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in vivo analytical study on the endogenous agonist for cannabinoid receptor by using a microdialysis technique

利用微透析技术对大麻素受体内源性激动剂进行体内分析研究

基本信息

批准号：
14572151
负责人：
FUKUSHIMA Takeshi
金额：
$ 2.18万
依托单位：
University of Shizuoka (2003-2004)The University of Tokyo (2002)
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

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572151/
关键词：
Anandamide DBD-COCl microdialysis DBD-COC 1 β-シクロデキストリンポリマーラット

项目摘要

This research project was carried out in Graduate School of Pharmaceutical Sciences, University of Tokyo and School of Pharmaceutical Sciences, University of Shizuoka.In 2002, this research was performed in Graduate School of Pharmaceutical Sciences, University of Tokyo. The aim of this research is to determine anandamide (AN) in rat brain microdialysis (MD) sample by using a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection using a fluorescence reagent, DBD-COCl, which can react with primary alcohol of AN. In order to increase recovery of AN from MD probe, β-cyclodextrin derivatives were added in the perfusate of MD. As a result, the addition of 2,3,6-O-trimethyl-β-CD (TriMeCD) afforded to increase the recovery of AN.In 2003-4, this research was performed in School of Pharmaceutical Sciences, University of Shizuoka. Next, extraction conditions for AN determination from rat brain MD sample were investigated. Among them, a mixed solvent of ethyl acetate and toluene (50/50) was effective for the extraction of AN, and further, the addition of TriMeCD gave a larger AN peak. This is resulted form the prevention of undesired AN adsorption to inner surface of the experimental bottle. By using a fluorescence spectrophotometer, the fluorescence intensity of DBD-CO-AN augmented with the increase of CH_3CN percentage in the solvent. Thus, when using a tandem series of an ODS column (250 x 4.6 mm i.d., 3 μm) with a mobile phase containing 95% CH_3CN, the detection sensitivity was approximately 4.5-fold greater than that in the previous HPLC method (detection limit: approximately 6 nM). In the proposed HPLC method, AN in rat brain MD sample was successfully detected after administration of AN to rats. Using the HPLC method developed in this term, the research on the brain AN will be progressed.

本研究项目由东京大学大学院药学研究科和静冈大学药学研究科共同实施，2002年在东京大学大学院药学研究科实施。采用柱切换高效液相色谱-荧光检测法，以能与花生四烯酸（AN）伯醇发生反应的DBD-COCl为荧光试剂，对大鼠脑微透析（MD）样品中的花生四烯酸（AN）进行检测。为了提高MD探针中AN的回收率，在MD灌流液中加入β-环糊精衍生物。2003年4月，静冈大学药学院进行了该研究。接着，研究了从大鼠脑MD样品中测定AN的提取条件。其中，乙酸乙酯和甲苯（50/50）的混合溶剂是有效的萃取AN，进一步，添加TriMeCD得到一个更大的AN峰。这是由于防止了不期望的AN吸附到实验瓶的内表面。用荧光分光光度计测得DBD-CO-AN体系的荧光强度随溶剂中CH_3CN含量的增加而增强。因此，当使用串联的ODS柱（250 × 4.6 mm i.d.，3 μm），移动的相为95%CH_3CN，检测灵敏度约为原HPLC法的4.5倍（检测限约为6 nM）。在所提出的高效液相色谱法，AN在大鼠脑MD样品成功地检测后，AN给药到大鼠。利用本课题建立的高效液相色谱分析方法，可进一步开展脑内硝酸铵的研究。