CN110075286A - 用于抗细胞内病原体的治疗的包含固体纳米颗粒和至少一种抗原的药物组合物 - Google Patents
用于抗细胞内病原体的治疗的包含固体纳米颗粒和至少一种抗原的药物组合物 Download PDFInfo
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Abstract
本发明涉及用于在患者中抗细胞内病原体的预防性治疗的药物组合物,所述组合物包含固体纳米颗粒和从所述病原体获得的至少一种抗原,其中所述固体纳米颗粒包含多孔阳离子‑多糖固体核心,该核心装载有至少一种阴离子磷脂并且所述核心不被任何磷脂层包围。
Description
本申请是中国发明专利申请(申请日:2013年9月13日;申请号: 201380048300.0(国际申请号:PCT/IB2013/002372);发明名称:用于抗细胞内病原体的治疗的包含固体纳米颗粒和至少一种抗原的药物组合物)的分案申请。
技术领域
本发明涉及药物组合物,其可用于预防性治疗由细胞内病原体等所导致的感染,所述病原体例如,病毒、细菌或寄生虫,且更具体是刚地弓形虫(Toxoplasma gondii)。
背景技术
刚地弓形虫(缩写为T.gondii)是弓形虫属中的一类寄生虫原虫。刚地弓形虫的终宿主(definitive host)是猫类,但是许多温血动物(鸟类、牛类、羊类还有人)可携带该寄生虫。刚地弓形虫的生命周期有两个阶段。生命周期的有性部分(球虫等)仅发生在猫类(无论是家猫还是野猫)中,这使得猫类成为该寄生虫的首要宿主(primary host)。第二阶段(生命周期的无性部分)可发生在其它温血动物中,该动物被称为中间宿主。
刚地弓形虫存在三种形式:
-速殖子,其始终在细胞内且在中间宿主的细胞中复制,所述速殖子形式不是污染物且当其在细胞外时可轻松地被破坏;
-缓殖子,其包含在囊肿中(主要是在中间宿主的免疫应答较低的肌肉和脑组织中);这种形式是经口污染物;
-子孢子,其包含在孢子形成的囊合子中,其为能够在中间宿主和终宿主外存活的形式;这种形式也是经口污染物。
弓形体病(刚地弓形虫是该疾病的致病因子)通常不严重且具自限性,但是却可能对胎儿(其母体在怀孕期间首次感染了该疾病)或者对免疫缺陷患者具有严重或甚至致命的影响。例如,在牛类和羊类中,弓形体病是流产的主要原因,因此会产生巨大的损失。
近来,已经有了这样的假定,认为弓形虫与精神分裂症具有一定程度的因果关系。这个假设依赖于弓形虫抗体的患病率以及精神分裂症发展之间的正相关。这项研究提供了在情绪和精神病症模型中弓形虫的累计行为影响值的一个实例(Yolken RH,Bachmann S,Rouslanova I,Lillehoj E,Ford G,Fuller Torrey et al.Antibodies to Toxoplasmagondii in individuals with first-episode schizophrenia.Clin.Infect.Dis.2001;32:842-44.)。
因此,本发明的一个目的是提供药物组合物,其能够提供疫苗为患者抵抗细胞内病原体,更具体是抵抗细胞内寄生虫(例如,原虫),更具体地是,刚地弓形虫。
发明内容
本发明涉及药物组合物,其包含作为活性成分的以下混合物:
-固体纳米颗粒,其包含多孔阳离子-多糖核心,该核心装载有至少一种阴离子磷脂并且在所述核心周围没有任何的磷脂层;
-从所述病原体(pathogenic agent)获得的至少一种抗原;和
-药学上可接受的溶剂。
更具体地,本发明涉及前述药物组合物,其用于在患者中抗细胞内病原体的预防性治疗或预防性治疗由细胞内病原体导致的疾病或感染。
根据本发明,术语“治疗”是指在疾病、病症或障碍的治疗或改善或预防中任何成功的标志,包括任何客观的或主观的参数,例如减缓、缓和、减轻症状或使患者对该疾病病症更加耐受,减缓恶化或衰退速度或者使得恶化的终点不那么使人虚弱。治疗或改善症状可基于客观的或主观的参数,包括医生的检查结果。
术语“治疗”还指患者体内的病原体数量的任何减少或是病原体的任何改变,即使是短暂的时间。当病原体具有多种形式(包括细胞外形式),病原体的至少一种形式的减少或改变均被认为是治疗效果。如果该病原体可能存在于患者体内的多个器官中,在至少一个器官中病原体数量的减少或病原体的改变均被认为是治疗效果。
因此,术语“治疗”包括给药本发明组合物以预防或推迟、减轻、或阻止或抑制与本文所述的疾病、病症或障碍有关的症状或病症的发展。术语“治疗性的”是指在受试者中至少部分减少、消除或预防该疾病、该疾病的症状或该疾病的副作用。
术语“预防性的”是指该治疗能够预防或推迟疾病的发作,或能够预防其临床表现或亚临床症状。
术语“药学上可接受的”是指在合理的医学判断范围内,适于与人和动物的组织接触而没有过度的毒性、刺激性、过敏反应或其他问题并发症的具有合理的益处/风险比的那些溶剂、化合物、原料、组合物或剂型。
根据本发明的一个方面,本发明涉及用作药物,尤其是用作使患者产生细胞免疫应答的免疫原性药物的前述组合物。但是,本发明不限于Th1免疫应答(细胞应答)。所述药物组合物还可诱导产生特异性抗体(体液应答)或诱导这两种应答。在本发明中,“免疫应答”是指由免疫系统、细胞免疫应答和 /或体液免疫应答引起的任何反应。这些反应包括生物体免疫系统响应抗原的活性的改变并且可包括,例如,抗体的产生、细胞介导的免疫的诱导、补体的激活或免疫耐受的发展。所述免疫应答可以是Th1应答或Th2应答或其混合。术语“免疫原性”是指直接或间接地诱导免疫应答的能力。
根据本发明,所述纳米颗粒核心装载有至少一种磷脂。因此,该固体核心是多孔的且其孔填充有磷脂或至少两种脂或磷脂的混合物。该纳米颗粒核心由阳离子多糖或至少两种阳离子-多糖的混合物构成。它还可由与所述阳离子多糖共聚或不与所述阳离子多糖共聚的任何其他化合物构成。
根据本发明,所述纳米颗粒核心未被任何磷脂层包围。磷脂层被定义为包含至少一种磷脂的层。
所述阳离子多糖可以是交联聚合物且可通过多糖与阳离子配体(例如季铵盐)之间的反应获得,所述多糖选自淀粉、葡聚糖、糊精和麦芽糖糊精。还可用伯胺、仲胺和叔胺。更具体地,所述阳离子多糖可由麦芽糖糊精和缩水甘油基三甲铵之间的反应获得。
所述阴离子磷脂可选自甘油磷脂且可以是,例如,二酰基磷脂酰甘油,例如二酰基磷脂酰丝氨酸、二酰基磷脂酰肌醇和二棕榈酰基磷脂酰甘油。
以下实验结果表明,至少就刚地弓形虫而言,本发明组合物诱导产生抵抗刚地弓形虫的特异性IFN-γ。就弓形虫病而言,已知的是,宿主抗性似乎是通过NK细胞和自适应T淋巴细胞合成IFN-γ而引发的。感染后,抗原呈递细胞合成TNF-α和IL-12,其诱导NK细胞分泌IFN-γ。IL-12和IFN-γ的联合作用诱导T辅助性前体(helper precursor)急剧分化成Th1淋巴细胞。然后这些CD4+T细胞合成大量的IFN-γ和IL-2。最终这两种细胞因子诱导CD8+T淋巴细胞的增殖和IFN-γ的分泌。因此,对刚地弓形虫感染的预防主要取决于细胞介导的免疫。因此,当母体已经用本发明组合物/疫苗治疗时,本发明组合物还能保护其胎儿。
根据本发明,所述细胞内病原体为细胞内病原体,其可以是病毒、细菌、分枝杆菌、真菌或寄生虫。将细胞内病原体定义为具有至少一种细胞内形式的任意微生物或大型生物。术语“致病的”是指能够引起患者或患者的子代 (progeny)或后代(offspring)的生理和/或心理上的变化的因素。
可引用以下作为细胞内病原体的实例:单纯疱疹病毒1和2、人乳头状瘤病毒、爱泼斯坦-巴尔病毒(Epstein-Barr virus)、巨细胞病毒、结核分枝杆菌、登革热病毒、HIV、人呼吸道合胞体病毒(RSV)、甲型肝炎病毒、乙型肝炎病毒和丙型肝炎病毒。
根据本发明,“寄生虫”是指从消耗所谓“宿主”的其他生物体中获益的任何微生物或大型生物。根据本发明,术语寄生虫包括大型寄生虫(代表性地有蠕虫)和微型寄生虫(代表性地有原虫),以及在它们生命周期过程中可能存在的所有形式。
当所述病原体为在其生命周期过程中具有细胞内形式的寄生虫时,则其可选自:顶复门细胞内寄生虫(apicomplexanintracellular parasites),例如刚地弓形虫、艾美耳球虫(Emeria spp)、新孢子虫(Neospora caninum)、肉孢子虫 (Sarcocystis spp)、疟原虫(Plasmodium spp)(例如恶性疟原虫(Plasmodium falciparum)、间日疟原虫(Plasmodiumvivax)、卵形疟原虫(Plasmodium ovale)、三日疟原虫(Plasmodium malariae)、诺氏疟原虫(Plasmodium knowlesi))和隐孢子虫(Cryptosporidium spp.)。它也可以选自:棘阿米巴原虫(Acanthamoeba spp.)、巴贝西虫(Babesia spp.)、结肠小袋虫(Balantidiumcoli)、芽囊原虫 (Blastocystis)、脆弱双核阿米巴(Dientamoebafragilis)、溶组织内阿米巴 (Entamoebahistolytica)、兰伯贾第虫(Giardia lamblia)、贝氏等孢子球虫(Isospora belli)、利什曼原虫(Leishmania spp.)、福氏耐格里原虫(Naegleriafowleri)、西伯鼻孢子虫(Rhinosporidiumseeberi)、阴道毛滴虫(Trichomonasvaginalis)、布氏锥虫(Trypanosomabrucei)和克氏锥虫(Trypanosomacruzi)。已知这些寄生虫至少是人和兽的寄生虫。
本发明术语“从病原体获得的抗原”可以是任何抗原或其混合物,其为已知的且已经用于获得患者对所述病原体的免疫应答。这些术语是指天然抗原或其混合物、重组抗原或其混合物、以及天然和重组抗原的混合物。所述抗原可为蛋白,尤其是表面蛋白。术语“抗原”是指能够通过其自身(直接地)诱导免疫应答的任何物质,和/或能够通过与一些免疫应答产物组合(一旦形成) 来诱导免疫应答的任何物质。
根据本发明,所述抗原可以包含在任何形式的病原体中。也可以使用活的、灭活的或减毒的病原体自身以提供所述抗原或其混合物。
所述抗原或抗原混合物可获自感染的细胞。然后将包含在这些感染的细胞中的细胞内病原体例如通过破坏细胞膜从而提取出来。根据裂解细胞膜所用的技术,所述病原体可同时灭活。
根据本发明的一个方面,所述抗原获自先前灭活的病原体。当所述病原体为寄生虫,尤其是可为速殖子的原虫时,所述抗原可获自所述速殖子,尤其是先前灭活的速殖子。
本发明人已经发现,刚地弓形虫的速殖子形式(其在细胞内,但不是污染物)提供了至少一种用于预防弓形体病的有效抗原。所述抗原可存在于混合物中,该混合物获自含有速殖子的感染的细胞。
术语“患者”是指温血动物(例如哺乳动物),其患有本文所述的一种或多种疾病和病症,或有患本文所述的一种或多种疾病和病症的可能。本文所用术语“患者”包括人和非人生物,且包括但不限于人、非人的灵长类、羊类(绵羊、山羊…)、犬类、猫类、鼠类、牛类、马类和猪类。所述患者还可以是鸟类。
本发明还涉及抗细胞内病原体的疫苗,其包含本发明药物组合物,且其还包含合适的赋形剂和/或合适的载体和/或合适的媒介物。
载体、赋形剂、稀释剂和/或佐剂根据给药方式进行选择。它们可选自:乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、微晶纤维素、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、聚乙二醇、纤维素、(无菌)水、甲基纤维素、羟苯甲酯和羟苯丙酯、滑石、硬脂酸镁、食用油、植物油和矿物油或其合适的混合物。所述药物组合物可任选地含有通常可用在药物制剂中的其它物质,例如润滑剂、湿润剂、乳化剂和助悬剂、分散剂、崩解剂、稳定剂、等渗剂、膨胀剂、填充剂、防腐剂、甜味剂、矫味剂、芳香剂、着色剂、抗菌剂和/或抗真菌剂例如对羟苯甲酸、三氯叔丁醇、苯酚、山梨酸、分散剂、流动调节剂、脱模剂等。
术语“疫苗”涉及能够预防由将来暴露于细胞内病原体所导致的感染的产品。
本发明组合物和/或疫苗可按照多种方式给药。例如,可将所述组合物/ 疫苗配制用于口服或局部或肠内或非胃肠外给药。术语“口服”、“肠内”、“肠内地”、“口服地”、“非胃肠外”、“非胃肠外地”等,是指通过一定途径或方式,将组合物沿消化道给药至个体。组合物“口服”给药途径的实例包括,但不限于,经口吞咽液体或固体形式的疫苗组合物、通过鼻空肠管或胃造口管给药疫苗组合物、十二指肠内给药疫苗组合物和直肠给药(例如使用栓剂)。
术语“局部给药”是指将药物施用到皮肤或粘膜的外表面(包括鼻、肺和口的表面膜),使得该药物穿过皮肤或粘膜外表面并进入到下面的组织。局部给药可导致将该药物有限地分布于皮肤和周围组织,或者,当该药物经血流离开该治疗区域时,可导致该药物的全身分布。
可将本发明组合物和/或疫苗配制用于鼻内给药。术语“鼻内给药”是指任意给药形式,该给药形式是以推入或其他方式将活性成分引入受试者的鼻道,从而使得该活性成分与鼻腔的呼吸上皮接触,然后从鼻腔内吸收进入体循环。鼻腔粘膜给药还可包括与嗅上皮接触,所述嗅上皮位于鼻腔顶部,介于中央的鼻中隔和各主鼻道外侧壁之间。
本发明还涉及纳米颗粒作为疫苗佐剂的用途,该纳米颗粒包含阳离子- 多糖固体核心,该核心是多孔的,装载有阴离子磷脂且在所述核心不被任何磷脂层包围。如下文所解释,在本发明组合物中所用的纳米颗粒可用作疫苗佐剂且至少与霍乱毒素同样有效。
可选择的多糖和/或磷脂如上文针对本发明药物组合物所述。
本发明还涉及在患者中引发抗细胞内病原体的免疫应答的方法,所述方法包括给予患者治疗量的本发明组合物或本发明疫苗。
根据本发明方法一个方面,所述免疫应答包括Th1应答。
本发明还涉及如前所述的药物组合物在制造抗细胞内病原体的疫苗中的用途。
根据本发明,本发明疫苗中所含抗原的量不限于确定的值。例如,所述组合物可包含5μg-1mg的抗原,更具体地包含10μg-1mg的抗原和例如 15μg-200μg的抗原。
本发明还涉及包含本发明疫苗和使用手册的试剂盒。
附图说明
图1显示了在经三次鼻内给药(每次给药间隔15天)DGNP、TE(总抗原提取物)、TE和NPS的混合物以及TE和DGNP的混合物所处理的Swiss小鼠的血清中的刚地弓形虫IgG抗体的ELISA分析,该分析分别在第一次给药当天、第一次给药后的第14天(第二次给药)、第一次给药后的第28天(第三次给药)以及第一次给药后最终的第42天进行;
图2显示了在培养的脾细胞上清液中的IFN-γ的ELISA分析,所述脾细胞获自第三次鼻内给药后如图1所述的处理组小鼠;
图3显示了由显微计数获得的小鼠脑中寄生虫的负载,其是在将图1所涉及的处理组小鼠的脑匀浆化之后且在刚地弓形虫囊肿感染后进行的,该结果是在刚地弓形虫感染后6周获得的。
图4显示了在分别经单独的TE、单独的DGNP、TE和DGNP的混合物、单独的霍乱毒素(CT)以及TE和CT的混合物的三次鼻内给药处理的CBA/J 小鼠血清中的弓形虫IgG抗体的体液分析结果,所述结果在第三次鼻内给药后的第14天获得,交叉符(x)是指小鼠,而破折号(-)是指各组小鼠所获得的平均值;将在用TE和DGNP的混合物以及用TE和CT的混合物分别处理的组中具有更强结果的小鼠所指代的交叉符圈出来;还将显示了前述各组的平均体液应答的小鼠所指代的交叉符圈出来。
图5显示了在图4所提及的且在第三次鼻内给药后的各组的培养的脾细胞上清液中的IFN-γ(pg/ml)的ELISA分析;
图6显示了由如图4所限定的在第三次鼻内给药后2周的各组小鼠所产生的脾细胞的IFN-γ生成,深灰色柱是指分别在TE-DGNP和TE-CT组中具有更强的体液应答的小鼠的IFN-γ生成,而浅灰色柱是指分别在TE-DGNP 和TE-CT组中具有平均体液应答的小鼠的IFN-γ生成;
图7显示了分别在未处理的小鼠组、三次鼻内给药单独的DGNP处理的小鼠组、三次鼻内给药单独的TE处理的小鼠组、三次鼻内给药TE和DGNP 混合物处理的小鼠组、三次鼻内给药单独的CT处理的小鼠组和三次鼻内给药TE和CT混合物处理的小鼠组的小鼠脑中的囊肿数,所述囊肿数是在第三次鼻内给药后2周获得的。
实验部分
纳米颗粒的制备
如前所述,多糖颗粒由美国药典麦芽糖糊精制备(Paillard等人,2010)。简而言之,将100g麦芽糖糊精溶于2N氢氧化钠中,同时在室温进行磁力搅拌。此外,加入1-氯-2,3-环氧丙烷(环氧氯丙烷)和缩水甘油基三甲基氯化铵(羟胆碱,阳离子配体)以制备阳离子多糖凝胶。然后将该凝胶用乙酸中和并在高压下在Minilab均化器(Rannie;APV Baker,Evreux,法国)中剪切。将得到的60nm多糖纳米颗粒在SGI Hi-flow系统(空心纤维模块(hollow fiber module):30UFIB/1S.6/40kDa;Setric Génie Industriel,法国)中超滤以除去低分子量的试剂和盐。所获得的纳米颗粒以下简称NPS。
一些前述NPS装载有阴离子磷脂。通过注射二棕榈酰-磷脂酰基甘油 (DPPG)的乙醇溶液,将阴离子磷脂装载到这些多孔NPS中。含有磷脂的所述多孔纳米颗粒以下简称DGNP。这些纳米颗粒的核心没有被任何磷脂层包围。
刚地弓形虫的总抗原提取物(TE)的合成和纯化
速殖子从连续分裂的感染的HFF细胞(人包皮成纤维细胞)获得。从一个 225cm2培养瓶中得到约1.108个速殖子,相当于200μg的TE。然后通过冷冻 /解冻循环进行速殖子的细胞裂解,采集、超声(2x 10mn,60W在冰中)并通过微量BCA方法评估蛋白的量。
TE是指根据上述操作从速殖子获得的产物。TE与纳米颗粒(NPS和 DGNP)组合用于小鼠免疫、Elisa涂覆和细胞再刺激试验。TE是多种抗原的混合物。
接种和NP和DGNP之间的激发方案-选择(challenge protocol-choice)
以体液和细胞应答的强度以及保护强度为基础,测定作为抗原载体最有效的纳米颗粒。
从Janvier(France)购买成年雌性Swiss和CBA/J小鼠,20-25g重合6-8 周龄。该动物实验符合法国政府的伦理与动物实验条例。
Swiss小鼠接受鼻内处理,以15天的时间间隔给药三次,单独给药TE(10 μg)和单独给药DGNP纳米颗粒(30μg)(作为对照组)或组合给药TE+NPS、 TE+DGNP(10μg的TE和30μg的NPS或DGNP)。
将各剂上述总提取物(TE)、纳米颗粒及其混合物在磷酸盐缓冲盐水(10 mM磷酸盐,140mM NaCl[PBS])中稀释成10μl的最终体积并用微量移液器将其滴入到未经麻醉的小鼠的鼻孔中(5μl/鼻孔)。在处理后1个月将经处理的小鼠经口感染50个76K弓形虫菌株的囊肿,然后进行随后6周的临床检查。
体液免疫应答研究
通过ELISA,将经处理的小鼠血清中的特异性弓形虫IgG进行量化。连续监测血清中抗弓形虫抗原的IgG的生成。结果如图1所示。
图1显示了光密度(DO),其在经处理的小鼠的血清中进行测定。所述光密度显示了抗刚地弓形虫的血清IgG的水平。DGNP是指在感染前单独用 DGNP处理的小鼠,TE是指在感染前单独用总抗原提取物(TE)处理的小鼠, TE-NPS是指在感染前用TE和NPS的混合物处理的小鼠,和TE-DGNP是指在感染前用TE和DGNP的混合物处理的小鼠。D0是指各处理组小鼠在处理前所测量的光密度。D14是指在第一次鼻内给药后第14天所测量的光密度,D28是指在第二次鼻内给药后第14天所测量的光密度和D42是指在第三次鼻内给药后第14天所测量的光密度。
如图1所示,在用TE-NPS或TE-DGNP免疫的小鼠组中,第二次鼻内给药后可检测到特异性的弓形虫IgG。由第三次鼻内给药引起的放大效应导致强烈地诱导IgG表达,但是在这两种纳米颗粒之间未观测到显著差异。在仅用TE处理的小鼠组中未检测到IgG。
细胞免疫应答研究:
为了研究细胞免疫应答,在第三次鼻内给药后第3周,对前述处理小鼠的经弓形虫刺激的脾细胞的上清液中的脾细胞细胞因子(一种疫苗功效的强免疫原性生物标记)进行分析。细胞因子(IFN-γ、IL-12、IL-10、IL-13、TNF-α、IL-5)通过ELISA进行定量。
由于T-细胞衍生的IFN-γ还是弓形体病中保护性免疫的重要标志,因此在弓形虫抗原刺激后可用ELI斑点分析测定IFN-γ。
为了研究在用TE-NPS、TE-DGNP、单独的TE或单独的纳米颗粒处理后所诱导的细胞免疫应答,对不同组的2只小鼠的脾的培养细胞的上清液评估了响应于TE再刺激(10μg.ml-1)的IFN-γ、IL-10和IL-12生成。
图2显示了获自上述处理组小鼠的培养的脾细胞上清液中的IFN-γ浓度 (pg/mL)。
标为DGNP的各柱对应的是仅用DGNP处理的小鼠。标为TE的各柱对应的是仅用TE处理的小鼠。标为TE-NPS的各柱对应的是用TE和NPS的混合物处理的小鼠。标为TE-DGNP的各柱对应的是用TE和DGNP的混合物处理的小鼠。
如图2所示,仅有一只用TE和NPS混合物免疫的小鼠通过脾细胞生成 IFN-γ(175pg/ml)从而响应于TE刺激。相对地,如图2所示,观测到用TE 和DGNP的混合物免疫的2只小鼠的脾细胞的特异性IFN-γ生成(分别是237 和375pg/ml)。因此,DGNP似乎对诱导细胞免疫应答更为有效。
用TE和DGNP的混合物处理的小鼠的脑中囊肿数的评估
在刚地弓形虫感染后第6周,将用TE和DGNP的混合物处理并接着用刚地弓形虫感染的小鼠处死,并采集它们的脑。
在感染后第6周,从存活小鼠采集脑并用研棒和研钵在5mL的RPMI 1640中匀浆。在显微镜下计数在各个脑匀浆中的囊肿(10次计数,每次10μl)。各组结果以平均值±SEM表示。该数据使用Mann-Whitney U检验(GraphPad prism软件)进行统计学分析(P<0.05)。其结果如图3所示。
图3显示了在刚地弓形虫感染前,仅用DGNP(称为DGNP)、仅用TE(称为TE)、用TE和NPS的混合物(称为TE-NPS)和用TE和DGNP的混合物(称为TE-DGNP)处理的小鼠脑中的囊肿数。
如图3所示,用TE和DGNP的混合物处理的小鼠所具有的囊肿显著少于分别用DGNP、TE以及TE和NPS的混合物处理的小鼠所具有的囊肿(分别是672、1333、1180和1072;p<0.05)。用TE和DGNP的混合物处理的小鼠脑中具有的囊肿数仅用其它处理的小鼠脑中具有的囊肿数的56%。这些数据表明,用TE和DGNP的混合物进行的处理(免疫)诱导细胞免疫应答,然后降低了脑中寄生虫的蔓延以及囊肿的形成。
根据所得结果,DGNP纳米颗粒用于以下免疫实验并用于比较DGNP和霍乱毒素(CT)的疫苗接种程序。
DGNP和霍乱毒素(CT)之间的比较
以15天的间隔,对6组各10只CBA/J小鼠分别进行三次鼻内给药TE(10 μg)、纳米颗粒(DGNP-30μg)、单独的霍乱毒素(CT–0.5μg)(定义为对照组)、 TE+DGNP的组合、TE+CT的组合(10μg的TE和30μg的DGNP或0.5μg 的CT)进行处理。该实验设计包括未处理组小鼠。
将各剂上述毒素、纳米颗粒和组合(混合物)在磷酸盐缓冲盐水(10mM磷酸盐,140mM NaCl[PBS])中稀释成10μl的最终体积并用微量移液器滴入未经麻醉的小鼠的鼻孔中(5μl/鼻孔)。已完成5次独立的实验。
体液应答分析:
在第三次鼻内给药后第14天,监测血清中抗弓形虫抗原的IgG生成。该实验方案参考图1进行描述。该结果描述于图4中。图4描述了经上述处理的小鼠所获得的血清光密度。所述特异性抗体效价是由产生光密度(OD) 的最高稀释液的倒数得出的,其光密度(OD)是以相同程度稀释的未处理小鼠血清的2.5倍。该结果表示为平均log效价和标准差(S.D)。
如图4所示,检测用TE和DGNP的混合物以及TE和CT的混合物处理的小鼠组的特异性的弓形虫IgG。如图4所示,这两组之间未观测到显著差异。在单独用TE、CT或DGNP免疫的小鼠组中,未检测到IgG。
细胞应答分析:
为了研究上述处理后所诱导的细胞免疫应答,用ELISA分析评估上述不同组的2只小鼠的脾的培养细胞的上清液中响应于TE再刺激的IFN-γ、 IL-10、IL-13、TNF-α、IL-5和IL-12的生成,而用Elispot评估IFN-γ。结果如图5所示。
图5显示了在前面定义的各组的两只小鼠的培养的脾细胞上清液中的 IFN-γ浓度(pg/mL)。
如图5所示,用TE和DGNP的混合物免疫的小鼠以及用TE和CT的混合物处理的小鼠均通过脾细胞生成IFN-γ从而响应于TE刺激。用TE和 DGNP的混合物免疫的小鼠产生超过400pg/mL的IFN-γ。用TE和CT处理的小鼠产生约1400pg/mL的IFN-γ。
此外,由脾细胞生成IFN-γ是在第三次鼻内给药后第2周进行测量的。该结果如图6所示。
图6显示了图4中的各组的两只小鼠的IFN-γ生成。对于用DGNP和 TE的混合物(DGNP-TE)处理的组以及用TE和CT的混合物(TE-CT)处理的组而言,得到了用圈起来的交叉符表示的小鼠的结果。如图6所示,由脾细胞生成IFN-γ在疫苗接种DGNP和TE的混合物的小鼠中有所增加。在任何所分析样品中未检测到IL-12、IL-13、TNF-α和IL-5的释放。
感染测试
将分别经三次鼻内给药单独的DGNP、单独的TE、TE和DGNP的混合物、单独的CT以及CT和DGNP的混合物处理的小鼠经口感染刚地弓形虫的76K品系的囊肿。未处理组的小鼠也被感染,作为对照组。
存活百分比
在感染80个囊肿后的30天中,追踪各组的存活。各组中所测试动物的总数为n=8。
除了对照组中的一只小鼠,其余小鼠均迅速表现出疾病的临床症状、体重减轻且在感染后的11天内死亡,而100%的疫苗接种了TE和DGNP的混合物的小鼠在超过30天的实验期中得以存活。
小鼠脑中的囊肿数
将各处理组的小鼠经口感染50个囊肿并在经口感染后1个月处死。通过测量小鼠脑的囊肿数来评估抗刚地弓形虫的保护作用(50个囊肿,三次实验)。在各组中所测动物的总数为n=8。该方案参考图3进行描述。该结果如图7所示。
图7显示了各组小鼠脑中的囊肿数。如图7所示,在感染刚地弓形虫之前用TE和DGNP的混合物处理的小鼠所具有的囊肿显著少于对照组小鼠所具有的囊肿(分别是611vs1980个囊肿;p<0.01),这表示70%的降低。用 TE和DGPN的混合物处理的小鼠组所具有的囊肿少于用TE和CT的混合物处理的小鼠所具有的囊肿。上述结果表明,疫苗接种DGNP和TE的混合物提供了长期的保护。
本发明涉及以下实施方案:
1.药物组合物,其包含作为活性成分的以下混合物:
-固体纳米颗粒,其包含多孔阳离子-多糖核心,该核心装载有至少一种阴离子磷脂并且在所述核心周围没有任何磷脂层;
-从细胞内病原体获得的至少一种抗原;和
-药学上可接受的溶剂。
2.实施方案1的药物组合物,其用于在患者中抗所述细胞内病原体的预防性治疗。
3.实施方案1的药物组合物,其用于实施方案2的用途,其中所述患者选自:人、非人的灵长类、羊类、犬类、猫类、鼠类、牛类、马类、猪类和鸟类。
4.实施方案1-3中任一项的药物组合物,其中所述阳离子多糖为交联聚合物且其获自多糖与至少一种阳离子配体间的反应,所述多糖选自淀粉、葡聚糖、糊精和麦芽糖糊精,而所述阳离子配体选自伯胺、仲胺、叔胺和季铵。
5.实施方案1-4中任一项的药物组合物,其中所述阴离子磷脂选自甘油磷脂,且更具体地为二酰基磷脂酰甘油例如二棕榈酰基磷脂酰甘油、二酰基磷脂酰丝氨酸或二酰基磷脂酰肌醇。
6.实施方案1-5中任一项的药物组合物,其中所述细胞内病原体选自病毒、细菌、分枝杆菌和真菌,且更具体地选自单纯疱疹病毒1和2、人乳头状瘤病毒、爱泼斯坦-巴尔病毒、巨细胞病毒、结核分枝杆菌、登革热、HIV、人呼吸道合胞体病毒(RSV)、甲型肝炎病毒、乙型肝炎病毒和丙型肝炎病毒。
7.实施方案1-5中任一项的药物组合物,其中所述细胞内病原体为细胞内寄生虫,且更具体地为选自以下的细胞内寄生虫:刚地弓形虫、艾美耳球虫、新孢子虫、肉孢子虫、疟原虫、隐孢子虫、棘阿米巴原虫、巴贝西虫、结肠小袋虫、芽囊原虫、脆弱双核阿米巴、溶组织内阿米巴、兰伯贾第虫、贝氏等孢子球虫、利什曼原虫、福氏耐格里原虫、西伯鼻孢子虫、阴道毛滴虫、布氏锥虫和克氏锥虫。
8.实施方案1-7中任一项的药物组合物,其中所述抗原包含在获自先前灭活的病原体的混合物中。
9.实施方案7或8的药物组合物,其中所述抗原获自所述病原体的速殖子形式。
10.抗细胞内病原体的疫苗,包含实施方案1-9中任一项的药物组合物,还包含合适的赋形剂和/或合适的载体和/或合适的媒介物。
11.根据实施方案10的疫苗,其中将所述疫苗配制用于鼻内给药。
12.固体纳米颗粒作为疫苗佐剂的用途,所述颗粒包含多孔阳离子-多糖核心,该核心装载有至少一种阴离子磷脂且在所述核心周围没有任何磷脂层。
13.一种在有此治疗需要的患者中引发抗细胞内病原体的免疫应答的方法,所述方法包括给药所述患者治疗量的实施方案1-9中任一项的组合物。
14.实施方案13的方法,其中所述组合物是鼻内给药的。
15.实施方案13或14的方法,其中所述免疫应答是Th1型应答。
16.试剂盒,其包含实施方案9或10的疫苗以及使用手册。
Claims (10)
1.药物组合物,其包含作为活性成分的以下混合物:
-固体纳米颗粒,其包含多孔阳离子多糖核心,该核心装载有至少一种阴离子磷脂并且在所述核心周围没有任何磷脂层;
-从细胞内病原体获得的至少一种抗原;和
-药学上可接受的溶剂。
2.权利要求1的药物组合物,其用于在患者中抗所述细胞内病原体的预防性治疗。
3.权利要求1或2的药物组合物,其中所述阳离子多糖为交联聚合物且其获自多糖与至少一种阳离子配体间的反应,所述多糖选自淀粉、葡聚糖、糊精和麦芽糖糊精,而所述阳离子配体选自伯胺、仲胺、叔胺和季铵。
4.权利要求1-3中任一项的药物组合物,其中所述阴离子磷脂选自甘油磷脂,且更具体地为二酰基磷脂酰甘油例如二棕榈酰基磷脂酰甘油、二酰基磷脂酰丝氨酸或二酰基磷脂酰肌醇。
5.权利要求1-4中任一项的药物组合物,其中所述细胞内病原体选自病毒、细菌、分枝杆菌和真菌,且更具体地选自单纯疱疹病毒1和2、人乳头状瘤病毒、爱泼斯坦-巴尔病毒、巨细胞病毒、结核分枝杆菌、登革热、HIV、人呼吸道合胞体病毒(RSV)、甲型肝炎病毒、乙型肝炎病毒和丙型肝炎病毒。
6.权利要求1-4中任一项的药物组合物,其中所述细胞内病原体为细胞内寄生虫,且更具体地为选自以下的细胞内寄生虫:刚地弓形虫、艾美耳球虫、新孢子虫、肉孢子虫、疟原虫、隐孢子虫、棘阿米巴原虫、巴贝西虫、结肠小袋虫、芽囊原虫、脆弱双核阿米巴、溶组织内阿米巴、兰伯贾第虫、贝氏等孢子球虫、利什曼原虫、福氏耐格里原虫、西伯鼻孢子虫、阴道毛滴虫、布氏锥虫和克氏锥虫。
7.抗细胞内病原体的疫苗,包含权利要求1-6中任一项的药物组合物,还包含合适的赋形剂和/或合适的载体和/或合适的媒介物。
8.固体纳米颗粒作为疫苗佐剂的用途,所述颗粒包含多孔阳离子多糖核心,该核心装载有至少一种阴离子磷脂且在所述核心周围没有任何磷脂层。
9.权利要求1-6中任一项的组合物在制备用于在有此治疗需要的患者中引发抗细胞内病原体的免疫应答的药物中的用途。
10.试剂盒,其包含权利要求7的疫苗以及使用手册。
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