WO2017096963A1 - 干扰素刺激基因(sting)激动剂在治疗阿尔茨海默症中的应用 - Google Patents

干扰素刺激基因(sting)激动剂在治疗阿尔茨海默症中的应用 Download PDF

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WO2017096963A1
WO2017096963A1 PCT/CN2016/096816 CN2016096816W WO2017096963A1 WO 2017096963 A1 WO2017096963 A1 WO 2017096963A1 CN 2016096816 W CN2016096816 W CN 2016096816W WO 2017096963 A1 WO2017096963 A1 WO 2017096963A1
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disease
alzheimer
sting
cgamp
agonist
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谭相石
向道凤
徐启明
袁红
谭瀛轩
张跃茹
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聊城市奥润生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide

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  • the invention belongs to the field of biomedical technology, and particularly relates to a STING agonist, including a cyclic dinucleotide cGAMP and a thio derivative thereof, in the anti-Alzheimer's disease and in preparing a drug for neurodegenerative diseases such as anti-Alzheimer's disease.
  • a STING agonist including a cyclic dinucleotide cGAMP and a thio derivative thereof
  • Neurodegenerative diseases are disease states in which cellular neurons of the brain and spinal cord are lost.
  • the brain and spinal cord are made up of neurons, which have different functions, such as controlling movement, processing sensory information, and making decisions.
  • Neurodegenerative diseases are caused by the loss of neurons or their myelin, which deteriorate over time to cause dysfunction.
  • Neurodegenerative diseases include: Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple Sclerosing disease, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, etc.
  • Alzheimer's disease is a progressive degenerative neurological degenerative disease. Clinically, it is characterized by memory impairment, aphasia, misuse, loss of recognition, visual spatial impairment, executive dysfunction, and personality and behavioral changes such as personality dementia. The etiology has not been known so far. Before the age of 65, the disease is called Alzheimer's disease; after the age of 65, the disease is called Alzheimer's disease. Among all dementias, the highest proportion of Alzheimer's disease is about 50 to 60%. Among them, women are more likely to suffer than men, and 11% of the population over the age of 65, 50% of those over the age of 80 will develop this disease. Its symptoms last for 1 to 25 years after the onset of the disease, and it will become more and more serious, leading to death.
  • Alzheimer's disease attacks the brain of a person, and it is not a normal aging phenomenon. Patients who get Alzheimer's disease will gradually lose their memory and have language and emotional barriers. When the disease becomes more and more serious, patients need day and night care in all aspects of life, so the lives of their relatives and friends are often greatly affected.
  • Alzheimer's disease is still a disease that cannot be cured. Alzheimer's disease was discovered in 1906. German neuroscientist Ayros Alzheimer first recorded images of Alzheimer's disease with microscopic images. Subtle changes in the brain. He showed a pathological anatomy of a female patient whose mentality became more and more chaotic in the years before her death. By pathological anatomy, he took the brain from the female patient The observed changes are called plaques and tangles, both of which block the brain's ability to communicate and communicate with each other.
  • Alzheimer's disease is mainly caused by protein deposition in the brain, which causes brain nerve cell death.
  • the current Alzheimer's disease research focuses on the starch precursor protein (APP), which is beneficial to destructive plaque in the brain. Formation.
  • APP starch precursor protein
  • researchers have shown that in addition to the starch precursor protein and the formation of plaques, the development of Alzheimer's disease may have other mechanisms of action.
  • causes of Alzheimer's Disease Before understanding Alzheimer's disease, pathologists have known that human cerebral cortex sometimes has rounded plaques, which can be seen under the microscope. These plaques are wrapped in fine fibers. Axon and dendrites. In 1853, the German pathologist Virchow said that these deposits were starch-like deposits, which were later studied as a protein molecule.
  • Starch deposition is derived from APP protein.
  • APP is a transmembrane protein consisting of a large N-terminal extracellular domain, a transmembrane domain, and a smaller C-terminal intracellular domain. It was found through analysis that the APP molecule can be divided into three highly conserved regions from the N-terminus to the C-terminus: E1 region, E2 region and C-terminal region. The researchers proposed and confirmed a new model of A ⁇ cytotoxicity: A ⁇ accelerates APP aggregation, enhances neuronal sensitivity, and promotes apoptosis through interaction with APP (the site of action is the A ⁇ region on APP).
  • a ⁇ binds directly and specifically to the A ⁇ region of APP located on the cell surface like a ligand to accelerate APP aggregation; then caspase or its similar enzyme is cleaved after Asp664 in the APP695 intracellular region, releasing The 31 amino acid residue polypeptide C31 at the C-terminus of the APP (Xinwei Cao, Thomas C. Südhof. Science. 6 July 2001: Vol. 293 no. 5527 pp. 115-120).
  • C31 is a potential cytotoxic molecule capable of interacting with various proteins such as intracellular Fe65 through the sequence "GYENPTY” motif (amino acid residues 681-687 in APP695), presumably associated with Fe65 and CP2/LSF/ LBP1 forms a ternary complex that induces expression of GSK-3 ⁇ , thereby causing apoptosis (Dennis J. Selkoe, Science 25 October 2002: Vol. 298 no. 5594 pp. 789-791).
  • GYENPTY amino acid residues 681-687 in APP695
  • AD Alzheimer's disease
  • Interferon gene stimulating protein is a transmembrane protein that usually forms a dimer in the 152-173 dimerization domain (DD) and is in a self-inhibiting state. When stimulated by a partial ligand (such as CDN), the molecular conformation changes and is activated, recruiting TANK-binding kinase 1 (TBK1) in the cytoplasm, mediating TBK1 phosphorylation of IRF3, causing interference Formation of interferon (IFN)- ⁇ and various other cytokines. The production of IFN ⁇ is a marker of STING activation (Yasuo Tanaka & Zhijian J. Chen. Sci Signal. 2012 Mar6; 5 (214)). The cyclic dinucleotide cGAMP is the only STING agonist discovered so far that both directly activates the murine source and activates the human STING protein.
  • An agonist/activator is a chemical or drug that binds to a protein molecule on a cell receptor or signal transduction pathway and produces the typical physiological potency of a natural substance.
  • the cyclic dinucleotide cGAMP a natural agonist of STING, is capable of inducing I-interferon production (X Cai, YH Chiu, ZJ Chen, Molecular cell, Volume 54, Issue 2, 24 April 2014, Pages 289-296).
  • the STING agonist/activator is a cyclic dinucleotide, including: cGAMP and its thio derivative, and also includes a cyclic dinucleotide composed of AMP or GMP.
  • STING agonist/activator can inhibit neuronal apoptosis, inhibit amyloid aggregation, improve cognitive ability, reduce AD brain inflammation, and have obvious anti-Alzheimer's effect.
  • anti-Alzheimer's disease drugs For the preparation of anti-Alzheimer's disease drugs.
  • dinucleotide cGAMP refers to 2'3'-cGAMP, C20H22N10O13P2.2NH4, and CAS No. 1441190-66-4.
  • GENE name is: TMEM173; GENE ID is: 340061; other names disclosed by STING include: Transmembrane Protein 173, ERIS, MITA, MPYS, NET23, SAVI, STING, hMITA, hSTING.
  • APP is a specific protein name, which, unless otherwise stated, is consistent with most public literature and NCBI databases, European gene databases. Its GENE name is: APP; GENE ID is: 351; other names disclosed by STING include: AAA, ABETA, ABPP, AD1I, CTFgamma, CVAP, PN-II, PN2. AD.
  • STING agonists, cyclodinucleotides, as referred to herein, include, but are not limited to, cGAMP and its sulfur-substituted derivatives, and combinations thereof.
  • cGAMP (cyclized-GMP-AMP) was synthesized by cyclized cGMP-AMP dinucleotide synthetase (cGAS) under the activation conditions of the literature after binding to DNA. The purity is above 98%. (Pingwei Li, et al., Immunity, 2013, 39(6), 1019-1031.). Thio cGAMP, c-di-AMP, and c-di-GMP were purchased from Sigma.
  • Example 2 Alzheimer's disease mouse model and STING agonist drug therapy
  • APPswe/PSEN1dE9 two-transgenic Alzheimer's disease model mice, 5 months old, weighing 24-26 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; C57BL/6 mice (5 months old) , weight 20-25), purchased from Shanghai SIPPR-BK Laboratory Animal Co., Ltd.
  • the experimental rats were divided into four groups, which were divided into AD model group, low-dose administration group, high-dose administration group and normal mouse control group, with 10 rats in each group.
  • Test drug name STING agonist.
  • Traits white powder.
  • Solvent normal saline.
  • Preparation method Prepare a solution of the desired concentration with a physiological saline solution before use.
  • the doses were 7.5 mg/kg/day and 15 mg/kg/day, respectively, and were continuously spread by intravenous diffusion using a slow-release pump (Osmotic pumps, 200 ⁇ l, Alza Corporation, Palo Alto, CA, USA) embedded in a rat back. Administration, 0.15 ⁇ l per hour, for 6 weeks.
  • a slow-release pump Osmotic pumps, 200 ⁇ l, Alza Corporation, Palo Alto, CA, USA
  • Example 3 Morris water maze to verify the cognitive ability of STING agonists in Alzheimer's disease mice effect.
  • Positioning navigation test The test lasted for 6 days and was scheduled to be trained 4 times a day for a fixed period of time.
  • the platform is placed in the first quadrant, and the mouse is placed facing the pool wall into the pool from any of the four starting points of the wall.
  • the free video recording system records the time and swimming path of the mouse to find the platform, and the four trainings put the mice into the water from four different starting points (different quadrants).
  • the experimenter guides it to the platform, rests on the platform for 10 seconds, and then conducts the next test.
  • Example 4 Inhibition of brain A ⁇ protein aggregation by Alzheimer's disease mice by STING agonist
  • This experiment is a thioflavine S staining experiment.
  • the experimental procedure is: taking mouse brain tissue, fixing, paraffin embedding, sectioning, xylene dewaxing, ethanol gradient dehydration, TBS washing three times, 0.3% thioflavin S (dissolved in 50%). Ethanol) was dripped on the tissue, incubated for 10 min at room temperature, washed three times with 50% ethanol, washed with TBS, dried in the air, mounted, and observed under a laser confocal microscope.
  • the STING activator cGAMP can significantly reduce the deposition of A ⁇ protein in the brain of AD rats, and can alleviate the deposition of A ⁇ protein, an important indicator of Alzheimer's disease.
  • Example 5 Inhibition of STING agonists on apoptosis of brain neurons in Alzheimer's disease mice
  • the TUNEL Apoptosis Kit (G3250 kit) was purchased from Promega. Mouse brain tissue, fixed, paraffin-embedded, sectioned, dewaxed with xylene, dehydrated with ethanol, washed with PBS, incubated with proteinase K for 10 min at room temperature, fixed with formaldehyde after washing with PBS, added to equilibration buffer and pre-equilibrated, then added. Incubation buffer (containing equilibration buffer, nucleoside mixture and rTdT enzyme), incubate at 37 ° C for 1 h in the dark, terminate the reaction, co-stain DAPI, dry, seal, and photograph with laser microscope.
  • Incubation buffer containing equilibration buffer, nucleoside mixture and rTdT enzyme
  • Apoptotic cells can be stained cyan by TUNEL staining kit, and nerve cells can be stained blue by DAPI staining. The more cyan, the more obvious the apoptosis, and vice versa.
  • the results of the experiment are shown in Fig. 3.
  • the results show that the use of STING activator cGAMP can inhibit the apoptosis of brain neurons in AD mice, and the neuronal apoptosis of the important disease indicators of Alzheimer's disease in model rats is inhibited. It is indicated that the STING activator has the effect of treating Alzheimer's disease.
  • Example 6 Effect of STING agonists on Alzheimer's disease-associated proteins and cytokines
  • mice were anesthetized, blood was taken from the eyelids, centrifuged at 3000 r/min for 15 min, and the supernatant was taken. The expression levels of IL-1 ⁇ , TNF- ⁇ , IL-4, IL-10 and IFN- ⁇ were detected by ELISA kit. The brain tissue of the mice was taken, and the supernatant was centrifuged, and the expression levels of IL-1 ⁇ , TNF- ⁇ , IL-4, IL-10 and IFN- ⁇ were detected by ELISA kit.
  • STING agonists can significantly reduce the expression of IL-1 ⁇ and TNF- ⁇ , and up-regulate the expression of anti-inflammatory factors IL-4, IL-10 and IFN- ⁇ . It has the function of regulating inflammatory cytokines related to Alzheimer's disease, and can reduce the inflammatory factors to the amount of normal mice (as shown in Fig. 4 and Fig. 5).
  • Example 7 Effect of STING agonist cGAMP on brain immune microglia and protective immune function in AD rats.
  • FIG. 1 Effect of STING agonist cGAMP on cognitive ability in Alzheimer's disease mice.
  • the results in the figure show that the STING agonist cGAMP can significantly improve the cognitive ability of mice.
  • Tg is the AD model group and Tg-cGAMP is the cGAMP administration group.
  • FIG. 1 Effect of STING agonist cGAMP on amyloid small peptide A ⁇ protein deposition in the brain of Alzheimer's disease mice.
  • the results showed that STING agonist cGAMP and the like can significantly inhibit the deposition of amyloid peptide A ⁇ protein in the brain of Alzheimer's model mice.
  • (B) Quantification of numbers and surface area of A ⁇ plaques (n 10; *p ⁇ 0.05)].
  • FIG. 3 Effect of STING agonist cGAMP as a drug on neuronal apoptosis in the brain of Alzheimer's disease mice.
  • the results show that the STING agonist cGAMP and the like can inhibit the apoptosis of nerve cells in the brain of Alzheimer's disease mice.
  • [cGAMP reduces apoptosis of neurons with dose dependence.
  • TUNEL assay of neurons in mice brains using the "In Suit Cell Death Detection Kit, Fluorescein" (Roche, Germany). Scale bar 100 ⁇ m].
  • Figure 4 Effect of STING agonist cGAMP on the expression of Alzheimer's disease-associated inflammatory factors.
  • the levels of interleukin (IL)-1 ⁇ and tumour necrosis factor alpha (TNF- ⁇ ) in the mice plasma (A) and brains (B). (n 10, *p ⁇ 0.05, **p ⁇ 0.01)].
  • Figure 5 Effect of STING agonist cGAMP on Alzheimer's disease-associated cytokine expression.
  • STING agonist cGAMP reduces AD mouse immune cells (microglia) and enhances protective immune function.
  • the results showed that STING agonist cGAMP can significantly reduce the total amount of glial cells in AD rats (ion-calcium binding adapter molecule (IBA-1) is a marker of microglia activity), inhibiting its M1 type small glue with proinflammatory function.
  • IBA-1 ion-calcium binding adapter molecule
  • M2 microglial cell activity interstitial cell activity enhanced the anti-inflammatory function (Il-4, CD206, Arginase -1 activation marker is M2), activation of protective immunity function (activated CD4 + T cells).

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Abstract

使用固有免疫通路中关键靶蛋白STING的激动剂cGAMP及其衍生物在制备抗阿尔茨海默症（AD）等神经退行性疾病药物中的应用。STING激动剂可以改善AD模型鼠认知功能，抑制AD鼠神经细胞凋亡，抑制AD鼠脑神经淀粉样蛋白沉积，降低AD鼠脑免疫小胶质细胞总数，降低AD鼠脑炎症，防止AD进展。

Description

干扰素刺激基因(STING)激动剂在治疗阿尔茨海默症中的应用 技术领域
本发明属于生物医药技术领域，具体涉及STING激动剂包括环二核苷酸cGAMP及其硫代衍生物等在抗阿尔茨海默症中以及在制备抗阿尔茨海默症等神经退行性疾病药物中的应用。
背景技术
神经退行性疾病是大脑和脊髓的细胞神经元丧失的疾病状态。大脑和脊髓由神经元组成，神经元有不同的功能，如控制运动，处理感觉信息，并作出决策。神经退行性疾病是由神经元或其髓鞘的丧失所致，随着时间的推移而恶化，以导致功能障碍。神经退行性疾病包括：阿尔茨海默症、肌肉萎缩性侧索硬化症、共济失调毛细血管扩张症、牛海绵状脑病、克雅二氏病、亨丁顿舞蹈症、小脑萎缩症、多发性硬化症、帕金森氏病、原发性侧索硬化、脊髓性肌萎缩症等。
阿尔茨海默病是一种起病隐匿的进行性发展的神经系统退行性疾病。临床上以记忆障碍、失语、失用、失认、视空间技能损害、执行功能障碍以及人格和行为改变等全面性痴呆表现为特征，病因迄今未明。65岁以前发病者，称早老性痴呆；65岁以后发病者称老年性痴呆。在所有的痴呆症中,以阿尔茨海默症所占的比率最高，约50～60％。其中,女性又比男性较易罹患,并且在65岁以上人口中有11％,80岁以上有50％会出现这种病症。它的症状自病发后会持续1～25年,而且会越来越严重,进而导致死亡。阿尔茨海默症会侵袭人的脑部,它并非正常的老化现象。得到阿尔茨海默症的病人会渐渐丧失记忆,并且出现语言和情绪上的障碍。当这个疾病越来越严重时,病患在生活各方面都需要他人日夜的照护,因此病患亲友的生活往往也跟著受到很大的影响。
目前阿尔茨海默症仍是一种无法根治的疾病，阿尔茨海默症发现于1906年,德国神经科学家爱罗斯.阿尔茨海默首次以显微镜取得的影像,来记录阿尔茨海默症患者脑部的细微变化。他展示一位女性病人的病理解剖图,这位女性的心智在她往生的前几年渐趋混乱。藉著病理解剖的方法,他把从这位女性病人脑部 所观察到的变化称为斑块和缠结,两者都会阻断脑部神经彼此沟通和传递讯息的功能。
阿尔茨海默症主要是由于蛋白质在脑部沈积而造成脑神经细胞死亡，目前的阿尔茨海默症研究集中在淀粉前体蛋白(APP)上，该蛋白有利于大脑中破坏性斑块的形成。研究人员已经证明除了淀粉前体蛋白和形成的斑块，影响阿尔茨海默症的发展也可能有其它作用机制的原因。阿尔茨海默症的病因在了解阿尔茨海默症之前,病理学家就已经知道人类的大脑皮质有时会出现圆形的斑块,在显微镜下可以看出这些斑块是由细纤维包裹的轴突和树突所组成。1853年,德国病理学家菲尔克(Virchow)称这些沈积物是类淀粉沈积,后来进一步研究得知它们是一种蛋白质分子。1984年,格列纳(Glenner)和翁(Wong)首先由阿尔茨海默症患者的脑膜血管中分离出类淀粉沉积。不久,马士德(Masters)和贝伦索(Beyrenther)等人也由老化斑块核心中分离出类淀粉沉积,它的分子量及氨基酸组成与格列纳等人所分离的类淀粉沉积相同。
淀粉沉积来源于APP蛋白。APP是一种跨膜蛋白，由一个较大的N端胞外区、跨膜区和一个较小的C端胞内区组成。经分析发现，APP分子从N端到C端，总体上可以划分为三个高度保守的区域：E1区域、E2区域和C端区域。研究者提出并证实了一个新的Aβ细胞毒性模型：Aβ通过与APP的相互作用(作用的位点为APP上的Aβ区域)，加速APP聚集，增强神经元的敏感性，促进细胞凋亡。具体过程是：首先，Aβ像配体一样直接且特异地与位于细胞表面的APP的Aβ区域相结合，加速APP聚集；然后caspase或其类似酶在APP695细胞内区域的Asp664后切割之，释放出APP C末端的31个氨基酸残基的多肽C31(Xinwei Cao,Thomas C.Südhof.Science.6July 2001:Vol.293no.5527pp.115-120)。C31是潜在的细胞毒性分子，能够通过其上的序列“GYENPTY”基序(APP695中第681—687位氨基酸残基)与细胞内Fe65等多种蛋白质相互作用，推测与Fe65和CP2/LSF/LBP1形成了三元复合物，后者诱导GSK-3β的表达，从而引起细胞凋亡(Dennis J.Selkoe,Science 25October 2002:Vol.298no.5594pp.789-791)。
最近的研究表明，大脑慢性炎症也是阿尔茨海默症(AD)的重要病理特征之一。基于消除大脑炎症的免疫抗炎治疗是防治AD病的理想治疗方法之一。 目前，没有治疗AD的特效药物，所有基于化解老年斑的金属离子螯合剂药物和阻止淀粉样蛋白产生的分泌酶抑制药物均治表不治本。近期的AD临床阶段的药物研究大多宣告失败。因此，基于加强保护性免疫和消除大脑炎症的免疫治疗药物具有巨大发展前景。
干扰素基因刺激蛋白(STING)是一种跨膜蛋白，通常在152-173位区域(dimerization domain，DD)交接形成二聚体并处于自我抑制状态。当受到部分配体(比如CDN)的刺激后分子构型发生变化并被激活，招募细胞质中的TANK结合激酶1(TANK-binding kinase 1，TBK1)，介导TBK1对IRF3的磷酸化，导致干扰素(interferon，IFN)-β和其它多种细胞素(cytokines)的形成。IFNβ的产生是STING活化的标志(Yasuo Tanaka&Zhijian J.Chen.Sci Signal.2012Mar6；5(214))。环二核苷酸cGAMP，是到目前为止发现的唯一一类既能直接激活鼠源又能激活人源STING蛋白的STING激动剂。
激动剂/激活剂是指能与细胞上受体或信号转导途径的蛋白分子相结合，并产生天然物质的典型生理效能的化学品或药物。环二核苷酸cGAMP，作为STING的天然激动剂，能够诱导I性干扰素产生(X Cai,YH Chiu,ZJ Chen,Molecular cell,Volume 54,Issue 2,24April 2014,Pages 289–296)。STING激动剂/激活剂为环二核苷酸，包括：cGAMP及其硫代衍生物,也包括由AMP或GMP构成的环二核苷酸等。
发明内容
本发明的目的在于提供STING激动剂/激活剂在治疗阿尔茨海默症中的应用，以在制备抗阿尔茨海默症等退行性疾病药物中的应用。
本发明实验研究表明，STING激动剂/激活剂可以抑制神经细胞的凋亡，抑制淀粉样蛋白聚集，提高认知能力，降低AD脑神经炎症，具有明显的抗阿尔茨海默症的作用，可用于制备抗阿尔茨海默症药物。
本文提及二核苷酸cGAMP，如不加特殊说明，均指2’3’－cGAMP，C20H22N10O13P2.2NH4,CAS号为1441190-66-4。
本文提及STING，为特定蛋白质名称，如不加说明，均与多数公开文献及NCBI数据库、欧洲基因数据库一致。其GENE名为：TMEM173；GENE ID为：340061；STING公开的其它命名包括：Transmembrane Protein 173,ERIS,MITA, MPYS,NET23,SAVI,STING,hMITA,hSTING。
本文提及APP，为特定蛋白质名称，如不加说明，均与多数公开文献及NCBI数据库、欧洲基因数据库一致。其GENE名为：APP；GENE ID为：351；STING公开的其它命名包括：AAA,ABETA,ABPP,AD1I,CTFgamma,CVAP,PN-II,PN2。AD。
本文提及的STING激动剂，环二核苷酸，包括但不限制于cGAMP及其硫取代衍生物以及它们的组合物。
具体实施方式
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好地阐述本发明，并不是用来限制本发明的范围。
实施例1：STING激动剂的制备
cGAMP的制备：cGAMP(环化-GMP-AMP)按文献方法在结合DNA后的活化条件下，由环化cGMP-AMP二核苷酸合成酶(cGAS)催化合成。纯度在98％以上。(Pingwei Li，et al.,Immunity,2013,39(6),1019-1031.)。硫代cGAMP、c-di-AMP、c-di-GMP均购自sigma公司。
实施例2：阿尔茨海默症小鼠模型及STING激动剂药物治疗
APPswe/PSEN1dE9(APP/PS1)二转基因阿尔茨海默症模型小鼠，5月龄，体重24-26g，购自北京维通利华实验动物技术有限公司；C57BL/6小鼠(5月龄，体重20-25该)，购自上海SIPPR-BK实验动物有限公司。实验鼠分四组，分AD模型组，低剂量给药组，高剂量给药组和正常鼠对照组，每组10只。受试药物名称：STING激动剂。性状：白色粉末。溶媒：生理盐水。配制方法：临用前用生理盐水溶液配制成所需浓度的溶液。给药剂量分别为：7.5mg/kg/天和15mg/kg/天，用包埋在鼠背的缓释泵(Osmotic pumps，200μl，Alza Corporation,PaloAlto,CA,USA)通过静脉连续扩散缓释给药，每小时0.15μl，给药6周。
实施例3：Morris水迷宫验证STING激动剂对阿尔茨海默症小鼠认知能力的 作用。
装置:圆形水池，直径1m，高50cm，水深30cm，池底白色，水温保持在23±2℃；池壁上标记四个等距离点N、E、S、W作为试验的起始点，分水池为四个象限，在第三象限中央放置平台(平台与池壁圆心距离相等)；没于水下1cm，使平台不可见。水池周围贴有丰富的参照线索(不同颜色三角形、四方形、圆、菱形置于各个象限)且保持不变，供小鼠用来定位平台。
定位航行试验:试验共历时6天，每天定于固定时间段训练4次。训练开始时，将平台置于第一象限，从池壁四个起始点的任一点将小鼠面向池壁放入水池。自由录像记录系统记录小鼠找到平台的时间和游泳路径，4次训练即将小鼠分别从四个不同的起始点(不同象限)放入水中。小鼠找到平台后或90秒内找不到平台(潜伏期记为90秒)，则由实验者将其引导到平台，在平台上休息10秒，再进行下一次试验。
空间探索试验：定位航行试验结束24h后，撤除站台。然后将小鼠由第三象限放入水中，记录小鼠在180s内的游泳路径，记录鼠在目标象限(第三象限)的停留时间和穿越原站台所在位置的次数，观察受试鼠的空间定位能力。在第三象限停留时间越长，代表小鼠能记住站台的位置，说明认知能力越好。实验结果如图1所示，结果表明：STING激活剂cGAMP对阿尔茨海默症小鼠的认知能力有增强作用。
实施例4：STING激动剂对阿尔茨海默症小鼠脑Aβ蛋白聚集的抑制作用
本实验为硫黄素S染色实验，实验流程为：取小鼠脑组织，固定，石蜡包埋，切片，二甲苯脱蜡，乙醇梯度脱水，TBS洗三次，0.3％硫黄素S(溶于50％乙醇)滴于组织上，室温孵育10min，50％乙醇洗三次，TBS清洗，阴干，封片，激光共聚焦显微镜下观察。通过硫黄素S染色，聚集的淀粉样小肽Aβ蛋白能被染成绿色，绿色斑点越多，代表脑内淀粉样小肽Aβ沉积越严重，阿尔茨海默症越严重，反之，若减少，代表阿尔茨海默症疾病减轻。实验结果如图2所示，STING激活剂cGAMP能够显著降低AD鼠脑内Aβ蛋白的沉积，能够减轻阿尔茨海默症疾病的重要指标Aβ蛋白的沉积。
实施例5：STING激动剂对阿尔茨海默症小鼠脑神经细胞凋亡的抑制作用
TUNEL凋亡试剂盒(G3250试剂盒)购自Promega公司。取小鼠脑组织，固定，石蜡包埋，切片，二甲苯脱蜡，乙醇梯度脱水，PBS洗涤，蛋白酶K室温孵育10min，切片PBS洗涤后甲醛固定，加入平衡缓冲液预平衡后洗涤，之后加入孵育缓冲液(内含平衡缓冲液，核苷混合物和rTdT酶)，37℃避光孵育1h，终止反应后共染DAPI，阴干，封片，激光显微镜拍照。凋亡的细胞能够通过TUNEL染色试剂盒被染成青色，神经细胞的能够通过DAPI染色染成蓝色，青色越多代表凋亡越明显，反之代表细胞凋亡被抑制。实验结果如图3所示，结果表明：使用STING激活剂cGAMP等后，能够抑制AD小鼠大脑神经细胞的凋亡，模型鼠的阿尔茨海默症疾病重要疾病指标神经细胞凋亡被抑制，说明STING激活剂具有治疗阿尔茨海默症的作用。
实施例6：STING激动剂对阿尔茨海默症相关蛋白及细胞因子的影响
小鼠模型、药物治疗建立参照实施例2，给药剂量分别是7.5mg/kg和15mg/kg，ELISA试剂盒购自R&D公司。分别麻醉小鼠，眼眶取血，3000r/min离心15min，取上清，用ELISA试剂盒检测IL-1β、TNF-α、IL-4,IL-10、IFN-γ细胞因子的表达量。取小鼠脑组织，离心取上清，用ELISA试剂盒检测IL-1β、TNF-α、IL-4,IL-10、IFN-γ细胞因子的表达量。结果表明：与AD疾病模型小鼠相比，STING激动剂能够显著降低促进炎症相关细胞因子IL-1β、TNF-α的表达，上调抗炎因子IL-4、IL-10、IFN-γ的表达，具有调控与阿尔茨海默症有关炎症细胞因子的功能，能将炎症因子降低至正常鼠的量(如图4、图5所示)。
实施例7.STING激动剂cGAMP对AD鼠脑免疫小胶质细胞和保护性免疫功能的影响。
小鼠模型、药物治疗建立参照实施例2，给药剂量分别是7.5mg/kg和15mg/kg，ELISA试剂盒购自R&D公司。取AD小鼠脑组织进行免疫组织化学分析和RT－PCR分析。结果显示(图6)，STING激动剂cGAMP能显著降低AD鼠脑小胶质细胞总量(离子钙结合适配器分子(IBA-1)是小胶质细胞活力的标志)，抑制其具有促炎功能的M1型小胶质细胞活性，增强抗炎功能的M2型小胶质细胞活性(Il－4、CD206、Arginase-1是M2活化标志)，激活保护性免疫 功能(激活CD4+T细胞)。
附图说明：
图1、STING激动剂cGAMP对阿尔茨海默症小鼠认知能力的影响。图中结果显示：STING激动剂cGAMP能够显著改善老鼠的认知能力。(Tg为AD模型组，Tg－cGAMP为cGAMP给药组)。[cGAMP treatment improved cognitive functions in AD mice models with dose dependence.Evaluation of spatial learning using the Morris water maze latency test after cGAMP treatment for 6weeks.There was significant difference between the Tg-cGAMP(7.5mg/kg)and Tg groups in terms of spatial learning(n＝10,*p<0.05,**p<0.01,Tg-cGAMP(7.5mg/kg)group vs.Tg group)with dose dependence(n＝10,*p<0.05,**p<0.01,Tg-cGAMP(15mg/kg)group vs.Tg-cGAMP(7.5mg/kg)group)].
图2、STING激动剂cGAMP对阿尔茨海默症老鼠脑内淀粉样小肽Aβ蛋白沉积的影响。结果显示STING激动剂cGAMP等均能显著抑制阿尔茨海默症模型老鼠脑内淀粉样小肽Aβ蛋白的沉积。[(A)Representative sections ofdifferent dose cGAMP treated mice brains versus control brains stained with Thioflavin S.(B)Quantification ofnumbers and surface area ofAβplaques(n＝10；*p<0.05)].
图3、STING激动剂cGAMP等作为药物对阿尔茨海默症老鼠脑内神经细胞凋亡的影响。图中结果表明：STING激动剂cGAMP等能够抑制阿尔茨海默症老鼠脑内神经细胞的凋亡。[cGAMP reduces apoptosis ofneurons with dose dependence.TUNEL assay ofneurons in mice brains using the“In Suit Cell Death Detection Kit,Fluorescein”(Roche,Germany).Scale bar＝100μm].
图4、STING激动剂cGAMP对阿尔茨海默症相关炎症因子表达的影响。
结果显示STING激动剂cGAMP能显著降低AD鼠脑炎症因子IL-1β和TNF-α的表达量。[The levels ofinterleukin(IL)-1βand tumour necrosis factor alpha(TNF-α)in the mice plasma(A)and brains(B).(n＝10,*p<0.05,**p<0.01)]。
图5、STING激动剂cGAMP对阿尔茨海默症相关细胞因子表达的影响。
结果显示STING激动剂cGAMP能显著促进AD鼠脑组织抗炎因子IL-4、IL-10和IFN-γ的表达量。
图6.STING激动剂cGAMP降低AD鼠免疫细胞(小胶质细胞)、增强保护性免疫功能。结果显示STING激动剂cGAMP能显著降低AD鼠脑小胶质细胞总量(离子钙结合适配器分子(IBA-1)是小胶质细胞活力的标志)，抑制其具有促炎功能的M1型小胶质细胞活性，增强抗炎功能的M2型小胶质细胞活性(Il－4、CD206、Arginase-1是M2活化标志)，激活保护性免疫功能(激活CD4+T细胞)。

Claims (8)

  1. STING激动剂/激活剂在抗阿尔茨海默症中的应用。
  2. STING激动剂/激活剂在制备抗阿尔茨海默症药物中的应用。
  3. STING激动剂/激活剂在制备抗阿尔茨海默症等抗神经退行性疾病药物中的应用。
  4. 根据权利要求1所述STING激动剂/激活剂在抗阿尔茨海默症中的应用，其特征在于：(1)提升认知能力；(2)抑制脑内淀粉样肽Aβ沉积；(3)抑制脑内神经细胞凋亡；(4)减轻脑神经炎症。
  5. 权力要求1所述的STING激动剂/激活剂，包括但不限制于2’3’－cGAMP及其硫取代衍生物以及它们的组合物，也包括由AMP或GMP构成的环二核苷酸。
  6. 权力要求2所述的STING激动剂/激活剂，包括但不限制于2’3’－cGAMP及其硫取代衍生物以及它们的组合物，也包括由AMP或GMP构成的环二核苷酸。
  7. 权力要求3所述的STING激动剂/激活剂，包括但不限制于2’3’－cGAMP及其硫取代衍生物以及它们的组合物，也包括由AMP或GMP构成的环二核苷酸。
  8. 根据权利要求2、3所述的STING激动剂按常规药剂学制成各种剂型，所述的剂型包括片剂、胶囊剂、颗粒剂、混悬剂、乳剂、溶液剂、糖浆剂或注射剂等中的一种或多种，采取口服或注射(包括静脉注射、静脉滴注、肌肉注射或皮下注射、或脑室直接给药等)中的一种或多种给药途径进行抗阿尔茨海默症及其直接相关的神经退行性疾病的预防或治疗。
PCT/CN2016/096816 2015-12-09 2016-08-26 干扰素刺激基因(sting)激动剂在治疗阿尔茨海默症中的应用 WO2017096963A1 (zh)

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