新型抗帕金森病寡肽前药库的建立与筛选

We propose to establish a library of thousands of L-DOPA-containing oligopeptides and then to discover new, highly effective anti-Parkinsonism prodrugs through screening tests of enzymatic degradation and Parkinson's disease cell and animal models. Parkinson's disease is a neurodegenerative disorder. Among numerous drugs, levodopa (L-DOPA) is the most efficacious one. However, due to its instability and low permeability across the blood-brain barrier (BBB), L-DOPA is taken in large amounts and causes severe damages. To search for a replacement, researchers have eyed on chemically modified L-DOPA prodrugs. A potential methodology is to incorporate L-DOPA into oligopeptides. Some L-DOPA-containing oligopeptides, e.g. H-Val-DOPA-OH, are more active than L-DOPA in reversing reserpine-induced catatonia. In 2010, Wang et al. reported a L-DOPA dipeptide with better oral bioavailability and slow dopamine-release property. Unfortunately, DOPA is easily oxidizable and its chemical manipulation requires proper protection of the catechol. Acetonide cyclization is proved an ideal strategy. However, Fmoc-DOPA(Acetonide)-OH, is highly costly and thus is not readily available. Therefore, until today only several tens of such prodrugs have been reported. To expedite the discovery of new anti-Parkinsonism drugs, we have recently developed a convenient method to synthesize Fmoc-DOPA(Acetonide)-OH and successfully applied it to make short DOPA-containing peptides. After applying the strategy, we also obtained a highly pure dopamine prodrug, which strongly withstands enzymatic degradation and readily permeates across BBB. Encouraged by the results, we plan to synthesize hundreds of grams of Fmoc-DOPA(Acetonide)-OH, enough to couple with various L-amino acids, D- amino acids, metabolites, fatty acids, or a combination thereof to produce a library of thousands of DOPA-containing oligopeptides. Afterwards, a series of experiments will be carried out to test them against enzymatic degradation in intestinal mucosal suspension and in liver homogenate. The promising ones will then be subjected to cell model tests, such as PC12 cells and SH-SY5Y cells. Finally, top candidates will be evaluated by animal models, including MPTP-induced rat model and 6-OHDA-induced SD mouse model, to identify the best anti-Parkinsonism L-DOPA-containing oligopeptide prodrugs.

建立一个完备的含多巴的寡肽库集，并对其进行一系列地多模型地筛选，极有可能发现新型高效的抗帕金森病的多巴前药。帕金森病是一种严重的神经疾病；左旋多巴一直是该病最有效的黄金标准。由于多巴的易降解性和低脂溶性，病人需要大量服食并深受其害。采取化学修饰的方法可望改善多巴的性能，例如含多巴的寡肽表现出很高的生物药效率。然而多巴易被氧化不易处理，其寡肽合成关键中间体 Fmoc-DOPA(Acetonide)-OH价格昂贵，至今文献仅报道了几十种该类前药。我们计划通过优化合成方法得到数百克此中间体，并采用基于Fmoc的固相多肽合成法，与左旋氨基酸、右旋氨基酸、Omega-3脂肪酸、代谢中间物等进行反应，合成五大类共数千种含多巴的寡肽库集。在此基础上，采用小肠粘膜悬浊液降解、肝匀浆降解，PCl2细胞法，SH-SY5Y细胞法等实验筛选出数种最优的寡肽；再采用药物致病的动物模型确定出抗帕金森病的最优寡肽前药。

帕金森病是全球第二大神经退行性疾病。现阶段药物治疗的最佳方案是由L-DOPA，多巴脱羧酶抑制剂（DDCI，如卡比多巴）和儿茶酚氧位甲基转移酶抑制剂（COMTI，如恩托卡朋）组成的复方制剂。其中，L-DOPA是抗帕金森病的金标准，但生物利用度太低（1%）；DDCI和COMTI通过抑制相关降解酶，提高L-DOPA利用度到约15%，但同时带来较大毒副作用。.探索一种新型的抗帕金森病药是本项目的核心内容。.含2-3个氨基酸的寡肽可以不被分解，直接透过小肠壁寡肽通道而被吸收。据此，本项目提出了把L-DOPA与氨基酸结合成寡肽作为前药，进入人体被降解后释放出L-DOPA的研究方案。同时，采用肝匀浆，自由基清除，PC12细胞等实验测定寡肽的稳定性，细胞毒性和神经保护作用，以初步评估寡肽前药的有效性和安全性。.借助实验室合成的缩丙酮保护的L-DOPA中间体，采用液相或固相合成法制备了一个包括近50个含多巴直链二肽、三肽和环二肽的前药库集。在SD大鼠肝匀浆中：直链二肽展现出不同的稳定性，其中DOPA-Met的半衰期很短(16 min), 而DOPA-Asp的最长(174 min)；环二肽比直链二肽更稳定，cyclo(DOPA-Ala)的半衰期最长（57 h）。对降解混合物进行HPLC分析，实验检测到直链二肽释放出L-DOPA，但环二肽未检测到。在DPPH自由基清除上，直链二肽DOPA-Glu的能力最强（IC50,4.2 μM），是L-DOPA的10倍，维C的9倍，抗心肌缺血药丹参素的4倍。PC12细胞毒性实验显示，含多巴直链二肽基本无细胞毒性，且有一定的促增殖作用。鱼藤酮诱导的帕金森病PC12细胞模型实验表明，cyclo（DOPA-Ser）有一定的神经保护作用。. 据此提出治疗帕金森病的群药思想：采用L-DOPA与营养小分子（氨基酸，脂肪酸等）结合形成群药库集，从中选取缓释前药（DOPA-Asp），DDCI（理论筛选中），COMTI,自由基清除剂（DOPA-Glu），神经保护剂（cyclo(DOPA-Ser))组成复方/群药制剂全面对抗帕金森病。另外，本项目建立了一种实验室快捷检测L-DOPA衍生物的分析方法，探索出一种更优的制备Fmoc-DOPA(Acetonide)-OH的方法。这些科研发现将极大促进L-DOPA相关化合物的合成与活性筛选，推进抗帕金森病药物研究快速发展。