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Development of cyclooxygenase-2 assay kits for new anti-inflammatory drugs

新型抗炎药环氧合酶2检测试剂盒的研制

基本信息

批准号：
07557014
负责人：
YAMAMOTO Shozo
金额：
$ 9.92万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Fatty acid cyclooxygenase (COX) initiates the synthesis of bioactive prostaglandins and thromboxanes, and has been considered as a target of anti-inflammatory drugs. The enzyme had been found in vesicular gland, platelets, stomach and various other animal tissues. In 1991 an isozyme of the enzyme (COX-2) was discovered, and found to be an inducible enzyme. Since COX-2 is induced rapidly and transiently by LPS and proinflammatory cytokines, more attentions have been directed to COX-2 rather than COX-1 in the development of anti-inflammatory drugs. The purpase of this project is to develop and establish practicallly convenient assay methods of the protein and mRNA of COX isozymes and the in vivo effect of anti-inflammatory drugs.1) Antibodies were raised with a COX-2 fragment with a specific sequence. COX-1 selective antibodies were found among monoclonal antibodies previously raised against the enzymes of bovine vesicular gland and human platelets. These antibodies for COX-1 and COX-2 could be applied to Western blottings of the enzymes.2) Amplification of mRNAs for COX-1 and COX-2 by RT-PCR was attempted to develop sensitive and convenient assays. Non-radioactive probes by the use of a fluorescent label are being prepared.3) For the assessment of in vivo effects of drugs, diseases models were investigated. Rat pleurisy induced by carrageenin was used for COX-2 studies. Non-treated (for COX-1) and LPS-treated (for COX-2) rats received arachidonic acid intravenously, and the changes of arachidonate metabolites in the blood were determined quantitatively upon administration of drugs.

脂肪酸环氧合酶（考克斯）是生物活性的环氧丙烷和血栓烷的合成的起始酶，被认为是抗炎药物的作用靶点。该酶存在于泡状腺、血小板、胃和多种动物组织中。1991年发现了该酶的同工酶（考克斯-2），并发现其为诱导酶。由于考克斯-2可被LPS和促炎细胞因子快速、短暂地诱导表达，因此在抗炎药物的开发中，考克斯-2的作用比考克斯-1更受关注。本课题的目的是发展和建立实用简便的考克斯同工酶蛋白和mRNA的检测方法及抗炎药物的体内效应。1）用特异性序列的考克斯-2片段制备抗体。考克斯-1选择性抗体中发现的单克隆抗体先前提出的对酶的牛水泡腺和人血小板。2）应用RT-PCR技术扩增考克斯-1和考克斯-2的mRNA，建立了灵敏、简便的检测方法。通过使用荧光标记的非放射性探针正在被研究。3）为了评估药物的体内效应，研究了疾病模型。将角叉菜胶诱导的大鼠胸膜炎用于考克斯-2研究。未处理的（对于考克斯-1）和LPS处理的（对于考克斯-2）大鼠静脉内接受花生四烯酸，并且定量测定给药后血液中花生四烯酸代谢物的变化。