Enantioselective Formation of Ibuprofen-S-Acyl-Glutathione in Vitro in Incubations of Ibuprofen with Rat Hepatocytes

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)-(−)-[3,3,3-2H3]- 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.