Assessment of human drug metabolism by expression of cytochrome P-450 in yeasts.

通过酵母中细胞色素 P-450 的表达评估人类药物代谢。

• 批准号: 03557113
• 负责人: KATO Ryuichi
• 金额: $ 9.73万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557113/
• 关键词: Cytochrome P-450; Gene expression; Human drug metabolism; P-450 mixture; 薬物代謝; 薬物代謝酵素; 異所的発現; ヒト薬物代謝能; チトクロムP-450

The purpose of this project is development of a new assessment method for human drug metabolism by using mixture of cytochrome P-450 expressed in yeast cells. We first cloned CYP3A4, a major human cytochrome P-450, and expressed it in yeast cells. On the other hand, we expressed human NADPH-cytochrome P-450 reductase and cytochrome b_5 in yeast cells. Then we improved the expression vectors to co-express different vectors in the same cells . We succeeded in co-expressing CYP3A4, NADPH-cytochrome P-450 reductase and cytochrome b_5 in the same cells and we observed high enzymic activities for several substrates. These results indicates further possible way to express sever al different cytochrome P-450 by this method.We, then, try to increase the yield of cytochrome P-450 in yeast cells changing by conditions of the incubation. Thus, we concluded that this approach is useful method for the assessment of human drug metabolism by using a mixture of human cytochrome P-450 expressed in yeast cells.

本研究的目的是利用酵母细胞表达的细胞色素P-450混合物，建立一种新的人体药物代谢评价方法。我们首先克隆了CYP 3A 4，一种主要的人类细胞色素P-450，并在酵母细胞中表达。另一方面，我们在酵母细胞中表达了人NADPH-细胞色素P-450还原酶和细胞色素b_5。然后，我们改进了表达载体，使不同的载体在同一细胞中共表达。我们成功地在同一细胞中共表达了CYP 3A 4、NADPH-细胞色素P-450还原酶和细胞色素b_5，并观察到对几种底物的高酶活性。这些结果为进一步利用该方法表达多种不同的细胞色素P-450提供了可能。因此，我们得出结论，这种方法是有用的方法，通过使用酵母细胞中表达的人细胞色素P-450的混合物来评估人类药物代谢。

项目成果

Chen,L.: "Hepatic microsomal hydroxylation in Japanese:in vitro evidence for rapid and slow metabolizers." Pharmacogenetics.

Chen,L.：“日语中的肝微粒体羟基化：快速和慢速代谢者的体外证据。”

Yasumori, T.: "Cytochrome P-450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers : Differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts." J. Biochem.109. 711-717 (1991)

Yasumori, T.：“人肝脏美芬妥因羟基化多态性中的细胞色素 P-450 human-2 (P-450IIC9)：酵母中表达的微异质 P-450IIC 物种之间底物和立体选择性的差异。”

YAZUMORI,T.: "Species differences in stereoselective metabolism of mephenytoin by cytochrome P450(CYP2C and CYP3A)." J.Pharmacol.Exp.Ther.264. 89-94 (1993)

YAZUMORI,T.：“细胞色素 P450（CYP2C 和 CYP3A）对美芬妥英立体选择性代谢的物种差异。”

Kato, R.: "Cytochrome P450 : Characterization of human P450IIC isozyme by using a yeast expression system." Methods Enzymol.206. 183-190 (1991)

Kato, R.：“细胞色素 P450：使用酵母表达系统表征人类 P450IIC 同工酶。”

安盛 俊雄: "ヒト肝P-450human-2とその変異型によるメフェニトイン.ヘキソバルビタールの立体選択的水酸化の比較" 臨床薬理. 22. 65-66 (1991)

Toshio Yasamori：“人肝 P-450 human-2 及其突变体对美芬妥英和己巴比妥的立体选择性羟基化的比较”《临床药理学》22. 65-66 (1991)。