新型毒蕈碱型M1受体激动剂的发现及其在Alzheimer症动物模型中的评价

发布时间：2018-09-01 14:54

【摘要】：随着人口老龄化程度的不断加剧,一些与大脑衰老相关的神经退行性疾病的流行程度和发病率不断攀升,如阿尔茨海默病(Alzheimer's Disease, AD)等,日益成为威胁人类健康的主要疾病之一,也给社会、经济和家庭带来沉重的负担。AD是最常见的痴呆症,从发现至今已经一个多世纪,人们对AD的成因仍不清楚,目前也没有能有效治疗AD的药物问世。目前FDA批准的用于AD的药物主要为基于胆碱能假说而来的胆碱酯酶抑制剂(Acetylcholinesterase inhibitor, AChE I),此类药物通过抑制乙酰胆碱(Acetylcholine, ACh)的降解来提高大脑中的ACh水平,从而缓解AD症状。但随着AD病情的加重和胆碱能神经元的逐步丢失,病人自身产生的ACh会严重降低,因此依赖于内源性ACh的AChE I的治疗效果也会逐渐丧失。另一方面,毒蕈碱型M1乙酰胆碱受体(Muscarinic M1acetylcholine receptor, M1mAChR)在AD病人大脑中的密度没有发生下降。而且大量研究表明,M1受体在AD的病理过程中扮演重要角色,是治疗AD的一个重要靶点。M1受体激动剂可以有效改善AD的多个典型症状,包括大脑内由于Ap前体蛋白(Amyloid precursor protein, APP)的错误剪切而造成的神经毒性蛋白Aβ的产生和沉积,tau蛋白的过度磷酸化,以及由于胆碱能神经元的丢失而造成的胆碱能系统的损伤等。一些M1受体激动剂先后进入临床实验,如Xanomeline在临床实验中已被证明能改善AD患者的认知能力和行为异常,但由于副作用较大而在三期临床实验中被淘汰。为了改进Xanomeline,以期获得活性更好、副作用更小的新型M1受体激动剂,我们与北京大学雷小平教授课题组合作,通过对Xanomeline的母核进行的系统的修饰和改造,获得了一系列Xanomeline衍生物,本文的工作由此展开。在内容上,本文共分为四章,从Xanomeline这个在三期临床被淘汰的药物出发,第一章描述了对Xanomeline系列衍生物的筛选和EUK1001的发现,并初步分析了EUK1001对APP的剪切的影响;第二章是在体实验部分,评价了EUK1001在AD动物模型前脑特异性早老素双敲除(PS cDKO)小鼠的治疗效果；第三章探讨了抗衰老蛋白klotho在PS cDKO小鼠神经退行性症状中的可能作用；第四章则综述了M1受体激动剂及klotho等的一些研究进展。具体为： 1、新型M1受体激动(?)-Xanomeline新型衍生物EUK1001的筛选利用基于报告基因技术的细胞筛选模型,对172个Xanomeline的衍生物进行了筛选,找到了18个活性化合物,其中包括活性最好的两个化合物EUK1001和EUK1002。与Xanomeline相比,EUK1001对M1受体的EC50更小,最大激活倍数更高。EUK1001和EUK1002的活性在瞬时转染了M1受体和报告基因的中国仓鼠卵巢细胞(Chinese hamster ovary cells, CHO)细胞中得到了确证。同时,克隆并在小鼠神经母细胞瘤细胞Neuro2A (N2a)中过表达了人源APP基因瑞典型突变体(APPsw),以此为AD细胞模型,发现EUK1001和EUK1002能通过激活M1受体促进具有神经保护作用的sAPPa的释放。从这些筛选结果和离体实验数据可以看出,EUK1001和EUK1002不仅可以比Xanomeline更有效地激活M1受体,还能促进APP的α-剪切,在调节AD的Ap病理途径中可能有一定潜力。 2、EUK1001对前脑特异性早老素双敲除(PS cDKO) AD小鼠模型部分神经退行性症状的改善随后的动物药理和毒理实验结果显示,EUK1001诱发的副作用次数明显少于Xanomeline,而EUK1002则没有得到改进。在本实验中,以早老素PS1/PS2前脑特异性双敲除(PS cDKO)小鼠为AD动物模型,从行为学、组织学和分子生物学等几个不同的水平和角度,分析了EUK1001对PS cDKO小鼠的神经退行性症状的影响。结果发现,经过3个月的慢性给药(0.5mg/kg/day)后,在新物体识别实验中,EUK1001可以显著改善PS cDKO小鼠受损的识别记忆能力,同时对大脑切片的分析结果显示,EUK1001能有效缓解由于神经元大量丢失而造成的脑萎缩,但在这两方面,同剂量的Xanomeline都没有表现出明显的改善效果。最后在分子水平的分析显示,EUK.1001和Xanomeline都能有效地降低PScDKO小鼠大脑内的tau蛋白过度磷酸化,但它们对PS cDKO小鼠升高的炎症反应都没有抑制作用。综合这些结果可以看出,EUK1001对PS cDKO小鼠的部分神经退行性症状有明显的改善作用,而且效果要优于同剂量的Xanomeline,具有进一步开发的潜力。 3、前脑特异性早老素双敲除(PS cDKO)小鼠中klotho表达的下降和血磷的升高 Klotho蛋白是一个重要的抗衰老因子,只在肾脏和大脑中高表达,但以循环因子的形式在全身多个部位发挥多种功能,如调节钙磷平衡,抗氧化应激,抗炎症等。Klotho水平的下降与衰老及多种疾病有关,但klotho在一些神经退行性疾病(如AD)中的作用尚未被阐明。在本实验中,我们分析了AD动物模型PS cDKO小鼠中klotho的表达以及若干与klotho功能相关的指标,如血磷、血钙和肾功能等,并且结合前人在该模型中的一些结果,初步探讨了klotho在PS cDKO小鼠的神经退行性症状中可能发挥的作用。结果发现,与同龄正常对照小鼠相比,12月龄PS cDKO小鼠肾脏klotho表达显著下降,血磷水平明显升高,血钙水平略微下调,但没有观察到明显的肾功能障碍,而在3月龄时,肾脏klotho水平未发生变化,血磷血钙亦与正常对照无异,但此时大脑klotho水平已发生下调。这些结果初步显示,klotho可能与PS cDKO鼠的症状有关,其表达水平的下降可能导致了血磷浓度的上升,但不足以引发肾功能障碍。此外,klotho作为一个抗氧化和抗炎症的因子,其表达水平的下降可能与PS cDKO小鼠氧化压力和炎症反应的升高有关,但需要进一步的实验证实。
[Abstract]:With the increasing aging of the population, the prevalence and incidence of some neurodegenerative diseases related to brain aging, such as Alzheimer's Disease (AD), have become one of the main diseases threatening human health, and have brought heavy burden to society, economy and family. It has been more than a century since the discovery of dementia. The cause of AD is still unknown, and there are no effective drugs to treat it.
Currently, FDA-approved drugs for AD are mainly cholinesterase inhibitors (AChE I) based on the cholinergic hypothesis. These drugs increase ACh levels in the brain by inhibiting the degradation of acetylcholine (ACh), thus alleviating AD symptoms. On the other hand, the density of muscarinic M1 acetylcholine receptor (M1mAChR) in the brain of AD patients has not decreased. And a large number of studies have shown that M1 receives. M1 receptor agonists can effectively ameliorate many typical symptoms of AD, including the production and deposition of neurotoxic protein A beta and the hyperphosphorylation of tau protein due to the wrong shearing of Ap precursor protein (APP) in the brain. Some M1 receptor agonists have been introduced into clinical trials, such as Xanomeline, which has been shown to improve cognitive and behavioral abnormalities in AD patients, but has been eliminated in phase III clinical trials due to its large side effects.
In order to improve Xanomeline and obtain a novel M1 receptor agonist with better activity and less side effects, we have obtained a series of Xanomeline derivatives through systematic modification and modification of the parent nucleus of Xanomeline in collaboration with Professor Lei Xiaoping of Peking University. In the first chapter, we describe the screening of Xanomeline derivatives and the discovery of EUK1001, and preliminarily analyze the effect of EUK1001 on the shearing of APP. In the second chapter, we evaluate the effect of EUK1001 on the forebrain specific presenilin double knockout (PS cDKO) in AD animal model in vivo. In the third chapter, the possible role of Klotho in the neurodegenerative symptoms of PS cDKO mice was discussed. In the fourth chapter, the research progress of M1 receptor agonists and Klotho was reviewed.
1, the screening of a new M1 receptor activator (-Xanomeline) new derivative EUK1001.
Using a cell screening model based on reporter gene technology, 172 derivatives of Xanomeline were screened and 18 active compounds were found, including the two most active compounds EUK1001 and EUK1002. Compared with Xanomeline, EUK1001 had smaller EC50 and higher maximum activation multiple for M1 receptor. The activities of EUK1001 and EUK1002 were transient. At the same time, we cloned and expressed a Swedish mutant of human APP gene (APPsw) in mouse neuroblastoma cell Neuro2A (N2a), which was used as AD cell model and found that EUK1001 and EUK1002 could be stimulated by stimulation. From these screening results and in vitro experimental data, we can see that EUK1001 and EUK1002 can not only activate M1 receptor more effectively than Xanomeline, but also promote alpha-shearing of APP, which may have some potential in regulating the pathological pathway of AP in AD.
2. EUK1001 improves some neurodegenerative symptoms in forebrain-specific presenilin double knockout (PS cDKO) AD mice
Subsequent animal pharmacological and toxicological experiments showed that the number of side effects induced by EUK1001 was significantly less than that induced by Xanomeline, while EUK1002 was not improved. The effects of EUK1001 on neurodegenerative symptoms in PS cDKO mice were analyzed. The results showed that EUK1001 significantly improved the impaired recognition and memory abilities in PS cDKO mice after 3 months of chronic administration (0.5mg/kg/day). The results of brain slices analysis showed that EUK1001 could effectively alleviate the impaired recognition and memory abilities in PS cDKO mice. The results showed that both EUK. 1001 and Xanomeline could effectively reduce tau protein hyperphosphorylation in the brain of PS cDKO mice, but both of them had increased inflammatory responses to PS cDKO mice. These results show that EUK1001 can obviously improve some neurodegenerative symptoms of PS cDKO mice, and the effect is better than that of Xanomeline at the same dose, which has potential for further development.
3, the decrease of Klotho expression and the increase of blood phosphorus in the forebrain specific PS cDKO mice.
Klotho protein is an important anti-aging factor, which is highly expressed only in the kidney and brain, but plays a variety of functions in the form of circulating factors in many parts of the body, such as regulating calcium and phosphorus balance, antioxidant stress, anti-inflammation and so on. In this experiment, we analyzed the expression of Klotho and some indexes related to the function of klotho, such as blood phosphorus, blood calcium and kidney function, in AD animal model PS cDKO mice, and combined with some previous results in this model, we preliminarily discussed the possible occurrence of Klotho in neurodegenerative symptoms of PS cDKO mice. The results showed that the expression of Klotho in kidney of 12-month-old PS cDKO mice was significantly lower than that of normal control mice, the level of serum phosphorus was significantly higher, and the level of serum calcium was slightly lower than that of normal control mice, but no obvious renal dysfunction was observed. These results preliminarily suggest that Klotho may be associated with the symptoms of PS cDKO mice. The decrease in Klotho expression may lead to an increase in serum phosphorus concentration but is not sufficient to induce renal dysfunction. Oxidative stress is associated with increased inflammatory response in mice, but further experimental confirmation is needed.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16;R-332

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