Abstract
Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity.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 μM), 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 μM) with (R)-(−)-FLX (10 μM) 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 μM) with GSH (10 mM) in buffer (pH 7.4, 37°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.
Footnotes
↵1 Current affiliation: BioMarin Pharmaceutical Inc., Novato, California.
↵2 Current affiliation: School of Pharmacy, University of California at San Francisco, San Francisco, California.
This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM36633].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.029371
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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- FLX
- flunoxaprofen, (S)-(+)-2-[2-(4-fluorophenyl)-1,3-benzoxazol-5-yl]propanoic acid
- NSAID
- nonsteroidal anti-inflammatory drug
- BNX
- benoxaprofen, (R,S)-2-[2-(4-chlorophenyl)-1,3-benzoxazol-5-yl]propanoic acid
- FLX-CoA
- FLX-S-acyl-CoA thioester
- FLX-1-O-G
- FLX-1-O-acyl glucuronide
- GSH
- glutathione
- FLX-SG
- FLX-S-acyl-glutathione thioester
- CBZ
- carbamazepine
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- MS
- mass spectrometry
- CID
- collision-induced dissociation
- HPLC
- high-performance liquid chromatography
- MRM
- multiple reaction monitoring
- I-CoA
- (R,S)-ibuprofen-acyl-CoA
- I-SG
- ibuprofen-S-acyl-glutathione
- P450
- cytochrome P-450
- AUC
- area under the curve.
- Received July 7, 2009.
- Accepted September 25, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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