzylic carbon-centered free radical intermediate which partitions between elimination (ortho-imine methide production) and recombination (alcohol formation) pathways. The benzyl alcohol intermediate, which was not analyzed for in the present studies, if formed could undergo dehydration to provide a reactive ortho-imine methide species. The ortho-imine methide intermediate then is proposed to react covalently with GSH forming DPAB-SG.

Key words: bioactivation, chemical toxicology, CYP3A, cytochrome P450 catalyzed oxidations, glutathione conjugates, hypersensitivity, idiosyncratic drug reactions, non-steroidal anti-inflammatory drugs