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Studies on the Metabolic Activation of Mefenamic Acid: Characterization of the Acyl-Linked Metabolite, Mefenamyl-Adenylate

  • Author(s): Horng, Howard Tony
  • Advisor(s): Benet, Leslie Z
  • et al.
Abstract

Mefenamic acid, (MFA), a nonsteroidal anti-inflammatory drug, is metabolized to MFA-1-O-acyl-glucuronide (MFA-1-O-Gluc), a chemically-reactive conjugate implicated in the formation of protein-adducts and the potential toxicity of the drug. We investigated the ability of mefenamic acid to become bioactivated to reactive metabolites that transacylate glutathione (GSH) forming MFA-S-acyl-glutathione (MFA-GSH) in vitro in incubations with rat hepatocytes. Mefenamic acid (100 µM) incubations, followed by LC-MS/MS analysis, led to the detection of MFA-GSH. The initial formation of MFA-GSH was rapid reaching a concentration of 1.7 ìM after 60-min of incubation. The product MFA-GSH was shown to be unstable in incubations with rat hepatocytes (t1/2 ~10 min). MFA-S-acyl-CoA (MFA-CoA) was undetectable until the 4-min time point, reaching a concentration of 45.6 nM at the 60-min time point. MFA-1-O-Gluc reached a Cmax of 42.2 ìM after 1-h of incubation. Co-incubation of MFA (10 µM, 10-min) with (-)-borneol (100 µM), an inhibitor of glucuronidation, led to a 91.1% decrease in MFA-1-O-Gluc formation, however no inhibition of MFA-SG formation was observed. By contrast, co-incubation with lauric acid (1 mM), an inhibitor of acyl-CoA formation, led to a 66.1% inhibition of MFA-GSH formation. Since these data does not completely explain the formation of MFA-GSH, we predicted that the intermediate MFA-acyl-adenylate (MFA-AMP) may be responsible in mediating the formation of MFA-GSH. MFA-AMP was detected in rat hepatocyte incubation extracts forming a concentration of 90.1 nM at the 20-sec time point. MFA-AMP was shown to be reactive with GSH, but ~10-fold less reactive than MFA-CoA, however, in the presence of GST, MFA-AMP mediated formation of MFA-GSH increased 6-fold. MFA-1-O-Gluc did not react with GSH to form MFA-GSH. MFA-GSH has also been shown to be reactive in itself toward N-acetyl-cysteine. ABT and temperature dependent inhibition of hepatocyte timecourse of formation incubations reveal that MFA-GSH formation is mediated by GST via MFA-AMP. These results demonstrate that mefenamic acid becomes bioactivated in vitro in rat hepatocytes to reactive transacylating derivatives into MFA-AMP and MFA-CoA that contribute to the transacylation of GSH forming MFA-GSH.

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