Xenobiotica ( IF 1.8 ) Pub Date : 2020-07-26 , DOI: 10.1080/00498254.2020.1775913 Bikash Dangi 1 , Nadezhda Y Davydova 1 , Nikita E Vavilov 2 , Victor G Zgoda 2, 3 , Dmitri R Davydov 1
Abstract
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We closely characterized 7-Dimethylamino-4-trifluromethylcoumarin (Coumarin 152, C152), a substrate metabolized by multiple P450 species, to establish a new fluorogenic probe for the studies of functional integration in the cytochrome P450 ensemble.
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Scanning fluorescence spectroscopy and LC/MS-MS were used to characterize the products of N-demethylation of C152 and optimize their fluorometric detection. The metabolism of C152 by the individual P450 species was characterized using the microsomes containing cDNA-expressed enzymes. C152 metabolism in human liver microsomes (HLM) was studied in a preparation with quantified content of eleven P450 species.
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C152 is metabolized by CYP2B6, CYP3A4, CYP3A5, CYP2C19, CYP1A2, CYP2C9, and CYP2C8 listed in the order of decreasing turnover. The affinities exhibited by CYP3A5, CYP2C9, and CYP2C8 were lower than those characteristic to the other enzymes.
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The presumption of additivity suggests the participation of CYP3A4, CYP2B6, and CYP2C19 to be 84, 8, and 0.2%, respectively. Contrary to this prediction, inhibitory analysis identified CYP2C19 as the principal C152-metabolizing enzyme.
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We thoroughly characterize C152 for the studies of drug metabolism in HLM and demonstrate the limitations of the proportional projection approach by providing an example, where the involvement of individual P450 species cannot be predicted from their content.
中文翻译:
在使用香豆素 152(一种多特异性细胞色素 P450 底物)的研究中揭示了人类微粒体药物代谢的非可加性。
摘要
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我们密切表征了 7-Dimethylamino-4-trifluromethylcoumarin(Coumarin 152, C152),一种由多种 P450 物种代谢的底物,以建立一种新的荧光探针,用于研究细胞色素 P450 集合中的功能整合。
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使用扫描荧光光谱和 LC/MS-MS 表征 C152 的 N-去甲基化产物并优化其荧光检测。使用含有 cDNA 表达酶的微粒体表征单个 P450 物种对 C152 的代谢。在具有 11 种 P450 种类的定量含量的制剂中研究了人肝微粒体 (HLM) 中的 C152 代谢。
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C152 被 CYP2B6、CYP3A4、CYP3A5、CYP2C19、CYP1A2、CYP2C9 和 CYP2C8 代谢,按代谢率递减的顺序列出。CYP3A5、CYP2C9 和 CYP2C8 表现出的亲和力低于其他酶的特征。
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可加性推定表明 CYP3A4、CYP2B6 和 CYP2C19 的参与率分别为 84、8 和 0.2%。与此预测相反,抑制分析将 CYP2C19 鉴定为主要的 C152 代谢酶。
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我们彻底表征了 C152 用于 HLM 中药物代谢的研究,并通过提供一个示例证明了比例投影方法的局限性,其中无法从其含量预测单个 P450 物种的参与。