2000 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 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), 医薬品総合研究, 所長(研究・開発担当) 上舘民夫北海道大学, 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	本研究では現在臨床応用されているCYPで代謝される医薬品の50%強の代謝反応に関与するチトクロームP450(CYP)分子種であるCYP3A4による薬物間相互作用をインビトロで効率的に予測するハイスループットシステム(HTS)構築を目指している.CYP3A4の代謝反応では,小胞体電子伝達成分のチトクロームb_5(b_5)が関与する例がしばしば観察されているが,現在までどのような医薬品の代謝反応にb_5が関わるのか明らかにされていない.しかしながら,CYP3A4による医薬品代謝を生体内により近い条件でインビトロで再現するためには,b_5の共存下で反応を行うべきことは避けられないと予想される.そこで本年度はまず,ヒトのb_5を大量に発現可能な大腸菌発現系の開発を目指し,大腸菌用の発現ベクターpCWに,当研究室で単離したヒトb_5cDNAを挿入し,b_5発現プラスミドを構築した.Westernブロッティングおよび,酸化-還元差スペクトルより,b_5の発現を確認した.ついでb_5が局在する膜画分を調製し,現在臨床応用されている約15種類の医薬品の,CYP3A4での代謝を,b_5非存在下とb_5存在下で比較検討した.検討した医薬品のうち,汎用されているCa拮抗薬であるジルチアゼム,抗うつ薬のイミプラミン,デシプラミン,アミトリプチリン,クロミプラミンなどの薬物は,b_5の共存によりその代謝活性が顕著に上昇したが,同じ抗うつ薬でもノルトリプチリンでは,b_5共存下でその代謝が40%まで抑制された.その他の医薬品については,b_5共存下非共存下でも代謝活性にほとんど差が認められなかった.以上のことから,CYP3A4のHTSを構築する場合,b_5共存下で反応を行うことは,CYP3A4が代謝を触媒する医薬品によってはその活性に顕著に影響することを明らかにした.今後はさらに多くの医薬品の代謝におけるb_5の影響を検討し,医薬品の構造-b_5要求性相関を明らかにすることで,生体内をより正確に予測できるHTS構築の基礎的情報を得る必要があると考えられる。

Research Products

(20 results)

All Other

All Publications (20 results)

[Publications] 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)
[Publications] Noritaka Ariyoshi et al.: "A Novel Single Nucleotide Polymorphism Altering Stability and Activity of CYP2A6."Biochemical Biophysical Research Communications. In press. (2001)
[Publications] 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)
[Publications] Noritaka Ariyoshi et al.: "Pharmacogenetics on cancer chemotherapy : from basic studies."Japanese Journal of Clinical Pharmacology and Therapeutics. In press. (2001)
[Publications] 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)
[Publications] 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)
[Publications] Noritaka Ariyoshi et al.: "Studies on the genetic polymorphism of CYP2A6."Drug Metabolism and Disposition. 15. 57-61 (2000)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[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. 269. 623-627 (2000)
[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-butanone (NNK) ."Biochemical Biophysical Research Communications. 269. 393-396 (2000)
[Publications] Ken-ichi Fujita et al.: "Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1) ."Life Science. 66. 1955-1967 (2000)
[Publications] Noritaka Ariyoshi et al.: "Handbook of Experimental Pharmacology"Springer-VerlagBerlin. (2001)
[Publications] 有吉範高: "月刊薬事2001年臨時増刊号"じほう. (2001)
[Publications] 鎌滝哲也: "薬物療法学"南江堂. (2001)
[Publications] 有吉範高: "月刊薬事3月臨時増刊号"じほう. 834 (2000)
[Publications] 鎌滝哲也: "薬物代謝学"東京化学同人. 282 (2000)

2000 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] 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)

[Publications] Noritaka Ariyoshi et al.: "A Novel Single Nucleotide Polymorphism Altering Stability and Activity of CYP2A6."Biochemical Biophysical Research Communications. In press. (2001)

[Publications] 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)

[Publications] Noritaka Ariyoshi et al.: "Pharmacogenetics on cancer chemotherapy : from basic studies."Japanese Journal of Clinical Pharmacology and Therapeutics. In press. (2001)

[Publications] 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)

[Publications] 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)

[Publications] Noritaka Ariyoshi et al.: "Studies on the genetic polymorphism of CYP2A6."Drug Metabolism and Disposition. 15. 57-61 (2000)

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

[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. 269. 623-627 (2000)

[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-butanone (NNK) ."Biochemical Biophysical Research Communications. 269. 393-396 (2000)

[Publications] Ken-ichi Fujita et al.: "Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1) ."Life Science. 66. 1955-1967 (2000)

[Publications] Noritaka Ariyoshi et al.: "Handbook of Experimental Pharmacology"Springer-VerlagBerlin. (2001)

[Publications] 有吉範高: "月刊薬事2001年臨時増刊号"じほう. (2001)

[Publications] 鎌滝哲也: "薬物療法学"南江堂. (2001)

[Publications] 有吉範高: "月刊薬事3月臨時増刊号"じほう. 834 (2000)

[Publications] 鎌滝哲也: "薬物代謝学"東京化学同人. 282 (2000)

鎌滝哲也北海道大学, 大学院・薬学研究科, 教授 (00009177)

[Publications] Noritaka Ariyoshi et al.: "Structural characterization of a new variant of the CYP2A6 gene (CYP2A61B) appparently diagnosed as heterozygotes of CYP2A61A and CYP2A6*4C."Pharmacogenetics. 10. 678-693 (2000)