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 µ g 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 µ M) 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.