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Analysis of C-3 epimerization mechanism in vitamin D_3 to develop a new osteoporosis drug

分析维生素D_3中C-3差向异构机制开发骨质疏松新药

基本信息

批准号：
14572030
负责人：
HIGASHI Tatsuya
金额：
$ 2.18万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

First, highly sensitive LC/MS methods for analysis of vitamin D_3 drug and metabolites were developed. Cookson-type reagents, which quantitatively react with vitamin D_3 compounds to form the Diets-Alder adducts, were employed to enhance their detection responses in APCI-MS. Pharmacokinetic study of 1α-hydroxyvitamin D_3 in humans was carried out by the LC/MS method combined with derivatization with a proton-affinitive Cookson-type reagent, DMEQTAD. Using this method, urinary vitamin D_3 metabolites in human under physiological conditions were also detected and characterized. Next, an electron-affinitive Cookson-type reagent, NPTAD, was applied to the assay of 25-hydroxyvitamin D_3 in human plasma using LC/electron capture APCI-MS. This method was superior to the commercially available RIA in sensitivity and sample throughput. Furthermore, a new derivatization reagent having the boronic acid as the reacting group was developed for the assay of 24,25-dihydroxyvitamin D_3 [24,25(OH)_2D_3].Second, studies on the C-3 epimerization in 24,25(OH)_2D_3 were performed. In consequence, we found the 3-epimer[3-epi-24,25(OH)_2D_3] is formed from 24,25(OH)_2D_3 by the catalysis of 3α-and β-hydroxysteroid dehydrogenase (HSD) and the HSD-catalyzed epimerization is reversible. In addition, 3-oxo-form was also isolated from the reaction mixture, which strongly suggested that the formation of the 3-epimer by HSD involves a dehydrogenation and reduction processes.In third study, osteoclastic and osteoblastic activities of some vitamin D_3 compounds were examined using a culture system of the goldfish scale. It was observed that 3-epi-24,25(OH)_2D_3 activated osteoblastic cell at 10^<-8>M, which was different from the effects of 24,25(OH)_2D_3 and active vitamin D_3, 1,25-dihydroxyvitamin D_3, in bone metabolism.

首先，建立了维生素D_3药物及其代谢产物的高灵敏LC/MS分析方法。本文采用Cookson型试剂DMEQ 3衍生化液相色谱-质谱联用技术研究了1α-羟基维生素D_3在人体内的药代动力学。应用该方法对人体生理条件下尿中维生素D_3代谢产物进行了检测和表征。然后，将Cookson型亲电子试剂NP 100应用于LC/EPCI-MS测定人血浆中25-羟基维生素D_3，该方法在灵敏度和样品通量方面均上级市售RIA。此外，还研制了一种新的以硼酸为反应基团的衍生试剂，用于24，25-二羟维生素D_3 [24，25（OH）_2D_3]的测定。结果表明，24，25（OH）_2D_3在3α-和β-羟基类固醇脱氢酶（HSD）的催化下生成3-差向异构体[3-epi-24，25（OH）_2D_3]，且HSD催化的差向异构反应是可逆的。此外，还从反应混合物中分离出3-氧代体，这有力地表明HSD形成3-差向异构体的过程涉及脱氢和还原过程。第三项研究用金鱼鳞片培养系统研究了某些维生素D_3化合物的成骨和成骨活性。10 μ M时，3-表-24，25（OH）_2D_3对成骨细胞有激活<-8>作用，这与24，25（OH）_2D_3和活性维生素D_3（1，25-dihydroxyvitamin D_3）对骨代谢的影响不同。