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

• 研究課題/領域番号: 11557175
• 研究種目: 基盤研究(B)
• 配分区分: 補助金
• 応募区分: 展開研究
• 研究分野: 生物系薬学
• 研究機関: 北海道大学
• 研究代表者: 鎌滝 哲也 北海道大学, 大学院・薬学研究科, 教授 (00009177)
• 研究分担者: 小林 智 協和発酵工業(株), 医薬総合研究所, 所長(研究・開発担当) ; 上館 民夫 (上舘 民夫) 北海道大学, 大学院・工学研究科, 教授 (70185990) ; 藤田 健一 北海道大学, 大学院・薬学研究科, 助手 (60281820) ; 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)
• 研究期間 (年度): 1999 – 2001
• 研究課題ステータス: 完了 (2001年度)
• 配分額: 8,200千円 (直接経費: 8,200千円); 2001年度: 1,300千円 (直接経費: 1,300千円); 2000年度: 4,500千円 (直接経費: 4,500千円); 1999年度: 2,400千円 (直接経費: 2,400千円)
• キーワード: P450 / ハイスループット / プレートリーダー / 阻害 / 薬物代謝 / 薬物相互作用 / 遺伝子組換え / 医薬品開発 / ハイスループットシステム / 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

研究概要

薬物相互作用の主な原因のひとつに,ヒトにおける主要な薬物代謝酵素であるチトクロームP450(CYP)の阻害が含まれる.そのため近年ではCYPを阻害する物質は医薬品の開発段階で除外される流れにある.この阻害作用の有無の予測を開発の初期に行うことができれば大幅な効率化につながると期待される.このことから,膨大な数の候補化合物をヒトCYPに対する影響を基準に絞りこむための系として,ハイスループットスクリーニング(HTS)システムによるヒトCYPの触媒活性測定法の開発が待たれている.ヒトCYPの触媒活性の測定を大規模に行うためにはヒトCYPが大量に必要とされる.そこで本研究では,大腸菌を宿主としたヒトCYPの大量発現系の樹立,およびそれを用いた薬物相互作用の大規模試験系の確立を目的とした.13種類のヒトCYP(CYP1A1,CYP1A2,CYP1B1,CYP2A6,CYP2B6,CYP2C8,CYP2C9,CYP2C19,CYP2D6,CYP2E1,CYP3A4,CYP3A5およびCYP3A7)をそれぞれ大腸菌に発現させることに成功した.大腸菌に発現したヒトCYPを用い,カルシウム拮抗薬であるニカルジピンが抗癌薬であるタキソールのCYP2C8による代謝を強力に阻害することを見出し,大腸菌に発現したCYPを用いて薬物相互作用を検討できることを示した.大腸菌に発現したヒトCYPをHTSに応用するため,これらの大腸菌を全容10Lの大量培養装置にて培養し,一度に大量のCYPを得ることを可能にした.我々はさらに,マイクロプレートを用いてクマリン7-水酸化酵素活性(CYP2A6)および7-エトキシレゾルフィン0-脱エチル化酵素活性(CYP1A1)を測定する系を確立した.これらの系を応用することによりヒトにおける薬物相互作用を短時間に,しかも大量に検討できるようになり,医薬品開発の効率化に貢献できると期待される.

研究成果

• [文献書誌] Fujita, K. et al.: "Predicting the Mutagenicity of Tobacco-related N-Nitrosamines in Humans Using Eleven Strains of Salmonella Typhimurium YG7108 Each Co-expressing a Form of Human Cytochrome P450 with NADPH-cytochrome P450 Reductase"Environ. Mol. Mutagen. 38. 339-346 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Fujita, K. et al.: "Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase"Environ. Mol. Mutagen. 38. 329-338 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Fujita, K. et al.: "Screening of organosulfur compounds as inhibitors of human CYP2A6"Drug Metab. Dispos. 29. 983-989 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Fujita, K. et al.: "Role of Human Cytochrome P450 (CYP) in the Metabolic Activation of N-alkylntrosamines : Application of Genetically Engineered Salmonella Typhimurium YG7108 Expressing Each Form of CYP Together with Human NADPH-cytochrome P450 Reductase"Mutat. Res.. 483. 35-41 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Ariyoshi N. et al.: "Comparison of the Levels of Enzymes Involved in Drug Metabolism between Transgenic or Gene-knockout and the Parental Mice"Toxicologic Pathology. 29. 161-172 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Ariyoshi N., et al.: "A single nucleotide polymorphism of CYP2B6 found in Japanese enhances catalytic activity by autoactivation"Biochem. Biophys. Res. Commun. 281. 1256-1260 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] 鎌滝 哲也: "人類とバイオーナノ・バイオへの期待-"東京テクノ・フォーラム21事務局、東京. 30 (2001)
  • 説明: 「研究成果報告書概要(和文)」より
• [文献書誌] Noritaka Ariyoshi, Yu-ichi Sawamura, Tetsuya Kamataki: "A novel single nucleotide polymorphism altering stability and activity of CYP2A6"Biochem. Biophys. Res. Commun.. 281(3). 810-814 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Marie-Anne Loriot, Sandra Rebuissou, Mikael Oscarson, Sylvie Cenee, Masami Miyamoto, Noritaka Ariyoshi, Tetsuya Kamataki, Denis Hemon, Philippe Beaune, Isabelle Stucker: "Genetic polymorphism of cytochrome P450 2A6 in a case control study on lung cancer in a French population"Pharmacogenetics.. 11. 39-44 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Ken-ichi Fujita, Tetsuya Kamataki: "Screening of organosulfur compounds as inhibitors of human CYP2A6"Drug Metab. Dispos.. 29. 983-989 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Jason R. Baker, Soisungwan Satarug, Paul E. Reilly, Robert J. Edwards, Noritaka Ariyoshi, Tetsuya Kamataki, Michael R. Moore, David J. Williams: "Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples"Biochem., Pharm.. 62(6). 713-721 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Shigeharu Muto, Ken-ichi Fujita, Yoshiyuki Yamazaki, Tetsuya Kamataki: "In hibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450"Mutat. Res.. 479. 197-206 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Mariko Murata, Masatoshi Watanabe, Mikio Yamanaka, Yoshinobu Kubota, Minako Nagao, Takahiko Katoh, Tetsuya Kamataki, Junichi Kawamura, Ryuichi Yatani, Taizo Shiraishi: "Genetic polymorphisms in cytochrome P450(CYP)1A1, CYP142, CYP2E1, glutathione S-transferase (GST) M1 and GSTT1 and susceptibility to prostate cancer in the Japanese population"Cancer Letters. 165. 171-177 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Ken-ichi Fujita, Kazuo Nakayama, Yoshiyuki Yamazaki, Kazuhiro Tsuruma, Masami Yamada, Takehiko Nohmi, Tetsuya Kamataki: "Construction of Salmonella typphimurium YG7108 stains each co-expressing a form of human cytochrome P450 with NADPH-cytochrime P450 reductase"Environ. Mol. Mutagen.. 38. 329-338 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Ken-ichi Fujita, Tetsuya Kamataki: "Predictiong the mutagenicity of tobacco-related N-nitrosamines in humans using eleven strains of Salmonella Typhimurium YG7108 each co-expressing a form of human cytochrome reductase"Environ. Mol. Mutagen.. 38. 339-346 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Ken-ichi Fujita, Tetsuya Kamataki: "Role of human cytochrome P450 (CYP) in the metaboloc activation of N-alkylntrosamines: Applications of genetically engineered Salmonella typhimurim YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase"Mutat. Res.. 483. 35-41 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Noritaka Ariyoshi, Susumu Imaoka, Kazuo Nakayama, Yoshiki Takahashi, Ken-ichi Fujita, Yoshihiko Funae, Tetsuya Kamataki: "Comparison of the levels of enzymes involved in drug metabolism between transgenic or gene-knockout and the parenatal mice"Toxicologic Pathology. 29. 161-172 (2001)
  • 説明: 「研究成果報告書概要(欧文)」より
• [文献書誌] Fujita, K. et al: "Predicting the Mutagenicity of Tobacco-related N-Nitrosamines in Humans Using Eleven Strains of Salmonella Typhimurium YG7108 Each Co-expressing a Form of Human Cytochrome P450 with NADPH-cytochrome P450 Reductase"Environ. Mol. Mutagen. 38. 339-346 (2001)
• [文献書誌] Fujita, K. et al: "Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase"Environ. Mol. Mutagen. 38. 329-338 (2001)
• [文献書誌] Fujita, K. et al: "Screening of organosulfur compounds as inhibitors of human CYP2A6"Drug Metab. Dispos. 29. 983-989 (2001)
• [文献書誌] Fujita, K. et al: "Role of Human Cytochrome P450 (CYP) in the Metabolic Activation of N-alkylntrosamines : Application of Genetically Engineered Salmonella Typhimurium YG7108 Expressing Each Form of CYP Together with Human NADPH-cytochrome P450 Reductase"Mutat. Res. 483. 35-41 (2001)
• [文献書誌] Ariyoshi N. et al: "Comparison of the Levels of Enzymes Involved in Drug Metabolism between Transgenic or Gene-knockout and the Parental Mice"Toxicologic Pathology. 29. 161-172 (2001)
• [文献書誌] Ariyoshi N., et al: "A single nucleotide polymorphism of CYP2B6 found in Japanese enhances catalytic activity by autoactivation"Biochem. Biophys. Res. Commun. 281. 1256-1260 (2001)
• [文献書誌] 鎌滝 哲也: "人類とバイオ-ナノ・バイオへの期待-"東京テクノ・フォーラム21事務局、東京. 30 (2001)
• [文献書誌] Noritaka Ariyoshi et al.: "A Single Nucleotide Polymorphism of CYP2B6 Found in Japanese Enhances Catalytic Activity by Auto activation."Biochemical Biophysical Research Communications. In press. (2001)
• [文献書誌] Noritaka Ariyoshi et al.: "A Novel Single Nucleotide Polymorphism Altering Stability and Activity of CYP2A6."Biochemical Biophysical Research Communications. In press. (2001)
• [文献書誌] Takeshi Ozeki et al.: "Cooperative regulation of the transcription of human dihydrodiol dehydrogenase (DD) 4/AKR1C4 gene by hepatocyte neclear factor (HNF) -4α/γ and HNF-1α."Biochemical Journal. In press. (2001)
• [文献書誌] Noritaka Ariyoshi et al.: "Pharmacogenetics on cancer chemotherapy : from basic studies."Japanese Journal of Clinical Pharmacology and Therapeutics. In press. (2001)
• [文献書誌] Taisei Mushiroda et al.: "Accumulation of the 1-methyl-4-phenylpyridinium ion (MPP+) in suncus (Suncus murinus) brain : Implication for flavin-containing monooxygenase activity in brain microvessels."Chemical Research in Toxicology. In press. (2001)
• [文献書誌] Loriot, M-A et al.: "Genetic polymorphism of cytochrome P450 2A6 in a case control study on lung cancer in a French population."Pharmacogenetics. 11. 39-44 (2001)
• [文献書誌] Noritaka Ariyoshi et al.: "Studies on the genetic polymorphism of CYP2A6."Drug Metabolism and Disposition. 15. 57-61 (2000)
• [文献書誌] Hirotaka Kushida et al.: "Development of a Salmonella tester strain sensitive to promutagenic N-nitrosamines : expression of recombinant CYP2A6 and human NADPH-cytochrome P450 reductase in S.typhimurium YG7108."Mutation Research. 471. 135-143 (2000)
• [文献書誌] Takashi Satoh et al.: "Studies on the interactions between drugs and estrogen. Analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli co-expressing human CYP3A4 with human NADPH-cytochrome P450 reductase."Analytical Biochemistry. 286. 179-186 (2000)
• [文献書誌] Noritaka Ariyoshi et al.: "Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) appparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C."Pharmacogenetics. 10. 678-693 (2000)
• [文献書誌] Taisei Mushiroda et al.: "The house musk shrew (Suncus murinus) : a unique animal with extremely low level of expression of mRNA for CYP3A and flavin-containing monooxygenase."Comparative Biochemistry & Physiology. 126. 225-234 (2000)
• [文献書誌] Hirotaka Kushida et al.: "Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase."Carcinogenesis. 21. 1227-1232 (2000)
• [文献書誌] 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. 269. 623-627 (2000)
• [文献書誌] 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-butanone (NNK) ."Biochemical Biophysical Research Communications. 269. 393-396 (2000)
• [文献書誌] Ken-ichi Fujita et al.: "Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1) ."Life Science. 66. 1955-1967 (2000)
• [文献書誌] Noritaka Ariyoshi et al.: "Handbook of Experimental Pharmacology"Springer-VerlagBerlin. (2001)
• [文献書誌] 有吉範高: "月刊薬事2001年臨時増刊号"じほう. (2001)
• [文献書誌] 鎌滝哲也: "薬物療法学"南江堂. (2001)
• [文献書誌] 有吉範高: "月刊薬事3月臨時増刊号"じほう. 834 (2000)
• [文献書誌] 鎌滝哲也: "薬物代謝学"東京化学同人. 282 (2000)
• [文献書誌] 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.
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Taisei Mushiroda, et al.: "The Suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation"Toxicology and Applied Pharmacology. In press.
• [文献書誌] Toshiyuki Kume, et al.: "Characterization of a novel variant (S145C/L311V) of 3-hydroxysteroid/dihidrodiol dehydrogenase in human liver"Pharmacogenetics. 9. 763-771 (1999)
• [文献書誌] Masami Miyamoto, et al.: "CYP2A6 gene deletion reduces susceptibility to lung cancer"Biochemical Biophysical Research Communications. 261. 658-660 (1999)
• [文献書誌] Michihiro Chida, et al.: "Genetic polymorphism of CYP2D6 in Japanese population"Pharmacogenetics. 9. 601-605 (1999)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Timothy Marcylo, et al.: "Protection against the bacterial mutagenicity of heterocyclic amines by purpurin, a natural anthraquinone pigment"Mutation Research. 444. 451-461 (1999)
• [文献書誌] Taisei Mushiroda, et al.: "The Suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation"Toxicology and Applied Pharmacology. (in press).
• [文献書誌] 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).
• [文献書誌] 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).
• [文献書誌] 鎌滝哲也: "薬物療法学"南山堂. in press
• [文献書誌] 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)
• [文献書誌] 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)