Bailey M J, Dickinson R G
Department of Medicine, University of Queensland, Brisbane, Australia.
Chem Res Toxicol. 1996 Apr-May;9(3):659-66. doi: 10.1021/tx960017o.
Carboxylate drugs usually form acyl glucuronide conjugates as major metabolites. These electrophilic metabolites are reactive, capable of undergoing hydrolysis, rearrangement, and covalent binding reactions to proteins. The last-mentioned property has the potential to initiate immune and other toxic responses in vivo. In this study, we compared the extent and pattern of covalent adduct formation in plasma and livers of rats dosed with the nonsteroidal anti-inflammatory drugs (NSAIDs) zomepirac (ZP) and diflunisal (DF), the hypolipidemic agent clofibric acid (CA), and the anti-epileptic agent valproic acid (VPA). These drugs form acyl glucuronides with diverse intrinsic reactivities (apparent first order degradation t 1/2 values of 0.5, 0.6, 3, and 60 h, respectively). Rats were dosed iv twice daily for 2 days (50 mg/kg for ZP, DF, and CA, 150 mg/kg for VPA). Chemical analysis of tissues obtained 6 h after the last dose revealed adduct concentrations of 0.31, 0.44, 0.28, and 0.05 micrograms of drug equivalents/mL of plasma and 2.21, 2.31, 0.96, and 0.96 micrograms of drug equivalents/g of liver for ZP, DF, CA and VPA treatments, respectively. For both plasma and liver, the higher concentrations of adducts were found with ZP and DF, which have the more reactive glucuronides. The low concentrations of VPA adducts found in plasma were in keeping with the very low reactivity of its glucuronide. In liver, however, VPA adducts achieved concentrations of the same order of magnitude as the other drugs and were accompanied by adducts of the (E)-2-en metabolite of VPA at 0.38 micrograms of VPA equivalents/g of liver. The liver data for VPA can be explained by an acyl CoA/beta-oxidation pathway of adduct formation in addition to that from acyl glucuronidation. Immunoblotting using rabbit polyclonal antisera raised against synthetic drug-protein adducts revealed major bands at 110, 140, and approximately 200 kDa in livers of ZP- and DF-treated rats. A fourth major band at 70 kDa in ZP-treated liver had the same apparent molecular weight as the only major band detected in CA-treated liver. A 140 kDa band was detected in liver tissue from VPA-treated rats, as well as several lower molecular weight bands. In plasma, the antisera specifically detected drug-modified serum albumin in samples from rats treated with ZP, DF, and CA, but not VPA. The results with this small series of carboxylate drugs suggested that (a) adduct concentrations in plasma but not liver could be related to acyl glucuronide reactivity, (b) while some modified proteins detected were common, the pattern of modification varied from drug to drug, and (c) caution should be exercised in attributing adduct formation exclusively to the acyl glucuronidation pathway.
羧酸盐类药物通常会形成酰基葡萄糖醛酸结合物作为主要代谢产物。这些亲电代谢产物具有反应活性，能够发生水解、重排以及与蛋白质的共价结合反应。最后提到的这种特性有可能在体内引发免疫及其他毒性反应。在本研究中，我们比较了给大鼠静脉注射非甾体抗炎药佐美酸（ZP）和双氯芬酸（DF）、降血脂药氯贝酸（CA）以及抗癫痫药丙戊酸（VPA）后，大鼠血浆和肝脏中形成共价加合物的程度和模式。这些药物形成的酰基葡萄糖醛酸具有不同的固有反应活性（表观一级降解半衰期值分别为0.5、0.6、3和60小时）。大鼠每天静脉注射两次，持续2天（ZP、DF和CA为50mg/kg，VPA为150mg/kg）。末次给药6小时后获取的组织化学分析显示，ZP、DF、CA和VPA处理组大鼠血浆中的加合物浓度分别为0.31、0.44、0.28和0.05微克药物当量/毫升，肝脏中的加合物浓度分别为2.21、2.31、0.96和0.96微克药物当量/克。对于血浆和肝脏而言，ZP和DF形成的加合物浓度更高，它们的葡萄糖醛酸反应活性更强。血浆中VPA加合物浓度较低，与其葡萄糖醛酸的极低反应活性相符。然而在肝脏中，VPA加合物达到了与其他药物相同数量级的浓度，并且伴有VPA的（E）-2-烯代谢产物的加合物，浓度为0.38微克VPA当量/克肝脏。VPA的肝脏数据可以通过除酰基葡萄糖醛酸化途径之外的酰基辅酶A/β-氧化加合物形成途径来解释。使用针对合成药物-蛋白质加合物制备的兔多克隆抗血清进行免疫印迹分析，结果显示ZP和DF处理组大鼠肝脏中有110、140和约200kDa的主要条带。ZP处理组肝脏中70kDa的第四条主要条带与CA处理组肝脏中唯一检测到的主要条带具有相同的表观分子量。在VPA处理组大鼠的肝脏组织中检测到一条140kDa的条带以及几条分子量较低的条带。在血浆中，抗血清在ZP、DF和CA处理组大鼠的样本中特异性检测到了药物修饰的血清白蛋白，但在VPA处理组中未检测到。这一小系列羧酸盐类药物的研究结果表明：（a）血浆而非肝脏中的加合物浓度可能与酰基葡萄糖醛酸的反应活性有关；（b）虽然检测到的一些修饰蛋白是常见的，但修饰模式因药物而异；（c）在将加合物形成完全归因于酰基葡萄糖醛酸化途径时应谨慎。
