CN101954065A - 注射用重组人血管内皮抑制素纳米粒组合物及制备方法 - Google Patents
注射用重组人血管内皮抑制素纳米粒组合物及制备方法 Download PDFInfo
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Abstract
本发明涉及一种注射用重组人血管内皮抑制素纳米粒组合物及其制备方法。本发明中重组人血管内皮抑制素纳米粒组合物可通过共价交联法、离子诱导法等,采用可生物降解的聚合物作为载体制备。本发明重组人血管内皮抑制素纳米粒组合物可制成注射液和冻干粉针,载药量高,粒径分布均匀,可供静脉注射,同时延缓重组人血管内皮抑制素的释放。
Description
技术领域
本发明涉及一种注射用重组人血管内皮抑制素纳米粒组合物及其制备方法。本发明重组人血管内皮抑制素纳米粒组合物,载药量高,粒径分布均匀,可制成注射液和冻干粉针供静脉注射用,同时延缓重组人血管内皮抑制素的释放。
背景技术
美国哈佛医学院的Folkman教授在20世纪60年代提出“肿瘤生长依赖血管生长”学说,即实体瘤在生长和转移过程中,血管的生成起到了至关重要的作用,此时肿瘤细胞会发出一些促进血管向肿瘤组织增生的细胞因子(aFGF,bFGF,VEGF等)。Folkman教授在上世纪七十年代又提出了用血管内皮抑素(Endostatin)来抑制肿瘤生长的“饿死肿瘤疗法”理论。O’Reilly教授于1997年从小鼠内皮细胞系EOMAD的培养液中分离得到了具有抑制血管内皮细胞生长的物质(血管内皮抑制素,Endostatin),其通过抑制肿瘤血管的生成能够显著地抑制多种原发性肿瘤的生长(O’Relly,M.S.,et al.Cell.1997,88:277-285)。但由于Endostatin在表达制备过程中存在着易于沉淀和复性困难等问题,限制了其在肿瘤患者中的大规模应用。
清华大学教授罗永章和以他为主的留美博士研发团队通过修饰人血管内皮抑制素的核苷酸编码序列,历经八年艰辛探索,生产出N末端带有9个附加氨基酸序列的重组人血管内皮抑制素(取名为:中文名:)(ZL 00107569.1),大大简化了纯化步骤,提高了产物的纯度(ZL 00107569.1),并成功解决了大规模复性的问题。所生产的重组人血管内皮抑制素由192个氨基酸构成,其氨基酸序列为:(M)GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK。
重组的人血管内皮抑制素Endostar保持内源性Endostatin的所有生物活性,没有因为附加N端序列而导致体内免疫原性,其注射液已经获得中国国家药品食品监督管理局的批准上市销售,临床上联合NP化疗方案用于非小细胞肺癌患者。但是作为外源性蛋白,重组的人血管内皮抑制素在体内很容易被免疫系统识别,进而被降解,因此体内半衰期很短。目前市场上销售的Endostar为普通注射液,规格为15mg/3ml/支,临床使用中患者需每天注射一次,连续14天为一个疗程,休息一周后,再继续下一疗程。由于频繁的注射给药,在生理上给患者带来了巨大的痛苦,同时给患者带来了巨大的经济压力。近期有研究发现动物体内小剂量持续给予Endostatin的药效要优于间歇性给药(MinoruKuroiwa,et al.J.Pediatr.Surg.2003,38:1499-1505;Oliver Kisker,et al.Cancer Res.2001,61:7669-7674)。Folkman报道采用连续皮下给药方式能够使rhEndostatin对小鼠Lewis肺癌的抑制率达到99%。因此,将Endostar开发成注射一次可以持续释药多日的长效制剂是必要的。
目前将半衰期短,分子量大,质量不稳定的蛋白药物制成长效制剂通常采用将药物制成缓释微球的形式,通过组成微球的聚合物材料在体内的缓慢降解从而达到药物在体内缓慢释放、吸收的目的。组成微球的聚合物材料主要是聚酯类,如常用的聚乳酸、聚羟基乙酸或两者的共聚物(聚乳酸-羟基乙酸,PLGA)。虽然微球技术拥有很多优点,但微球生产过程非常复杂,一般需要专门化的和无菌的生产设备;生产过程中用到有机溶剂,生产低溶剂残留量的产品通常很困难,且易破坏的药物可能在加工过程中降解(或损失活性);微球所用材料对质量要求较高,价格比较昂贵;开发这些复杂的产品具有挑战性,特别是资金和/或时间的限制,因而在实际的开发过程中面临着诸多困难。
纳米粒是指粒径为10~1000nm的给药体系。由于人体最小毛细管的直径大约为4μm,因此直径小于1μm的纳米粒很容易通过这些毛细管,从而通过非胃肠道途径给药可以达到缓释和在特定组织或靶部位释药的目的。纳米粒作为蛋白质类药物的载体是近来研究的热点。纳米粒制备工艺相对简单,所用载体材料主要为天然聚合物,来源广泛,使用过程中可避免使用有机溶剂。大量研究表明纳米粒有助于选择更为方便的给药途径,保护蛋白的活性,降低毒副作用,延长半衰期,起到缓释、靶向的作用(Calonge P,et al.Pharm Res.1996,13(2):311-315;Kawashima Y,et al.J Controlled Release.1999,62(1-2):279-287)。
发明内容
本发明的目的是提供一种注射用重组人血管内皮抑制素纳米粒组合物及制备方法,将重组人血管内皮抑制素制备成纳米粒,供静脉注射,可以延缓药物释放,从而降低患者生理上的痛苦,提高患者的临床顺应性。
本发明的注射用重组人血管内皮抑制素纳米粒组合物包括可生物降解的聚合物,生物相容性助剂和注射用重组人血管内皮抑制素。
本发明所述的可生物降解的聚合物系指在生物体内能被降解或酶解,生成的小分子物质被机体吸收并排出体外的聚合物。该聚合物为合成的聚合物或天然聚合物,合成的聚合物包括聚乳酸、聚羟基乙酸、聚乳酸-羟基乙酸共聚物、聚己内酯、聚β-羟基丁酸、聚磷酸酯、聚羧酸酐,天然聚合物包括白蛋白、胶原蛋白、纤维蛋白原、明胶、海藻酸盐、纤维素、壳聚糖及其衍生物、右旋糖酐、透明质酸盐、淀粉。优选的,本发明中可生物降解的聚合物主要是天然聚合物壳聚糖或壳聚糖的衍生物,其中壳聚糖衍生物包括羧甲基壳聚糖、羧基壳聚糖、羟烷基壳聚糖、酰基化壳聚糖、磺化壳聚糖、壳聚糖季铵盐的一种或多种。更优选的,可生物降解的聚合物分子量范围在10000至500000道尔顿,重量占组合物重量的50%-80%。
本发明中所述生物相容性助剂包括化学交联剂、表面活性剂、阴离子大分子或凝聚剂。其中化学交联剂可以是戊二醛或三聚磷酸钠。其中表面活性剂可以是聚乙二醇、聚氧乙烯-聚氧丙烯嵌段共聚物、山梨醇脂肪酸酯、泊洛沙姆、聚山梨酯、脂肪酸山梨坦(司盘类)、辛基苯基聚氧乙烯醚或聚氧乙烯脂肪酸酯。其中阴离子大分子可以是羧甲基纤维素钠、丙烯酸树脂、海藻酸钠或DNA。其中凝聚剂可以是硫酸钠、氯化钠或硫酸铵。
本发明中所述重组人血管内皮抑制素为重组人血管内皮抑制素Endostar,重组人血管内皮抑制素重量占组合物重量的1%wt至50%wt,优选20%wt至35%wt。
本发明中注射用重组人血管内皮抑制素纳米粒组合物通过已知方法制备,包括共价交联法、离子诱导法、大分子复合法、去溶剂化法、反相胶束法。
本发明中重组人血管内皮抑制素纳米粒组合物可制成注射液或冻干粉针。
本发明中所述冻干粉针是在注射剂中加入药学上可接受的赋形剂后喷雾干燥或冷冻干燥得到。所述的赋形剂为下述一种或其中任意两种或两种以上混合的多元醇,包括甘露醇、乳糖、海藻糖、葡萄糖、蔗糖、麦芽糖、山梨醇或果糖。
考虑从静脉注射用药的安全性,本发明制备的重组人血管内皮抑制素纳米粒的粒径分布为10-1000nm。
本发明的重组人血管内皮抑制素纳米粒组合物可以延长重组人血管内皮抑制素的释放。
本发明的优点主要在于:
第一本发明组合物中可生物降解的材料主要是天然聚合物壳聚糖或壳聚糖的衍生物,在许多无机或有机酸的水溶液中都可以溶解,可以避免制备过程中使用有机溶剂(如聚乳酸-羟基乙酸需使用二氯甲烷等有机溶剂溶解),破坏蛋白活性;
第二与微球等给药系统相比,本发明中的组合物工艺路线简单可行,成本低廉;制备条件温和,对蛋白活性影响很小。
第三本发明得到的组合物释放时间可控制在7天,且释放完全。
因此,将重组人血管内皮抑制素制备成纳米粒制剂不仅能保护蛋白不会失活,并能实现缓释,降低毒副作用,达到长效、安全的目的,该剂型的研究将改善患者的痛苦,提高患者的临床顺应性。
附图说明
图1实施例4的重组人血管内皮抑制素纳米粒组合物粒径分布图。
图2实施例5的重组人血管内皮抑制素纳米粒组合物释放曲线。
具体实施方式
本发明通过下列实施例作更详细的描述,但本发明的保护范围不局限于此。
实施例一
称取分子量为100000道尔顿的羧基壳聚糖100mg,溶于50ml司盘-80溶液中。以蒸馏水配制5mg/ml的硫酸钠溶液4ml。将含30mg Endostar醋酸盐缓冲液(pH5.5)加入壳聚糖溶液中,磁力搅拌下缓慢滴加硫酸钠溶液,反应30分钟,100W功率下超声5分钟,10000转/分条件下离心30分钟,分离纳米粒。将所得纳米粒重新分散于50ml水中,加入1g果糖,冷冻干燥,即得重组人血管内皮抑制素纳米粒。所得纳米粒载药量为17.1%。
实施例二
称取分子量为200000道尔顿的羧甲基壳聚糖500mg,溶于50ml 1%的醋酸溶液中。以蒸馏水配制5mg/ml的海藻酸钠溶液20ml。将含200mg Endostar醋酸盐缓冲液(pH5.5)加入壳聚糖溶液中,磁力搅拌下缓慢滴加海藻酸钠溶液,反应60分钟,将上述溶液在4摄氏度,10000转/分条件下离心30分钟,分离纳米粒。将所得纳米粒重新分散于50ml水中,加入1g蔗糖,冷冻干燥,即得重组人血管内皮抑制素纳米粒。所得纳米粒载药重为20.7%。
实施例三
称取分子量为150000道尔顿的壳聚糖250mg,溶于50ml 1%的醋酸溶液中,得到5mg/ml的壳聚糖溶液。以蒸馏水配制2.5mg/ml的三聚磷酸钠溶液20ml。将含100mgEndostar磷酸盐缓冲液(pH5.5)和10ml聚乙二醇加入壳聚糖溶液中,磁力搅拌下缓慢滴加三聚磷酸钠溶液,反应10分钟,100W功率下探头超声2分钟,将上述溶液在4摄氏度,10000转/分条件下离心30分钟,分离纳米粒。将所得纳米粒重新分散于50ml水中,加入1.5g海藻糖,冷冻干燥,即得重组人血管内皮抑制素纳米粒。所得纳米粒载药量为22.1%。
实施例四
称取分子量为100000道尔顿的壳聚糖250mg,溶于50ml 1%的醋酸溶液中。以蒸馏水配制5mg/ml的三聚磷酸钠溶液10ml。将含100mg Endostar醋酸盐缓冲液(pH5.5)加入壳聚糖溶液中,磁力搅拌下缓慢滴加三聚磷酸钠溶液,反应5分钟,100W功率下探头超声2分钟。将上述溶液在4摄氏度,10000转/分条件下离心30分钟,分离纳米粒。将所得纳米粒重新分散于50ml水中,加入1.0g海藻糖,冷冻干燥,即得重组人血管内皮抑制素纳米粒。所得纳米粒载药量为19.8%。
将冻干后的纳米粒用蒸馏水复溶,用nano-ZS90马尔文粒径仪测定纳米粒粒径。所得重组人血管内皮抑制素纳米粒粒径分布均匀,平均粒径为192nm,粒径分布见图1。
实施例五
称取分子量为100000道尔顿的壳聚糖100mg,溶于50ml 1%的醋酸溶液中,得到2mg/ml的壳聚糖溶液。以蒸馏水配制2mg/ml的三聚磷酸钠溶液10ml。将含80mgEndostar醋酸盐缓冲液(pH5.5)加入壳聚糖溶液中,磁力搅拌下缓慢滴加三聚磷酸钠溶液,反应60分钟,100W功率下超声2分钟。将上述溶液在4摄氏度,10000转/分条件下离心30分钟,分离纳米粒。将所得纳米粒重新分散于50ml水中,加入1.5g甘露醇,冷冻干燥,即得重组人血管内皮抑制素纳米粒。所得纳米粒载药量为34.9%。
称取重组人血管内皮抑制素纳米粒100mg,置透析袋内,加入pH值为7.4的磷酸盐缓冲液5ml,扎紧袋口后置于盛有(37±0.5)℃、pH值为7.4的磷酸盐缓冲液50ml的恒温振荡器中,转速100转/分。定时取样5ml,同时补加等量同温介质,释放的重组人血管内皮抑制素含量采用BCA方法(试剂盒来自于Thermo Scientific,名为BCATMProtein Assay Kit)测定,释放曲线见图2。
Claims (10)
1.一种注射用重组人血管内皮抑制素纳米粒组合物,其特征在于包括:
a)壳聚糖或壳聚糖的衍生物;
b)生物相容性的助剂;和
c)重组人血管内皮抑制素。
2.根据权利要求1所述的纳米粒组合物,其特征在于所述壳聚糖衍生物包括羧甲基壳聚糖、羧基壳聚糖、羟烷基壳聚糖、酰基化壳聚糖、磺化壳聚糖、壳聚糖季铵盐的一种或多种。
3.根据权利要求1所述的纳米粒组合物,其特征在于所述的生物相容性的助剂包括化学交联剂、表面活性剂、阴离子大分子或凝聚剂。
4.根据权利要求3所述的纳米粒组合物,其特征在于所述的化学交联剂包括戊二醛或三聚磷酸钠。
5.根据权利要求3所述的纳米粒组合物,其特征在于所述的表面活性剂包括聚乙二醇、聚氧乙烯-聚氧丙烯嵌段共聚物、山梨醇脂肪酸酯、泊洛沙姆、聚山梨酯、脂肪酸山梨坦、辛基苯基聚氧乙烯醚或聚氧乙烯脂肪酸酯。
6.根据权利要求3所述的纳米粒组合物,其特征在于所述的阴离子大分子包括羧甲基纤维素钠、丙烯酸树脂、海藻酸钠或DNA。
7.根据权利要求3所述的纳米粒组合物,其特征在于所述的凝聚剂包括硫酸钠、氯化钠或硫酸铵。
8.根据权利要求1所述的纳米粒组合物,其特征在于所述的重组人血管内皮抑制素为重组人血管内皮抑制素Endostar。
9.根据权利要求1所述的纳米粒组合物,其特征在于所述的重组人血管内皮抑制素重量占组合物重量的1%wt至50%wt。
10.根据权利要求1-9中任一项所述的纳米粒组合物,其特征在于所述的纳米粒组合物可制成注射液或冻干粉针。
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CN102039108A (zh) * | 2011-01-13 | 2011-05-04 | 广州市戴文高分子材料科技有限公司 | 一种香精微胶囊的制备方法 |
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CN101953796A (zh) * | 2010-07-20 | 2011-01-26 | 江苏先声药物研究有限公司 | 一种注射用重组人血管内皮抑制素壳聚糖纳米粒的制备方法 |
CN102039108A (zh) * | 2011-01-13 | 2011-05-04 | 广州市戴文高分子材料科技有限公司 | 一种香精微胶囊的制备方法 |
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