title: Enantioselective formation of ibuprofen-S-acyl-glutathione in vitro in incubations of ibuprofen with rat hepatocytes.
abstract: 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 microM) 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 microM 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-(2)H3]- 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 microM) glucuronidation with (-)-borneol (100 microM) 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 microM 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.
authors.affiliation: Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Inc., Kalamazoo, Michigan 49007-4940, USA. grillo@amgen.com
authors.forename: Mark P
authors.initials: MP
authors.lastname: Grillo
authors.forename: Fengmei
authors.initials: F
authors.lastname: Hua
mesh_terms: D000818:Animals; D001892:Camphanes / Q000494:pharmacology; D002853:Chromatography, Liquid; D005978:Glutathione / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism*; D007052:Ibuprofen / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007850:Lauric Acids / Q000494:pharmacology; D008297:Male; D013058:Mass Spectrometry; D008968:Molecular Conformation; D010421:Pentanoic Acids / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013237:Stereoisomerism; D013463:Sulfuric Acid Esters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014635:Valproic Acid / Q000494:pharmacology
chemical_list: D001892:Camphanes; D007850:Lauric Acids; D010421:Pentanoic Acids; D013463:Sulfuric Acid Esters; C534677:ibuprofen-S-acyl-glutathione; C030358:lauric acid; D014635:Valproic Acid; C005566:pivalic acid; D005978:Glutathione; C022871:isoborneol; D007052:Ibuprofen