DEVELOPMENT OF THE MODEL SYSTEM FOR DRUG METABOLISM STUDIES

在药物发现和开发阶段，需要构建一个反映体内代谢的模型系统，用于临床前药物代谢研究。此外，迫切需要一种高度可靠的方法来分析生物体液中的痕量化合物。1)建立了基于负离子电喷雾电离(ESI)-MS的胆汁酸3-葡萄糖醛酸苷定量分析方法。通过记录负离子可以获得最高的灵敏度(200fmoL)，所获得的质谱图由强分子离子[M-H]^-和双电荷离子[M-2H]^&lt；2-gt；表征，根据C-24处的酸性部分的pKa值，这些负离子的比例明显地受到添加到柔和相中的盐的酸性成分的影响。该技术在…中的应用更多的证明了它的用处，主要是在肝病的诊断上。2)为了获得电喷雾离子质谱仪测定溶液中不易电离的中性和非极性物质的灵敏度和特异度，研究了生理条件下固定化重组磺基转移酶反衍生化为胆汁酸24-葡萄糖醛酸苷的活性物质。以脱氢表雄酮为模型化合物，在37ﾟC的腺苷-3‘-磷酸-5’-磷酸存在下与固定化酶孵育60min，实现了定量衍生化。在负离子ESI-MS模式下，硫酸盐的光谱显示一个准分子离子[M-H]^-作为干扰峰。3)为了建立一个用于解释药物代谢和处置预测的模型体系，将含有细胞色素P-450酶的大鼠肝微粒体部分非共价固定在固定的人工膜上，并通过跟踪雌二醇的体外羟化来研究酶的活性。该方法被发现在药物的I期代谢图谱和机制研究中很有用。较少

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