Gamma-glutamyltranspeptidase-mediated degradation of diclofenac-S-acyl-glutathione in vitro and in vivo in rat.
Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac-S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic gamma-glutamyltranspeptidase (gamma-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic gamma-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for gamma-GT in vivo in rat and in vitro with commercially available gamma-GT enzyme. We also examined the ability of D-SG to inhibit gamma-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the gamma-GT-mediated D-SG degradation product diclofenac-N-acyl-cysteinylglycine (D- N-CG), where a total of approximately 8 microg was excreted 6 h postadministration. When D-SG (100 microM) was incubated with gamma-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where approximately 94 microM D- N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of gamma-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the gamma-GT enzyme. Results from theses studies indicate that D-SG is a substrate for gamma-GT; however, the conjugate may not contribute significantly to the decrease in gamma-GT activity reported to occur in vivo in rat.
Grillo
Mark P
MP
Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Inc., Kalamazoo, Michigan 49007-4940, USA. grillo@amgen.com
Hua
Fengmei
F
March
Kristi L
KL
Benet
Leslie Z
LZ
Knutson
Charles G
CG
Ware
Joseph A
JA
D000818:Animals; D001646:Bile / Q000378:metabolism; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D004151:Dipeptides; C495780:diclofenac-S-acyl-glutathione; D004008:Diclofenac; C028505:cysteinylglycine; D005723:gamma-Glutamyltransferase; D005978:Glutathione