CN116850189B - 普拉曲沙在制备治疗hsv所致疾病的药物中的用途 - Google Patents
普拉曲沙在制备治疗hsv所致疾病的药物中的用途 Download PDFInfo
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Abstract
本发明提供了普拉曲沙在制备治疗HSV所致疾病的药物中的用途以及相应方法和药物组合物；本发明的研究证明，普拉曲沙在极低的用量下对HSV‑1的抗病毒活性和安全性即优于传统抗病毒药物阿昔洛韦。
Description
发明领域
本发明属于抗病毒药物技术领域，具体地，本发明提供了普拉曲沙在制备治疗HSV所致疾病的药物中的用途以及相应方法和药物组合物。
背景技术
单纯疱疹病毒(Herpes simplex virus，HSV)属于人类疱疹病毒科α亚科，是一种有包膜的球形病毒。分单纯疱疹病毒Ⅰ型(Herpes simplex virusⅠ，HSV-1)和单纯疱疹病毒Ⅱ型(Herpes simplex virusⅡ，HSV-2)。都是致病性病毒，可引起疱疹性脑炎、疱疹性角膜炎、生殖器疱疹或新生儿脑炎，至今无药可根治，也没有疫苗预防感染。
单纯疱疹病毒性角膜炎(Herpes simplex keratitis，HSK)是由HSV-1引起的感染型眼角膜疾病，严重时致盲。人类是这种病毒的唯一天然宿主，自然状态下人类感染HSV-1的情况非常普遍，据统计，世界上50％-80％人是HSV-1的携带者，每年新增150万由感染了HSV病毒引起的角膜炎患者，其中4万患者视力损坏或失明。
人类是HSV-1病毒的唯一天然宿主，且被HSV-1感染的情况非常普遍。大多数人为携带者，一旦感染HSV-1，病毒将就终身潜伏在神经元中，在机体免疫力低下时可能受到HSV-1的攻击引发炎症，其中，HSV-1感染眼角膜引起病毒性角膜炎症状。HSV-1引起的HSK是世界上角膜瘢痕形成和角膜混浊致盲的主要原因。现如今，临床上治疗HSK的药物首选是阿昔洛韦(Acyclovir，ACV)、更昔洛韦(Ganciclovir)等类似的广谱性抗病毒药物，此外还有糖皮质激素、干扰素、环孢素等用于辅助治疗；目前尚在开发的此类药物主要是阿昔洛韦的脂质体、胶束等新剂型(药物治疗单纯疱疹病毒基质型角膜炎的研究进展，眼科学报，2022年，第37卷，第8期)。轻症患者初次使用抗病毒药物时时抗病毒效果很好，但只能短时抑制患病部位的HSV-1的复制，无法清除病毒，而且对于潜伏在神经中的病毒毫无对策，从而疾病复发。此外，患者长期使用广谱性抗病毒药物易产生耐药性的副作用，所以影响后续的疾病治疗效果。HSK重症患者需要通过角膜移植手术恢复视力，但是角膜供体资源少，不能解决所有患者的移植需求，另外，角膜移植后也有疾病复发的可能。所以急需寻找新的化合物和新的治疗靶点来解决以上问题。
抗叶酸类药物不仅是一类广谱、高效、低毒的化疗药物，也具有抵抗细菌和寄生虫感染的作用，被广泛应用于临床疾病的治疗。2009年，第四代抗叶酸剂普拉曲沙(Folotyn)被FDA批准在美国上市，用于治疗急性淋巴细胞白血病。进一步地研究表明普拉曲沙对新冠病毒和流感病毒也有抑制效果。但目前国内外没有关于普拉曲沙抑制HSV-1或HSV-2的研究报道，也没有普拉曲沙在制备预防和/或治疗单纯疱疹病毒感染的疾病的药物中的应用与报道，此外也没有普拉曲沙抑制阿昔洛韦耐药单纯疱疹病毒株感染的应用与报道。
发明内容
针对上述问题，为了扩展普拉曲沙的用途，为HSV治疗提供新的选择，一方面，本发明提供了普拉曲沙在制备治疗HSV所致疾病的药物中的用途。
进一步地，所述HSV为HSV-1。
进一步地，普拉曲沙抑制HSV的复制。
进一步地，所述HSV所致疾病为角膜炎、龈口炎、脑炎、生殖系统感染、新生儿疱疹或者HSV感染所致宫颈癌。
另一方面，本申请提供了治疗HSV所致疾病的药物，所述药物中包括普拉曲沙。
进一步地，所述药物中普拉曲沙为唯一有效成分。
进一步地，所述药物为口服或注射剂，优选注射剂。
进一步地，所述药物为外用制剂，优选滴眼剂、膏剂、霜剂、洗液、凝胶剂。
进一步地，所述药物包含药学上可接受的辅料。
另一方面，本发明提供了非治疗性抑制HSV增殖或者抑制HSV感染的方法，所述方法包括施用普拉曲沙或者包含普拉曲沙的药物的步骤。
本发明中的名词普拉曲沙，可以与Folotyn、Pralatrexate/PDX交替使用，表示同一含义，即CAS号为146464-95-1的化合物。
本发明中所述非治疗性方法，包括但不限于用于科研的方法以及用于抑制非致病性隐形感染的方法。
除普拉曲沙外，本发明的药物中不排斥其他治疗HSV感染的药物，这些药物包括但不限于阿昔洛韦。
本领域技术人员可以根据具体疾病的需要和药剂领域的技术情况为普拉曲沙选用适合的已知或研究中的剂型，这些剂型不限于上述剂型。剂型中可以常规选用已知或研究中的辅料，包括但不限于溶剂、助溶剂、稳定剂、pH调节剂、包衣剂、填充剂、抗氧化剂、防腐剂等。
普拉曲沙(Folotyn)在极低的用量下对HSV-1的抗病毒活性和安全性优于传统抗病毒药物阿昔洛韦(ACV)。在HFF细胞中，Folotyn显著抑制HSV-1F strain复制，IC50＝10nM，CC50>1000nM，SI＞100。因此，Folotyn对HSV-1的抗病毒活性非常出色，且在治疗剂量下安全性高，适宜用作抗病毒剂。
附图说明
图1为PDX抑制HSV-1活性和复制的效果。
图2为PDX子代病毒增殖的效果。
图3为PDX和ACV对HSV-1/153的IC50测定。
图4为PDX和ACV抑制HSV-1/153gD-1蛋白表达的效果。
图5为PDX和ACV对HSV-1/blue的IC50测定。
图6为PDX和ACV抑制HSV-1/blue gD-1蛋白表达的效果。
图7为PDX在角膜上抑制HSV-1感染实验中实验鼠体重变化。
图8为PDX在角膜上抑制HSV-1感染实验中实验临床症状评分。
图9为PDX在角膜上抑制HSV-1感染实验中实验鼠角膜表面损伤情况照片。
图10为PDX在角膜上抑制HSV-1感染实验中实验鼠荧光素染色数据。
图11为PDX在角膜上抑制HSV-1感染实验中实验鼠角膜完整性和厚度观察。
图12为PDX在角膜上抑制HSV-1感染实验中实验鼠泪液中子代病毒感染情况。
图13为Folotyn抑制巨细胞病毒增殖的效果。
具体实施方式
下面结合具体实施例详述本发明。以下实施例仅做展示用，本发明的保护范围由权利要求限定，不局限于以下实施例。
实施例1Folotyn浓度依赖性抑制HSV-1复制
指数增长的HFF细胞接种于96孔细胞培养板中，使得每个孔中含有1×104个细胞，并在1 00uL 10％ FBS的DMEM培养基中培养。培养条件为37℃恒温、恒湿及5％ CO2。24小时后，将-80℃保存的HSV-1McKrae病毒颗粒于冰上解冻，并按MOI＝1的滴度感染HFF细胞。2小时后采用DMSO稀释Folotyn储存液，使其在细胞培养基中的浓度为0nM、0.46nM、1.37nM、4.12nM、12.35nM、37.04nM,111.11nM,333.33nM和1000nM，且保证DMSO在培养基中的含量低于1％。继续培养48小时，采用TRizol裂解细胞收获RNA。采用qPCR方法检测HSV-1的gD-1的mRNA表达的情况。发现FOLOTYN处理能够显著抑制HSV-1的复制(图1)。TCID50方法测上清子代病毒增殖情况，在Vero细胞上测试TCID50，发现Folotyn在4.12nM,12.35nM和37.04nM可以显著降低子代病毒的增殖(图2)。
此外，申请人尝试了Folotyn抑制疱疹病毒科人巨细胞病毒(HCMV)的效果：HFF(人包皮成纤维细胞被巨细胞病毒感染后，加入1uM普拉曲沙处理感染细胞，并未见显著抑制巨细胞病毒编码基因IE1，UL44和pp150表达(如图13所示)。结果显示：1uM普拉曲沙处理会促进巨细胞病毒编码基因IE1和UL44表达，且对pp150表达不影响。说明普拉曲沙无法干预巨细胞病毒增殖。此外，如果加入1um普拉曲沙处理HSV-1/153和HSV-/blue感染的细胞，可以显著抑制HSV-1病毒蛋白gD-1表达，效果优于1uM ACV(如图4和6)。
实施例2Folotyn体外抗耐药病毒HSV-1/153和HSV-1/blue感染效果优于ACV
向HSV-1/153感染的HFF细胞中分别添加不同浓度的Folotyn(0～1000nM)或ACV(1000nM)进行干预。采用CCK8方法，噬斑方法和WB方法观察Folotyn和ACV处理对HSV-1/153gD-1mRNA和gD-1蛋白的影响，结果发现Folotyn的半数毒性浓度(CC50)为大于1000nM，ACV的CC50＞1000nM(图3)。Folotyn对HSV-1/153的半数抑制浓度(IC50)为37nM，而ACV对HSV-1/153的IC50为＞1000nM(图3)。1000nM Folotyn抑制HSV-1/153gD-1蛋白表达的效果优于ACV(图4)。
向HSV-1/blue感染的HFF细胞中分别添加不同浓度的Folotyn(0～1000nM)或ACV(1000nM)进行干预。采用CCK8方法，噬斑方法和WB方法观察Folotyn和ACV处理对HSV-1/blue抗病毒效果和gD-1蛋白的影响，结果发现Folotyn的半数毒性浓度(CC50)为大于1000nM，ACV的CC50＞1000nM(图5)。FOLOTYN对HSV-1/blue的半数抑制浓度(IC50)为10.08nM，而ACV对HSV-1/blue的IC50为＞1000nM(图5)。1000nM Folotyn抑制HSV-1/bluegD-1蛋白表达的效果优于ACV(图6)。
实施例3Folotyn在体内阻碍HSV-1感染
选取6周龄雄性BALB/C小鼠，采用戊巴比妥钠进行腹腔注射麻醉后，充分暴露右眼。在体式显微镜下用1mL注射器针头在角膜上做“井”字划痕。然后立即感染HSV-1McKrae(1×107PFU)，并通过轻柔上下眼睑辅助HSV-1感染。4小时后，采用Folotyn(溶解于10％DMSO+40％ PEG300+5％ Tween80+45％ PBS)1μg/小鼠，结膜下注射治疗。每隔1日1次，连续3次。从第0天到第7天收集并记录小鼠的体重信息(图7)。从第0天到第7天收集并记录小鼠临床症状疾病打分(图8)。于第7天用钴蓝光进行角膜荧光素染色来确定角膜表面的损伤情况(图9和图10)，用光学相关断层扫描(OCT)来观察角膜的完整性和厚度(图11)。用TCID50测定小鼠泪液中子代病毒增殖的情况，结果发现，Folotyn治疗可以显著抑制HSV-1在小鼠角膜中的感染(图12)。
Claims (8)
1.普拉曲沙在制备治疗单纯疱疹病毒Ⅰ型所致疾病的药物中的用途，所述单纯疱疹病毒Ⅰ型所致疾病为角膜炎。
2.根据权利要求1所述的用途，其中普拉曲沙抑制HSV的复制。
3.根据权利要求1所述的用途，其中所述药物中普拉曲沙为唯一有效成分。
4.根据权利要求1所述的用途，其中所述药物为口服或注射剂。
5.根据权利要求4所述的用途，其中所述药物为注射剂。
6.根据权利要求1所述的用途，其中所述药物为外用制剂。
7.根据权利要求6所述的用途，其中所述药物为滴眼剂、膏剂、霜剂、洗液、凝胶剂。
8.根据权利要求1-7任一项所述的用途，其中所述药物包含药学上可接受的辅料。
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