title: Mechanistic studies on the bioactivation of diclofenac: identification of diclofenac-S-acyl-glutathione in vitro in incubations with rat and human hepatocytes.
abstract: Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to diclofenac-1-O-acyl glucuronide (D-1-O-G), a chemically reactive conjugate that has been implicated as playing a role in the idiosyncratic hepatoxicity associated with its use. The present studies investigated the ability of diclofenac to be metabolized to diclofenac-S-acyl-glutathione thioester (D-SG) in vitro in incubations with rat and human hepatocytes and whether its formation is dependent on a transacylation-type reaction between D-1-O-G and glutathione. When diclofenac (100 microM) was incubated with hepatocytes, D-SG was detected in both rat and human incubation extracts by a sensitive LC-MS/MS technique. The initial formation rate of D-SG in rat and human hepatocyte incubations was rapid and reached maximum concentrations of 1 and 0.8 nM, respectively, after 4 min of incubation. By contrast, during incubations with rat hepatocytes, the formation of D-1-O-G increased over 30 min of incubation, reaching a maximum concentration of 14.6 microM. Co-incubation of diclofenac (50 microM) with (-)-borneol (400 microM), an inhibitor of glucuronidation, led to a 94% decrease in D-1-O-G formation, although no significant decrease in D-SG production was observed. Together, these results indicate that diclofenac becomes metabolically activated in vitro in rat and human hepatocytes to reactive acylating derivatives that transacylate glutathione forming D-SG, but which is not solely dependent on transacylation by the D-1-O-G metabolite. From these results, it is proposed that reactive acylating metabolites of diclofenac, besides D-1-O-G, may be significant in the protein acylation that occurs in vivo and therefore also be important with regard to the mechanism(s) of diclofenac-mediated idiosyncratic hepatotoxicity.
authors: Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Inc., Kalamazoo, Michigan 49007-4940, USA. mark.grillo@msn.com
authors: Mark P
authors: MP
authors: Grillo
authors: Fengmei
authors: F
authors: Hua
authors: Charles G
authors: CG
authors: Knutson
authors: Joseph A
authors: JA
authors: Ware
authors: Chunze
authors: C
authors: Li
mesh_terms: D000215:Acylation; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D001711:Biotransformation; D001892:Camphanes / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D020719:Glucuronides / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013997:Time Factors
chemical_list: C487366:1-O-(2-((2',6'-dichlorophenyl)amino)phenylacetyl)glucopyranuronic acid; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001892:Camphanes; D020719:Glucuronides; C495780:diclofenac-S-acyl-glutathione; D004008:Diclofenac; D005978:Glutathione; C022871:isoborneol