Dmd048645 111..121

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), ibuprofenN-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 mg excreted 10-hours postadministration, respectively). I-N-CG is proposed to be formed from the degradation of I-SG by g-glutamyltranspeptidase (g-GT)-mediated cleavage of the g-glutamyl group, leading to an unstable ibuprofenS-acyl-cysteinylglycine (I-S-CG) intermediate that undergoes spontaneous S to N intramolecular rearrangement. Then, dipeptidasemediated cleavage of glycine from I-N-CG leads to the formation of I-N-C. Treatment of racemic I-SG (100 mM) in vitro with commercially available bovine kidney g-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 g-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 g-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 amidelinked conjugates, respectively.