CN113712986A - 一种Azacytidine在制备抗病毒药物中的应用 - Google Patents
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Abstract
本发明公开一种Azacytidine在制备抗病毒药物中的应用，其特征在于，所述的病毒为病程短的病毒。Azacytidine可以有效抑制病毒的复制，并且降低免疫风暴对人体造成的伤害，从而让人体有足够的产生抗体的时间，从而提高病人的治愈率。
Description
本发明专利申请是分案申请。原案的申请号是202010612522.2，申请日是2020年6月 30日，发明名称是：一种Azacytidine在制备抗病毒药物中的应用。
技术领域
本发明涉及抗病毒药物领域，更具体地涉及一种Azacytidine在制备抗病毒药物中的应用。
背景技术
病毒是一种个体微小，结构简单，只含一种核酸(DNA或RNA)，必须在活细胞内寄生并以复制方式增殖的非细胞型生物。病毒是一种非细胞生命形态，它由一个核酸长链和蛋白质外壳构成，病毒没有自己的代谢机构，没有酶系统。因此病毒离开了宿主细胞，就成了没有任何生命活动、也不能独立自我繁殖的化学物质。它的复制、转录、和转译的能力都是在宿主细胞中进行，当它进入宿主细胞后，它就可以利用细胞中的物质和能量完成生命活动，按照它自己的核酸所包含的遗传信息产生和它一样的新一代病毒。
由于病毒个体较小，有的能使bai人和猪发生胃肠炎；有的寄生在人的扁桃腺中，本身缺乏繁殖能力，但在侵入扁桃腺中另一种辅助病毒——腺病毒的帮助下，也能繁殖致病菌。在微小RNA病毒中，有能造成小儿麻痹症的脊髓灰质炎病毒，有可引起心肌炎、脑膜炎、流行性胸肌痛、咽喉气管炎及胃肠炎等诸多病毒，还有如鼻病毒可引起伤风感冒，口蹄疫病毒可造成牛、猪、羊等家畜口腔发炎及蹄部溃烂，并往往导致大规模死亡。
当病毒进入人体后，很容易在人体的呼吸道中复制，并跟随口腔的分泌物进行直接传播，从而形成大规模感染。
病毒所致疾病大多是人类的主要传染病，病毒可侵犯不同组织器官，感染细胞引起疾病。由病毒引起的常见疾病有：
①流行性疾病:流行性感冒、普通感冒、麻疹、腮腺炎、小儿麻痹症、传染性肝炎、小儿麻痹；
②慢性感性:乙型肝炎、艾滋病(AIDS)
③潜伏感染:疱疹性角膜炎、性病疱疹病毒。
根据抗病毒药物的作用机制，可将目前的抗病毒药物分为以下几类：[1]
穿入和脱壳抑制剂，DNA多聚酶抑制剂，逆转录酶抑制剂，核苷类，非核苷类，蛋白质抑制剂，神经氨酸酶抑制剂，广谱抗病毒药：利巴韦林、干扰素。
发明内容
本发明的目的在于提供一种已知药物在抗病毒中的应用。
根据以上目的，首先提供一种Azacytidine(又称5-Azacytidine)在制备抗病毒药物中的应用，所述的病毒为病程短的病毒，这里所说的病程较短的病毒，包括感染后马上致病使人快速死亡的病毒，也包括经历一定的无病状的潜伏期，发病后会使人快速死亡的病毒。
更具体地，所述的抗病毒药物为抑制病毒复制的药物。
更具体地，所述的抗病毒药物为降低细胞因子风暴的药物。
更具体地，降低抗病毒药的毒副作用
更具体地，所述病毒是依赖RNA为基因载体，或者RNA作为中间代谢物的病毒。
更具体地，所述的病毒为冠状病毒。
更具体地，所述的冠状病毒为新型冠状病毒。
更具体地，所述的病毒为SARS病毒、MERS病毒或nCoV-2019病毒。
再提供一种Azacytidine在制备治疗非典型性肺炎的药物中的应用，所述的非典型性肺炎为由于病毒引起的肺炎。
更具体地，所述的抗病毒药物为减缓急性病毒病程发展的药物。
更具体地，所述的抗病毒药物人体使用计量1-10000mg/m2.
病毒感染，病毒感染以后伤害细胞，有文章表明非典的nsp1蛋白可以剪切细胞的RNA，从而导致细胞的RNA降解从而损伤细胞；病毒感染导致血液里面IgG增加，血液里面免疫细胞过度反应发生免疫风暴，而免疫风暴往往是最致命的，这个机理和埃博拉类似；病毒进程比较快，人体来不及生产相应抗体，机体已经呼吸衰竭，所以抢救的途径往往是以呼吸机为主。
艾滋病药物可以在初期抑制非典的复制，但是因为艾滋病是靶向药物，对于非靶向的非典有多大效果，可能还有待研究考证。目前的研究是绝大部分的艾滋病和流感药物对于新冠无效。Glead发布remdesivir作为核苷类似物可以终止非典病毒RNA合成，但是目前结果也是无效。原因主要分为两点，第一RNA聚合酶有保真性，所以对于核苷类似物都有比较强的识别作用，导致不会整合进入病毒的RNA，例如利巴韦林。其次就是绝大部分蛋白酶抑制剂也是靶向药物，所以蛋白结构特异性也非常高，所以对于新冠起效难。
而且目前治疗细胞因子风暴，又称免疫风暴，主要使用糖皮质激素，而过量使用糖皮质激素会造成严重的后遗症，比如股骨头坏死，肺部纤维化，以及其他的后遗症，在2003年的 SARS疫情下尤其明显。
Azacytidine(下称Aza)治疗新冠的机理
1.Aza是一个高度近似的核苷类似物，目前真核细胞的所有聚合酶都无法特异拒绝Aza 的整合。
2.Aza作为一个核苷类似物，可以高度有效的整合进入细胞的RNA。
3.整合进入病毒的RNA的里面可以有效的打乱病毒高速翻译的机理，从而减缓病毒感染
4.同时整合进入细胞本身的RNA，也有可能会起到防止病毒切割宿主RNA的作用，从而保护细胞被破坏。
5.这个药物同时也可以通过整合Aza进入免疫细胞RNA，从而减缓免疫细胞的反应，从而减弱免疫风暴。
综上所述，通过研究发现，Azacytidine可以有效抑制病毒的复制，并且降低免疫风暴对人体造成的伤害，从而让人体有足够的产生抗体的时间，从而提高病人的治愈率。
附图说明
图1为实施例1的老鼠在免疫风暴下的存活曲线。
图2为实施例1的细胞因子TNF-a的表达量。
图3为实施例2的病毒感染数量。
图4为实施例2的对照回归曲线。
图5为实施例3的不同药物浓度下细胞因子(肿瘤坏死因子,白细胞介素-6)的表达量
具体实施方式
下面结合附图对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1Azacytidine抑制免疫风暴
对BALB/cj老鼠注射PDL1进行免疫风暴造模，让老鼠产生严重免疫性风暴。
然后对BALB/cj老鼠同时注射PDL1和Azacytidine，按照实验人员描述，老鼠免疫风暴状态明显缓解。最后统计由于免疫风暴死亡的老鼠数量也显著下降，如图1所示，老鼠在免疫风暴下的存活曲线。PD-L1处理组老鼠，因为免疫风暴快速死亡。PD-L1处理组老鼠，注射azacytidine以后，免疫风暴明显缓解，生存曲线明显延长。
测量了肺部组织里面的免疫风暴指示因子TNF-a，如图2所示，发现注射了Azacytidine 的老鼠肺部免疫风暴指示因子TNF-a浓度显著降低。
因此认为Azacytidine具备抑制免疫风暴的作用，可以缓解因为非典后期引起的免疫风暴，而且不会产生比如糖皮质激素的后遗症。
实施例2Azacytidine抑制病毒复制
实验步骤：
BHK21-hACE2细胞铺板，96孔板每孔2X104个细胞，使其12小时后密度可达到70％-80％；
小分子药物的稀释：
Aza(2mM)进行以下稀释：10ul Aza(2mM)+90ul DMEM使其终浓度为0.2mM，取0.2mM的样品50ul+150ul DMEM作为梯度1，取50ul梯度1+100ul DMEM作为梯度2，依次3倍稀释，共8个梯度；
Chloroquine-NIDVD(20mM)进行以下稀释:10ul Chloroquine-NIDVD(20mM)+990ulDMEM使其终浓度为0.2mM，后稀释方法与Aza(2mM)的稀释方法相同，共8个梯度；
取各个梯度稀释的药品80ul+20ul rVSV-SARS-CoV-2(3X10e5pfu/ml)混匀后37°孵育1h；
吸弃BHK21-hACE2细胞上清，加入100ul第3步孵育后的样品：药品80ul+20ulrVSV-SARS-CoV-2；
12h后拍照观察记录阳性细胞数；
计算，分析。
如图3所示，通过数据可以看见在药物低浓度的情况下，很多病毒可以感染此细胞，所以绝大部分细胞会表达荧光的蛋白，而当随着药物浓度提高以后，药物抑制了病毒的感染或者病毒表达复制，从而在高浓度药物的情况下，可以看见病毒的感染明显减少，荧光数量明显降低。因此可以明显看见药物Aza和氯喹在新冠病毒抑制上面有着相同的效果。
将荧光数量减少比例百分数作为抑制效率(inhibition rate)作为病毒感染的一个指数与药物的浓度进行画图。通过研究发现此药物和氯喹在抑制新冠病毒上的作用以及浓度是近似且相近的，如图4所示。
Mock为空白对照组(DMSO)。
inhibition rate＝(空白对照荧光数量-药物浓度下的荧光数量)/空白对照荧光数量(y 轴)
药物浓度(x轴)
如图4所示，根据以上做出的回归曲线。发现Aza和氯喹在抑制病毒感染效率上相近。化合物的细胞毒性通过Cell Counting Kit去分析细胞活力和状态，将细胞的活力和药物浓度作图，发现随着药物浓度提高，细胞活力下降，证明药物的毒性随着浓度上升。
在2.5-5之间Aza的细胞毒性略高于氯喹，但是在高浓度大于5的时候Aza的细胞毒性低于氯喹，证明Aza在高浓度情况下细胞活力优于氯喹且毒性较低。
实施例3免疫风暴检测
293T细胞瞬时转染S蛋白(S蛋白质粒)，24h后消化铺板24孔板，每孔2*105个细胞。24h后加入S-CART和不同浓度的Aza药物(1uM,5uM,10uM)。S蛋白可以刺激对应的S-cart 产生免疫风暴，免疫风暴相关因子分泌进入上清。48h后收上清。通过ELISA检测上清中的细胞因子(肿瘤坏死因子,白细胞介素-6)，从而判定免疫风暴的严重程度。
如图5所示，横轴表示药物浓度，纵轴表示检测到细胞因子的信号强度(指示浓度)。可以明显看见，在高浓度5uM以上，上清中的细胞因子IL6和TNFa明显下降了，说明Aza有明显可以抑制免疫风暴的作用。
以上结合附图对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
Claims (6)
1.一种Azacytidine在制备抗病毒药物中的应用，其特征在于，所述的病毒为病程短的病毒。
2.根据权利要求1所述的应用，其特征在于，所述的抗病毒药物为抑制病毒复制的药物。
3.根据权利要求1所述的应用，其特征在于，所述的病毒为冠状病毒。
4.根据权利要求2所述的应用，其特征在于，所述的冠状病毒为新型冠状病毒。
5.根据权利要求1所述的应用，其特征在于，所述的病毒为SARS病毒、埃博拉病毒、MERS病毒或nCoV-2019病毒。
6.根据权利要求1所述的应用，其特征在于，所述的抗病毒药物为减缓急性病毒病程发展的药物。
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| AMR H SAWALHA等: "Epigenetic dysregulation of ACE2 and interferon-regulated genes might suggest increased COVID-19 susceptibility and severity in lupus patients", 《CLIN IMMUNOL》 * |
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