γ-Glutamyltranspeptidase-Mediated Degradation of Diclofenac-S-acyl-glutathione in Vitro and in Vivo in Rat
γ-Glutamyltranspeptidase-Mediated Degradation of Diclofenac-S-acyl-glutathione in Vitro and in Vivo in Rat
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DOI：
10.1021/tx800073p
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发表时间：
2008-10-01
影响因子：
4.1
通讯作者：
Ware, Joseph A.
中科院分区：
文献类型：
--
作者：
Grillo, Mark P.;Hua, Fengmei;Ware, Joseph A.
已知双氯芬酸是一种非甾体类药物，在大鼠体内以及体外大鼠和人肝细胞中代谢为化学反应性中间体，可转酰GSH形成双氯芬酸-S-酰基-谷胱甘肽（D-SG）。最近，据报告，双氯芬酸给药大鼠导致肝γ-谷氨酰转肽酶（γ-GT）（一种细胞外小管膜酶）活性显著降低。由于研究表明，D-SG是一种化学反应转酰物种，排泄到大鼠胆汁中，我们建议，D-SG在肝脏中形成的可能是一个底物，和潜在的抑制剂，肝γ-GT。本实验进行调查的能力，D-SG是一个底物γ-GT在大鼠体内和体外与市售的γ-GT酶。我们还检测了D-SG在体外抑制γ-GT的能力。因此，对Diclavac给药大鼠（200 mg/kg，iv）胆汁提取物的LC-MS/MS分析显示，存在γ-GT介导的D-SG降解产物Diclavac-N-酰基-半胱氨酰甘氨酸（D-N-CG），给药后6 h共排泄约8 μ g。当D-SG（100 μ M）与γ-GT（1单位/mL）孵育时，GSH加合物以线性时间依赖性方式降解，孵育20分钟后形成类似于94 μ M的D-N-CG。透析研究表明D-SG对γ-GT的抑制是完全可逆的。进一步的抑制研究表明，D-SG是γ-GT酶的竞争性抑制剂。这些研究的结果表明，D-SG是γ-GT的底物;然而，结合物可能不会显著降低大鼠体内报告的γ-GT活性。
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 similar to 8 mu g was excreted 6 h postadministration. When D-SG (100 mu M) was incubated with gamma-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where similar to 94 mu M 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.