CN113171440A - 一种生物蛋白敷料组合物及其制备方法 - Google Patents
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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Abstract
本发明涉及一种生物蛋白敷料组合物及其制备方法,属于抗肿瘤药物制备领域,目的在于开发出一种稳定性好、疗效好的抗HPV病毒感染的组合物及其制备方法,技术方案是公开了一种生物蛋白敷料组合物,包括马来酰化β‑酪蛋白、卡拉胶、β‑葡聚糖、甘油、醋酸氯己定、丙酮酸钠的磷酸盐缓冲液。本发明还公开了该组合物的制备方法。本发明技术效果为,该组合物用于预防及治疗HPV感染引起的宫颈病变、生殖道疣以及治疗后的复发,安全有效预防宫颈癌的发生。
Description
技术领域
本发明属于抗肿瘤药物制备领域,具体涉及一种生物蛋白敷料组合物及其制备方法。
背景技术
人乳头瘤病毒(HPV)是一种属于乳多空病毒科的乳头瘤空泡病毒A属,是球形DNA病毒,能引起人体皮肤黏膜的鳞状上皮增殖,目前已分离出130多种,不同的型别引起不同的临床表现,根据侵犯的组织部位不同有不同的临床表现。这是一种古老的病毒,广泛存在于自然界中,对人体黏膜的上皮细胞具有特异嗜性,对其他动物无致病性。这种病毒与人类多种良、恶性肿瘤都有关系,HPV长期的持续性感染是导致宫颈细胞病变和致癌的主要原因,近年来研究成果表明在病毒感染的早期,通过切断病毒与细胞表面的病毒颗粒受体(如糖胺多糖、低密度脂蛋白等)的识别或直接结合到病毒上阻止病毒进入宿主细胞。因此,如何开发出一种稳定性好、疗效好的抗HPV病毒感染的组合物及其制备方法成为发明人亟待解决的问题。
发明内容
本发明的首要目的是提供一种生物蛋白敷料组合物,其能够预防和治疗女性人乳头瘤病毒感染。
本发明提供的一种生物蛋白敷料组合物,其特征在于该组合物包含按质量百分含量计的下列组分:
马来酰化β-酪蛋白: 0.005~2%,
卡拉胶: 0.1~2%,
β-葡聚糖: 0.1 ~ 2%,
甘油: 1~5%,
醋酸氯己定: 0.01~0.2%,
含丙酮酸钠的磷酸盐缓冲液:调节PH值为4.5~6.5。
本发明还提供了上述组合物的制备方法,包括如下操作步骤:
a)将β-酪蛋白中加入硼酸钠或磷酸钠缓冲液,搅拌溶解,得到β-酪蛋白溶液;
b)将β-酪蛋白溶液冷却到-10℃~5℃之间,将马来酸酐乙腈溶液滴加到β-酪蛋白溶液中,用氢氧化钠溶液维持体系pH7.5~10.0,继续搅拌得到混合溶液;
c)将上述混合溶液用pH6.5~8.0的磷酸盐缓冲盐水溶液透析,放置10~24小时,得到透析液;
d)将上述透析液用0.22μm微孔滤膜过滤除菌,得到马来酰化β-酪蛋白溶液;
e)将卡拉胶和β-葡聚糖在水中搅拌溶胀得到溶液A;
f)将甘油和醋酸氯己定溶解到纯水中,加到溶液A中,冷却至室温,加入马来酰化β-酪蛋白溶液,搅拌均匀,得到溶液B;
g)搅拌下,溶液B中加入含有丙酮酸钠的磷酸盐缓冲液,调节pH4.5~6.5,加水至100g,搅匀,即得组合物。
其中步骤a)中硼酸钠缓冲液的浓度为0.05~0.5M,pH值为8.0~9.5。
其中步骤 b)中氢氧化钠溶液浓度为0.1~5M。
其中步骤 b)中马来酸酐乙腈溶液浓度为0.2~2M。
其中步骤g)丙酮酸钠的磷酸盐缓冲液的配制方法为氯化钠5~12g,氯化钾0.05~1g,磷酸氢二钠0.5~2.0g,磷酸二氢钾0.1~0.5g,氯化钙0.05~0.5g,六水合氯化镁0.05~0.5g溶于1L纯化水中,加入丙酮酸或丙酮酸钠调节pH到4.0~7.0。仅改变缓冲液中各组分配比或简单增删组分种类,也在本专利保护范围。
由上述方法制备的组合物,可用于预防和治疗女性人乳头瘤病毒感染。
本发明得到的组合物,主要是作用在病毒感染的早期,通过切断病毒与细胞表面的病毒颗粒受体(如糖胺多糖、低密度脂蛋白等)的识别或直接结合到病毒上阻止病毒进入宿主细胞。可以有效地结合HPV病毒颗粒衣壳蛋白L1蛋白上带正电荷氨基酸的富集区,而该区域是与HPV的细胞受体硫酸乙酰肝素蛋白多糖(HSPG)结合的关键区域。本发明组合物与L1的结合阻断了HPV与HSPG的结合,从而阻断HPV感染靶细胞。HPV长期的持续性感染是导致宫颈细胞病变和致癌的主要原因, 本发明组合物阻断降低了子代HPV病毒对宫颈基底层细胞的反复感染几率,因此,控制了HPV的感染进程,从而能够有效地阻止宫颈癌的发生。本发明组合物除了能有效防控HIV和HPV感染,也能抑制其他多种病原体进入靶细胞。
此外阴道pH值受到多重因素的影响,正常女性阴道pH值(3.8~4.5)的维持主要依靠阴道内乳杆菌分解糖原产生的乳酸,当阴道发生感染引起内环境发生变化,pH升高,本发明制备的电荷修饰蛋白PI小于3.5,低于正常或感染阴道pH,在这样的环境下才能保证电荷修饰蛋白带大量负电荷。另外丙酮酸钠、钙、镁的磷酸盐缓冲液具有细胞营养性和安全性,葡聚糖对于慢性宫颈炎伴HPV感染患者的治疗作用,卡拉胶的负静电荷可以和带负电荷的蛋白更稳定的凝胶,能够结合、包裹HPV病毒。选择的卡拉胶除了可将失活的HPV吸附、包裹排出体外,本身还具有广谱的抗病毒活性,并对女性人乳头瘤病毒具有抑制效果,因此本发明组合物适用于阻断HPV感染和传播;用于预防及治疗HPV感染引起的宫颈病变、生殖道疣以及治疗后的复发,安全有效预防宫颈癌的发生。
具体实施方式
下面用具体实施例来进一步说明本发明的内容,但并不以任何方式意味着对本发明进行限制。下列实施例中所用方法如无特别说明均为常规方法。
实施例1
取300mgβ-酪蛋白,加入0.2 M硼酸钠缓冲液(pH 8.5) 30 ml,搅拌,溶解得到β-酪蛋白溶液。冰浴搅拌20分钟,滴加1ml 1M马来酸酐乙腈溶液,用1M氢氧化钠溶液维持体系pH8.5。将反应混合溶液用磷酸盐缓冲液(pH7.4)透析,放置24小时。用0.22um微孔滤膜过滤除菌,得到马来酰化β-酪蛋白溶液,冷藏干燥保存备用。
实施例2
一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料,包括如下重量百分数的组分:马来酰化β-酪蛋白0.05%,卡拉胶0.5%,β-葡聚糖0.5%,甘油2%,醋酸氯已定0.02%,含丙酮酸钠的磷酸盐缓冲液调节pH值到4.5,余量纯化水。
实施例3
一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料及制备方法,包括如下重量百分数的组分:马来酰化β-酪蛋白0.05%,卡拉胶0.5%,β-葡聚糖0.5%,甘油2%,醋酸氯已定0.02%,含丙酮酸钠的磷酸盐缓冲液调节pH值到6.5,余量纯化水。
实施例4
一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料及制备方法,包括如下重量百分数的组分:马来酰化β-酪蛋白0.05%,卡拉胶1.0%,β-葡聚糖1.0%,甘油2%,醋酸氯已定0.05%,含丙酮酸钠的磷酸盐缓冲液调节pH值到4.5,余量纯化水。
对比例
一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料及制备方法,包括如下重量百分数的组分:马来酰化β-酪蛋白0.05%,卡拉胶1.0%,β-葡聚糖1.0%,甘油2%,醋酸氯已定0.05%,余量纯化水。
经过对实施例2~实施例4和对比例样品的加速条件留样(40℃/75%RH)0、3、6个月后,检查项目性状(澄明粘稠胶体)是否变化、pH、蛋白含量汇总列表如下:
对比例中没有磷酸盐缓冲液稳定pH,留样过程中,pH不稳定,会影响样品稳定性;实施例4比实施例2样品粘度更大,此类辅料pH以接近正常阴道为宜,粘度不宜过大,所以选用实施例2进行病毒减载效果试验。
实施例5 体外抗HPV感染效果
由于目前病毒所没有保存有HPV病毒毒株,而H8 细胞株因含有HPV16 型 E6和E7基因,且将其接种到裸鼠不能形成肿瘤,可作为体外研究宫颈HPV 感染的细胞模型。本实验以H8 细胞作为靶细胞,旨在研究一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料对HPV的减载活性。
细胞培养——将H8细胞株(中国医学科学院基础医学研究所提供)在含有10%胎牛血清的DMEM 高糖培养基中进行常规传代培养。
取浓度为2.5×105/ml的对数生长期的H8细胞接种于96孔板中,每孔200μl。实验分为敷料组、阴性对照组(H8细胞接种,不加敷料)、空白敷料组(无H8细胞,仅加入等体积培养液和敷料)、空白对照组(无加入等体积培养液),其中敷料组和空白辅料组设敷料1倍、10倍、100倍、1000倍稀释加入。每组均设3个复孔。分别培养1、2、4、8 h 后,分别取样染色,在高倍显微镜下观察细胞团聚(静电聚集)状态。
实验结果为辅料组10倍、100倍敷料稀释细胞团聚(静电聚集)明显,其他组无细胞团聚。
实验说明电荷修饰β-酪蛋白的凝胶敷料对HPV有减载活性,减载机制为静电吸引聚集。
实施例6
一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料,包括如下重量百分数的组分:马来酰化β-酪蛋白0.1%,卡拉胶0.5%,β-葡聚糖0.5%,甘油2%,醋酸氯已定0.02%,含丙酮酸钠的磷酸盐缓冲液调节pH值到4.5,余量纯化水。
实施例7
一种抗HPV感染的电荷修饰β-酪蛋白的凝胶敷料,包括如下重量百分数的组分:马来酰化β-酪蛋白0.02%,卡拉胶0.5%,β-葡聚糖0.5%,甘油2%,醋酸氯已定0.02%,含丙酮酸钠的磷酸盐缓冲液调节pH值到4.5,余量纯化水。
以上所述仅是本发明的优选实施方式,在不脱离本发明原理的前提下,还可以对其作出若干同等变换和替代,这些同等变换和替代也应视为属于本发明的保护范围。
Claims (6)
1.一种生物蛋白敷料组合物,其特征在于该组合物包含按质量百分含量计的下列组分:
马来酰化β-酪蛋白: 0.005~ 2%,
卡拉胶: 0.1~2%,
β-葡聚糖: 0.1 ~ 2%,
甘油: 1~5%,
醋酸氯己定: 0.01~0.2%,
丙酮酸钠的磷酸盐缓冲液:调节PH值为4.5~6.5。
2.权利要求1所述的组合物的制备方法,其特征在于包括如下操作步骤:
a)将β-酪蛋白中加入硼酸钠或磷酸钠缓冲液,搅拌溶解,得到β-酪蛋白溶液;
b)将β-酪蛋白溶液冷却到-10℃~5℃之间,将马来酸酐乙腈溶液滴加到β-酪蛋白溶液中,用氢氧化钠或其它碱金属溶液维持体系pH7.5~10.0,继续搅拌得到混合溶液;
c)将上述混合溶液用pH6.5~8.0的磷酸盐缓冲盐水溶液透析,放置10~24小时,得到透析液;
d)将上述透析液用微孔滤膜过滤除菌,得到马来酰化β-酪蛋白溶液;
e)将卡拉胶和β-葡聚糖在水中搅拌溶胀得到溶液A;
f)将甘油和醋酸氯已定溶解到纯水中,加到溶液A中,冷却至室温,加入马来酰化β-酪蛋白溶液,搅拌均匀,得到溶液B;
g)搅拌下,溶液B中加入含有丙酮酸钠的磷酸盐缓冲液,调节pH4.5~6.5,加水至100g,搅匀,即得组合物。
3.根据权利要求2所述的制备方法,其特征在于,其中步骤a)中硼酸钠缓冲液的浓度为0.05~0.5M,pH值为8.0~9.5。
4.根据权利要求2所述的制备方法,其特征在于,其中步骤 b)中氢氧化钠或其它碱金属溶液浓度为0.1~5M。
5.根据权利要求2所述的制备方法,其特征在于,其中步骤 b)中马来酸酐乙腈溶液浓度为0.2~2M。
6.根据权利要求2所述的制备方法,其特征在于,其中步骤g)中丙酮酸钠的磷酸盐缓冲液的配制方法为氯化钠5~12g,氯化钾0.05~1g,磷酸氢二钠0.5~2.0g,磷酸二氢钾0.1~0.5g,氯化钙0.05~0.5g,六水合氯化镁0.05~0.5g溶于1L纯化水中,加入丙酮酸或丙酮酸钠调节pH到4.0~7.0。
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