Interaction of γ-glutamyltranspeptidase with ibuprofen-S-acyl-glutathione in vitro and in vivo in human.
Ibuprofen is metabolized to chemically reactive acyl glucuronide and S-acyl-CoA metabolites that are proposed to transacylate glutathione (GSH) forming ibuprofen-S-acyl-GSH (I-SG) in vivo. Herein, we report the detection of novel metabolites of ibuprofen, namely ibuprofen-N-acyl-cysteinylglycine (I-N-CG), ibuprofen-N-acyl-cysteine (I-N-C), and the mercapturic acid conjugate, ibuprofen-S-acyl-N-acetylcysteine (I-S-NAC), in urine from an ibuprofen-dosed volunteer. Thus, analysis of ibuprofen-dosed (Advil, 800 mg, Pfizer, Madison, NJ) human urine extracts by sensitive liquid chromatography tandem mass spectrometric detection resulted in the identification of I-N-CG, I-N-C, and I-S-NAC derivatives as minor metabolites (6.0, 1.7, and 0.2 µg excreted 10-hours postadministration, respectively). I-N-CG is proposed to be formed from the degradation of I-SG by γ-glutamyltranspeptidase (γ-GT)-mediated cleavage of the γ-glutamyl group, leading to an unstable ibuprofen-S-acyl-cysteinylglycine (I-S-CG) intermediate that undergoes spontaneous S to N intramolecular rearrangement. Then, dipeptidase-mediated cleavage of glycine from I-N-CG leads to the formation of I-N-C. Treatment of racemic I-SG (100 µM) in vitro with commercially available bovine kidney γ-GT (0.1 units/ml) in buffer at pH 7.4 and 37°C resulted in its complete degradation, yielding (R)- and (S)-I-N-CG after 15 minutes of incubation. In vitro enzyme kinetic studies with bovine kidney γ-GT incubated separately with (R)- and (S)-I-SG isomers revealed no enantioselective degradation. Results from these studies provided evidence that ibuprofen is metabolized in human to reactive transacylating-type intermediates that react with GSH, forming I-SG thioester that, following degradation by γ-GT and dipeptidase enzymes and following S to N intramolecular rearrangement, leads to the urinary excretion of the I-N-CG and I-N-C amide-linked conjugates, respectively.
Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA. grillo@amgen.com
Mark P
MP
Grillo
Michelle Tadano
MT
Lohr
Smriti
S
Khera
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007052:Ibuprofen / Q000031:analogs & derivatives* / Q000378:metabolism; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D053719:Tandem Mass Spectrometry; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
C534677:ibuprofen-S-acyl-glutathione; D005723:gamma-Glutamyltransferase; D005978:Glutathione; D007052:Ibuprofen