DEVELOPMENT OF NOVEL ALTERNATIVE METHODS FOR THE PREDICTION OF DRUG METABOLISM IN HUMANS

我们已经成功地建立了稳定表达人NAT 1、NAT 2、CYP 1A 2、CYP 2 E1、CYP 3A 4和CYP 3A 7的细胞系。我们检查了IQ和MeIQx在单独或联合表达人CYP 1A 2、NAT 1和NAT 2的细胞中的细胞毒性和致突变性。结果得出以下结论：（a）单独使用每种酶均未激活任一化合物产生细胞毒性和致突变代谢物;（B）CYP 1A 2与NAT 2联合可有效激活化合物;（c）CYP 1A 2与NAT 1共同表达对致突变激活无效。表达CYP 2 E1的细胞显示7-乙氧基香豆素OMICRON-脱乙基酶活性。此外，这些细胞在细胞毒性试验中显示出比亲本细胞更高的对DMN的敏感性。表达CYP 3A 4和CYP 3A 7的细胞对黄曲霉毒素B_1的敏感性高于亲本细胞。这些细胞系提供了一个新的有价值的小组，研究人类代谢的外源性物质的能力，直接 ...更多信息 与P450催化的代谢有关。(2)我们建立了几个携带CYP 3A 7转基因的转基因小鼠品系，在所有6个转基因品系中，M2小鼠在肾脏、小肠和皮肤等多种组织中表达CYP 3A 7基因，而M10小鼠在肝脏和睾丸中表达CYP 3A 7转基因，在mRNA和蛋白水平上。以咪达唑仑（MZ）为底物，对转基因小鼠在药物代谢中的潜在应用进行了评价。通过腹膜内注射给予M10系成年小鼠该药物导致小鼠血清中1 '-OH-MZ和4-OH-MZ的水平显著升高。使用成年M10小鼠肝微粒体的动力学研究表明，与非转基因小鼠相比，MZ 1 '-羟基化的Vmax较高，Km值相同。这些结果表明，在这些转基因小鼠中表达的CYP 3A 7对MZ具有正常的催化活性，其特征与人肝脏中的类似。少

We have succeeded in establishing cell lines which stably expressed human NATI,NAT2, CYP1A2, CYP2E1, CYP3A4 and CYP3A7. We examined the cytotoxicity and mutagenicity of IQ and MeIQx in the cells expressing human CYP1A2, NAT1, and NAT2 alone or in combinations. The results led to the following conclusions : (a) no activation to produce cytotoxic and mutagenic metabolites from either compound was achieved by each enzyme alone ; (b) CYP1A2 in combination with NAT2 efficiently activated the compounds ; (c) expression of CYP1A2 togather with NAT1 was not effective for mutagemic activation. The cells expressing CYP2E1 showed 7-ethoxycoumarin OMICRON-deethylase activity. Moreover, these cells showed higher sensitivity to DMN in cytotoxicity assay as compared to parental cells. The cells expressing CYP3A4 and CYP3A7 were more sensitive to aflatoxin B_1 than parental cells. These cell lines provide a new valuable panel for studying the human metabolism of xenobiotics with an ability to directly … More relate to P450-catalyzed metabolism.(2) We established several lines of transgenic mice harboring a CYP3A7 transgene.Of all the six transgenic lines established, M2 mice expressed the CYP3A7 gene in many tissues such as in the kidney, small intestine, and skin, etc, while M10 mice expressed the CYP3A7 transgene in the liver and testis, at both mRNA and protein levels. The potential application of this transgenic mice in drug metabolism was assessed using midazolam (MZ) as a substrate. Administration of this drug through intraperitoneal injection into adult mice of line M10 resulted in significantly higher levels of both 1'-OH-MZ and 4-OH-MZ in mouse serum. Kinetic study using liver microsomes from adult M10 mice indicated a higher Vmax with the same Km values in 1'-hydroxylation of MZ as compared with nontransgenic mice. These results indicate that CYP3A7 expressed in these transgenic mice possesses normal catalytic activities towards MZ with characteristics similar to those in the human livers. Less