Bioactivation of phencyclidine in rat and human liver Microsomes and recombinant p450 2B enzymes: Evidence for the formation of a novel quinone methide intermediate
Bioactivation of phencyclidine in rat and human liver Microsomes and recombinant p450 2B enzymes: Evidence for the formation of a novel quinone methide intermediate
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DOI：
10.1021/tx700145k
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发表时间：
2007-10-01
影响因子：
4.1
通讯作者：
O'Donnell, John P.
中科院分区：
文献类型：
--
作者：
Driscoll, James P.;Kornecki, Katherine;O'Donnell, John P.
关于麻醉剂苯环利定（PCP）的心理副作用可能是由PCP或其活性代谢物与大脑中关键大分子的不可逆结合引起的这一假设导致了许多旨在表征PCP生物活化途径的体外研究。本文所述的研究扩展了目前对五氯苯酚代谢的了解，并提供了关于五氯苯酚以前未知的代谢活化途径的详细信息。在与补充有NADPH和GSH的人和大鼠肝微粒体以及重组P450 2B酶一起孵育后，通过LC/MS/MS分别检测到具有547和482 Da MH+离子的两种巯基缀合物。处置34，375-383]还观察到在PCP与用NADPH和GSH强化的大鼠P450 2131和兔P450 2134同种型孵育中具有547 Da MH+的GSH缀合物1。I的分子量与生物活化途径一致，该途径涉及巯基亲核试剂与PCP的推定2，3-二氢吡啶代谢物的Michael加成，该代谢物通过母体化合物中哌啶环的四电子氧化获得。在482 Da处具有MH+离子的新型GSH加合物2的质谱表明了独特的PCP生物活化途径，该途径涉及PCP中苯环的初始邻位或对位羟基化，然后自发分解为哌啶和亲电子醌甲基化物中间体，该中间体与GSH反应后产生加合物2。酶促生成的2的LC保留时间和质谱特性与通过GSH与合成对羟基PCP在磷酸盐缓冲液（pH 7.4，37 ℃）中反应获得的参比标准品相同。对合成产生的2的H-1 NMR和C-13-无畸变极化转移增强（DEPT）NMR光谱研究表明，可能保留了对羟基苯基和环己基环的结构完整性，并且GSH添加的位点是连接两个支架的苄基碳。加入双重P450 2C 19/P450 2B 6抑制剂（+）-N-3-苄基尼凡诺后，人微粒体中2的形成减少。与这一发现相一致，重组P450 2136和P450 2C 19均催化五氯苯酚生物活化至2。在不存在GSH的情况下，合成的对羟基PCP经历快速分解（t（1/2）类似于5.2 min），得到对羟基苯基环己烷醇和对羟基苯基环己烯，推测是通过醌甲基化物中间体。总的来说，我们的研究结果对合成的p-hydroxyPCP容易降解，产生亲电醌甲基化物中间体能够与亲核试剂，包括谷胱甘肽和水反应，表明一个固有的不稳定性的推定酚PCP代谢物。因此，如果在体内酶促形成，对羟基五氯苯酚可能不需要进一步代谢来释放醌甲基化物，然后醌甲基化物可以与大分子反应。据我们所知，这是第一次报告的醌甲基反应中间体获得的人肝微粒体代谢的五氯苯酚。
The hypothesis that the psychological side effects associated with the anesthetic, phencyclidine (PCP) may be caused by irreversible binding of PCP or its reactive metabolite(s) to critical macromolecules in the brain has resulted in numerous in vitro studies aimed at characterizing pathways of PCP bioactivation. The studies described herein extend the current knowledge of PCP metabolism and provide details on a previously unknown metabolic activation pathway of PCP. Following incubations with NADPH- and GSH-supplemented human and rat liver microsomes and recombinant P450 2B enzymes, two sulfhydryl conjugates with MH+ ions at 547 and 482 Da, respectively, were detected by LC/MS/MS. Shebley et al. [(2006) Drug Metab. Dispos. 34, 375-383] have also observed the GSH conjugate 1 with MH+ at 547 Da in PCP incubations with rat P450 2131 and rabbit P450 2134 isoforms fortified with NADPH and GSH. The molecular weight of I is consistent with a bioactivation pathway involving Michael addition of the sulfhydryl nucleophile to the putative 2,3-dihydropyridinium metabolite of PCP obtained via a four-electron oxidation of the piperidine ring in the parent compound. The mass spectrum of the novel GSH adduct 2 with an MH+ ion at 482 Da was suggestive of a unique PCP bioactivation pathway involving initial ortho- or para-hydroxylation of the phenyl ring in PCP followed by spontaneous decomposition to piperidine and an electrophilic quinone methide intermediate, which upon reaction with GSH yielded adduct 2. The LC retention times and mass spectral properties of enzymatically generated 2 were identical to those of a reference standard obtained via reaction of GSH with synthetic p-hydroxyPCP in phosphate buffer (pH 7.4, 37 degrees C). H-1 NMR and C-13-distortionless enhancement by polarization transfer (DEPT) NMR spectral studies on synthetically generated 2 suggested that the structural integrity of the p-hydroxyphenyl and cyclohexyl rings likely was preserved and that the site of GSH addition was the benzylic carbon joining the two scaffolds. The formation of 2 in human microsomes was reduced upon addition of the dual P450 2C19/P450 2B6 inhibitor (+)-N-3-benzylnirvanol. Consistent with this finding, both recombinant P450 2136 and P450 2C19 catalyzed PCP bioactivation to 2. In the absence of GSH, synthetic p-hydroxyPCP underwent rapid decomposition (t(1/2) similar to 5.2 min) to afford p-hydroxyphenylcy- clohexanol and p-hydroxyphenylcylohexene, presumably via the quinone methide intermediate. Overall, our findings on the facile degradation of synthetic p-hydroxyPCP to yield an electrophilic quinone methide intermediate capable of reacting with nucleophiles, including GSH and water, suggest an inherent instability of the putative phenolic PCP metabolite. Thus, if formed enzymatically in vivo, p-hydroxyPCP may not require further metabolism to liberate the quinone methide, which can then react with macromolecules. To our knowledge, this is the first report of a quinone methide reactive intermediate obtained in human-liver microsomal metabolism of PCP.