候选药物PN404抗血小板聚集作用机制初步研究

Herein,we plan to accomplish an antiplattelet mechanism investegation of action about PN404 candidate(Figure 1).To develop novel antiplatelet aggregation drugs which has independent intellectual property rights works for our country’s long-term development program. Platelet aggregation is the main cause of cardiovascular and cerebrovascular diseases such as atherosclerosis，mioeardial infarction and stroke. Antiplatelet aggregation drugs have a good clinical application in cardiovascu1ar disease，however still with hemorrhage side effects and the antiplatelet mechanisms of some new drugs action were also unknown.Thus further investigation the mechanism are necessary.Since 1996 we have been used to delve into looking for new antiplatelet drugs resemble the structure features of Picotamide through remarking its two side chains on 1 and 3 positions. Antiplatelet drug Picotamide(Figure2), is a derivative of 4-methoxyisophthalic acid, which influences the platelet prostaglandin pathway by inhibiting TXA2 synthesis; furthermore, at equivalent concentrations, and it competitively interferes with TXA2 action by antagonism with specific platelet TXA2 receptor.According to the antiplatelet mechanism and principles of isostere & bioisosterism, 350 novel compounds were designed and synthesized. 60 compounds of which have been found to exhibit significant antiplatelet activities in activity screening in vitro, and espicially one of which, candidate PN404 has the strongest pharmacological effects in our previous pharmacodynamics tests and its pharmacokinetics has been completed. PN404 was chosen as a candidate drug in this application to study on its mechanism of action, including influencing metabolism of rachidonic acid enhancing the release of cyclic adenylic acid within the platelet depressing glycoprotein receptor，inhibiting the agonist of special platelet membrane and receptor. These researches will help us to make clear of the pharmacophore,pharmacological action, specify and some biotransformation laws of PN404 and to know the reasons of PN404 having antiplatelet activities, how affects of interaction between PN404 and receptors, possible therapeutic action and untoward or adverse reaction of PN404. So that we could look into and further probe structure-activity relationships of PN404 and other compounds as above．Besides, publishing papers,applying patents and posting our students will also be our projects. We hope what we do could provide scientific basis for our later research in pharmacological tests & clinical application stages and make our own contribution to improve whole people's overall level of medicine in our country.

研发具有自主知识产权的创新药物符合国家长期发展战略。血小板聚集是引发多种心脑血管疾病的重要因素，抗血小板药物在治疗心血管疾病中有良好应用，但仍存在出血等不良反应，一些新药作用机制也不明确。课题组通过对抗血小板药物吡考他胺进行结构改造制得350个新的结构类似物，经体外抗血小板活性粗筛发现了60个高活性化合物，其中15个化合物的LD50﹥5000mg/kg。选出其中3个进行的药效学研究表明，候选药物PN404各项指标最高，均优于吡考他胺和阿司匹林。本申请将从影响花生四烯酸代谢途径、促进血小板内环腺苷酸释放和血小板胞浆游离钙浓度等方面，对PN404的抗血小板作用机制进行初步研究，明确其具有抗血小板活性的原因和主要作用环节、可能的的靶点类型及特异性归属，比较其与吡考他胺作用机制的异同性。为今后药理研究及临床应用提供依据。为提高我国全体人民的用药水平做出贡献。

对PN404的作用机制研究，重点在花生四烯酸代谢系统方面；在PN404促进血小板内环腺苷酸（cAMP）释放方面和对血小板胞浆游离钙浓度影响方面只进行初步研究。.机制初步研究实验结果表明，与吡考他胺类似，PN404属于抑制花生四烯酸代谢的药物，通过抑制COX-1活性，降低TXA2的生成而发挥抗血小板聚集和抗血栓作用；在抗血小板聚集方面具有多重作用机制。.1.备选药物PN404作用机制初步研究结果.在花生四烯酸代谢系统方面，研究表明，PN404能抑制环氧酶的生成，能抑制TXB2的生成，可促进PGI2生成（PGI2与TXA2是竞争关系），即PGI2/TXA2也升高，相对减少TXA2的量，从而抑制花生四烯酸诱导的血小板聚集及血栓，在抗血小板聚集方面表现出多重作用机制。.在促进血小板内环腺苷酸释放方面，PN404在相应剂量组中对cAMP浓度无影响。.对血小板胞浆游离钙浓度影响方面，试验表明，PN404可降低血小板内钙离子浓度，有一定的钙拮抗作用。.2.备选药物PN404作用机制初步研究结论.PN404具有以下多重作用机制：.1)有显著抑制由ADP诱导的血小板聚集作用；.2)能显著抑制由花生四烯酸(AA)诱导的血小板聚集作用；.3)能显著抑制胶原诱导的血小板聚集作用；.4)能抑制TXA2的生成；.5)能促进PGI2的生成； .6)能抑制COX-1的生成。.7)有一定的钙拮抗作用，可降低血小板内钙离子浓度，使血管舒张。.8)可能没有促进血小板内cAMP释放的作用。.3. PN404和吡考他胺作用机制的异同点.相同点：.①均能抑制ADP、AA和胶原诱发的血小板聚集； .②均能拮抗TXA2受体的作用；.③能促进PGI2的合成与释放；.④能抑制TXA2合成酶，从而抑制TXA2的合成；.⑤有一定的钙拮抗作用，可降低血小板内钙离子浓度。.不同点：吡考他胺能提高血小板内cAMP含量，具有促进血小板内cAMP释放的作用；PN404没有促进血小板内cAMP释放的作用。.4. 关于PN404和吡考他胺等与L929细胞相互作用实验.实验表明吡考他胺总体上对L929细胞有更小影响或更低毒性。.关于机制更加全面的结论有待于进行更加深入和系统的研究。研究结果共获得中国发明专利授权1项，申请1项；发表论文3篇（发表1篇；已接收2篇）；培养硕士研究生8名（2014年3月毕业3人，另有5名继续做后续相关工作）。