Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: Identification of diclofenac-S-acyl-glutathione in rat bile

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 muM) with GSH (10 mM) at pH 7.4 and 37 degreesC showed a linear time-dependent formation of diclofenac-S-acyl-glutathione (D-SG, 3 muM/h) through 60 min of incubation, reaching a maximum of 3.7 muM after 2 h of incubation. The major reaction that occurred was acyl migration of D-1-O-G (t(1/2), 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 muM) was compared with D-1-O-G (100 muM) 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 similar to1% 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.