title: Characterization of the acyl-adenylate linked metabolite of mefenamic Acid.
abstract: Mefenamic acid, (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug (NSAID), is metabolized into the chemically reactive conjugates MFA-1-O-acyl-glucuronide (MFA-1-O-G) and MFA-S-acyl-CoA (MFA-CoA), which are both implicated in the formation of MFA-S-acyl-glutathione (MFA-GSH) conjugates, protein-adduct formation, and thus the potential toxicity of the drug. However, current studies suggest that an additional acyl-linked metabolite may be implicated in the formation of MFA-GSH. In the present study, we investigated the ability of MFA to become bioactivated into the acyl-linked metabolite, mefenamyl-adenylate (MFA-AMP). In vitro incubations in rat hepatocytes with MFA (100 μM), followed by LC-MS/MS analyses of extracts, led to the detection of MFA-AMP. In these incubations, the initial rate of MFA-AMP formation was rapid, leveling off at a maximum concentration of 90.1 nM (20 s), while MFA-GSH formation increased linearly, reaching a concentration of 1.7 μM after 60 min of incubation. In comparison, MFA-CoA was undetectable in incubation extracts until the 4 min time point, achieving a concentration of 45.6 nM at the 60 min time point, and MFA-1-O-G formation was linear, attaining a concentration of 42.2 μM after 60 min of incubation. In vitro incubation in buffer with the model nucleophile glutathione (GSH) under physiological conditions showed MFA-AMP to be reactive toward GSH, but 11-fold less reactive than MFA-CoA, while MFA-1-O-G exhibited little reactivity. However, in the presence of glutathione-S-transferase (GST), MFA-AMP mediated formation of MFA-GSH increased 6-fold, while MFA-CoA mediated formation of MFA-GSH only increased 1.4-fold. Collectively, in addition to the MFA-1-O-G, these results demonstrate that mefenamic acid does become bioactivated by acyl-CoA synthetase enzyme(s) in vitro in rat hepatocytes into the reactive transacylating derivatives MFA-AMP and MFA-CoA, both of which contribute to the transacylation of GSH and may be involved in the formation of protein adducts and potentially elicit an idiosyncratic toxicity.
authors: Department of Bioengineering and Therapeutic Sciences, University of California-San Francisco, CA, USA.
authors: Howard
authors: H
authors: Horng
authors: Leslie Z
authors: LZ
authors: Benet
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000215:Acylation; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D008297:Male; D008528:Mefenamic Acid / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
chemical_list: D000214:Acyl Coenzyme A; D000894:Anti-Inflammatory Agents, Non-Steroidal; D008528:Mefenamic Acid; D000249:Adenosine Monophosphate; D005978:Glutathione
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