Abstract
Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to a reactive acyl glucuronide that has been proposed to mediate toxic adverse drug reactions associated with its use. In the present study, we examined the ability of diclofenac acyl glucuronide (D-1-O-G) to transacylate glutathione (GSH) in vitro in buffer and in vivo in rats. Thus, in vitro reactions of D-1-O-G (100 μM) with GSH (10 mM) at pH 7.4 and 37°C showed a linear time-dependent formation of diclofenac-S-acyl-glutathione (D-SG, 3 μM/h) through 60 min of incubation, reaching a maximum of 3.7 μM after 2 h of incubation. The major reaction that occurred was acyl migration of D-1-O-G (t1/2, 54 min) to less reactive isomers. The D-SG thioester product was shown to be unstable by degrading primarily to 1-(2,6-dichlorophenyl)indolin-2-one and by hydrolysis to diclofenac. After administration of diclofenac to rats (200 mg/kg), bile was collected and analyzed for D-SG by liquid chromatography-tandem mass spectrometry. Results indicated the presence of D-SG, which was confirmed by coelution with synthetic standard and by its tandem mass spectrum. When the reactivity of D-SG (100 μM) was compared with D-1-O-G (100 μM) in vitro in reactions with N-acetylcysteine (NAC, 10 mM), results showed the quantitative reaction of D-SG with NAC after 30 min of incubation, whereas only ∼1% of D-1-O-G reacted to form diclofenac-S-acyl-NAC at the same time point. Results from these studies indicate that GSH reacts with D-1-O-G in vitro, and presumably in vivo, to form D-SG, and that the product D-SG thioester is chemically more reactive in transacylation-type reactions than the D-1-O-G metabolite.
Footnotes
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↵1 Abbreviations used are: D-1-O-G, diclofenac-1-O-acyl glucuronide; UGT, uridine diphosphate glucuronosyltransferase; GSH, glutathione; D-SG, diclofenac-S-acyl-glutathione thioester; D-SCoA, diclofenac-S-acyl-CoA thioester; CBZ, carbamazepine; β-glucuronidase, β-D-glucuronide glucuronosohydrolase; D-SNAC, diclofenac-S-acyl-N-acetylcysteine thioester; HPLC, high-performance liquid chromatography; SVC, superior vena cava; ACN, acetonitrile; LC-MS/MS, liquid chromatography-tandem mass spectrometry; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; NAC, N-acetylcysteine; CID, collision-induced dissociation; MRM, multiple reaction monitoring; SRM, selected reaction monitoring.
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A preliminary account of this work was presented at the XIVth World Congress of Pharmacology (IUPHAR) in San Francisco, CA in July 2002.
- Received February 4, 2003.
- Accepted July 28, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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