山乌龟亚属中阿朴菲类生物碱抗心律失常构效关系及给药系统减毒的研究

Multiple ion channel block is one of the main development direction of new anti-arrhythmic drug. In our previous research, Crebanine (Cre), an Aporphine(Ap) alkaloid, which extracted from plants of Subgen. Tuberiphania Lo et M. Yang showed high anti-arrhythmic activity and tendency of multi-channel block, but also obvious toxicity. In order to utilize reasonably plant resource of this subgenus and to discovery the valuable anti-arrhythmic new drugs, and in view of some of the Ap alkaloids have the same activity as Cre and aiming directly at the high activity and obvious toxicity of Cre, this project is to carry out: ① Screening the nature Aps and analyze the structure-function relationship. Ap alkaloids that has been extracted (Crebanine, Dicentrine, Isocorydine, etc.) are used to carry the antiarrhythic pharmacological test and acute-term toxicity test; the method of patch clamp and other methods are used to survey the effect of the Ap alkaloids to calcium channel, potassium channels and sodium channel of cardiac muscle and explore the antiarrhythic mechanism. ② Comparing the antiarrhythic activity and toxicity of Cre among different administration routes and dosage form（iv, p.o. and transdermal）and between Cre and its nanoparticle delivery carrier, and to explore approaches and feasibility for activity enhancment (or remaining) and toxicity reduction. It is expected that the results could provide the basis for screening antiarrhythic lead compounds from Aps, explaining the antiarrhythic mechanism, directing structural modification and exploring methods of reducing the toxicity.

多离子通道阻滞作用是抗心律失常新药研发的方向之一。前期研究发现山乌龟亚属植物中提取的阿朴菲类Aporphine(Ap)成分克班宁（Crebanine，Cre）有较强抗心律失常活性与多通道阻滞趋势，但毒性较大。为合理利用山乌龟亚属植物资源，发现有价值的抗心律失常新药，基于多个Aps成分均有类似活性，针对Cre活性高毒性大的问题，本项目拟开展：① 筛选天然Ap类成份，展开构效关系分析。利用提取分离到的多种Ap类成分（Cre、Dicentrine、Isocorydine等），进行抗实验性心律失常药效学和急性毒性试验；采用膜片钳等法观察其对心肌钙、钾与钠通道的作用，探讨作用机制；② 比较不同给药途径与剂型（静注、口服、经皮）及纳米粒载药对Cre活性与毒性的影响，探索其增（或保）效减毒的途径与可行性。研究可为从Ap类中筛选抗心律失常先导物、阐释作用机制、指导结构修饰与探索降低毒性方法提供依据与思路。

为分析构效关系并筛选高效低毒活性成分或适合剂型，对阿朴菲类生物碱进行提取分离、结构修饰、毒性与药理试验、剂型改造研究。从云南地不容（Stephania yunnanensis Lo）等3个千金藤属植物中分离鉴定了28化合物，其中2个新化合物。对克班宁Cre、异可利定Isoc、千金藤Ste进行B环开环、溴代、甲基化、乙酰化、季铵化、去氢和还原等结构修饰，共获得21个目标化合物；采用氯仿诱发小鼠室颤模型初筛36种成分的活性，结果Cre、荷包牡丹碱Dic、Isoc、青风藤碱Sino，以及3-溴代克班宁等6个合成产物能显著地降低小鼠室颤发生率（P<0.01或0.05,以下同）；小鼠急性毒性试验求得9个化合物与4种Cre微粒制剂、Cre灌胃等的LD50，显示Cre溴代物及纳米粒毒性明显减小；活性比较①对BaCl2致大鼠心律失常，Cre、Dic、Isoc、Ste、荷包牡丹酮、波尔定、荷叶碱、罗默碱、3个Cre溴代物、N-甲基克班宁、去氢千金藤及Sino恢复窦性心律作用显著；② Cre、Isoc、波尔定、Ste、去氢千金藤碱及Sino明显增加乌头碱致大鼠室性心律失常用量；③ Cre、Dic与Isoc能缩短肾上腺素致豚鼠心律失常的持续时间；④ Isoc、Ste，Sino均能降低缺血及再灌注期间大鼠心肌缺血范围与心肌梗死范围；Cre和Sino对hERG通道、IKs、IKu。T-type通道、L-type通道、Nav1.5通道等6个钾、钙与钠通道均有抑制作用，提示通过多靶点调控对抗心律失常。构效关系分析认为阿朴菲A环上C1,2亚甲二氧基、D环取代基数目、溴取代、B环开环或N-季铵化、C环6a-H等均与其抗心律活性及毒性明显相关；研究了Cre经皮贴剂与4种微粒给药制剂的制备方法与质量评价；Cre灌胃与Cre纳米粒静注对氯仿、BaCl2与乌头碱致心律失常，Cre贴剂对BaCl2模型均有显著作用；Cre溶液灌胃吸收快，AUC0-∞=0.333mg/L*h，Tmax=0.25h，Cmax=7.73mg/L；而贴剂经皮缓释作用显著，AUC0-∞=1.968mg/L*h；Tmax=10.333h，Cmax=204.5mg/L，；Cre-PELGE纳米粒有明显延效作用（t1/2β 290.3min）。研究为在阿朴菲类生物碱中发现抗心律失常先导化合物与制剂方法增效减毒提供了实验依据与思路。