1999 Fiscal Year Annual Research Report

新薬開発を飛躍的に効率化するハイスループットスクリーニングシステムの開発研究

Research Project

Project/Area Number	11557175
Research Category	Grant-in-Aid for Scientific Research (B)
Research Institution	Hokkaido University
Principal Investigator	鎌滝哲也北海道大学, 大学院・薬学研究科, 教授 (00009177)
Co-Investigator(Kenkyū-buntansha)	有吉範高北海道大学, 大学院・薬学研究科, 助教授 (00243957) 高橋芳樹北海道大学, 大学院・薬学研究科, 助手 (80292019) 藤田健一北海道大学, 大学院・薬学研究科, 助手 (60281820) 小林智協和発酵工業(株), 医療総合研究所, 所長(研究・開発担当) 上館民夫北海道大学, 大学院・工学研究科, 教授 (70185990)
Keywords	ハイスループットシステム / 薬物代謝 / チトクロームP450 / CYP3A4 / 大腸菌発現系 / チトクロームb_5 / 薬物間相互作用 / 水溶性ポリマー
Research Abstract	CYP3A4は、成人肝に発現する最も主要なP450分子種であり、現在臨床応用されている医薬品の50%強の代謝に関与するため、ハイスループットシステム(HTS)構築の最初の標的酵素としては最適である。HTS構築のためには、酵素源は大量かつ迅速に調整する必要があるため、ヒトCYP3A4を短時間に大量発現できる大腸菌発現系が最適と考えられるが、大腸菌にはCYPが酵素活性を発現するために必須なNADPH-cytochrome P450還元酵素(OR)がない。そこで本年度はCYP3A4とORを同時に大腸菌内に発現できる発現系を樹立した。発現した酵素は酵素活性を有しており、CYP3A4の基質薬物を用いて速度論的解析を行って、ヒト肝ミクロゾームでの結果と比較した結果、HTSに応用するために充分な酵素活性を示した。またCYP3A4はその活性発現にチトクロームb_5(b_5)を要求する場合があり、肝臓での活性をより正確に反映するためには、b_5の添加を考慮する必要がある。したがってb_5の高効率な精製法を確立した。この場合、b_5は、動物種間でアミノ酸配列がきわめて良く保存されており、機能的にもほとんど差がないことを当研究室ですでに明らかにしているため、ヒト肝ミクロゾームから精製する必要はなく、他の動物種の肝臓で代用した。非イオン性の界面活性剤と水溶性ポリマーの相分配を利用して、従来行われていた数本のカラム操作を省くことによって、精製にかかる時間短縮と回収率の向上に成功した。精製したb_5は-80℃で長時間安定に保存できるため、HTSのツールに有用であると考えられる。

Research Products
(20 results)

All Other

All Publications (20 results)

[Publications] Eri Inoue, et al.: "Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of Cytochrome P450"Biochemical Biophysical Research Communications. In press.
[Publications] Yasumasa Sasaki, et al.: "Cooperative regulation of CYP2C12 gene expression by signal transducer and activator of transcription 5(STAT5) and liver-specific factor in female rats"Journal of Biological Chemistry. 274. 37117-37124 (1999)
[Publications] Tomonori Tateishi, et al.: "Analysis of the CYP2D6 gene in relation to dextromethorphan O-demethylation capacity in Japanese population"Clinical Pharmacology and Therapeutics. 65. 570-575 (1999)
[Publications] Miki Nakajima, et al.: "Genetic polymorphism in the 5"-flanking region of human CYP1A2 gene : effect on the CYP1A2 inducibility in humans"Journal of Biochemistry. 125. 803-808 (1999)
[Publications] Ken-ichi Nunoya, et al.: "CYP2A6 gene deletion responsible for the in vivo polymorphic metabolism of (+)-cis-3,5-dimethy1-2-(3-pyridyl)thiazolidine-4-one hydrochloride in humans"Journal of Pharmacology and Experimental Therapeutics. 289. 437-442 (1999)
[Publications] Michihiro Chida, et al.: "A new variant CYP2D6 allele (CYP2D6^★21) with a single insertion in exon 5 in a Japanese population associated with a poor metabolizer phenotype"Pharmacogenetics. 9. 287-293 (1999)
[Publications] Taisei Mushiroda, et al.: "The Suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation"Toxicology and Applied Pharmacology. In press.
[Publications] Toshiyuki Kume, et al.: "Characterization of a novel variant (S145C/L311V) of 3-hydroxysteroid/dihidrodiol dehydrogenase in human liver"Pharmacogenetics. 9. 763-771 (1999)
[Publications] Masami Miyamoto, et al.: "CYP2A6 gene deletion reduces susceptibility to lung cancer"Biochemical Biophysical Research Communications. 261. 658-660 (1999)
[Publications] Michihiro Chida, et al.: "Genetic polymorphism of CYP2D6 in Japanese population"Pharmacogenetics. 9. 601-605 (1999)
[Publications] Ken-ichi Nunoya, et al.: "Homologous unequal cross-over within the human CYP2A gene cluster as a mechanism for the deletion of entire CYP2A6 gene associated with the poor"Journal of Biochemistry. 126. 402-407 (1999)
[Publications] Taisei Mushiroda, et al.: "Sensitive assay of trimethylamine N-oxide in liver microsomes by head-space gas chromatography with flame thermionic detection"Journal of Chromatography. 734. 319-323 (1999)
[Publications] Michihiro Chida, et al.: "Detection of three genetic polymorphisms in the 5'-flanking region and the intron 1 of human CYP1A2 in the Japanese population"Japanese Journal of Cancer Research. 90. 899-902 (1999)
[Publications] Timothy Marcylo, et al.: "Protection against the bacterial mutagenicity of heterocyclic amines by purpurin, a natural anthraquinone pigment"Mutation Research. 444. 451-461 (1999)
[Publications] Taisei Mushiroda, et al.: "The Suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation"Toxicology and Applied Pharmacology. (in press).
[Publications] Erin Schuetz, et al.: "The glucocorticoid receptor is essential for induction of cytochrome P4502B by steroids but not for drug or steroid induction of CYP3A or P450 reductase"Drug Metabolism and Disposition. (in Press).
[Publications] Taisei Mushiroda, et al.: "A high level expression of CYP2A in the lung of the Suncus (Suncus murinus) and its role in the Activation of 4-(methylntirosamino) -1-(3-pyridyl)-1-"Biochemical Biophysical Research Communications. (in press).
[Publications] 鎌滝哲也: "薬物療法学"南山堂. in press
[Publications] 有吉範高: "別冊医学のあゆみ消化器疾患"医歯薬出版株式会社. 562 (1999)
[Publications] 中山佳都夫: "別冊医学のあゆみ消化器疾患"医歯薬出版株式会社. 562 (1999)

1999 Fiscal Year Annual Research Report

新薬開発を飛躍的に効率化するハイスループットスクリーニングシステムの開発研究

Principal Investigator

鎌滝 哲也 北海道大学, 大学院・薬学研究科, 教授 (00009177)

Research Products

[Publications] Eri Inoue, et al.: "Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of Cytochrome P450"Biochemical Biophysical Research Communications. In press.

[Publications] Yasumasa Sasaki, et al.: "Cooperative regulation of CYP2C12 gene expression by signal transducer and activator of transcription 5(STAT5) and liver-specific factor in female rats"Journal of Biological Chemistry. 274. 37117-37124 (1999)

[Publications] Tomonori Tateishi, et al.: "Analysis of the CYP2D6 gene in relation to dextromethorphan O-demethylation capacity in Japanese population"Clinical Pharmacology and Therapeutics. 65. 570-575 (1999)

[Publications] Miki Nakajima, et al.: "Genetic polymorphism in the 5"-flanking region of human CYP1A2 gene : effect on the CYP1A2 inducibility in humans"Journal of Biochemistry. 125. 803-808 (1999)

[Publications] Ken-ichi Nunoya, et al.: "CYP2A6 gene deletion responsible for the in vivo polymorphic metabolism of (+)-cis-3,5-dimethy1-2-(3-pyridyl)thiazolidine-4-one hydrochloride in humans"Journal of Pharmacology and Experimental Therapeutics. 289. 437-442 (1999)

[Publications] Michihiro Chida, et al.: "A new variant CYP2D6 allele (CYP2D6^★21) with a single insertion in exon 5 in a Japanese population associated with a poor metabolizer phenotype"Pharmacogenetics. 9. 287-293 (1999)

[Publications] Taisei Mushiroda, et al.: "The Suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation"Toxicology and Applied Pharmacology. In press.

[Publications] Toshiyuki Kume, et al.: "Characterization of a novel variant (S145C/L311V) of 3-hydroxysteroid/dihidrodiol dehydrogenase in human liver"Pharmacogenetics. 9. 763-771 (1999)

[Publications] Masami Miyamoto, et al.: "CYP2A6 gene deletion reduces susceptibility to lung cancer"Biochemical Biophysical Research Communications. 261. 658-660 (1999)

[Publications] Michihiro Chida, et al.: "Genetic polymorphism of CYP2D6 in Japanese population"Pharmacogenetics. 9. 601-605 (1999)

[Publications] Ken-ichi Nunoya, et al.: "Homologous unequal cross-over within the human CYP2A gene cluster as a mechanism for the deletion of entire CYP2A6 gene associated with the poor"Journal of Biochemistry. 126. 402-407 (1999)

[Publications] Taisei Mushiroda, et al.: "Sensitive assay of trimethylamine N-oxide in liver microsomes by head-space gas chromatography with flame thermionic detection"Journal of Chromatography. 734. 319-323 (1999)

[Publications] Michihiro Chida, et al.: "Detection of three genetic polymorphisms in the 5'-flanking region and the intron 1 of human CYP1A2 in the Japanese population"Japanese Journal of Cancer Research. 90. 899-902 (1999)

[Publications] Timothy Marcylo, et al.: "Protection against the bacterial mutagenicity of heterocyclic amines by purpurin, a natural anthraquinone pigment"Mutation Research. 444. 451-461 (1999)

[Publications] Taisei Mushiroda, et al.: "The Suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation"Toxicology and Applied Pharmacology. (in press).

[Publications] Erin Schuetz, et al.: "The glucocorticoid receptor is essential for induction of cytochrome P4502B by steroids but not for drug or steroid induction of CYP3A or P450 reductase"Drug Metabolism and Disposition. (in Press).

[Publications] Taisei Mushiroda, et al.: "A high level expression of CYP2A in the lung of the Suncus (Suncus murinus) and its role in the Activation of 4-(methylntirosamino) -1-(3-pyridyl)-1-"Biochemical Biophysical Research Communications. (in press).

[Publications] 鎌滝哲也: "薬物療法学"南山堂. in press

[Publications] 有吉範高: "別冊 医学のあゆみ 消化器疾患"医歯薬出版株式会社. 562 (1999)

[Publications] 中山佳都夫: "別冊 医学のあゆみ 消化器疾患"医歯薬出版株式会社. 562 (1999)

鎌滝哲也北海道大学, 大学院・薬学研究科, 教授 (00009177)

[Publications] 有吉範高: "別冊医学のあゆみ消化器疾患"医歯薬出版株式会社. 562 (1999)

[Publications] 中山佳都夫: "別冊医学のあゆみ消化器疾患"医歯薬出版株式会社. 562 (1999)