Flunoxaprofen

Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(-)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereoselective metabolism of (R)-(-)- and (S)-(+)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(-)- or (S)-(+)-FLX (100 microM), both FLX-CoA and FLX-SG were detected by sensitive liquid chromatography-tandem mass spectrometry techniques. However, these derivatives were observed primarily from (R)-(-)-FLX incubation extracts, for which the formation rates of FLX-CoA and FLX-SG were rapid, reaching maximum concentrations of 42 and 2.8 nM, respectively, after 6 min of incubation. Incubations with (S)-(+)-FLX over 60 min displayed 8.1 and 2.7% as much FLX-CoA and FLX-SG area under the concentration versus time curves, respectively, compared with corresponding incubations with (R)-(-)-FLX. Coincubation of lauric acid (1000 microM) with (R)-(-)-FLX (10 microM) led to the complete inhibition of FLX-CoA formation and a 98% inhibition of FLX-SG formation. Reaction of authentic (R,S)-FLX-CoA (2 microM) with GSH (10 mM) in buffer (pH 7.4, 37 degrees C) showed the quantitative formation of FLX-SG after 3 h of incubation. Together, these results demonstrate the stereoselective transacylation of GSH in hepatocyte incubations containing (R)-(-)-FLX, which is consistent with bioactivation by stereoselective (R)-FLX-CoA formation.

Benoxaprofen (BNX), a nonsteroidal anti-inflammatory drug (NSAID) that was withdrawn because of hepatotoxicity, is more toxic than its structural analog flunoxaprofen (FLX) in humans and rats. Acyl glucuronides have been hypothesized to be reactive metabolites and may be associated with toxicity. Both time- and concentration-dependent glucuronidation and covalent binding of BNX, FLX, and ibuprofen (IBP) were determined by exposing sandwich-cultured rat hepatocytes to each NSAID. The levels of glucuronide and covalent protein adduct measured in cells followed the order BNX > FLX > IBP. These results indicate that 1) BNX-glucuronide (G) is more reactive than FLX-G, and 2) IBP-G is the least reactive metabolite, which support previous in vivo studies in rats. The proportional increases of protein adduct formation for BNX, FLX, and IBP as acyl glucuronidation increased also support the hypothesis that part of the covalent binding of all three NSAIDs to hepatic proteins is acyl glucuronide-dependent. Moreover, theses studies confirmed the feasibility of using sandwich-cultured rat hepatocytes for studying glucuronidation and covalent binding to hepatocellular proteins. These studies also showed that these in vitro methods can be applied using human tissues for the study of acyl glucuronide reactivity. More BNX-protein adduct was formed in sandwich-cultured human hepatocytes than FLX-protein adduct, which not only agreed with its relative toxicity in humans but also was consistent with the in vitro findings using rat hepatocyte cultures. These data support the use of sandwich-cultured human hepatocytes as an in vitro screening model of acyl glucuronide exposure and reactivity.