Approach for in vivo protein binding of 5-n-butyl-pyrazolo[1,5-a]pyrimidine bioactivated in chimeric mice with humanized liver by two-dimensional electrophoresis with accelerator mass spectrometry.
Approach for in vivo protein binding of 5-n-butyl-pyrazolo[1,5-a]pyrimidine bioactivated in chimeric mice with humanized liver by two-dimensional electrophoresis with accelerator mass spectrometry.
复制标题
通过加速器质谱二维电泳在具有人源化肝脏的嵌合小鼠中生物激活的 5-n-丁基-吡唑并[1,5-a]嘧啶的体内蛋白质结合方法。
DOI：
10.1021/tx900323a
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发表时间：
2010
影响因子：
4.1
通讯作者：
S. Ohta
中科院分区：
文献类型：
--
作者：
H. Yamazaki;S. Kuribayashi;Tae Inoue;C. Tateno;Yasufumi Nishikura;K. Oofusa;D. Harada;S. Naito;T. Horie;S. Ohta
一种潜在的镇痛药5-正丁基-7-（3,4,5-三甲氧基苯甲酰氨基）吡唑[1,5-a]嘧啶的药物开发被叫停，因为它对人类的肝毒性作用有限，无法从动物或体外研究中预测。由于人细胞色素P450 1A2在体外形成共价结合代谢物的有趣能力，本研究在静脉给药给人或大鼠肝脏嵌合小鼠后，研究了谷胱甘肽偶联物的体内形成和模型化合物5-正丁基-吡唑啉[1,5-a]嘧啶的共价结合。经静脉给药后，嵌合小鼠血浆或肝脏中放射性标记的5-正丁基吡唑[1,5-a]嘧啶迅速分布和消除。然而，在静脉给药后0.17-24小时，肝脏中检测到类似的共价结合。放射- lc分析显示，人源化肝脏嵌合小鼠在血浆和肝脏中优先给予3-羟基化代谢物及其谷胱甘肽偶联物。相反，具有大鼠肝脏的嵌合小鼠体内有一些大鼠特有的代谢物。用加速器质谱法对嵌合小鼠体内放射性标记的肝脏蛋白进行电泳分析发现，具有生物活性的5-正丁基吡唑[1,5-a]嘧啶非特异性地结合到多种微粒体蛋白上，包括人P450 1A2以及人肝脏嵌合小鼠肝脏中的细胞质蛋白。这些结果表明，肝毒性模型化合物5-正丁基-吡唑啉[1,5-a]嘧啶在人源化嵌合小鼠体内被人肝微粒体P450 1A2激活为活性中间体，并且可以相对非特异性地结合P450 1A2等生物分子和其他蛋白质。
Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.