Short Communication A Novel Bioactivation Pathway for 2-[2-(2,6-Dichlorophenyl) aminophenyl]ethanoic Acid (Diclofenac) Initiated by Cytochrome P450-Mediated Oxidative Decarboxylation

Diclofenac (2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid), 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 that 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-dichloro-phenylamino)benzyl-S-thioether glutathione (DPAB-SG) in incubations with rat liver microsomes (RLMs) and human liver microsomes (HLMs) fortified with NADPH and GSH. Thus, after incubation of diclofenac (50 M) with liver microsomes (1 mg protein/ml), the presence of DPAB-SG was detected in both RLM and HLM incubation extracts by liquid chromatography-tandem mass spectrometry techniques. The formation of DPAB-SG was NADPH-, concentration-, and time-dependent. Coincubation of diclofenac (10 M) with ketoconazole (1 M), an inhibitor of cytochrome P450 (P450) 3A4, with HLMs led to a 75% decrease in DPAB-SG formation. However, in contrast, coincubation with the P450 2C9 inhibitor sulfaphenazole (10 M) or the P450 2D6 inhibitor quinidine (40 M) led to a 1.9and 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 that partitions between elimination (o-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 o-imine methide species. The o-imine methide intermediate then is proposed to react covalently with GSH, forming DPAB-SG. Diclofenac (Fig. 1), a nonsteroidal antiinflammatory drug used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain (Small, 1989), is metabolically activated by cytochrome P450 to chemically reactive species that react with endogenous nucleophiles such as glutathione (GSH) and protein (Tang, 2003). Adverse reactions to diclofenac include enteropathy (common with longterm use), acute and chronic hepatitis (15% of patients have increased plasma transaminase levels), hemolytic anemia, and fatal anaphylaxis (i.e., IgEor IgG-mediated immune response). Reactive intermediates have been proposed to play a role in the rare, but sometimes severe, toxicity associated with use of the drug (Boelsterli, 2003). Diclofenac is metabolized by cytochromes P450 3A4 and 2C9 by oxidation of the aromatic ring to 5and 4 -hydroxylated products, respectively, which can undergo further P450-mediated oxidation to quinone imine-type-reactive metabolites that react covalently with GSH-forming conjugates readily detected by LC-MS/MS techniques (Tang et al., 1999a). The most recent reported glutathione conjugate of diclofenac, namely 2-(2,6-dichlorophenylamino)benzyl-S-thioether glutathione (DPAB-SG), was detected from high-resolution accurate mass LTQ-Orbitrap mass spectrometric analysis of extracts from rat and human hepatocyte incubations with diclofenac and from extracts of diclofenac-dosed rat bile (Teffera et al., 2008). In that same report, 2-(2,6-diclorophenylamino)benzoic acid was also identified as a metabolite of diclofenac excreted in rat bile and formed in rat and human hepatocyte incubations. However, in that report, 2-(2,6-diclorophenylamino)benzoic acid and DPAB-SG were not detected in extracts from rat and human liver microsomal preparations fortified with NADPH and GSH. The present studies were designed to examine more closely the ability of cytochrome P450 to catalyze the NADPHand GSH-dependent metabolism of diclofenac, leading to the formation of DPAB-SG. We propose that oxidative decarboxylation of diclofenac could be mediated by P450 catalysis. The decarboxylation of arylacetic acidand 2-arylpropionic acid-containing drugs by cytochrome P450 has been reported (Komuro et al., 1995). Accordingly, we propose that diclofenac may undergo a similar P-450-mediated oxidative decarboxylation, leading to a transient 2-(2,6-dichlorophenylamino)benzyl-carbon-centered free radical that could either recombine with hydroxyl radical-forming 2-(2,6dichlorophenylamino)benzyl alcohol that undergoes dehydration, leading to the o-imine methide, or undergo further oxidation, leading directly to the o-imine methide reactive intermediate (Fig. 1). Reaction of the o-imine methide intermediate at the benzylic position with GSH then forms DPAB-SG conjugate as proposed by Teffera et al. (2008). Materials and Methods Materials. Diclofenac sodium, carbamazepine, ketoconazole, sulfaphenazole, quinidine, and glutathione were purchased from Sigma-Aldrich (St. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.021287. ABBREVIATIONS: diclofenac, 2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid; GSH, glutathione; P450, cytochrome P450; LC-MS/MS, liquid chromatography-tandem mass spectrometry; DPAB-SG, 2-(2,6-dichloro-phenylamino)benzyl-S-thioether glutathione; RLM, rat liver microsome; HLM, human liver microsome; CID, collisionally induced dissociation; ACN, acetonitrile; CBZ, carbamazepine. 0090-9556/08/3609-1740–1744$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 9 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 21287/3372211 DMD 36:1740–1744, 2008 Printed in U.S.A. 1740 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from