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

在药物发现和开发阶段，需要构建反映体内代谢的模型系统用于临床前药物代谢研究。此外，迫切需要一种用于分析生物流体中痕量化合物的高度可靠的方法。因此，我们采用液相色谱-质谱（LC-MS）联用技术和固定化药物代谢酶进行了全面的研究。1）建立了胆汁酸3-葡萄糖醛酸苷的负离子电喷雾质谱（ESI-MS）定量分析方法。通过记录负离子可以获得最大灵敏度（200 fmol），并且所获得的质谱的特征在于强分子离子[M-H]^-与双电荷离子[M-2 H]^<2->，并且这些负离子的比例显著地受添加到移动相中的盐的酸性组分的影响，根据在C-24处的酸性部分的pKa值。应用这一技术 ...更多信息 证明其有用性，主要是在肝脏疾病的诊断。该方法还应用于与大鼠肝葡萄糖醛酸转移酶孵育混合物中胆汁酸24-葡萄糖醛酸苷的表征。2）为了获得ESI-MS测量溶液中不易电离的中性和非极性物质的灵敏度和特异性，本文研究了在生理条件下利用固定化重组磺基转移酶反向衍生化ESI活性物质的方法。在负离子电喷雾质谱模式下，硫酸根离子的光谱显示准分子离子[M-H]^-为参考峰。3）建立了一个用于预测药物代谢和处置的模型体系，将含有细胞色素P-450酶的大鼠肝微粒体组分非共价固定在固定化人工膜上，并通过体外雌二醇羟化反应研究了酶的活性。该方法被认为是有用的I相代谢谱和药物的机制研究。少

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。

Junichi Goto: "Separation and characterization of carboxyl-linked glucuronides of bile acids in incubation mixture of rat livermicrosomes." Steroids. (in press). (1998)

Junichi Goto：“大鼠肝微粒体孵育混合物中胆汁酸羧基连接的葡萄糖苷酸的分离和表征。”