Abstract

Zomepirac (ZP), a nonsteroidal anti-inflammatory drug that was withdrawn from use, is metabolized to zomepirac-1-O-acyl-glucuronide (ZP-1-O-G), a chemically reactive conjugate that has been implicated in the toxicity of the drug. In the present studies, we investigated the ability of ZP to become bioactivated to reactive metabolites that transacylate glutathione (GSH) forming ZP-S-acyl-glutathione thioester (ZP-SG) in vitro and in vivo in rat. When ZP (100 μM) was incubated with rat hepatocytes, ZP-SG was detected in incubation extracts by a sensitive selected reaction monitoring liquid chromatography/tandem mass spectrometry (LC/MS-MS) technique. The initial formation rate of ZP-SG was rapid and reached a maximum concentration of 0.24 ± 0.03 nM after 4 min of incubation, then decreased, in a fairly linear fashion, to 0.07 ± 0.03 nM after 60 min of incubation. The product ZP-SG (1 μM) was shown to be unstable by undergoing rapid hydrolysis (apparent half-life ∼0.8 min) in incubations with rat hepatocytes. After administration of ZP to a male Sprague-Dawley rat (100 mg/kg i.p.), bile was collected and analyzed for ZP-SG by LC/MS-MS. Results indicated the presence of ZP-SG in bile (6.7 μg excreted after 6 h of collection), which was confirmed by coelution with synthetic standard and by its tandem mass spectrum. Together, these results demonstrate that ZP becomes metabolically activated in vitro in rat hepatocytes and in vivo in rat to reactive acylating derivative(s), such as ZP-1-O-G, that transacylate GSH forming ZP-SG. Finally, we propose that ZP-SG thioester could be used as a marker derivative for mechanistic studies on the bioactivation of the drug.