CN111759841A - 一种用匹多莫德在抗新型冠状病毒感染中的应用 - Google Patents
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Abstract
本发明涉及用于抗新型冠状病毒感染治疗的药物和用途,具体涉及含有(R)‑3‑[(S)‑(5‑氧代‑2‑吡咯烷基)羰基]‑四氢噻唑‑4‑羧酸(匹多莫德,英文名Pidotimod)治疗药物。通过匹多莫德抑制病毒主蛋白酶活性作用,达到抗新型冠状病毒感染的目的。匹多莫德在治疗新型冠状病毒感染中做抑制剂。
Description
技术领域
本发明属于医药技术领域,具体涉及一种用匹多莫德在抗新型冠状病毒感染中的应用。
背景技术
人类免疫缺陷新型冠状病毒(新型冠状病毒)感染以及相关疾病是全球范围内主要的公共健康问题。人类免疫缺陷新型冠状病毒1型(新型冠状病毒-1) 编码至少三种新型冠状病毒复制所必需的酶:逆转录酶(RT)、蛋白酶(Prt)和整合酶(Int)。尽管靶向逆转录酶和蛋白酶的药物已经被广泛使用并且也显示出具有疗效,特别是当联合应用时,但是毒性和耐药性菌株的出现使其应用受到限制(Palella等人,N.Engl.J.Med.(1998)338:853-860;Richman,D.D. Nature(2001)410:995-1001)。水解酶参与新冠病毒在人体内的复制作用,通过抑制该水解酶的作用可以达到治疗新冠肺炎的目的。但是,新冠患者体内由于受病毒的刺激导致巨噬细胞M1型过度激活,分泌过多的促炎因子和趋化因子,而 M2型巨噬细胞激活受到抑制,导致炎症损伤组织得不到修复,形成正反馈循环,进而突破阈值而失控,最终形成细胞因子风暴。因此,治疗新冠肺炎不仅需要抑制病毒在体内的复制,同时需要逆转诱发炎症风暴的关键细胞因子,阻断部分单核/巨噬细胞的M1极化及其信号传导,促进单核/巨噬细胞的M2极化,降低并修复炎症反应对病人肺组织和多器官的损伤。
目前针对新冠肺炎无特效抗病毒药物,治疗主要是对症治疗和支持治疗,对症治疗就是患者出现低氧血症就补充氧气,水电解质失衡就纠正水电解质平衡;支持治疗就是维持及帮助机体的功能的治疗,比如补充水分电解质维生素及营养类物质。针对自身免疫对肺组织的攻击,使用激素(例如糖皮质激素) 抑制免疫避免进一步肺组织损害,但这样同时压制了免疫应答使得免疫系统难以完成病毒感染病程中关键的自身抗体形成,并且大量使用激素产生较大的副作用。
匹多莫德商品名为万适宁,英文名为Pidotimod,化学名为(R)-3-[(S)-(5-氧代-2-吡咯烷基)羰基]-四氢噻唑-4-羧酸,分子式为C9H12N2O4S,分子量为244.26,化学结构式如图。匹多莫德(美国专利号US09446027B2、US09408833B2)结构如图4所示。
匹多莫德原是为免疫促进剂,对非特异免疫反应和特异免疫反应均有促进作用。能加强巨噬细胞及中性粒细胞的吞噬活性,提高其趋化性。激活自然杀伤细胞,促进有丝分裂原引起的淋巴细胞增殖,使免疫功能低下的辅助性T细胞(CD4+)与抑制性T细胞(CD8+)的比值升高,恢复正常。通过刺激白介素-a和 r-干扰素促进细胞免疫反应。
发明内容
针对上述问题本发明提供了一种用匹多莫德在抗新型冠状病毒感染中的应用。
为了达到上述目的,本发明采用了下列技术方案:
一种用匹多莫德在抗新型冠状病毒感染中的应用:通过匹多莫德抑制病毒主蛋白酶活性作用,达到抗新型冠状病毒感染的目的。
进一步,所述匹多莫德在治疗新型冠状病毒感染中做抑制剂。
进一步,所述匹多莫德为颗粒剂、口服液、散剂、片剂或胶囊剂。
再进一步,所述片剂为普通片和分散片。
进一步,所述匹多莫德的化学名为(R)-3-[(S)-(5-氧代-2-吡咯烷基)羰基]-四氢噻唑-4-羧酸,分子式为C9H12N2O4S,分子量为244.268。
与现有技术相比本发明具有以下优点:
本发明发现匹多莫德不仅可以提高免疫力,更为重要的是我们发现匹多莫德在体内、体内抗病毒试验中,均可以在一定浓度范围可有效的抑制新冠病毒所致的病毒性肺炎,与模型对照组比较,差异显著。
附图说明
图1是匹多莫德浓度与细胞毒性量效关系曲线示意图;
图2是匹多莫德对新冠病毒的抑制作用示意图;
图3是匹多莫德体内治疗量效曲线示意图;
图4是匹多莫德的结构式。
具体实施方式
一种用匹多莫德在抗新型冠状病毒感染中的应用,通过匹多莫德抑制病毒主蛋白酶活性作用,达到抗新型冠状病毒感染的目的。匹多莫德在治疗新型冠状病毒感染中做抑制剂。匹多莫德为颗粒剂、口服液、散剂、片剂(普通片、分散片)或胶囊剂。
本发明经山西医科大学基础医学部所作的药效学研究报告表明,匹多莫德在体内外均具有明显的抗新冠病毒的作用。
具体报告内容如下:
目的:对匹多莫德体内外抗新冠病毒作用进行药效检测。
方法:采用组织细胞培养法、血凝法和建立流行性感冒动物模型,对不同浓度的匹多莫德进行体内外抗新冠病毒药效学研究。
结果:匹多莫德体外有明显的抑制新冠病毒的作用,对新冠病毒COVID-19 有明显的延缓致细胞病变作用。体内抗新冠病毒试验结果显示,匹多莫德可有效抑制新冠病毒所致小鼠病毒性肺炎。
结论:匹多莫德在体内外均具有明显的抗冠病毒的作用。
一、试验材料
1.药物
1)匹多莫德分散片:国药准字H20060718(处方药),为本实验待测药物。生产厂家:北京金城泰尔制药有限公司。用于体内外抗病毒试验。
2)利巴韦林注射液,100mg/支,由天津药业集团新郑股份有限公司,批号:020205,国药准字XF19990698号,作为本次试验阳性对照药物。
2.细胞株
Hep-2(喉癌细胞)购于卫生部药品生物制品检定所。
Hela(宫颈癌细胞)本教研室保存。
3.毒株
新冠病毒株购于中国科学院某病毒所。
4.主要试剂
培养基 IMDM为美国Sigma产品。
胎牛血清 长春生物制品研究所产品。
胰蛋白酶日本生产上海化学试剂厂分装。
5.实验动物
Swiss小鼠雌雄各半,3~4W龄,16~18g,雌雄分别分组,由山西医科大学实验动物中心提供。医动字第11-5218号。
二、方法
1.细胞准备
Hep-2、Hela传代细胞培养3~5d,使之成片,界线清晰,立体感及折光度强时,用胰酶消化,待细胞面出现针尖样小孔,吸尽消化液,取数毫升培养液吹散细胞,计数,用培养液稀释至约5×107/L后,接种于96孔培养板内,待细胞长成单层。
2.药物毒性测定
细胞毒性试验:将匹多莫德稀释为0.125g/L,0.0625g/L,0.0312g/L,0.0156 g/L,0.00781g/L,0.0039g/L,0.00195g/L,0.00097g/L等浓度。阳性药利巴韦林稀释为0.125g/L,0.0625g/L,0.03125g/L等浓度。将上述用培养液稀释好的不同浓度的药品滴加于Hep-2、Hela单层细胞上,每孔0.2mL,37℃5%CO2孵箱培养,每天镜下观察细胞病变(CPE),求出药液致细胞毒性,计算药物对细胞毒性浓度,最大无毒浓度(TC0),50%中毒浓度(TC50)。
药物对动物毒性实验将不同剂量药物分组给药,观察急性毒性,根据最大耐受量(MTD)和LD0计算出匹多莫德小鼠体内试验药物用量为:6mL/kg,12 mL/kg,24mL/kg,相当于0.0015g/kg、0.003g/kg、0.006g/kg三个浓度。分别连续给药6天。记录发病或死亡日期和数量,取标本进一步检测。阳性对照药利巴韦林用量为0.13g/kg。
3.药物对病毒致细胞病变作用的影响
在Hep-2和Hela细胞上分别加不同浓度的病毒液,吸附1h后洗去病毒液,加入无毒界限药液,每一病毒稀释度各加3复孔,同时设病毒对照,细胞对照,阳性药物对照组。置37℃CO2培养箱内培养,每天在倒置显微镜下观察病变,连续7d,记录各孔病变情况。计算出抑制细胞病毒的感染量,细胞病变抑制率,最小有效浓度(MTC),半数有效浓度(IC50),90%有效浓度(IC90)和治疗指数 (TI)。实验重复三次。
4.病毒TCID50的测定
将病毒进行10倍稀释10-1,10-2,10-3......10-6不同稀释度,并将各种稀释度的病毒感染Hep-2和Hela细胞,用96孔塑胶板单层培养。观察细胞病变,测定各病毒半数致细胞病变剂量(TCID50)。
5.结果判定及计算方法
以能使50%细胞孔发生CPE的最高稀释度作为终点,用karber法计算病毒滴度。
TCID50:50%组织细胞感染量
XM:病毒最高浓度稀释度的对数
d:稀释度系数(倍数)的对数
∑pi:每个稀释度病变百分数的总和
6.体内抗新冠病毒试验
1)病毒毒力测定:乙醚麻醉小鼠,滴鼻感染,每鼻孔不同稀释度病毒0.03ml,记录死亡百分数,计算LD50。
2)药物毒性测定:小鼠急性毒性和亚急性毒性,计算LD50及LD0。
3)抗新冠病毒试验:小鼠随机分组,10只/组,于感染前一天开始腹腔注射给药,连续5d,对照组腹腔注射相同体积的生理盐水。在乙醚浅麻醉下滴鼻感染病毒鼠肺适应株FM1,15L D50,感染后96h解剖,取肺称重,逐个算出肺指数值,与对照组比较,进行组间t检验。统计学处理,计算t值和P值。
4)肺病变组织学和病理学检查:取各试验组鼠肺、固定、包埋、切片染色、镜检等,具体方法略。
5)血凝试验检测鼠肺新冠病毒增殖和抗原滴度:动物分组、药物剂量、给药方法、病毒感染同前。感染病毒72h后,脱颈处死小鼠,解剖取肺,组织匀浆器研磨,生理盐水制成10%的肺组织悬液,离心取上清,倍比稀释,按0.2mL/ 孔滴于滴定板上后,每孔加入0.2mL1%鸡红细胞悬液,混匀,置室温30min,观察记录血凝滴度。以红细胞凝集(++)时为終点,并以悬液稀释倍数表示其滴度。
三、试验结果
1.病毒的TCID50
log TCID50=-2+0.5-(100+100+50)/100=-4
2.药物毒性测定
1).药物对细胞毒性测定结果
匹多莫德对Hep-2和Hela细胞最大无毒浓度(TCO)为0.0040g/L,半数毒性浓度(TC50)为0.017g/L,具体量效关系见下图。利巴韦林稀释为0.0625g/L以上稀释时对Hep-2和Hela细胞均无毒性作用。
匹多莫德浓度与细胞毒性量效关系曲线见图1
2).动物毒性测定结果
该药的半数致死量按最大给药浓度和小鼠的最大静脉注射容积无法求出半数致死剂量(LD50),求得最大耐受量(MTD)大于1000g生药/kg。
3.匹多莫德对病毒致细胞病变保护作用结果
在感染新冠病毒的情况下,匹多莫德使用浓度为0.0040g/L,匹多莫德对病毒的抑制其致细胞病变作用结果见表1;
表1
注:-表示无细胞病变;±表示有延缓细胞病变作用;+表示1/4以下的细胞病变;++表示1/4~1/2的细胞有病变;+++表示1/2~3/4的细胞有病变; ++++表示3/4以上的细胞有病变。
2).肺病变组织学和病理学检测结果
病毒性肺炎模型组镜下可见:支气管、细支气管壁、肺泡壁等肺间质充血、水肿以及淋巴细胞、单核细胞浸润,肺泡壁增宽,肺泡呈炎症反应。匹多莫德治疗各组鼠肺病变明显减轻,部分肺组织形态结构正常,最佳治疗剂量为 0.006g/kg。
3).小鼠肺组织中病毒抗原滴度测定---血凝试验结果
血凝试验结果详见表3。
表3匹多莫德对新冠病毒性肺炎小鼠肺组织血凝滴度的影响
从上表可见,匹多莫德治疗(0.006g/kg、0.003g/kg、0.0015g/kg)组,鼠肺中新冠病毒滴度明显低于病毒对照组。最佳治疗剂量为0.006g/kg、0.003g/kg。匹多莫德体内治疗量效曲线见图3,EC50=0.0195g/kg,匹多莫德对病毒的体内治疗指数为:
TI=MTD/EC50=10204
结论:匹多莫德无论在体外还是体内均有明显的抑制新冠病毒的作用,对新冠病毒有明显的延缓细胞病变作用。匹多莫德体内抗病毒试验结果显示,在一定浓度范围可有效的抑制新冠病毒所致的病毒性肺炎,与模型对照组比较,差异显著。关于匹多莫德的抗新冠病毒的作用机理有待进一步深入研究。
本发明说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。尽管上面对本发明说明性的具体实施方式进行了描述,以便于本技术领的技术人员理解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保护之列。
Claims (4)
1.一种用匹多莫德在抗新型冠状病毒感染中的应用,其特征在于:通过匹多莫德抑制病毒主蛋白酶活性作用,达到抗新型冠状病毒感染的目的。
2.根据权利要求1所述的一种用匹多莫德在抗新型冠状病毒感染中的应用,其特征在于:所述匹多莫德在治疗新型冠状病毒感染中做抑制剂。
3.根据权利要求1所述的一种用匹多莫德在抗新型冠状病毒感染中的应用,其特征在于:所述匹多莫德为颗粒剂、口服液、散剂、片剂或胶囊剂。
4.根据权利要求3所述的一种用匹多莫德在抗新型冠状病毒感染中的应用,其特征在于:所述片剂为普通片和分散片。
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