P-糖蛋白抑制载体调控丹参素前药设计及脑靶向给药的研究

Danshensu (3-(3,4-dihydroxyphenyl) lactic acid) has many biological activities such as antithrombotic property, resisting free radical damage and inhibiting intracellular calcium concentration overload. However, it has short half-life time and great difficulty in penetrating the blood brain barrier to reach the brain, which limits its application in the treatment of cerebrovascular diseases. P-glycoprotein (P-gp) plays an inhibiting role in Danshensu transportation in brain. Pretreated with P-gp inhibitor or reducing the expression of P-gp may increase Danshensu concentrations in the brain. In this subject, the brain targeting design strategy of P-gp inhibition is introduced into Danshensu prodrug design. It is proposed to modulate the P-gp affinity and pharmacokinetic characteristics of the prodrug by P-gp inhibition carrier, therefore brain targeting delivery of Danshensu is achieved. We choose borneol, tetramethyl pyrazine, paeonol as P-gp inhibition carrier to design a series of Danshensu prodrug; at the same time some computional chemistry methods such as P-gp molecular docking are applied to exclude some poor molecules and the others are further analyzed and screened, the better prodrug molecules are synthesized and characterized. On this basis, the study on the metabolism in vitro and in vivo, brain targeting effect, activity evaluation of the prodrugs are carried out. The study sought to find the optimal Danshensu prodrug with prolonged half-life time, retained or enhanced biological activities and improved brain targeting effect, and explore the P-gp inhibition carrier modulation mechanism on brain targeting delivery of the prodrug.

丹参素具有抗血栓形成、抗自由基、抑制细胞内[Ca2+]i升高等作用；但由于其体内半衰期短、难以穿透血脑屏障到达脑部，限制了其作为脑血管病治疗药物的用途。P-糖蛋白(P-gp)对丹参素跨越血脑屏障转运至脑具有抑制作用，合用P-gp抑制剂或降低P-gp表达均可提高脑组织的丹参素浓度。本课题将P-gp抑制的靶向策略引入丹参素的前药设计，拟通过P-gp抑制载体调控前药分子与P-gp的亲和性及其药代特性，达到脑靶向给药的目的。本课题中我们选择冰片、川芎嗪、丹皮酚三类分子作为P-gp抑制载体设计一系列结构多样性的丹参素前药；同时拟采用P-gp的分子对接等计算化学方法对设计的前药分子进行排除与初筛，并对筛选出的较优分子进行化学合成与结构鉴定。在此基础上，进行体内外代谢、靶向性、活性评价等研究，试图找出半衰期延长、活性保留或增强并且脑靶向性提高的丹参素前药，并探索P-gp抑制载体对前药脑靶向性的调控机制。

丹参素(Danshensu, DSS)化学稳定性差、血脑屏障（Blood brain barrier, BBB）透过率低；同时，P-糖蛋白(P-glycoprotein, P-gp)对DSS跨越BBB转运至脑亦具有抑制作用。合用P-gp抑制剂或降低P-gp表达均可有效提高DSS脑部分布，为此，本项目拟将P-gp抑制的靶向策略引入丹参素脑靶向前药设计。.实验室前期研究已证实：吡嗪结构在银杏内酯衍生物PGB中充当P-gp调节因子；PGB对大鼠脑组织P-gp表达及外排的抑制作用有利于其实现脑部靶向。因此，在DSS靶向前药设计中，我们优先将四甲基吡嗪及其药效团作为载体引入，并设计一系列衍生物。通过对14个典型衍生物（DT1-DT14）P-gp亲和力计算、脑靶向相关参数预测，筛选得到3个较优分子（DT1、DT3和DT4）。.DT1、DT3和DT4在大鼠体外血浆和脑匀浆代谢研究表明：DT3、DT4降解均经历DT3/DT4→DT1→DSS过程，DT3在血浆中降解相对DT1和DT4有所减缓，而在脑匀浆中可快速代谢出DSS。接下去，大鼠体内给予DT3和丹参素钠（SDSS）后，血浆中DT3代谢性质得以改善，其DT3→DSS半衰期和平均驻留时间均比SDSS延长（MRT: 46.177 vs. 9.412 min; t1/2: 29.823 vs. 7.571 min）。脑匀浆中DT3→DSS分布量较SDSS有较大提高（AUC: 13.701 vs. 2.783 mg/L×min），脑靶向指数为18.66；且脑内半衰期和平均驻留时间均延长，消除速率减小，这表明DT3具有明显脑靶向和缓释作用。.此外，我们也对丹参素-吡嗪酰胺类衍生物(D001-D011)进行了体外抗血小板聚集、抗PC12细胞损伤评价及小鼠脑缺血损伤保护研究，证实了D002 和D003对脑损伤具有较优保护作用。.冰片在脑靶向给药中具有BBB促透和P-gp 抑制双重作用，本课题通过大鼠体内给药证实了丹参素冰片酯（DBE）及丹参素钠-冰片联合用药（SDSS-B）的脑靶向作用与其有效降低大鼠海马组织P-gp表达水平（P< 0.01，与Control或SDSS组对比）有直接联系。.本课题研究证实了P-gp抑制载体如吡嗪、冰片在调控丹参素脑靶向给药中具有重要作用，同时为其它药物的脑靶向衍生物设计提供参考。