CN109260457B - 一种透明质酸-白介素10复合物、其制备方法及其应用 - Google Patents
一种透明质酸-白介素10复合物、其制备方法及其应用 Download PDFInfo
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Abstract
本发明属于生物医学技术领域,具体地说,涉及一种透明质酸‑白介素10复合物、其制备方法及其应用。所述的透明质酸‑白介素10‑复合物具有式Ⅰ所示的结构式:
Description
技术领域
本发明属于生物医学技术领域,具体地说,涉及一种透明质酸-白介素10复合物、其制备方法及其应用。
背景技术
透明质酸(hyaluronate,HA)是细胞外基质的组分之一,具有多种作用,如保护细胞,影响细胞迁移、增殖、分化以及通过反馈作用调节合成细胞的合成能力等等,因此在组织培养中也起到关键性的作用(Schagemann JC et al.,Biomaterials,2010,31(10):2798-2805;Masashi A et al.,Journal of Biomedical Materials Research Part A,2005,75(2):494-499.)。在神经组织工程方面,HA具有良好的神经相容性且能利于神经的再生与重建,因此在支架的形成、支架结构及改善生物性能方面发挥着重要作用。研究表明,HA明显影响着细胞的迁移,其过程为:透明质酸→构成基质→细胞开始移动→开始合成透明质酸酶→HA开始降解→细胞移动停止→细胞聚集(Neufang KF et al.,Rontgenblatter,1989,42(4):180-186.)。
透明质酸是一种多糖,为细胞外基质的重要组成部分,尤其大分子透明质酸具有抗炎症和抗纤维化的功能,并且对于维持人体正常的肺部功能起着非常重要的作用。
同时,HA水溶液具有极高的保湿性、粘弹性、润滑性和生物相容性和生物降解性,在医药、组织工程以及临床中得到广泛应用。然而,天然HA对强酸、强碱、热及透明质酸酶敏感,在体内停留时间短、易扩散、易降解等特性,限制了它的应用,因此在实际应用中还需对HA进行修饰改性。
白介素10被认为是一种抗炎症细胞因子,可以抑制炎症细胞因子的分泌。但其在体内短暂的半衰期(仅几分钟)极大地影响了它的临床应用。
发明内容
本发明要解决的技术问题在于克服现有技术的不足,提供一种透明质酸-白介素10复合物。该复合物可以在体内慢慢释放白介素10,使其在体内的功效从单一存在时的几分钟延长到至少7天,大大提高和发挥了白介素10抗炎症和抗纤维化的功能,为其在临床医学中发挥相应的作用打下了坚实的基础。
为解决上述技术问题,本发明采用如下技术方案:
一种透明质酸-白介素10复合物,其中,所述的透明质酸-白介素10复合物具有式Ⅰ所示的结构式:
白介素10被认为是一种抗炎症细胞因子,可以抑制炎症细胞因子的分泌。但其在体内短暂的半衰期(仅几分钟)极大地影响了它的临床应用。透明质酸是一种多糖,为细胞外基质的重要组成部分,尤其大分子透明质酸具有抗炎症和抗纤维化的功能,并且对于维持人体正常的肺部功能起着非常重要的作用。
本发明提供了一种透明质酸-白介素10复合物,该复合物是透明质酸的开环改性产物与白介素10形成的,透明质酸经糖环开环,形成醛基封端的透明质酸衍生物,醛基和白介素10的N端结合,形成透明质酸和白介素10的连接物,即透明质酸-白介素10复合物。该复合物可以在体内慢慢释放白介素10,使其在体内的功效从单一存在时的几分钟延长到至少7天,大大提高和发挥了白介素10抗炎症和抗纤维化的功能,为其在临床医学中发挥相应的作用打下了坚实的基础。
进一步的,所述的结构式中,10<m<500,10<n<300,10<l<200。
本发明还提供所述的透明质酸-白介素10复合物的制备方法。
所述的制备方法包括如下步骤:
1)将HA-ALD与白介素10混合,在NaBH3CN存在下在酸性条件下反应,得到中间体Ⅰ,反应式如下:
2)向所得的中间体Ⅰ中再加入肼基甲酸乙酯,在NaBH3CN存在下在酸性条件下反应,得到式Ⅰ所示的透明质酸-白介素10复合物,反应式如下:
上述制备方法中,步骤1)中所述的HA-ALD和白介素10的摩尔比为1-10:1,优选1-5:1。
上述制备方法中步骤2)中所述的中间体Ⅰ与肼基甲酸乙酯的摩尔比为1:1-10,优选1:1-5,更优选1:2。
上述制备方法中,步骤1)和步骤2)中所述的酸性条件为pH5.5的条件下。
上述制备方法中,步骤1)和步骤2)中NaBH3CN的用量为HA-ALD质量的1-10%。
本发明中,HA-ALD为二醛基封端的HA开环改性产物,即在一定氧化剂作用下,透明质酸糖环开环,形成醛基封端的透明质酸衍生物,从而引入更多化学修改点。HA-ALD的制备可参照现有技术的方法进行。
本发明还进一步提供一种组合物,所述的组合物含有本发明所述的透明质酸-白介素10复合物。
本发明中,所述的组合物为药物组合物,也可以为美容组合物。
进一步的,所述的组合物还含有硫酸乙酰肝素。
进一步的,所述的硫酸乙酰肝素与透明质酸-白介素10复合物的质量比为1%~99%:99%~1%。
进一步的,所述的组合物还含有辅料。
进一步的,所述的辅料为药学上可接受的辅料或化妆品用辅料。
进一步的,所述的组合物制备成药学上可接受的制剂或化妆品领域常用的剂型;
优选,所述的药学上可接受的制剂为口服制剂、注射制剂、涂抹制剂、擦剂或喷雾剂;
所述的化妆品领域常用的剂型为霜剂、膏剂或水剂。
所述的组合物在制备成药学上可接受的制剂时,按照本领域的公知常识加入药学上可接受的辅料,按照药学领域常规的方法制备。
所述的组合物在制备成化妆品领域常用的剂型时,可按照化妆品领域常用的方法添加化妆品用辅料制备成霜、乳等。
本发明还提供所述的透明质酸-白介素10的连接物或所述的组合物在制备治疗纤维化或皮肤病药物方面的应用;
优选,所述的纤维化为肺纤维化,所述的皮肤病为皮肤疤痕。
本发明通过药效学试验,惊喜地发现,本发明的透明质酸-白介素10复合物(HH-10)能够减少皮肤疤痕的形成,减少小鼠肺部纤维化,减少小鼠肺部炎症反应,提高小鼠存活率。
采用上述技术方案后,本发明与现有技术相比具有以下有益效果:
本发明所提供的透明质酸-白介素10复合物可以在体内慢慢释放白介素10,使其在体内的功效从单一存在时的几分钟延长到至少7天,大大提高和发挥了白介素10抗炎症和抗纤维化的功能,为其在临床医学中发挥相应的作用打下了坚实的基础。
同时通过药效学试验,惊喜地发现,本发明的透明质酸-白细胞介素10复合物(HH-10)能够减少皮肤疤痕的形成,减少小鼠肺部纤维化,减少小鼠肺部炎症反应,提高小鼠存活率。
下面结合附图对本发明的具体实施方式作进一步详细的描述。
附图说明
图1-a是透明质酸-白介素10复合物的凝胶渗透色谱图;
图1-b是透明质酸的凝胶渗透色谱图;
图2-a是白介素10的核磁共振图;
图2-b是透明质酸的核磁共振图;
图2-c是透明质酸和白介素10连接后的HH-10的核磁共振图;
图3是分别用生理盐水、IL-10和HH-10处理后的小鼠皮肤剖面图;
图4为疤痕面积比较图;
图5为小鼠存活率试验结果图;
图6为气管肺泡灌洗实验显示HH-10减少小鼠肺部炎症反应试验结果图;
图7为Ashcroft Score显示HH-10减少小鼠肺部纤维化的效果图;
图8为Sircol Assay显示HH-10减少小鼠肺部组织中胶原蛋白含量的效果图;
图9为HH-10减少小鼠肺部组织细胞中的细胞凋亡的效果图;
图10为Trichrome Staining显示HH-10减少小鼠肺部组织中胶原蛋白的聚集和沉积的效果图;
需要说明的是,这些附图和文字描述并不旨在以任何方式限制本发明的构思范围,而是通过参考特定实施例为本领域技术人员说明本发明的概念。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对实施例中的技术方案进行清楚、完整地描述,以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1、透明质酸-白介素10复合物
1)将HA-ALD与白介素10混合,其中HA-ALD与白介素10的摩尔比为1:1,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的1%,得到中间体Ⅰ,反应式如下:
2)向所得的中间体Ⅰ中再加入肼基甲酸乙酯,其中中间体Ⅰ与肼基甲酸乙酯的摩尔比为1:2,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的1%,得到式Ⅰ所示的透明质酸-白介素10复合物,反应式如下:
对所制得的式Ⅰ所示的透明质酸-白介素10复合物的结构进行了如下结构确证:
所制得的透明质酸-白介素10复合物凝胶渗透色谱图(Gel PermeationChromatograpy)见图1-a所示,检测波长为280nm。与图1-b中的透明质酸比较,透明质酸和白介素10连接后,由于连接物分子量增加,减少了其在色谱柱内滞留时间,即滞留时间从透明质酸的18.3分钟减少到了连接物的13.3分钟,证实了连接反应的进行和连接物的存在。
图2-a、图2-b和图2-c显示了透明质酸和白介素10连接前后的核磁共振对比图,峰型的变化显示了连接反应过程中化学键的变化。
所得的透明质酸-白介素10复合物的分子量为100000~2000000道尔顿。
实施例2、透明质酸-白介素10复合物
1)将HA-ALD与白介素10混合,其中HA-ALD与白介素10的摩尔比为10:1,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的10%,得到中间体Ⅰ,反应式如实施例1所示:
2)向所得的中间体Ⅰ中再加入肼基甲酸乙酯,中间体Ⅰ与肼基甲酸乙酯的摩尔比为1:1,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的10%,得到式Ⅰ所示的透明质酸-白介素10复合物,反应式如实施例1所示:
对所制得的式Ⅰ所示的透明质酸-白介素10复合物的结构进行了如实施例1所示的结构确证,其结果与实施例1相似。所得的透明质酸-白介素10复合物的分子量为1500000~20000000道尔顿。
实施例3、透明质酸-白介素10复合物
1)将HA-ALD与白介素10混合,其中HA-ALD与白介素10的摩尔比为5:1,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的5%,得到中间体Ⅰ,反应式如实施例1所示:
2)向所得的中间体Ⅰ中再加入肼基甲酸乙酯,中间体Ⅰ与肼基甲酸乙酯的摩尔比为1:5,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的5%,得到式Ⅰ所示的透明质酸-白介素10复合物,反应式如实施例1所示:
对所制得的式Ⅰ所示的透明质酸-白介素10复合物的结构进行了如实施例1所示的结构确证,其结果与实施例1相似。所得的透明质酸-白介素10复合物的分子量为10000~200000道尔顿。
实施例4、透明质酸-白介素10复合物
1)将HA-ALD与白介素10混合,其中HA-ALD与白介素10的摩尔比为2.5:1,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的5%,得到中间体Ⅰ,反应式如实施例1所示:
2)向所得的中间体Ⅰ中再加入肼基甲酸乙酯,中间体Ⅰ与肼基甲酸乙酯的摩尔比为1:10,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的5%,得到式Ⅰ所示的透明质酸-白介素10复合物,反应式如实施例1所示:
对所制得的式Ⅰ所示的透明质酸-白介素10复合物的结构进行了如实施例1所示的结构确证,其结果与实施例1相似。所得的透明质酸-白介素10复合物的分子量为800000~20000000道尔顿。
实施例5、透明质酸-白介素10复合物
1)将HA-ALD与白介素10混合,其中HA-ALD与白介素10的摩尔比为3:1,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的3%,得到中间体Ⅰ,反应式如实施例1所示:
2)向所得的中间体Ⅰ中再加入肼基甲酸乙酯,中间体Ⅰ与肼基甲酸乙酯的摩尔比为1:2.5,在NaBH3CN存在下在pH5.5条件下反应,NaBH3CN的用量为HA-ALD质量的3%,得到式Ⅰ所示的透明质酸-白介素10复合物,反应式如实施例1所示:
对所制得的式Ⅰ所示的透明质酸-白介素10复合物的结构进行了如实施例1所示的结构确证,其结果与实施例1相似。所得的透明质酸-白介素10复合物的分子量为2000000~20000000道尔顿。
实施例6、药物组合物
将实施例1制得的透明质酸-白介素10复合物与硫酸乙酰肝素以1%:99%的质量比混合,得到所述的药物组合物。
实施例7、药物组合物
将实施例1制得的透明质酸-白介素10复合物与硫酸乙酰肝素以99%:1%的质量比混合,得到所述的药物组合物。
实施例8、药物组合物
将实施例1制得的透明质酸-白介素10复合物与硫酸乙酰肝素以50%:50%的质量比混合,得到所述的药物组合物。
实施例9、化妆品
将实施例6制得的药物组合物添加适量的化妆品用辅料,如水性基质制成化妆品领域常用的面霜。
实施例10、药物
将实施例7制得的药物组合物添加药学上可接受的辅料,如填充剂等,制成药学上常见的片剂。
试验例1、疤痕修复方面的应用
图3是小鼠皮肤剖面图,皮肤伤口愈合后的疤痕可以用疤痕的面积大小和在皮肤表层有否凹陷来显示。图3的上图展示皮肤损伤用生理盐水处理,愈合后的疤痕大小为两箭头之间的面积,且产生凹陷;图3的中图显示单独用白介素10处理小鼠皮肤损伤,疤痕面积有所减小,凹陷减轻;图3的下图显示,用HH-10(透明质酸-白介素10复合物)处理,疤痕面积减少很多,局部无凹陷产生。
图4为疤痕面积比较,HH-10比较生理盐水和白介素10都显示出在减少疤痕面积上有显著性差别,说明了它在治疗皮肤疤痕上的作用。
试验例2、肺纤维化方面的应用
方法:50只小鼠随机分为5组,每组10只。所有小鼠先经历气管注射博来霉素。从第二天起,其中四组小鼠分别用生理盐水、白介素10、透明质酸和HH-10(预防组)鼻内吸入(1unit/kg),每天一次,连续7天。另外一组小鼠(治疗组),从博来霉素气管注射后的第7天起,做小鼠鼻内吸入HH-10,每天一次,连续7天。我们称这一组小鼠为治疗组,因为博来霉素注射后7天,小鼠的肺纤维化已经形成,这时用HH-10处理时为了观察HH-10对于肺纤维化的治疗作用。
博来霉素注射后的21天,本发明检查各组小鼠的存活率及其他指标。
图5显示,HH-10减少了由博来霉素诱发小鼠肺纤维化而导致的死亡率。横坐标显示博来霉素注射后的天数;纵坐标显示小鼠的存活率。sham用生理盐水处理,其它分别为用HH-10处理、只用透明质酸和只用白介素10处理,以及HH-10(T)显示治疗组小鼠的存活率。
从图5可以看出,比较sham曲线,HH-10提高了小鼠21天的存活率,显示了对肺纤维化的预防作用;对比sham曲线,治疗组曲线显示了HH-10在纤维化已经形成后的作用,显示了一定的治疗意义。
气管肺泡灌洗实验是在动物实验和临床检验中常用的测试肺部炎症反应的实验。由于肺纤维化起源于肺泡局部的炎症反应,而肺泡灌注液中细胞数量越多表明炎症反应越严重。图中纵坐标表示灌注液中细胞数量。图6中HH-10处理的小鼠,包括预防组(prevention)和治疗组(treatment),两个HH-10柱图所示,肺部炎症反应比较PBS对照组有显著降低,显示了HH-10的抗炎作用。
Ashcroft评分是经典的肺纤维化的评估指标,评分为0到8,越高越严重。主要根据肺组织切片中观察到的肺组织结构,肺间质厚度,蜂窝状结构以及炎症细胞的浸润程度来评分的。图7中显示,和PBS对照组比较,预防和治疗组中,HH-10显著地减少了肺纤维化的程度。
胶原蛋白含量是检测纤维化的重要指标。胶原蛋白在损伤部位的沉淀和堆积是纤维化的主要原因。图8中纵坐标显示胶原蛋白含量。图8中两个HH-10柱图显示与PBS对照组动物比较,HH-10在预防和治疗组中显著性地减少了小鼠肺组织中胶原蛋白的含量。
细胞凋亡是指为维持内环境稳定,机体受损组织细胞自主的死亡,是对受损组织的自我保护。在肺纤维化的过程中细胞凋亡的数量代表了炎症反应和纤维化的程度。图9中显示,在HH-10处理的小鼠肺组织中细胞凋亡的数量显著性地降低。
三色染色法染色小鼠肺组织切片。这是预防组的小鼠肺切片比较。图10中,比较PBS对照组,HH-10处理的小鼠切片显示了正常的肺泡组织,极少的胶原蛋白沉积和肺间质的变厚以及炎症细胞的浸润。单一的白介素10和透明质酸也一定程度地减少了组织纤维化,但HH-10作用最强。
以上所述仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本发明的技术人员在不脱离本发明技术方案范围内,当可利用上述提示的技术内容作出些许变动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明方案的范围内。
Claims (11)
2.根据权利要求1所述的透明质酸-白介素10复合物,其特征在于,所述的结构式中,10<m<500,10<n<300,10<l<200。
4.一种组合物,其特征在于,所述的组合物含有权利要求1所述的透明质酸-白介素10复合物。
5.根据权利要求4所述的组合物,其特征在于,所述的组合物还含有硫酸乙酰肝素。
6.根据权利要求5所述的组合物,其特征在于,所述的硫酸乙酰肝素与透明质酸-白介素10复合物的质量比为1%~99%:99%~1%。
7.根据权利要求6所述的组合物,其特征在于,所述的组合物还含有辅料。
8.根据权利要求7所述的组合物,其特征在于,所述的辅料为药学上可接受的辅料或化妆品用辅料。
9.根据权利要求8所述的组合物,其特征在于,所述的组合物制备成药学上可接受的制剂或化妆品领域常用的剂型。
10.根据权利要求9所述的组合物,其特征在于,所述的药学上可接受的制剂为口服制剂、注射制剂、涂抹制剂、擦剂或喷雾剂;
所述的化妆品领域常用的剂型为霜剂、膏剂或水剂。
11.一种权利要求1所述的透明质酸-白介素10复合物或权利要求4-9任意一项所述的组合物在制备治疗肺纤维化或皮肤疤痕药物方面的应用。
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Denomination of invention: A hyaluronic acid interleukin-10 complex, its preparation method and application Granted publication date: 20230613 Pledgee: Zheshang Bank Co.,Ltd. Suzhou high tech Industrial Development Zone sub branch Pledgor: GMAXX BIOLOGICS Ltd. Registration number: Y2024990000115 |