Search

Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac- S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic γ-glutamyltranspeptidase (γ-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic γ-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for γ-GT in vivo in rat and in vitro with commercially available γ-GT enzyme. We also examined the ability of D-SG to inhibit γ-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the γ-GT-mediated D-SG degradation product diclofenac- N-acyl-cysteinylglycine (D- N-CG), where a total of ∼8 μg was excreted 6 h postadministration. When D-SG (100 μM) was incubated with γ-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where ∼94 μM D- N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of γ-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the γ-GT enzyme. Results from theses studies indicate that D-SG is a substrate for γ-GT; however, the conjugate may not contribute significantly to the decrease in γ-GT activity reported to occur in vivo in rat. [ABSTRACT FROM AUTHOR]

Ibuprofen is metabolized to chemically reactive ibuprofen-1- O-acyl-glucuronide (I-1- O-G) and ibuprofen- S-acyl-CoA (I-CoA) derivatives, which are proposed to mediate the formation of drug−protein adducts via the transacylation of protein nucleophiles. We examined the ability of ibuprofen to undergo enantioselective metabolism to ibuprofen- S-acyl-glutathione thioester (I-SG) in incubations with rat hepatocytes, where I-CoA formation is known to be highly enantioselective in favor of the ( R)-(−)-ibuprofen isomer. We proposed that potential enantioselective transacylation of glutathione forming I-SG in favor of the ( R)-(−)-isomer would reveal the importance of acyl-CoA formation, versus acyl glucuronidation, in the generation of reactive transacylating-type intermediates of the drug. Thus, when ( R)-(−)- and ( S)-(+)-ibuprofen (100 μM) were incubated with hepatocytes, the presence of I-CoA and I-SG was detected in incubation extracts by LC-MS/MS techniques. The formation of I-CoA and I-SG in hepatocyte incubations with ( R)-(−)-ibuprofen was rapid and reached maximum concentrations of 2.6 μM and 1.3 nM, respectively, after 8−10 min of incubation. By contrast, incubations with ( S)-(+)-ibuprofen resulted in 8% and 3.9% as much I-CoA and I-SG formation, respectively, compared to that in corresponding incubations with the ( R)-(−)-isomer. Experiments with a pseudoracemic mixture of ( R)-(−)-[3,3,3- 2H 3]- and ( S)-(+)-ibuprofen showed that >99% of the I-SG detected in hepatocyte incubations contained deuterium and therefore was derived primarily from ( R)-(−)-ibuprofen bioactivation. Inhibition of ( R)-(−)-ibuprofen (10 μM) glucuronidation with (−)-borneol (100 μM) led to a 98% decrease in I-1- O-G formation; however, no decrease in I-SG production was observed. Coincubation with pivalic, valproic, or lauric acid (500 μM each) was shown to lead to a significant inhibition of I-CoA formation and a corresponding decrease in I-SG production. Results from these studies demonstrate that the reactive I-CoA derivative, and not the I-1- O-G metabolite, plays a central role in the transacylation of GSH in incubations with rat hepatocytes. [ABSTRACT FROM AUTHOR]