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 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 ( 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 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.