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

开发预测人体药物代谢的新替代方法

• 批准号: 06557124
• 负责人: KAMATAKI Tetsuya
• 金额: $ 5.31万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557124/
• 关键词: Alternatives; P450; Transgenic mouse; CYP3A7; p53-Knockout mouse; Cell expression system; Hepatocyte; アフラトキシンB_1; p53欠失マウス; 薬物代謝酵素; チトクロームP450; 大量発現系; N-アセチルトランスフェラーゼ; バキュロウイルス発現系; in vitro 毒性試験

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

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

项目成果

K. Nakamura et al.: "CYP2D6 is a principal cytochrome P450 responsible for metabolism of the histamine H1 antagonist promethazine in human liver microsomes." Pharmacogenetics. 6. 449-457 (1996)

K. Nakamura 等人：“CYP2D6 是负责人肝微粒体中组胺 H1 拮抗剂异丙嗪代谢的主要细胞色素 P450。”

Y. Takahashi et al.: "Expression of aryl hydrocarbon receptor (AhR) and Ah receptor nuclear translocator (Arnt) in adult rabbits known to be non-responsive to cytochrome P450 1A1 (CYP1A1) inducers." Eur. J. Biochem.242. 512-518 (1996)

Y. Takahashi 等人：“已知对细胞色素 P450 1A1 (CYP1A1) 诱导剂无反应的成年兔子中芳基碳氢化合物受体 (AhR) 和 Ah 受体核转位子 (Arnt) 的表达。”

Hisashi Hashimoto et al.: "Fetus-specific CYP3A7 and adult-specific CYP3A4 expressed in Chinese hamster CHL cells have similar capacity to activate carcinogenic mycotoxins." Cancer Res.55. 787-791 (1995)

Hisashi Hashimoto 等人：“中国仓鼠 CHL 细胞中表达的胎儿特异性 CYP3A7 和成人特异性 CYP3A4 具有相似的激活致癌霉菌毒素的能力。”

Tsutomu Sakuma et al.,: "Efficient complementary DNA directed expression of human fetal liver cytochrome P450(CYP3A7) in insect cells using baculovirus." Biochem. Mol. Biol. Intern.35. 447-455 (1995)

Tsutomu Sakuma 等人：“使用杆状病毒在昆虫细胞中高效互补 DNA 指导表达人胎肝细胞色素 P450 (CYP3A7)。”

Y. Li et al.: "Establishment of transgenic mice carrying human fetus-specific CYP3A7." Arch. Biochem. Biophys.329. 235-240 (1996)

Y. Li 等人：“携带人类胎儿特异性 CYP3A7 的转基因小鼠的建立。”