DEVELOPMENT OF THE MODEL SYSTEM FOR DRUG METABOLISM STUDIES

药物代谢研究模型系统的开发

• 批准号: 08457618
• 负责人: GOTO Junichi
• 金额: $ 1.02万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457618/
• 关键词: LC; MS; Bile Acid; Immobilized enzyme; Glucuronyltransferase; Derivatization; Ester glucuronide; グルクロニルトランスフェラーゼ; 光学異性薬物; エレクロスプレーイオン化; 高速液体クロマトグラフィー

Upon the phase of a drug discovery and development, construction of a model system reflecting the invivo metabolism for pre-clinical drug metabolism studies is desired. Moreover, a highly reliable method for the analysis of trace compounds in biological fluids is urgently needed. Therefore, we have been carried out on a comprehensive investigation by the technique of liquid chromatography-mass spectrometry (MS) and an immobilzed drug metabolizing enzyme.1) A quantitative analytical method based on negative ion electrospray ionization (ESI) -MS for bile acid 3-glucuronides has been developed. Maximum sensitivity (200 fmol) could be achieved by recording the negative ion and mass spectra obtained were characterized by an intense-molecular ion [M-H]^- with a doubly charged ion [M-2H]^<2->, and the ratio of these negative ions were markedly influenced by the acidic component of salt added to a moble phase, acccording to a pKa value of an acidic moiety at C-24. Application of this technique … More demonstrate its usefullness, principally in the diagnosis of liver disease. The method has also applied to characterization of bile acid 24-glucuronides in incubation mixture with rat liver glucuronyltransferase.2) For Obtaining sensitivity and specificity in ESI-MS for measuring the neutral and nonpolar substances that cannot be easily ionized in solution, reversed derivatization into ESI active substance by emloying immobilized recombinant sulfotransferase under physiological conditions has been studied The derivatization using dehydroepiandrosterone as a model compound was achieved quantitatively by incubation with the immobilized enzyme in the prsence of adenosine 3'-phosphate 5'-phosophosulfet at 37 ﾟC for 60 min. The spectrum of the sulfate exhibited a quasimolecular ion [M-H]^- as an inrence peak under the negative ion ESI-MS mode.3) To creat a model system that are being used for the interpretation of prediction of drug metabolism and disposition, rat liver microsomal fractions containing cytoihrome P-450 enzymes have been noncovalently immobilized on an immobilized artificial membrane, and the enzyme activity was investigated by following the in vitro hydroxylation of estradiol. The method was found to be useful in phase I metabolic profiling and mechanistic studies of drugs. Less

在药物发现和开发的阶段，需要建立一个模型系统，该模型系统反映了临床前药物代谢研究的现象代谢。此外，迫切需要一种高度可靠的方法来分析生物流体中的痕量化合物。因此，我们已经通过液相色谱 - 质量光谱法（MS）的技术进行了全面研究，并开发了一种基于负离子电喷雾电离（ESI）-MS的胆汁酸3-葡萄糖醛酸的基于负离子电化电离（ESI）-MS的定量分析方法。可以通过记录负离子和获得的质谱来实现最大敏感性（200 FMOL），其特征是强烈分子离子[M-H]^ - 具有双电荷离子[M-2H]^^<2->，这些负离子的比率显着影响到盐的酸性成分。该技术的应用……更多证明其有用性，主要是在肝病的诊断中。该方法还应用于与大鼠肝肝戊醛酮lutrylylansferase孵育混合物中胆酸24-葡萄糖醛酸的表征2）研究使用脱氢表雄酮作为模型化合物的衍生化是通过与固定的3'-磷酸腺苷5'-磷酸腺苷5'-磷酸磷酸酶在37°C下进行定量实现的，持续60分钟。 The spectrum of the sulfate exhibited a quasimolecular ion [M-H]^- as an inrence peak under The negative ion ESI-MS mode.3) To create a model system that are being used for the interpretation of prediction of drug metabolism and disposition, rat liver microsomal fractions containing cytoihrome P-450 enzymes have been noncovalently immobilized on an immobilized artificial membrane,并通过遵循雌二醇的体外羟基化研究了酶活性。发现该方法在I期代谢分析和药物的机理研究中很有用。较少的

项目成果

Sigeo Ikegawa: "Stereoisomeric bio-inversion of(25R)-and(25S)-3α,7α,12α-trihydroxy-5β-cholestanonic acid CoA thioesters in rat liver perosisme." Enantiomer. (in Press). (1998)

Sigeo Ikekawa：“大鼠肝脏 Perosisme 中 (25R)-和 (25S)-3α,7α,12α-三羟基-5β-胆甾烷酸 CoA 硫酯的立体异构生物转化”（出版）。

shigeo Ikegawa, Kenji Matsuura, Takehiro Sato, Ni Made Ria Isriyanti, Toshifumi Niwa, Shinichi Miyairi, Hideo Takashina, Youichi Kawashima and Junichi Goto: "Enantioselective immunoaffinity extraction for simultaneous determination of optically active buf

shigeo Ikekawa、Kenji Matsuura、Takehiro Sato、Ni Made Ria Isriyanti、Toshifumi Niwa、Shinichi Miyairi、Hideo Takashina、Youichi Kawashima 和 Junichi Goto：“用于同时测定光学活性 buf 的对映选择性免疫亲和萃取

Shigeo Ikegawa: "Separation and detection of bileacid 3-glucuronides in humanurine by liquid chromatography/electrospray ionization-mass spectrometry" Biomed. Chromatogr.10・6. 313-317 (1996)

Shigeo Ikekawa：“通过液相色谱/电喷雾电离-质谱法分离和检测人尿中的胆汁酸3-葡萄糖醛酸”Biomed.10・6（1996）。

Shigeo Ikegawa: "Separatory determination of bileacid 3-sulfates by liquid chromatography/electrospray ionization-mass spectrometry" J. Mass Spectrometry. (in press).

Shigeo Ikekawa：“通过液相色谱/电喷雾电离-质谱法分离测定胆汁酸 3-硫酸盐”J. 质谱法。

Shigeo Ikegawa, Naoaki Murao, Takashi Motoyama, Takamitsu Yanagihara, Toshifumi Niwa and Junichi Goto: "Separation and detection of bile acid 3-glucuronides in h umanurine by liquid chromatography/electrospray ionization-massspectrometry." Biom ed.Chromat

Shigeo Ikekawa、Naoaki Murao、Takashi Motoyama、Takamitsu Yanagihara、Toshifumi Niwa 和​​ Junichi Goto：“通过液相色谱/电喷雾电离质谱法分离和检测人尿中的胆汁酸 3-葡萄糖醛酸苷。”