CN117957009A - 预防及治疗严重特殊传染性肺炎(covid-19)及长期性严重特殊传染性肺炎(long covid)的方法及组合物 - Google Patents
预防及治疗严重特殊传染性肺炎(covid-19)及长期性严重特殊传染性肺炎(long covid)的方法及组合物 Download PDFInfo
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Abstract
一种预防及治疗COVID‑19(SARS‑CoV‑2)冠状病毒感染的组合物,其包含一医药上可接受组合的葡萄子萃取物、针叶樱桃萃取物、橄榄叶萃取物、万寿菊萃取物、绿茶萃取物、石榴萃取物、酵母β‑葡聚糖及大豆萃取物。一种预防及治疗COVID‑19(SARS‑CoV‑2)冠状病毒感染的方法。
Description
技术领域
本发明是关于一种预防及治疗哺乳类动物的抗病毒感染,特别是严重特殊传染性肺炎(COVID-19)(SARS-CoV-2感染)及长期性严重特殊传染性肺炎(Long COVID)的方法及组合物。
背景技术
自从2019年底一种名为SARS-CoV-2的新型冠状病毒爆发以来,已迅速成为大流行,世界卫生组织宣布其为紧急问题。临床特征显示,患者出现短期肺炎[1],且重症患者发展成多重器官衰竭[2],冠状病毒引起发炎反应及相关的淋巴球减少症[3]。SARS-CoV-2棘蛋白与ACE2受体的附着促使病毒进入、复制、新病毒体形成,接着释放出新的病毒颗粒,其引起发炎失调及细胞激素风暴[4]。COVID-19重症患者遭受细胞激素风暴,造成致命的ARDS,其发病率为41.8%,或不可逆的肺纤维化[5]。此类不受控的全身性过度发炎可由两种主要介质调节:铁蛋白及miRNA。先前的出版品已显示,重症患者的铁蛋白含量增加[6],且其等miRNA的概貌与细胞激素的高度表达密切相关[7,8]。尽管确定了COVID-19进展及潜在机制,但仅使用了11种紧急治疗方法且仅有2种核可药物[9]。目前所有这些药物皆针对单一标靶或一作用机制而设计,例如,瑞德西韦(Remdesivir)为一种抗病毒复制药物,而巴瑞克替尼(Baricitinib)与托珠单抗(Tocilizumab)为免疫调节剂。然而,SARS-CoV-2诱发复杂传讯,其阻断单一功能并导致治疗效率不佳。包括阿尔法(α)、贝他(β)、伽玛(γ)、德尔塔(δ)及奥米克戎(ο)等新突变株的出现,其传播与感染率比原始SARS-CoV-2高出50%,引起人们对疫苗功效的担忧[10]。因此,发掘出多功能药物备受关注。
希望开发出一种有效的疗法或治疗剂以治疗及/或预防Covid-19及/长期性Covid。
发明内容
本发明在提供一种预防及治疗Covid-19(亦即,SARS-CoV-2的感染)及长期性COVID的方法及组合物。
在一方面,本发明提供一种治疗或预防个体的Covid-19及长期性Covid的方法,其包含投予该个体一治疗上有效量的草本组合物,其包含葡萄子萃取物、针叶樱桃萃取物、橄榄叶萃取物、万寿菊萃取物、绿茶萃取物、石榴萃取物、酵母β-葡聚糖及大豆萃取物的混合物。
在另一方面,本发明提供一种治疗或预防Covid-19及/或长期性Covid的组合物,其包含葡萄子萃取物、针叶樱桃萃取物、橄榄叶萃取物、万寿菊萃取物、绿茶萃取物、石榴萃取物、酵母β-葡聚糖及大豆萃取物的混合物。
根据本发明,本发明的草本组合物包含从草本组合物分离的活性成分,包括槲皮素、橙皮苷、金雀异黄酮、大豆异黄酮苷素及白藜芦醇。
在本发明之一实例中,本发明的组合物(在下文中称为“草本组合物”)分别包含10重量%至50重量%的葡萄子萃取物、5重量%至30重量%的针叶樱桃萃取物、5重量%至30重量%的橄榄叶萃取物、1重量%至20重量%的万寿菊萃取物、1重量%至20重量%的绿茶萃取物;1重量%至20重量%的石榴萃取物;1重量%至20重量%的酵母β-葡聚糖;以及1重量%至20重量%的大豆萃取物,以草本组合物的总种量为基准。
在本发明之一特定实例中,本发明的草本组合物为由全球预防医学生技股份有限公司(Global Preventive Medicine Biotech Co.Ltd)(中国台湾)及/或基诺华生技公司(Geninova Biotech Co.,Ltd)(美国)提供的Virofree TM产品,其分别包含约250mg的葡萄子萃取物、约180mg的针叶樱桃萃取物、约160mg的橄榄叶萃取物、约90mg的万寿菊萃取物、约80mg的绿茶萃取物;80mg的石榴萃取物;约80mg的酵母β-葡聚糖;以及约80mg的大豆萃取物,分别以Virofree产品的总重量(1000mg)为基准。
在本发明中,本发明的草本组合物可以常用或标准方法配制,以获得常用的医药剂型,包括丸剂、锭剂、胶囊剂、饮剂或糖浆,并与一医药上可接受载体组合。
在本发明的另一实例中,草本组合物(Virofree)可制备成膳食形式,其包括锭剂、胶囊剂、胶剂、粉剂、饮剂及能量棒。
在另一方面,本发明提供一种根据本发明的草本组合物的用途,用于制造一治疗或预防COVID-19及长期性COVID的补充剂或组合物或医药上组合物。
本发明将通过下列实例进一步说明。然而,应理解的是,下列实例仅旨在说明的目的,且不应解释为在实践时局限本发明。
附图说明
当结合附图阅读时,将更好理解前述发明内容以及以下对本发明的详细描述。为了说明本发明,在附图中显示出目前较佳的具体实施例。
图1显示生物资讯学分析,以确定本发明的草本组合物(亦即,Virofree)在治疗COVID-19上的潜在作用机制;其中(A)ARDS患者的基因集GSEA富集图包括显著下调的基因(log2FC<-1.5,调整的p值<0.05);负富集评分(NES)意指通过药物处理使下调基因富集;(BD)显示在BEAS-2B细胞中低剂量(66.67μg/ml)Virofree的总转录本概貌的生物资讯学分析;(B)源自DEG的KEGG富集分析的前10个途径;颜色强度表示p值,且条形的长度表示输入DEG与基因集之间的重叠基因比率(基因计数);(C)前30个基因发生学(GO)项目富集,颜色表示项目类别,包括生物过程(BP)、细胞成分(CC)及分子功能(MF),且条形的长度表示DEG与GO项目之间的重叠基因数;(D)由DisGeNET/DO数据库预测的相关疾病;该网络说明了前15种疾病之间的关联性,其中红色突显了与COVID-19相关的症状;统计结果列于表3与表4中。(EF)低剂量与高剂量的Virofree处理的总转录本概貌的PCA分析;(E)低剂量与高剂量的Virofree处理的总转录本概貌的PCA图;在PCA分析之前预先处理基因表达概貌以消除批次效应;(F)有助于各主成分(PC)的基因的负荷评分:正负荷评分表示基因表达与PC的的间存在正相关;且负负荷评分表示基因表达与PC之间存在负相关。
图2显示本发明的草本组合物(Virofree)可通过诱发miR148b-5p、抑制M pro及抑制细胞激素风暴而潜在减少病毒复制;(A)在以33.3μg/ml的Virofree(n=3)处理24小时后,通过qRT-PCR测量miR-148b-5p表达量。所有数据皆以平均值±SD表示;(B)测定并显示抑制重组SARS-CoV-2M pro的Virofree IC 50(μg/ml);(C)在处理PMA分化的THP-1细胞6或24小时后,收集细胞培养基,接着以ELISA测量细胞激素释放的量。在分化的THP-1细胞中,单独处理LPS100ng/ml被视为阳性对照组(n=3)。[所有数据皆以平均值±SD表示。以单因子ANOVA进行统计分析,*或**:分别显著不同于相应的对照组,其中p<0.05或0.01]。
图3显示Virofree针对三聚体棘蛋白结合至ACE2的变体特异性;(AE)衍生自(A)野生型/原始株、(B)变体α、(C)变体β、(D)变体γ、(E)变体δ及(F)变体ο的三聚体棘蛋白系用于基于ELISA的棘蛋白与hACE2结合试验;阳性条件代表三聚体棘蛋白与hACE2的完全结合活性;所用的抑制剂为野生型棘RBD抗体(10μg/ml)。数据代表平均值±SEM(n=4)。当相较于相应的阳性组的结合效率时,*、**或***分别表示与相应对照组样品的显著差异,其中p<0.05、0.01或0.001。
图4显示Virofree在基于细胞的试验中可干扰SARS-CoV-2-棘介导的结合及合胞体(syncytium)形成;(A)通过LDH细胞毒性试验验证Virofree的细胞毒性。以指定量的Virofree处理BHK-21与Calu-3细胞,并通过LDH试验测定Virofree的细胞毒性;对照组代表无Virofree处理的组别,用作阴性对照组;以处理Triton-100作为阳性对照组。数据以平均值±SD表示;误差杠表示SD。ns表示相较于对照组不显著。**p<0.01,****p<0.0001。(B-C)将EGFP及野生型(B)或δ(C)棘共表达的BHK21细胞添加至Calu-3细胞中,并在4℃下培养1小时以进行棘-ACE2结合;在培养4小时(野生型)或2小时(δ)后,在对照组中形成大型的萤光多核细胞,表示棘介导的合胞体形成;柱形图描绘各组的结合与融合效率(n=3);(D)Calu-3细胞以0、0.67、2mg/ml的Virofree处理5小时(n=3)。(E-G)Virofree在基于细胞之中和试验中可阻断表达ACE2的HEK293T细胞中表达SARS-CoV-2棘蛋白的假病毒的感染;Virofree的IC 50在假病毒中和试验中对野生型(E)、δ(F)或ο(G)SARS-CoV-2的进入的抑制效果(n=3)。所有数据皆以平均值±SD表示。*p<0.05,**p<0.01,***p<0.001,****p<0.0001。
图5显示Virofree在THP-1-衍生的巨噬细胞中具有减少不稳定铁池及保护细胞免于铁依赖型细胞死亡(ferroptosis)的潜力;(A)THP-1-衍生的巨噬细胞分别接受6或24小时的不同浓度的Virofree处理。制备全细胞溶胞产物并进行西方墨点分析。GAPDH用作内部对照组(n=3);(B)THP-1-衍生的巨噬细胞在爱拉斯汀(erastin)(10μM)存在下分别接受6或24小时的不同浓度的Virofree处理。制备全细胞溶胞产物并进行西方墨点分析。GAPDH用作内部对照组(n=3)。
图6显示Virofree抑制TGF-β1诱发的α-SMA与ECM蛋白表达;将LL29细胞与2.5ng/ml的TGF-β1和不同浓度的Virofree共同处理48小时。经由西方墨点分析,定量结果显示Virofree对α-SMA、纤维接合素及N-钙黏蛋白表达的抑制效果(n=3)。所有数据皆以平均值±SD表示。*p<0.05,**p<0.01,***p<0.001。
图7显示Virofree为COVID-19的候选治疗性疗法;病毒通过复制过程侵入宿主细胞可造成细胞激素风暴及多重器官衰竭。铁摄取为在COVID-19患者中观察到的症状之一,其可导致铁依赖型细胞死亡,促进病毒感染。在本研究中,通过抑制野生型与多个变体的SARS-COV-2棘的病毒进入与融合,Virofree可作为铁依赖型细胞死亡抑制剂及抗病毒剂,其系经由降低ACE2与TMPRSS2蛋白表达、抑制病毒蛋白酶活性、增加mir-148b、靶向细胞激素风暴以及体外下调纤维化蛋白。模糊图说明降低或抑制(以BioRender.com产生)。
图8显示分析管道的概况;(A)BEAS2B在以66.67μg/ml或500μg/ml的Virofree处理6小时后,萃取RNA以进行次世代定序,并分析实质差异表达量及富集分析;所述平台,包括四个数据库,基因发生学(GO)、KEGG、疾病发生学(DO)及GSEA,可通过大数据分析显示Virofree的作用机制。Virofree的显著差异表达基因的鉴定。BEAS-2B细胞以66.67μg/ml(B)或500μg/ml的Virofree(C)处理,并萃取RNA以进行次世代定序;相较于对照组及计算的log2倍数变化(x轴),以火山图显示出代表上调/下调基因的正值/负值。y轴显示Virofree与对照组之间的显著变化。截止值表示1.5log2倍数变化;详细资讯列于表2中。
图9显示通过生物资讯学分析,不同剂量的Virofree具有类似机制;从大数据分析可知高剂量(500μg/ml)的Virofree的高度潜在疾病机制;(A)柱形图显示DEG列表中KEGG富集分析的前10个途径;颜色的强度表示p值,且条形的长度表示DEG与途径之间的重叠基因比率(基因计数);(B)柱形图显示源自基因发生学(GO)之前30个高Virofree的途径;颜色意指基因的分类,例如生物过程(BP)与细胞成分(CC);条形的长度表示DEG与GO项目之间的重叠基因数;分析结果列于(表4);(CD)比较低剂量(66.67μg/ml)的Virofree与高剂量(500μg/ml)的Virofree对基因表达量及相关的疾病的影响;(C)4个基因集中的常见DEG的相交文氏图,其中2个基因(LDLR与MSMO1)在两剂量下皆上调以及4个基因(CYP1B1、TIPARP、SLC25A37及RIPK4)在两剂量下皆下调。然而,STC2的表达量在低剂量与高剂量之间发生变化;阈值为1.5log2倍数变化且q值<0.01。(D)发明人将两个浓度之间的疾病结果相交,并在两种处理中显示5种常见疾病;尤其是,肺动脉高血压为Virofree成为治疗ARDS的候选药物的要素之一。
图10显示处理Virofree的细胞毒性;在Virofree处理24小时后,收集BEAS2B细胞,并通过SRB试验测量存活率(n=3)。所有数据皆以平均值±SD表示。
图11显示Virofree可通过诱发let-7a-5p而潜在减少病毒复制;在以333μg/ml的Virofree处理24小时后,通过qRT-PCR测量let-7a-5p表达量(n=3)。所有数据皆以平均值±SD表示。以t检定进行统计分析。*:显著不同于相应的各别对照组,其中p<0.05。
图12显示Virofree针对δRBD棘蛋白结合至ACE2的抑制活性;衍生自变体δ的RBD棘蛋白系用于基于ELISA的棘蛋白与hACE2结合试验;阳性条件代表三聚体棘蛋白与hACE2的完全结合活性;所用的抑制剂为野生型棘RBD抗体(10μg/mL)。数据代表平均值±SEM(n=4)。当相较于阳性组的结合效率时,**或***分别表示与相应对照组样品的显著差异,其中p<0.01或0.001。
图13显示Virofree在THP-1-衍生的巨噬细胞中减少不稳定铁池及保护细胞免于铁依赖型细胞死亡的量化结果;(A)THP-1-衍生的巨噬细胞分别接受6或24小时的不同浓度的Virofree处理;GAPDH用作内部对照组(n=3);(B)THP-1-衍生的巨噬细胞在爱拉斯汀(10μM)存在下分别接受6或24小时的不同浓度的Virofree处理。GAPDH用作内部对照组(n=3)。所有数据皆以平均值±SD表示。*、**、***或****分别表示与相应的对照组样本的显著差异,其中p<0.05、0.01、0.001或0.0001。
图14显示由NAC介导的ROS含量的降低减少了由Virofree介导的xCT的表达量;THP-1-衍生的巨噬细胞以5mM的NAC预处理1小时,接着分别以100μg/ml的Virofree处理6或24小时;制备全细胞溶胞产物并进行西方墨点分析。β-肌动蛋白用作内部对照组。
图15显示野生型和表达δ棘的BHK21与表达ACE2的Calu-3细胞的合胞体形成的时间进程试验;将EGFP及野生型(上图)或δ(下图)棘共表达的BHK21细胞添加至Calu-3细胞中,并在37℃下培养1、2或4小时;在对照组中形成大型的萤光多核细胞,表示棘介导的合胞体形成。
图16显示如图6所示的西方墨点的量化及统计分析。
图17显示Virofree改进了BLM诱发的ARDS大鼠的肺功能;(A)处理Virofree的实验流程图;(B)动脉血氧饱和度(SpO 2)及(C)呼吸速率(BPM:每分钟呼吸次数)。
图18显示处理Virofree的分化的THP-1细胞的细胞毒性;PMA分化的THP-1细胞在有或无LPS100ng/ml时以Virofree处理。在24小时后,进行MTS试验以测量细胞存活力;将无任何处理的组别视为对照组(n=3)。
图19显示如图5所示的原始西方墨点的完整未裁剪图像。
图20显示如图6所示的原始西方墨点的完整未裁剪图像。
在以下的描述中阐述本发明的一或多个实施例的细节。本发明的其他特征或优点将从以下几个具体实施例的详细描述以及所附权利要求中变得显而易见。
具体实施方式
除非另有定义,本文使用的所有技术及科学术语具有与熟悉本发明所属领域人员通常理解的相同含义。
本发明通过以下实施例进一步说明,期提供做为例示而不限制本发明。
如本文所用,“一”冠词是指一个或多于一个(即,至少一个)的冠词语法对象。举例来说,“一个成分”是指一个成分或多于一个成分。
本发明提供一种预防及治疗个体的Covid-19(SARS-CoV-2的感染)及长期性Covid的方法及组合物,其包含投予该个体一治疗上有效量的组合物,其包含葡萄子萃取物、针叶樱桃萃取物、橄榄叶萃取物、万寿菊萃取物、绿茶萃取物、石榴萃取物、酵母β-葡聚糖及大豆萃取物的混合物。
术语“长期性COVID”意指在COVID-19的典型康复期后持续存在长期后果的状况。其亦称为COVID-19后症候群、COVID-19后状况、COVID-19的急性后遗症(PASC)或慢性COVID症候群(CCS),长期性COVID几乎可影响每种器官系统,其后遗症包括呼吸系统疾病、神经系统与神经认知疾病、精神健康疾病、代谢疾病、心血管疾病、肠胃道疾病、肌肉骨骼疼痛及贫血。广泛的症状包括疲劳、不适、头痛、呼吸短促、嗅觉缺失(嗅觉丧失)、嗅觉倒错(气味扭曲)、肌肉无力、低烧及认知功能障碍。症状的确切性质及经历长期症状的人数尚不清楚。
在本发明中,生物资讯学分析平台提出了能通过多种生物学功能(包括病毒复制、细胞激素风暴及ARDS)抑制此疾病的药物。通过扫描数据库中1200个化合物的概貌[11],草本组合物(Virofree)经分析发现为COVID-19病理机制的完全阻断剂。本发明证实,通过基因集富集分析(GSEA),Virofree有望逆转SARS-CoV-2感染特征[12]。根据本发明的临床观察,确认Virofree具有药理学性质,包括预防及治疗流感、放疗/化疗的二次治疗、降低哮喘发作频率、抗发炎、激活肿瘤细胞凋亡、抑制癌症转移、受损基因修复。此外,通过另一个大数据系统,其整合数据库(KEGG[16]、基因发生学(GO)[17]及疾病发生学(DO)[18,19]),分析Virofree的生物学途径。本发明强烈确定Virofree与铁依赖型细胞死亡、细胞激素、miRNA及ARDS相关,这些作用机制常与COVID-19一起被报导。因此,本发明证实Virofree能介导SARS-CoV-2诱发的各种病理信号,并提供全面的抑制作用,以缓解患者感染COVID-19后的症状。
可确定的是,Virofree为一种治疗性组合物,其可调节细胞激素分泌并影响其他标靶途径以全面性地阻断病毒感染。另一标靶为miRNA,其在细胞过程中发挥关键调节剂作用。根据Gonzalo等人[20]和Kedzierski等人[21]的报告,两个团队在COVID-19住院患者中检测到miRNA表达列表。在两项研究中,有四种常见的miRNA可调节发炎反应(miR-150-5p、miR-148a-3p)及病毒感染(miR-92a-3p、miR-491-5p)。根据先前的研究,let-7a与miR-148b预测靶向SARS-CoV-2基因体的不同区域[22]。亦可通过压制病毒M pro活性而抑制病毒复制,M pro可切割开读框(ORF)1上的两个重叠的多蛋白(pp)1a与pp1ab,以生成更多的正链基因体RNA 。
由于病毒复制过程需要铁[40],巨噬细胞吸收铁而降低病毒铁生体可用率,以抑制病毒复制[41]。然而,巨噬细胞中过量的铁累积导致潜在氧化损伤的磷脂,最终引发特定形式的程序性细胞死亡,称为铁依赖型细胞死亡[42]。胱胺酸/麸胺酸反向运输蛋白xCT与麸胱甘肽过氧化酶4(GPX4)介导针对铁依赖型细胞死亡的细胞机制,以进行谷胱甘肽生合成及抗氧化剂防御[43]。其表明,了解与靶向巨噬细胞可能有助于提高COVID-19治疗的功效。上面的描述改进了铁代谢、免疫系统及ARDS之间的相关性。Virofree的最后一个假设功能改变了铁代谢的稳定性,并导致抑制发炎反应,以治疗COVID-19的ARDS。
根据本发明,通过破坏病毒棘与宿主ACE2受体之间的结合与融合,提供了一种预防及治疗COVID-19(SARS-CoV-2的感染)的有前景方法。
由于病毒复制过程需要铁[40],巨噬细胞吸收铁而降低病毒铁生体可用率,以抑制病毒复制[41]。然而,巨噬细胞中过量的铁累积导致潜在氧化损伤的磷脂,最终引发特定形式的程序性细胞死亡,称为铁依赖型细胞死亡[42]。胱胺酸/麸胺酸反向运输蛋白xCT与麸胱甘肽过氧化酶4(GPX4)介导针对铁依赖型细胞死亡的细胞机制,以进行谷胱甘肽生合成及抗氧化剂防御[43]。其表明,了解与靶向巨噬细胞可能有助于提高COVID-19治疗的功效。上面的描述改进了铁代谢、免疫系统及ARDS之间的相关性。Virofree的最后一个假设功能改变了铁代谢的稳定性,并导致抑制发炎反应,以治疗COVID-19的ARDS。
通过电脑模拟分析证实,本发明的草本组合物,称为“Virofree”,逆转了COVID-19与ARDS的遗传特征。
在本发明的实例中,生化学验证显示,Virofree可经由基于细胞的假型病毒试验而破坏野生型和δ-变体棘蛋白与ACE2的结合及其合胞体的形成,以及抑制数个变体重组棘(尤其是δ和ο)与ACE2之间的结合。
此外,本发明证实,Virofree提高miR-148b-5p含量、抑制SARS-CoV-2(M pro)的主要蛋白酶及减少LPS诱发的TNF-α释放。
本发明亦防止发生在SARS-CoV-2患者的导致铁依赖型细胞死亡的细胞铁累积。此外,本发明的Virofree能降低体外肺部纤维化相关蛋白的表达量。
此外,本发明提供一种对抗COVID-19的草本组合物,其突显了Virofree对SARS-CoV-2δ和ο变体的进入的抑制效果。
草本组合物可使用本领域技术人员已知的任何标准技术或常用方法配制。
针对本发明浓缩物的制备,使用本领域常用的常规方法或任何标准方法将组分混合在一起。在一实例中,将组分的粉末原料混合以获得均匀混合物。针对保存目的,浓缩物可进行巴氏杀菌或冻干。
本发明的草本组合物可为可用的液体形式或干燥形式。本发明的草本组合物可以常见方式加工成胶囊剂、锭剂、颗粒剂或粉剂。在本发明之一实例中,将草本组合物制备成胶囊剂,其可通过下列步骤的方法获得:将粉末形式的组分混合以获得混合物,接着将混合物封装。
在本发明的另一实例中,草本组合物可配制或制备成饮料,或浓缩物,其系通过将粉剂、锭剂或颗粒剂溶解在水、冷饮或热饮(如果汁或茶)中。
本发明以下列实施例进一步说明本发明的技术特征,但是不用以限制本发明的范畴。
实施例
实施例1 Virofree的制备
Virofree系由Geninova Biotech Inc.提供,基本上由下列组成:250mg的葡萄子萃取物、180mg的针叶樱桃萃取物、160mg的橄榄叶萃取物、90mg的万寿菊萃取物、80mg的绿茶萃取物、80mg的石榴萃取物、80mg的酵母β-葡聚糖、80mg的大豆萃取物,以药剂的总重量(1000mg)为基准。活性成分系从植物萃取物中分离,包括槲皮素、橙皮苷、金雀异黄酮、大豆异黄酮苷素及白藜芦醇。药剂系制备成胶囊剂形式,其系通过下列方法获得:将粉末形式的组分混合以获得混合物,接着将混合物封装。
实施例2
方法:
1.细胞培养
THP-1为衍生自急性单核细胞白血病患者非粘黏人类单核细胞株(ATCC,#TIB-202),其购自Bioresource Collection and Research Center,并培养在Roswell ParkMemorial Institute(RPMI)(Gibco)1640中,其中补充10mM HEPES、10%胎牛血清(FBS)、1%青霉素-链霉素及50μMβ-巯乙醇,并置于37℃的5%二氧化碳加湿环境的培养皿中。细胞在3-4天后连续传代。在实验方面,使用第8代以前的细胞株。
BEAS-2B为人类正常支气管上皮细胞株(ATCC,#CRL-9609),其培养在RPMI培养基中,其中补充10% FBS(Invitrogen)、1% PSA、1%非必需胺基酸及2mM L-麸胺酸(Invitrogen)。细胞维持在37℃与5% CO 2的细胞培养箱中,每3-4天传代一次。在实验方面,使用早期传代的细胞株(第6代以前)。
LL29为衍生自特发性肺纤维化肺组织的人类肺纤维母细胞细胞株(ATCC,#CCL-134)。LL29细胞系培养在含有15% FBS(Invitrogen)与1% PSA的Ham's F12K培养基中。细胞系培养在37℃的5% CO 2环境中,每3-4天进行胰蛋白酶化。在实验方面,使用早期传代的细胞株(第8代以前)。
幼仓鼠肾(BHK)-21细胞为衍生自幼仓鼠肾的纤维母细胞细胞株(ATCC,#CCL-10),且Calu-3细胞为衍生自肺腺癌患者的人类上皮肺细胞株(ATCC,#HTB-55),是培养于Dulbecco's Modified Eagle Medium(DMEM,Gibco)中,其中补充10% FBS与1×青霉素/链霉素溶液。BHK-21与Calu-3细胞系培养在37℃的5% CO 2环境中,且每2天与3-4天分别进行胰蛋白酶化。在实验方面,使用早期传代的细胞株(第8代以前)。
2.磺基玫瑰红B(sulforhodamine B)比色试验
磺基玫瑰红B(SRB)试验是用于基于测量细胞蛋白含量的细胞密度测定。本文所述方法已优化成针对96孔培养盘中的粘黏细胞进行化合物毒性筛选。细胞以每孔2,000个细胞接种16-20小时,接着以不同浓度的不同药物处理24小时。移去培养基,且细胞以磷酸盐缓冲液(PBS)轻轻洗涤两次,并在4℃下以冷的10%三氯乙酸(w/v)(SIGMA)固定1小时。在固定后,培养盘以水洗涤两次并风干。细胞在室温下以每孔100μl的0.1%(w/v,在1%乙酸中)SRB溶液染色1小时,接着以1%乙酸(AVANTOR)洗涤两次。在风干后,将100μl的20mM Tris-碱添加至各孔中,并在光学密度(OD)540nm下读取。
3.定量反转录聚合酶链反应(PCR)分析
将衍生自人类正常支气管上皮的BEAS-2B细胞用于检测影响呼吸道感染机制的生物试剂。为了评估Virofree对let-7a与miR-148表达的效果,在药物处理前24小时,将1×106个BEAS-2B细胞接种在10-cm的培养皿中。随后,在处理24小时后收集细胞。试剂系用于总RNA萃取,且RNA样品储存在-80℃中。使用定量反转录PCR(qRT–PCR)进行let-7a与miR-148b表达miRNA含量的量化,其中以U54作为内部对照组。使用即时PCR引子进行扩增,包括hsa-let-7a-5p(5'-GCCTGAGGTAGTAGGTTGTATAGTTA-3')、hsa-miR148b-5p(5'-AAGUUCUGUUAUACACUCAGGC-3')及U54(人属)(5'-GGTACCTATTGTGTTGAGTAACGGTGA-3')等特定正向序列。qRT–PCR系使用PhalanxmiRNA One/>Profiling(Phalanx BiotechGroup)进行。
4.细胞激素测定试验
以THP-1细胞株作为细胞模型。THP-1细胞通过50ng/ml的佛波醇12-十四酸-13-乙酯(PMA)(SIGMA;P1585)分化24小时。在以不含RPMI的血清洗涤非粘黏细胞后,以100ng/ml的脂多糖(LPS)(SIGMA;L2654)用作刺激剂以模拟发炎状况,并以单独处理100ng/ml LPS分化的THP-1细胞作为阳性对照组。在存在或不存在LPS的情况下以药物处理细胞,并在37℃下培养6或24小时。随后,收集细胞培养基并储存在-20℃。使用酵素结合免疫吸附分析法(ELISA)检测处理细胞所释放的TNF-α含量。按照制造商的方案(Invitrogen,Thermofisher;#88-7346),在Nunc平底96孔培养盘(Invitrogen,ThermoFisher;#442402)中使用人类TNF-α未涂覆的ELISA套组分析上清液。以Infinite200Pro OD读数器测量OD值,其使用Tecan i-control程式,在450nm与570nm波长下测量。
5.抑制M pro活性及测定半最大抑制浓度(IC 50)
为了测定M pro活性抑制能力,Virofree在酵素-受质试验中用作抑制剂,其中Mpro在PBS中的萤光胜肽受质(Abz-TSAVLQSGFRK-Dnp)的切割位点切割。Virofree在30℃下的PBS中与萤光胜肽受质一起培养3分钟,接着添加蛋白酶并在30℃下平衡3分钟。使用发光分光计(PerkinElmer LS50B),通过在321nm下激发,检测在423nm下的萤光发射[24]。由下列方程式获得IC50值
其中v为在不同浓度的培养抑制剂[I]下的速度,且v0为无抑制剂培养的初始速度,而n为希尔(Hill)常数。
6.细胞-细胞融合
人类肺癌Calu-3细胞,用作接受者细胞,首先以每孔1×10 6个细胞接种在12孔培养盘中,以形成单层细胞。BHK-21细胞以每孔4x10 5个细胞接种在6孔培养盘中,并使用Lipofectamine2000,以1:5的比率将EGFP与棘质体(原始株或δ变体)进行转染。在24小时后,通过将1ml的细胞分离缓冲液(含有5mM EDTA的PBS)添加至各孔中而收取EGFP-棘BHK细胞以分离完整细胞,并重新悬浮在无血清DMEM(Gibco)中。将表达EGFP与棘基因的转染的BHK-21细胞用作供体细胞;在单层Calu-3细胞中共培养,用作标靶细胞,进行存在或不存在Virofree处理下的细胞-细胞接触,并在4℃下培养1小时。在1小时后,以PBS洗去未结合的细胞,并替换成生长培养基。使用倒立式萤光显微镜(Olympus IX70),在五个随机视野中撷取EGFP阳性细胞的初始图像,其代表结合效率。通过计数附着于Calu-3细胞的EGFP阳性BHK-21细胞的初始数量,量化对照组与Virofree处理组中的EGFP阳性BHK-21细胞与Calu-3细胞的结合效率。将对照组中的EGFP细胞的数量定义为具有100%的结合效率。因此,通过以对照组标准化的结合效率百分比,确定Virofree对结合效率的效果。随后,这些细胞进行相应的处理,接着在37℃下将野生型另外培养4小时或将δ变体另外培养2小时,接着撷取EGFP阳性细胞的五个视野的随机选择图像,以评估融合效率。使用ImageJ,通过量化这些图像中EGFP阳性细胞的扩增区域而计算合胞体细胞的形成。将对照组中的EGFP阳性区域的初始至4小时(野生型)或2小时(δ)的倍数变化视为100%融合效率。根据下列方程式计算Virofree对合胞体形成的效果:
7.假型病毒中和试验
在Opti-MEM中,通过以所需浓度的系列稀释化合物培养野生型(G-SARS-CoV2-假病毒WT,LumiSTAR)、δ(G-SARS-CoV2-假病毒,B.1.617.2,LumiSTAR)或ο假病毒(G-SARS-CoV2-假病毒,B.1.1.529,LumiSTAR)以进行中和试验。将稳定表达人类ACE2基因的HEK-293T细胞(1×10 4个)接种在黑色μCLEAR平底96孔培养盘(Greiner Bio-oneTM)各孔的50μL的Opti-MEM(Gibco)中,且细胞在37℃与5% CO2下培养过夜。隔天,将各化合物以3倍系列稀释在Opti-MEM中,并在37℃下以SARS-CoV-2假型慢病毒培养1小时。将测试药物稀释3次,直至最低浓度为1μg/ml。将病毒-化合物混合物移至293T/ACE2细胞培养盘,最终感染复数(multiplicity of infection)为0.1。随后,在感染后16小时,将培养基更换成新鲜的DMEM(补充10% FBS、100U/ml青霉素/链霉素),且细胞继续另外培养56小时。感染的细胞在37℃下培养72小时后,在ImageXpress Micro Confocal High-Content Imaging System(Molecular Devices)中将细胞GFP萤光量化。
8.图像及IC50拟合
在37℃下培养72小时后,感染的细胞在37℃下以DAPI染色20分钟。随后,使用ImageXpress Micro Confocal High-Content Imaging System(Molecular Devices)检测GFP阳性细胞与总细胞核。使用20×水浸物镜取得原始图像(5×5个位点,总共25个位点),接着通过适当设定进行处理与拼接。量化各孔的总细胞(由核染色表示)与GFP阳性细胞。所有的分析皆使用MetaXpress细胞评分模组进行,其计数阳性细胞及各位置的总细胞数。在细胞评分分析后,利用Lumi-Vcal(LumiSTAR惯用分析软体)处理原始数据。通过将GFP阳性细胞除以总细胞数而确定转导率。通过以PBS处理的对照组的感染率进行药物处理组的感染率的标准化,以获得相对转导率。基于PBS处理的对照组诱发的0%抑制的假设而获得抑制百分比。使用Prism 8(GraphPad)绘制相对抑制率与药物浓度的曲线。以非线性回归方法[25]确定表达50%的GFP(IC50)的药物浓度。各药物以三重复方式测试。所有的SARS-CoV-2假病毒中和试验皆在BSL-2设施中进行。
9.酵素结合免疫吸附分析法
使用ELISA进行一项额外的实验,以评估Virofree干扰三聚体SARS-CoV-2的野生型(原始株)或变体(α、β、γ、δ、ο)棘蛋白及SARS-CoV-2的δ变体棘蛋白RBD结构域与生物素化人类ACE2重组蛋白的结合功效。首先,在96孔培养盘的各孔中涂覆100μl的棘蛋白(500ng/ml;cat.GTX135972-pro,GeneTex,中国台湾台北),其稀释于由碳酸钠(15mM)、碳酸氢钠(35mM),pH 9.6组成的涂覆缓冲液中,并在4℃下过夜。随后,涂覆的培养盘以由含有0.05%(v/v)Tween-20的PBS(pH 7.4)组成的洗涤缓冲液洗涤两次,接着在37℃下以250μl的由0.5%(w/v)牛血清白蛋白组成的阻断缓冲液阻断1.5小时。将培养盘洗涤三次,接着将溶于稀释缓冲液的100μl测试药物或抑制剂(10μg/ml;cat.GTX635791,GeneTex,中国台湾台北)添加至培养盘中,并在37℃下培养1小时。将100μl的生物素化人类ACE2蛋白(10ng/ml;cat.AC2-H82E6;ACRO Biosystems,OX,UK)添加至各孔中,并在37℃下另外培养1小时。将棘蛋白与ACE2受体在不含药物或抑制剂下的结合视为阳性对照组。随后,培养盘以洗涤缓冲液洗涤三次,接着添加溶于稀释缓冲液的100μl卵白素-HRP共轭体(100ng/ml;cat.GTX30949,GeneTex,中国台湾台北),并在37℃下培养1小时。随后,洗涤培养盘,并在37℃的光保护下以每孔200μl的TMB受质培养20分钟。随后,添加50μl的终止溶液以终止反应,并使用微盘读数器(Cytation 5,BioTek,美国佛蒙特州)检测450nm处的吸光度。
10.西方墨点分析
细胞在指定时间下暴露于不同的处理,细胞在冰上以裂解缓冲液裂解。通过以12,000g离心10分钟清除细胞溶胞产物。通过十二基硫酸钠-聚丙烯醯胺凝胶电泳分离溶胞产物,并将蛋白转移至聚二氟亚乙烯薄膜上。在以溶于Tris缓冲盐液的5%脱脂奶粉阻断后,薄膜以所需的一次抗体(FPN(Novus Biologicals,NBP1-21502,1:1000)、FTH-1(Cellsignaling,4393S,1:1000)、GPX4(Abcam,ab125066,1:1000)、TFRC(Cell signaling,13208s,1:1000)、xCT(Cell signaling,17681s,1:1000)、GAPDH(GeneTex,GTX100118,1:10,000)、α-SMA(Abcam,ab5694,1:1000)、纤维接合素(Santa Cruz,sc-9068,1:1000)、N-钙黏蛋白(BD,610920,1:1000)、β-肌动蛋白(GeneTex,GTX109639,1:10000))培养过夜。随后,将膜与适当的二次抗体一起培养。使用增强化学发光(ECL)法将免疫反应条带可视化,并通过Luminescence Imaging system(LAS 4000TM,Fuji Photo Film Co.Ltd)捕获。
11.RNAseq及数据挖掘
以RNeasy Mini套组(Qiagen)萃取BEAS-2B细胞中两个浓度的Virofree与PBS对照组样品的总RNA。使用次世代定序-RNAseq(Biotools Microbiome Research Center Inc.)检测各样品的mRNA表达量。生物资讯学管道如图8的概述。总转录本反应剖析使用两个不同的浓度,包括66.67μg/ml(低剂量)与500μg/ml(高剂量)。经由edgeR(v3.8.1)使用“M值的修剪平均值”将原始读取计数标准化[26,27],并使用MARS方法(具有随机抽样模型的基于MA图的方法(MA-plot-based method with Random Sampling model))通过DEGseq套装软体(v1.40.0)进行生物学上不重复的差异表达基因(DEG)分析[28]。
12.基因集富集分析
GSEA为一计算方法,用于确定一组预定义的基因是否在两个表型(治疗与不治疗)之间显示出统计学上的显著差异[29]。GSEA的目标在于确定基因集成员是否倾向于出现在排序的基因列表的顶部(或底部)。排序列表系基于两个表型之间的差异基因表达。GSEA针对各基因集的科莫-斯米诺(Kolmogorov-Smirnow)统计量指派一个富集评分,接着基于其等的大小将评分标准化。正评分表示基因集富集在排序列表的顶部,而负评分表示基因集富集在排序列表的底部。最后,基于标准化的富集评分,生成以排列为主的错误发现率,以表明富集评分的重要性。使用来自MSigDB v.7.2的C2与C7基因集集合进行GSEA。COVID-19与ARDS相关之特征为从MSigDB与基因表达综合(GEO)数据库中检索到的疾病状况中的上调或下调的基因集。通过limma套装软体分析从GEO检索到的GSE76293微阵列。进行GSEA分析,其使用指标之类别比将基因排序,1000种排列的基因集排列类型。
13.主成分分析(PCA)
通过sva套装软体调整两剂Virofree处理的基因表达概貌,以消除批次效应[30]。PCA分析系经由R语言的prcomp进行。
14.建立博莱霉素诱发的ARDS大鼠模型
6周大的雄性Sprague-Dawley大鼠以舒泰(Zoletil)20–40mg/kg与甲苯噻嗪(Xylazine)5-10mg/kg(腹腔注射)进行麻醉,之后通过气管注射投予溶于200μl PBS的5mg的博莱霉素/250g体重(Nippon Kayaku Co.,Ltd.),接着置于左侧60°处90分钟。动物随机接受下列治疗:(a)BLM组(n=1)大鼠以气管注射5mg BLM,并于第8天牺牲。从第1天至第7天,每天两次口服投予大鼠0.5ml的盐液。(b)BLM+Virofree(n=2)大鼠以气管注射接受5mgBLM,并于第8天牺牲。从第1天至第7天,每天两次口服投予大鼠150mg Virofree/0.5ml盐液。剂量系基于体表面积的动物等效剂量计算而确定[31]。
16.统计分析
数据以平均值±SD或SEM表示。通过单因子ANOVA,将数据与各实验中相应的对照组进行比较,接着进行Dunnett事后检定,其使用Prism 8(GraphPad)。将p值<0.05视为具有统计学上显著性。
结果:
1.基因集富集分析显示由Virofree诱发的COVID-19的反向特征。
为了筛选COVID-19候选治疗,分析不同药物治疗的总转录本反应概貌,以确定一给定药物是否可逆转疾病特征基因集。COVID-19、CRS及ARDS的特征在于基因在疾病条件中上调或下调,且反向特征意指基因在疾病条件中增加/减少,其可通过药物处理而减少/增加。
Blanco-Melo等人描述了COVID-19特征[32],相较于其他呼吸道病毒,SARS-CoV-2感染诱发的基因上调或下调的幅度更大,其表明COVID-19的独特特征。将这些基因集整合至MSigDB数据库中进行GSEA分析。结果显示,处理Virofree可逆转A549细胞中的SARS-CoV-2感染特征,其中通过处理,由SARS-CoV-2感染而上调的基因被下调(标准化的富集评分(NES)<0)且下调的基因被升高(NES>0)(表1)。此外,本发明对表达ACE2的细胞似乎具有更大的效果。
表1:在COVID-19病理学相关的基因集中处理Virofree的总转录本概貌的富集分析。
CRS:细胞激素释放症候群,俗称细胞激素风暴
GSEA分析亦表明Virofree降低CRS的效果。CD4+T细胞中的IL-6传讯减少(NES=-2.41,q值=0.003)且以干扰素-β处理的支气管上皮细胞中的下调基因增加(NES=1.62,q值<0.25),其表明了处理Virofree可减少IL-6并增加干扰素-β分泌(表1)。通过使用ARDS特征的GSEA分析,亦检查Virofree对ARDS的效果。ARDS特征系由源自ARDS患者(GSE76293)的血液多核嗜中性球(PMN)中的显著上调/下调的基因达成。阳性NES(NES=1.87,q值=0.01)表明Virofree可增进ARDS患者的下调基因(表1,图1A)。GSEA分析显示,Virofree可通过通过抑制IL-6及激活干扰素-β传讯途径以减少CRS而逆转COVID-19特征,以减少ARDS相关联的COVID-19。
2.大数据分析表明铁依赖型细胞死亡与miRNA为Virofree治疗COVID-19的潜在标靶。
通过药物处理基因表达剖析,可显示出Virofree的潜在作用机制。针对处理的转录反应,剖析了BEAS-2B细胞中低剂量(66.67μg/ml)的Virofree处理。处理低剂量Virofree会产生6个上调基因与8个下调基因(表2)。KEGG富集显示,Virofree功能与癌症的铁依赖型细胞死亡、HIF-1传讯途径、类固醇代谢及miRNA相关(图1B)。此外,GO显示了介导铁离子恒定性、固醇代谢过程、病毒受体活性及格形蛋白涂覆凹窝(clathrin-coated pit)的30个主要生物途径(图1C)。Virofree总转录本反应概貌经由DisGeNET数据库与疾病特征而相关联,其中前10个相关疾病包括铁代谢疾病、气道阻塞、肺动脉高血压及特发性肺高血压(图1D)。GO结果显示,高剂量Virofree(500μg/ml)证实microRNA调节(图9A),而KEGG柱形图显示治疗与转形生长因子-β的反应和氧含量的反应(图9B)相关联。一般而言,其表明了Virofree介导的细胞铁离子恒定性、miRNA及病毒感染传讯,以抑制COVID-19症状。
表2:高剂量与低剂量Virofree处理反应概貌的显著上调与下调基因。低剂量(66.67μg/ml)显示6个上调与8个下调的显著差异表达基因。表中列出了高剂量(500μg/ml)的前10个DEG。
DEG:差异表达基因。
已比较了两种不同Virofree剂量的治疗对基因表达量及相关疾病的效果(表3与表4)。在两种药物浓度下,2个DEG上调且4个DEG下调;然而,当Virofree的剂量增加时,STC2为唯一表达量发生显著变化的基因(图9C)。通过较高剂量的Virofree,约700个基因被诱发或被抑制,且这些DEG在较低剂量的Virofree下未显示,意指对照组与处理组之间无差异。低剂量与高剂量的Virofree可干扰5种常见的相关疾病,分别为肺动脉高压、心肌缺血、肺高血压(原发性,1,遗传性出血性毛细血管扩张症)、特发性肺高血压及脂肪性肝炎(图9D)。
表3:多个数据库中的低剂量Virofree的预测途径与疾病的详细列表。
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表4:多个数据库中的高剂量Virofree的预测途径与疾病的详细列表。
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此外,进行PCA分析以比较低剂量与高剂量Virofree(图1E-F)的总转录本反应。PC1主要可描述高剂量处理的总转录本概貌,而PC2可描述低剂量处理的总转录本概貌(图1E)。各主成分(PC)上的基因负荷评分可证实该基因在PC中的分布,或换言之,该基因在样品中的表达模式。正负荷评分表示基因表达与PC呈正相关。在PC1的情况下,由于高剂量Virofree处理的表达概貌在阳性PC1中富集化,因此在PC1中具有正负荷评分的基因倾向于上调,类似于PC2与低剂量的Virofree处理反应概貌。负负荷评分表示负相关,因此,具有负负荷分数的基因可能被下调。更高幅度的(正或负)负荷评分证实对该PC的影响更大。两个PC的负荷评分分布如图1F所示。一般而言,与铁恒定性相关的基因(包括编码xCT的FTH1与SLC7A11)在两种Virofree剂量中皆上调,其表明预防铁依赖型细胞死亡的效果。有趣的是,相较于对照组,两种Virofree剂量的IL6亦下调,表明Virofree在阻断IL-6传讯途径的具有前景的效果,以抑制细胞激素风暴。此外,纤维接合素(FN1)表达量可能会随着高剂量Virofree处理而降低,表明了对纤维化的抑制效果。这些基因表达的改变在后续实验中得到验证。
3.Virofree显著上调miRNA标靶以影响SARS-CoV-2
由于上调let-7a-5p与miR-148b-5p的表达可能对治疗COVID-19有益[33],因此研究了Virofree对这些miRNA的表达的效果。此外,在PC1的PCA分析中具有正负荷评分的前100个基因在含有miRTarBase let-7a-5p标靶的基因集中亦显示显著富集(p值=0.002;利用CPDB进行分析)。
首先,通过在96孔培养盘中以不同浓度处理BEAS-2B细胞24小时并使用SRB试验评估细胞存活力,以测定Virofree的最高安全剂量。处理的细胞维持超过80%的存活率(图10)。qRT-PCR结果显示,低剂量的Virofree(33.3μg/ml)可显著增加3.71倍的miR-148b-5p表达(图2A),而高剂量的Virofree(333μg/ml)可增加1.63倍的let-7a-5p表达(图11)。这些结果表明,Virofree可以剂量非依赖性方式上调标靶的miRNA的表达。
4.Virofree有效抑制SARS-CoV-2 M pro的切割
通过位于nsp5上的M pro对SARS-CoV-2多蛋白pp1a与pp1ab进行蛋白水解切割,可释出nsp5-16与nsp4的羧基(C)端,其功能为病毒复制所需[34]。SARS-CoV-2M pro为针对COVID-19的治疗斡旋的有前景标靶。因此,含有介于nsp4与nsp5之间切割位点的胜肽分别在N端与C端处以萤光团(Abz)及其淬灭体(Dnp)进行标记,以测量SARS-CoV-2M pro的蛋白酶活性。检查重组SARS-CoV-2M pro活性的抑制,其是在使用萤光探针的蛋白酶活性试验中以Virofree作为抑制剂。结果显示,Virofree可抑制M pro活性,其中IC 50为19.71±43.94μg/ml(图2B),其表明该药物可抑制SARS-CoV-2M pro活性。
5.Virofree可抑制巨噬细胞中的细胞激素风暴
为了量化TNF-α的释放量,其为血浆急性期COVID-19患者中检测到的最丰富的细胞激素之一[35],在处理Virofree后6或24小时收集细胞培养基以进行ELISA。将单独处理LPS刺激剂视为对照组。Virofree在LPS存在下显示出其抑制TNF-α的潜力。较高剂量的Virofree在两个时间点皆证实有更好的抑制效果,而较低剂量仅能在6小时的时间点略微降低TNF-α分泌含量(图2C)。这些数据表明,Virofree可通过抑制TNF-α释放而部分减少细胞激素风暴。
6.Virofree中断三聚体SARS-CoV-2棘蛋白野生型(原始株)或变体(α、β、γ、δ及o)与生物素化人类ACE2重组蛋白的结合
为了研究Virofree对SARS-CoV-2 S蛋白与ACE2之间的结合是否具有直接抑制效果,进行了体外生化学结合ELISA试验,其使用重组SARS-CoV-2 S蛋白与ACE2,并检查Virofree在1、2、4及8mg/ml剂量下的抑制效果。Virofree抑制了大约25%-50%的所有五种三聚体棘蛋白病毒株与ACE2的结合效率(图3A-F)。其中,Virofree似乎对δ与ο病毒株的棘蛋白比对其他的更有效(图3E与F)。亦观察到Virofree对δ变体棘蛋白的RBD的抑制活性与对三聚体形式具有类似效力(图12)。野生型棘RBD抗体(10μg/ml)似乎对ο变体棘蛋白无效。这些发现表明,Virofree可直接破坏ACE2与源自多种变体(包括ο)的SARS-CoV-2 S蛋白的交互作用。
7.Virofree经由基于细胞的试验抑制SARS-CoV-2棘蛋白与ACE2受体的结合及其合胞体的形成。
在感染细胞膜上表达的病毒蛋白可与相邻幼稚细胞上的受体交互作用,造成细胞-细胞融合及合胞体形成[36]。受体依赖性合胞体的形成系通过细胞膜中的SARS-CoV-2棘(S)蛋白触发[37-39]。因此,为了进一步阐明Virofree的抗SARS-CoV-2活性,测量Virofree对BHK-21细胞(其中共表达野生型或δ-变体SARS-CoV-2棘蛋白与EGFP)与Calu-3细胞(具有内源性hACE2受体表达)之间结合效率与融合效率的作用。BHK-21细胞与Calu-3细胞的结合表明SARS-CoV-2 S蛋白与ACE2受体的交互作用。此外,合胞体形成系由BHK-21与Calu-3细胞之间的膜融合引起。
为了证实Virofree对BHK-21与Calu-3细胞的细胞毒性,两种细胞处理不同浓度(0.2至2.0mg/ml)的Virofree。处理Triton-100以作为阳性对照组,而对照组代表非处理组。LDH试验清楚显示,相较于对照组,处理Virofree未导致显著细胞死亡,表明Virofree对BHK-21细胞或Calu-3细胞无细胞毒性(图4A)。在图4B中,相较于对照组,在各种Virofree处理组中,BHK-21细胞与Calu-3细胞的结合效率无显著差异。在培养4小时后,在对照组中形成具有扩展的绿色萤光信号的多核细胞,表明棘介导的合胞体形成。
此外,通过处理Virofree,野生型棘介导的合胞体形成呈现剂量依赖性显著降低。尽管SARS-CoV-2棘δ变体在BHK-21细胞中表达,但相比之下,处理Virofree不仅导致BHK-21细胞与Calu-3细胞的结合减少,还显著抑制合胞体形成(图4C)。西方墨点试验显示,处理Virofree剂量依赖性降低ACE2与TMPRSS2表达(图4D)。通过基于细胞的假病毒中和试验进一步证实Virofree的融合-阻断作用。结果显示,Virofree可抑制具有野生型(图4E)、δ(图4F)或ο(图4G)的SARS-CoV-2棘的假病毒的进入,其中IC 50分别为46.50、57.71及42.40μg/ml。
8.Virofree防止巨噬细胞发生氧化介导的毒性及铁依赖型细胞死亡。
为了研究处理Virofree的THP-1-衍生的巨噬细胞中参与铁恒定性的蛋白表达,在THP-1巨噬细胞与不同浓度的Virofree培养后收集细胞,接着通过西方墨点试验检测蛋白表达量。在以Virofree治疗后,THP-1巨噬细胞中的FTH1与xCT的表达呈现剂量依赖性增加(图5A)(图13)。结果表明,Virofree可具有通过增加xCT与FTH1的表达量而增进预防巨噬细胞中的铁依赖型细胞死亡的潜力。Virofree针对铁依赖型细胞死亡的保护作用与机制值得进一步研究。预处理N-乙醯基半胱胺酸(NAC)可显著降低xCT表达,其表明升高的ROS含量可诱发xCT表达以保护细胞(图14)。为了进一步确定Virofree是否可作为铁依赖型细胞死亡抑制剂,使用爱拉斯汀,并观察Virofree是否可挽救爱拉斯汀在细胞中诱发的铁依赖型细胞死亡。根据西方墨点结果,Virofree可挽救运铁素(ferroportin(FPN))与抗氧化酶GPX4(图5B)下调的表达量,其表明Virofree可预防THP-1巨噬细胞中的爱拉斯汀诱发的铁依赖型细胞死亡。
9.Virofree可抑制TGF-β1诱发的α-SMA与ECM蛋白的表达。
COVID-19感染可导致ARDS,并可能导致肺纤维化及终生后遗症[40]。TGF-β1在纤维性肺病的发病机制中扮演重要角色,其系通过促进纤维母细胞分化成肌纤维母细胞[41],并刺激ECM成分的合成,最终导致异常纤维化[42]。为了证实Virofree对纤维化的抑制效果,LL29细胞以TGF-β1与Virofree处理48小时。结果表明,Virofree可以剂量依赖性方式显著抑制TGF-β1诱发的α-SMA蛋白表达。此外,通过以Virofree 1,000μg/ml处理后,N-钙黏蛋白表达量显著降低。此外,发明人观察到LL29细胞以Virofree1,000μg/ml处理后,TGF-β1诱发的纤维接合素蛋白表达量降低(图6、图9)。这些数据表明Virofree对TGF-β1诱发的纤维化具有抑制效果。
总之,Virofree呈现出靶向SARS-CoV-2的多种功能。Virofree(1)通过中断不同亚型的棘与ACE2之间的蛋白结合以及降低ACE2和TMPRSS2蛋白表达而阻断病毒进入,(2)通过增加miR-148b含量及抑制M pro活性而减少病毒复制,(3)通过抑制LPS诱发的TNF-α产生而减少细胞激素风暴,(4)预防THP-1巨噬细胞的铁依赖型细胞死亡,以及(5)体外减少TGF-β1诱发的纤维化。发明人的研究表明,Virofree为一用于进一步的COVID-19临床前与临床研究δ与ο变体的出现及其等的传播的有前景草本药剂(图7)。
讨论
截至2021年9月30日,FDA已公布了640多个处于规划阶段的药物开发项目,470多个经过审查的试验,11个治疗授权紧急使用,以及仅1个批准的治疗。瑞德西韦(Remdesivir)为首个也是唯一被批准靶向抗病毒复制的药物。数个较大的世代研究收集了有关瑞德西韦结果的观察数据,报导了在疾病早期阶段的死亡风险较低及临床改善[43,44],但并非所有住院患者的总体死亡率有获益,且COVID-19重症患者的反应无显著差异[45]。其他治疗策略为免疫调节剂(巴瑞克替尼(JAK抑制剂)、托珠单抗(IL-6受体阻断剂))及棘蛋白单株抗体(如卡瑞单抗(Casirivimab)/依德单抗(Imdevimab)、巴尼单抗(Bamlanivimab)、埃特司韦单抗(Etesevimab)),其对一些患者的治疗效果存在局限[46]。更不用说正在进行的临床试验,洛匹那韦(lopinavir)-利托那韦(ritonavir)、静脉注射利巴韦林(ribavirin)、乌米芬韦(umifenovir)、皮质类固醇或干扰素-α-2b并未报告一致的临床益处[47]。最近,ο已出现在COVID-19的大流行中,其具有更高的传播性及病毒结合亲和力。重要的是,大多数其他ο突变的影响仍未知,导致缺失与突变的整个组合对病毒行为及对自然和疫苗介导的免疫易感性的影响的高度不确定性。再感染病例的增加与ο之中存在的免疫逃脱突变一致[48]。截至目前,仅尼马瑞韦(Nirmatrelvir)在体外对ο有效[49]。根据先前的出版品,SARS-CoV-2感染的病理过程始于病毒棘蛋白与人类ACE2受体的结合,最终进入细胞、复制、形成新的。随后,蛋白酶被切割成两个小型多胜肽片段(pp1a与pp1b)以进行病毒复制与转录[50,51]。随后,通过冠状病毒主要蛋白酶(M pro)的蛋白水解,包装新病毒颗粒的成分并释放功能性病毒多胜肽[51,52]。这些后面的因素触发细胞激素含量并使铁浓度失调,其导致铁依赖型细胞死亡及胎儿ARDS症候群。一般而言,控制SARS-CoV-2致病性的机制不止一种,目前的治疗皆针对单一生物学功能,其将忽略其他病毒致病的信号。因此,有必要发现一完全阻断SARS-CoV-2感染的具有多种功能的新颖药物。
大数据分析通过将药物反应概貌与疾病特征和作用机制连结,为药物开发提供一系统性及全面性的方法。经由GSEA针对COVID-19特征的化合物参考概貌的大规模筛选表明,Virofree(一种安全且健康的草本药剂)为潜在的候选药物之一(图1A)。富集分析显示,Virofree可针对多个COVID-19治疗标靶。最高排名的KEGG与GO富集途径包括铁离子恒定性、铁离子结合及铁依赖型细胞死亡(图1B-D)。在几项流行研究中,COVID-19患者具有异常低的血清铁含量(<7.8μmol/L)与高铁蛋白血症(hyperferritinemia)[53,54],其中铁失调与超过负荷可刺激ROS生成,以损害器官并导致发炎激活。HIF-1(缺氧诱导因子-1α)传讯途径在病毒感染与促炎反应中扮演重要角色[55,56],在处理Virofree的基因表达概貌中显著富集化(图1B),表明药物在此途径上的作用以抑制IL-6产生,其与PCA负荷评分一致(图1F),且最终抑制细胞激素风暴。GO富集表明Virofree可介导格形蛋白涂覆凹窝[57]及病毒受体活性,其与病毒-人类进入交互作用有关[58]。相比之下,经由DO的疾病关联性显示许多COVID-19症状或疾病在Virofree治疗的上限范围内,包括铁代谢疾病、肺动脉高血压、特发性肺高血压、气道阻塞及普通感冒。一般而言,Virofree可通过不同机制调节SARS-CoV-2的综合病理机制,且发明人的结果与先前的研究和临床证据一致。最近,探索了不同的SARS-CoV-2变体,使得治疗更困难且复杂。可进一步分析Virofree反应概貌及这些变体特征,以确定适合的治疗方案。
实验验证证明,Virofree增加BEAS-2B细胞株(正常支气管上皮细胞)中let-7a-5p与miR-148b-5p的表达。let-7家族减弱病毒感染的多样性,例如,流感病毒的毒力中断,其与肺炎和ARDS症状相关联[33]。Sardar等人[59]提出let-7a靶向SARS-CoV-2的非结构蛋白,且Chauhan等人[60]报导其他家族成员可调节TMPRSS2。作为免疫调节剂,let-7a可结合至TLR4 mRNA股以阻断下游MyD88与NF-κB传讯,包括驱动先天免疫和促炎反应(TNF-α激活)的IL-6[61]。同时,miR-148a(TLR3的标靶miRNA)在促炎反应中扮演另一种调节剂的角色,其亦调节IKK/NF-κB途径、MyD88及细胞激素(IFN-α、IFN-β、IL-6、IL-8)[62]。通过生物资讯学,一些研究预测miR-148a为靶向SARS-CoV-2基因体的高潜力候选者[63,64]。尽管miR-148b-5p、let-7a-5p及细胞激素产生之间的调节并不详尽,但是Virofree刺激了这两种miRNA的表达量(图2A与图11)并降低TNF-α(图2C)。在上述的证据中,Virofree证实了抑制SARS-CoV-2病理机制的多种功能(miRNA与细胞激素)。
此外,Virofree显示出两个额外的功能性质,包括抑制M pro以阻止病毒复制及中断SARS-COV-2 S蛋白结合至ACE2以阻断病毒进入。SARS-CoV-2复制基因编码两个重叠性多蛋白pp1a与pp1ab,其等为病毒复制与转录所需。通过蛋白水解过程,主要通过M pro(也称为3C样蛋白酶),从多蛋白释出功能性多胜肽。M pro在11个保守性位点处消化多蛋白。由于M pro在病毒生命周期中的功能重要性以及在人体中缺乏密切相关的同源物[65-67],使得M pro被视为抗病毒药物设计的诱人目标。酵素试验显示,Virofree可抑制M pro活性,其IC50为19.71±43.94μg/ml(图2B)。
基于ELISA的三聚体棘-ACE2结合试验证实,Virofree可阻断ACE2与数个棘蛋白(包括野生型、α、β、γ、δ及o变体)之间的结合(图3)。此外,结果亦表明,Virofree针对棘蛋白的抑制效果可能与棘蛋白的RBD有关。在这些变体棘蛋白之中,δ与ο变体显示出Virofree最敏感的抑制作用(图3E、F)。
未观察到野生型棘RBD抗体针对ο变体棘蛋白的抑制活性。此观察结果与最近的报告一致,其显示COVID疫苗预防ο的作用较弱[68]。因此,相较于靶标特异性RBD抗体,Virofree似乎对衍生自各种变体的棘蛋白呈现出更广效。细胞-细胞融合试验的结果显示,通过处理Virofree,可抑制δ棘与ACE2的结合(图4C),其与基于ELISA的三聚体棘-ACE2结合试验的结果一致(图3E)。
此外,SARS-CoV-2棘介导的合胞体形成可能意指病毒诱发的细胞融合促进病毒基因体递输至邻近细胞[69]。在检查已故COVID-19患者的组织病理学肺切片时,观察到普遍存在含有2-20个核的合胞体细胞,其表明合胞体形成与严重致病性之间的相关性。基于这些观察结果,使用棘介导的细胞融合以验证针对SARS-CoV-2感染的药物可为一有效策略[70]。进行野生型与δ棘蛋白的时程实验(图15)。相较于野生型,δ病毒株在初始结合步骤中显示出更高的结合能力,并在后续的时间点中显示出更快的融合动力学。δ变体在2小时内几乎完全融合;相比之下,野生型的细胞融合百分比仍相当低。因此,发明人已说明了δ变体在2小时而非4小时的时间点的融合结果(图4B)。
细胞-细胞融合试验的结果阐明了处理Virofree对棘介导的细胞-细胞融合及假病毒感染的抑制作用(图4B、4C及4E),其意味着Virofree不仅可中断变体棘与ACE2的结合,还可影响宿主蛋白酶参与的SARS-CoV-2膜融合。在ELISA与细胞-细胞融合试验中,相较于野生型S蛋白,δ变体对处理Virofree的敏感性更高。由于δ病毒株的棘介导的合胞体形成比其他SARS-CoV-2变体的更强[71],因此可敏感地抑制δ棘与ACE2的结合的Virofree将成为针对δ变体感染的有效药物。此外,西方墨点试验显示,处理Virofree可以剂量依赖性方式降低TMPRSS2表达(图4D),其表明Virofree对SARS-CoV-2感染的抑制可能是由于TMPRSS2的表达降低,其可切割SARS-CoV-2 S蛋白以触发SARS-CoV-2与宿主细胞的膜融合而促进病毒进入[72]。总之,这些发现表明,Virofree可通过两种不同机制抑制病毒结合与进入:抑制ACE2与TMPRSS2表达,以及直接中断SARS-CoV-2 S蛋白结合至ACE2。
此外,最近的研究亦显示棘蛋白的病理角色不仅在于促进肺血管重塑与血管内皮细胞功能障碍,还在于导致肺动脉高血压[73]。ACE2作用为响应棘蛋白刺激的受体[74]。因此,Virofree抑制棘蛋白与ACE2的结合除了预防病毒感染之外,亦可有助于肺保护。
此外,如图2所示,抑制病毒复制与细胞激素风暴似乎是细胞内事件。此外,中断棘蛋白/ACE2交互作用(图3)与减弱病毒感染(图4)为细胞外事件。发明人推测,此种标靶成分的差异可能是由于抑制病毒复制、细胞激素风暴及减弱病毒感染的所需剂量的差异所致。
COVID-19患者的发炎状况亦与铁代谢失调相关联[75]。然而,细胞内过量的铁可促进ROS产生。系统xc–,胱胺酸/麸胺酸反向运输蛋白(含有SLC7A11与SLC3A2次单元的跨膜蛋白复合体),由于谷胱甘肽合成而增加,以抑制细胞ROS产生[76]。细胞内铁,其通过摄取与代谢而保持相对平衡,可通过FPN而输出细胞外或经由铁蛋白以Fe(III)储存,以防止细胞内由过量游离铁引起的氧化压力。SARS-CoV-2感染会促进细胞中的铁累积,从而促进铁依赖型细胞死亡且最终导致器官衰竭。还有证据表明,SARS-CoV-2可通过特定机制[78]而感染参与铁代谢与发炎反应的巨噬细胞[77],其表明巨噬细胞为有吸引力的治疗标靶。实验结果显示,Virofree可增进巨噬细胞中的FTH1与xCT(SLC7A11)的表达量,并可逆转由处理爱拉斯汀(铁依赖型细胞死亡诱导剂)引起的FPN与GPX4表达量下调[79],其证实了Virofree防止巨噬细胞发生铁依赖型细胞死亡的潜力。另一发现为,根据生物资讯学分析及实验结果,Virofree下调细胞中的TFRC表达量(图5)。
在一项研究中亦报导了TFRC直接与SARS-CoV-2棘蛋白交互作用而介导病毒进入,亦表明了TFRC为SARS-CoV-2进入细胞的替代受体[80]。目前,未有ACE2抑制剂对COVID-19患者有益,并强调TFRC为有前景的抗COVID-19标靶。
COVID-19重症患者中的高血清含量的发炎性细胞激素和升高的铁蛋白量与疾病严重程度、发炎及铁离子代谢紊乱相关[81,82]。严重SARS-CoV-2感染的主要症状包括严重肺炎与ARDS。在SARS-CoV-2感染后发现肺损伤,包括广泛间质性与肺泡性发炎性浸润、肺泡中隔增厚、血管充血及肺水肿,其在一些患者中可能与不可逆的肺纤维化的发展相关联[83]。先前的世代研究亦表明,在SARS-CoV-2感染后有将近87%的COVID-19患者出现肺纤维化[84]。TGF-β1在纤维性肺病的致病性中扮演重要角色,其系通过促进纤维母细胞分化成肌纤维母细胞[41],并刺激ECM成分的合成,最终导致异常纤维化[42]。TGF-β1诱发的纤维化LL29细胞以Virofree处理48小时表明,Virofree可显著抑制TGF-β1诱发的α-SMA与N-钙黏蛋白的蛋白表达,并显著减少纤维接合素(图6、图16)。
为了更好地模拟患者的状况,使用BLM诱发的ARDS大鼠模型以研究Virofree的效果。发明人的初步观察显示,处理Virofree可通过增加动脉血氧饱和度及降低呼吸频率而改进ARDS大鼠的状况(图17)。由于高含量的TNF-α参与了ARDS的进展,因此抗TNF-α已被视为此疾病的关键疗法之一[85]。在初步体内结果中,Virofree能减少TNF-α分泌量、体外纤维化相关的蛋白表达(图6、图16),并改进了ARDS大鼠的生理指标,其表明Virofree为潜在的抗COVID-19与抗纤维化治疗。需要对其他大鼠进行进一步的研究,以确认其治疗ARDS的机制。
最近,有数种治疗COVID-19的治疗药物已获批紧急使用授权,例如尼马瑞韦/利托那韦(Pfizer)或莫纳皮拉韦(Molnupiravir)(Merck)。然而,其等仅可分别靶向病毒M pro或破坏RNA复制。由于突变病毒株的出现比药物开发速度更快,因此有必要发现及发展广效药物,以靶向由SARS-CoV-2引起的多种危险因子。
促炎细胞激素通过诱导型一氧化氮合成酶(iNOS)而诱导大量的一氧化氮(NO)形成,而抑制NO产生的化合物具有抗发炎效果。Virofree中的一些成分包括类黄酮,例如槲皮素、橙皮苷、金雀异黄酮、大豆异黄酮苷素及白藜芦醇,其等引发强的抗氧化与抗发炎效果[13-15]。据报导,槲皮素、金雀异黄酮及大豆异黄酮苷素可抑制iNOS蛋白和NO产生。这些成分亦能抑制NF-kB的激活,NF-kB系由TNF-α激活[86]并触发IL-6的激活[14]。此外,橙皮苷已证明对COVID-19有益,系因其免疫调节作用及抗病毒活性,造成抑制SARS-CoV-2M pro[87]。槲皮素为另一种对SARS-CoV-2M pro具有抗病毒效果的类黄酮[88]。因此,由具有抗发炎与抗病毒性质的类黄酮组成的Virofree可能具有作为针对COVID-19的候选草本药剂的潜力。
总之,数据表明,Virofree可靶向各种阶段的病毒进入与复制,尤其是针对δ与ο变体,以及下列后果,包括铁依赖型细胞死亡、细胞激素风暴、ARDS及肺纤维化(图7)。这些发现强调,在没有针对新变种的特异性与有效性药物的情况下,Virofree可作为多功能草本药剂而减少SARS-CoV-2感染并减轻感染后并发症的潜力。
虽本发明提供如上较佳实施例,但并非旨在限制本发明。任何本发明所属技术领域中具有通常知识者可在不脱离本发明的精神和范围的情况下,允许进行修改和修饰。因此,本发明的保护范围应依由随后所附的申请专利范围所定义。
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Claims (15)
1.一种治疗或预防一个体COVID-19及长期性COVID的方法,其包含给予该个体服用治疗有效量的一草本组合物,该草本组合物包含葡萄子萃取物、针叶樱桃萃取物、橄榄叶萃取物、万寿菊萃取物、绿茶萃取物、石榴萃取物、酵母Β-葡聚糖及大豆萃取物的混合物。
2.如权利要求1所述的方法,其中该草本组合物包含从植物萃取物分离的活性成分,其包括槲皮素、橙皮苷、金雀异黄酮、大豆异黄酮苷素及白藜芦醇。
3.如权利要求1所述的方法,其中该草本组合物分别包含10重量%至50重量%的葡萄子萃取物、5重量%至30重量%的针叶樱桃萃取物、5重量%至30重量%的橄榄叶萃取物、1重量%至20重量%的万寿菊萃取物、1重量%至20重量%的绿茶萃取物;1重量%至20重量%的石榴萃取物;1重量%至20重量%的酵母Β-葡聚糖;以及1重量%至20重量%的大豆萃取物,分别以该草本组合物的总重量为基准。
4.如权利要求1所述的方法,其中该草本组合物分别包含约250mg之葡萄子萃取物、约180mg的针叶樱桃萃取物、约160mg的橄榄叶萃取物、约90mg的万寿菊萃取物、约80mg的绿茶萃取物;约80mg的石榴萃取物;约80mg的酵母Β-葡聚糖;以及约80mg的大豆萃取物,分别以该草本组合物的总重量(1000mg)为基准。
5.如权利要求1所述的方法,其中该草本组合物在每次发生时独立地投予约500mg至约1500mg,每天约1至3次。
6.如权利要求1所述的方法,其中该草本组合物系配制成常见医药剂型,其包括丸剂、锭剂、胶囊剂、饮剂及糖浆,并与一医药上可接受载体组合。
7.如权利要求1所述的方法,其中该草本组合物系制备成膳食形式,其包括锭剂、胶囊剂、胶剂、粉剂、饮剂及能量棒。
8.一种用于治疗或预防一个体COVID-19及长期性COVID的草本组合物,其包含葡萄子萃取物、针叶樱桃萃取物、橄榄叶萃取物、万寿菊萃取物、绿茶萃取物、石榴萃取物、酵母Β-葡聚糖及大豆萃取物的混合物。
9.如权利要求8所述的草本组合物,其包含从植物萃取物分离的活性成分,其包括槲皮素、橙皮苷、金雀异黄酮、大豆异黄酮苷素及白藜芦醇。
10.如权利要求8所述的草本组合物,其中该草本组合物包含10重量%至50重量%的葡萄子萃取物、5重量%至30重量%的针叶樱桃萃取物、5重量%至30重量%的橄榄叶萃取物、1重量%至20重量%的万寿菊萃取物、1重量%至20重量%的绿茶萃取物;1重量%至20重量%的石榴萃取物;1重量%至20重量%的酵母Β-葡聚糖;以及1重量%至20重量%的大豆萃取物,分别以该草本组合物的总重量为基准。
11.如权利要求8所述的草本组合物,其中该草本组合物包含约250mg之葡萄子萃取物、约180mg的针叶樱桃萃取物、约160mg的橄榄叶萃取物、约90mg的万寿菊萃取物、约80mg的绿茶萃取物;约80mg的石榴萃取物;约80mg的酵母β-葡聚糖;以及约80mg的大豆萃取物,分别以该草本组合物的总重量(1000mg)为基准。
12.如权利要求8所述的草本组合物,其中该草本组合物在每次发生时独立地投予约500mg至约1500mg,每天约1至3次。
13.如权利要求8所述的草本组合物,其中该草本组合物系配制成常见医药剂型,其包括丸剂、锭剂、胶囊剂、饮剂及糖浆,并与一医药上可接受载体组合。
14.如权利要求8所述的草本组合物,其中该草本组合物系制备成膳食形式,其包括锭剂、胶囊剂、胶剂、粉剂、饮剂及能量棒。
15.一种如权利要求8-14中任一项所述的草本组合物用于制备治疗或预防COVID-19及长期性COVID的补充剂或组合物或医药组合物的用途。
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