CN113577299B - 一种ros响应性的单抗类药物口服纳米粒及其制备方法 - Google Patents
一种ros响应性的单抗类药物口服纳米粒及其制备方法 Download PDFInfo
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- CN113577299B CN113577299B CN202110586387.3A CN202110586387A CN113577299B CN 113577299 B CN113577299 B CN 113577299B CN 202110586387 A CN202110586387 A CN 202110586387A CN 113577299 B CN113577299 B CN 113577299B
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Abstract
本发明公开了一种ROS响应性的单抗类药物口服纳米粒及其制备方法。该单抗类药物口服纳米粒由如下方法获得:以具有对活性氧响应性断裂能力的酮缩硫醇交联剂将单抗药物交联形成纳米复合物,再将透明质酸通过化学键及静电吸附作用包裹于纳米复合物表面,得到所述ROS响应性的单抗类药物口服纳米粒。本发明通过将透明质包裹在纳米复合物表面,可以增强载药复合物的胃内稳定性。此外,交联剂可以在肠道炎症处特异性降解,从而释放游离药物。本发明的ROS响应性的单抗类药物口服纳米粒具有减少不良反应,提高蛋白药物的载药量和包封率,同时实现炎症靶向定位释放等优点。
Description
(一)技术领域
本发明涉及一种ROS响应性的单抗类药物口服纳米粒及其制备方法,属于药物制剂技术领域。
(二)背景技术
炎症性肠病是一种慢性非特异性肠道炎症疾病。患者肠道会出现炎症、糜烂、溃疡和大出血等,还会产生各种严重的并发症,危及生命。迄今为止,炎症性肠病还不能被治愈;因此,需要终生使用药物来缓解症状。目前,治疗炎症性肠病的药物,包括5-氨基水杨酸衍生物、皮质类固醇、免疫抑制剂,以及新型生物类大分子抗体药物(抗肿瘤坏死因子抑制剂(TNF-α),整合素拮抗剂等)。新型生物类大分子抗体药物,在临床应用中显示了良好的治疗效果,应用越来越广泛。但是,新型生物类大分子抗体药物通常采用注射方式给药,这种全身给药方式在药物缺乏对靶部位的选择性作用下致使全身免疫系统受到抑制,从而产生一系列不良反应。如果能通过口服给药的手段,将抗体药物直接转运至肠道炎症部位发挥疗效,则可以避免药物全身给药引起的不良反应。
如何实现抗体药物的口服给药以及在炎症部位的定位释放,是一个巨大的挑战。一方面,大分子药物易被消化道中的酸碱环境破坏而失去活性。另一方面,炎症部位在肠道的分布通常是不连续的分布。常规的口服给药方式,只有一小部分药物可能到达炎症部位,降低了药效,增加了副作用。研究表明,在炎症性肠病的病患者中,肠道炎症区域的粘膜活性氧(ROS)浓度要比健康粘膜组织高10-100倍,这种差异可以用于药物在炎症区域的定位释放,减少药物对正常黏膜组织的影响。
目前,市场上尚未见到针对炎症性肠病的,抗体靶向口服纳米制剂,也未见到类似药物制剂的研究报道。因此,开发这类药物不仅有重要的临床意义,也有广阔的市场前景。
(三)发明内容
基于现有技术中存在的缺陷和市场的需求,本发明提供了一种ROS响应性的单抗类药物口服纳米粒及其制备方法。
为了解决上述技术问题,本发明采用的技术方案是:
一种ROS响应性的单抗类药物口服纳米粒,由如下方法制备获得:以具有对活性氧(ROS)响应性断裂能力的酮缩硫醇交联剂将单抗药物交联形成纳米复合物,再将透明质酸通过化学键及静电吸附作用包裹于纳米复合物表面,得到所述炎症靶向的单抗类药物口服纳米粒;所述酮缩硫醇交联剂为二(4-硝基苯基)-C,C'-二甲基硫代二碳酸二乙酯(交联剂)、或二(4-硝基苯基)-C,C'-二甲基硫代二碳酸丙酯(交联剂)、或其同系物。
本发明通过具有对活性氧响应性断裂能力的交联剂,将抗体药物交联形成性能优良的纳米复合物,再将透明质酸修饰到抗体复合物表面,形成纳米粒。该纳米粒的交联剂可在炎症部位高浓度活性氧条件下断裂,释放出游离的抗体药物。本发明最终得到了具有高载药量和包封率,且具有炎症靶向性能的单抗类药物的口服纳米粒。
优选的,所述单抗药物为抗TNF-α单抗药物,或抗整合素抗体。
优选的,所述透明质酸为分子量范围为10~100kDa的氨基化透明质酸,透明质酸与单抗物质的量之比为1~10:1。
本发明还涉及制备所述ROS响应性的单抗类药物口服纳米粒的方法,所述方法包括如下步骤:
(1)纳米复合物的制备:取酮缩硫醇交联剂用DMSO溶解,缓慢滴加到单抗的碳酸盐缓冲液中,室温反应过夜;
(2)纳米粒的制备:取透明质酸溶液,滴加到步骤(1)反应液中,室温反应过夜,透析除去未反应的杂质,葡聚糖凝胶柱除去未反应的单抗,即可得到所述的单抗类药物口服纳米粒。所述纳米粒的粒径为100nm~500nm。
所述酮缩硫醇交联剂二(4-硝基苯基)-C,C'-二甲基硫代二碳酸二乙酯(交联剂)可由如下方法制备获得:
(1)取β-巯基乙醇、氟化钾和冰醋酸加入到圆底烧瓶中,于80℃搅拌反应16h,反应结束后,向反应液中加入适量纯水,用适量乙酸乙酯萃取后,收集有机相,将有机相先后用饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤,取有机层用无水硫酸钠干燥,旋干,得乙酸巯基乙酯(产物1)。
(2)称取产物1、2,2-二甲氧基丙烷和对甲苯磺酸于圆底烧瓶中,加入甲苯和5A分子筛,室温搅拌反应24h,反应结束后,除去溶剂,进行硅胶柱层析,收集洗脱液,旋干,得5,5-二甲基-4,6-二硫-壬二酸二甲酯(产物2)。
(3)取产物2和氢氧化钾适量,加入1,4-二氧六环,室温反应,反应结束后,减压浓缩,除去1,4-二氧六环,用稀盐酸调节pH至酸性,乙酸乙酯萃取,无水硫酸钠干燥,旋干,得5,5-二甲基-4,6-二硫-壬二醇(产物3)。
(4)取产物3和二(对硝基苯)碳酸酯,置于圆底烧瓶中,加入适量无水乙腈,搅拌溶解,再逐滴加入无水三乙胺,于室温下搅拌反应12h。反应结束后,将反应液旋转蒸发,使溶剂完全旋干。向其中加入适量二氯甲烷,用纯水洗涤3次,收集有机相用无水硫酸钠干燥,浓缩,过硅胶柱,收集洗脱液旋干,得到所述酮缩硫醇交联剂二(4-硝基苯基)-C,C'-二甲基硫代二碳酸二乙酯。
涉及反应式如下:
优选的,步骤(2)中透明质酸与单抗物质的量之比为1~10:1。步骤(2)中交联剂与单抗物质的量之比为10~30:1。
优选的,所述单抗为英夫利昔单抗、阿达木单抗、赛妥珠单抗或维多珠单抗。所述单抗还可以替换为其它具有免疫调节或抗炎症性质的抗体药物、水溶性蛋白、多肽等。
本发明的有益效果主要体现在:(1)本发明ROS响应性的单抗类药物口服纳米粒可通过口服给药的给药方式,将抗体药物转运至肠道炎症部位,可以避免药物全身给药的不良反应,同时增加患者的顺应性。(2)本发明方法得到的单抗类药物口服纳米粒,利用药物自身的交联性能,没有使用载体材料,可以大大提高抗体药物的载药量和包封率。(3)本发明方法制备的ROS响应性单抗类药物口服纳米粒,利用肠道炎症组织活性氧的浓度远远高于健康组织这一特性,制备的交联剂,使载药的纳米粒在肠道炎症部位定位释放,从而实现药物在肠道炎症部位的靶向功能。
(四)附图说明
图1交联剂的1H-NMR表征。
图2交联剂的高效液相色谱表征。
图3交联剂对活性氧响应性断裂实验。
图4为英夫利昔单抗纳米粒的粒径图。
图5为不同给药组小鼠给药后的结肠长度。
图6为不同给药组小鼠给药后肠道组织中TNF-α的浓度。
(五)具体实施方式
通过下列实例对本发明的内容做进一步说明,但本发明的保护范围,不限于此。
实施例1:
本实例为采用本发明提供出的方法制备ROS响应性的英夫利昔单抗口服纳米粒。
制备步骤:
1.交联剂的合成
称取-巯基乙醇(0.5g,6.39mmoL)和氟化钾(KF,0.45g,7.81mmoL)加入到圆底烧瓶中,加入10.3mL冰醋酸,于80℃搅拌反应16h,反应结束后,向反应液中加入适量纯水,用适量乙酸乙酯萃取3次后收集有机相,将有机相先后用饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤,取有机层用无水硫酸钠干燥,旋干,得透明油状液体乙酸巯基乙酯(产物1)。
称取产物1(0.2g,1.68mmoL)、2,2-二甲氧基丙烷(0.07g,0.67mmoL)和对甲苯磺酸(0.144g,0.84mmoL)于圆底烧瓶中,加入4.16mL甲苯和1.67mg 5A分子筛,室温搅拌反应24h,反应结束后,除去分子筛,减压浓缩,除去溶剂,进行硅胶柱层析,洗脱液为正己烷:乙酸乙酯=7:1,收集洗脱液,旋干,得透明油状液体为5,5-二甲基-4,6-二硫-壬二酸二甲酯(产物2)。
取产物2(0.055g,0.196mmoL)和氢氧化钾(0.05g,0.885mmoL),加入1,4-二氧六环2mL、1mL水,室温反应16h,反应结束后,减压浓缩,除去1,4-二氧六环,加入少量纯水,用稀盐酸调节pH至酸性,乙酸乙酯萃取,无水硫酸钠干燥,旋干,得无色稠状液体为5,5-二甲基-4,6-二硫-壬二醇(产物3)。
称取产物3(0.05g,0.255mmoL)和二(对硝基苯)碳酸酯(0.233g,0.765mmoL),置于圆底烧瓶中,加入适量无水乙腈,搅拌溶解,再逐滴加入1.5mL无水三乙胺,于室温下搅拌反应12h。反应结束后,将反应液于45℃下减压旋转蒸发,使溶剂完全旋干。向其中加入适量二氯甲烷,用纯水洗涤3次,收集有机相用无水硫酸钠干燥,浓缩,乙酸乙酯:石油醚=1:1过硅胶柱,收集洗脱液旋干,得白色固体粉末—二(4-硝基苯基)-C,C'-二甲基硫代二碳酸二乙酯(产物4,交联剂)。
2.交联剂的表征
取适量交联剂溶于0.6mL氘代DMSO中,超声助溶,溶解后转移至配套核磁管中,于核磁共振波谱仪中测定。结果如图1所示,δ=1.62ppm(e)为-CH3-的特征峰,δ=3.01ppm(d)为-CH2-的特征峰,δ=4.40ppm(c)为碳酸酯键旁边的-CH2-的特征峰,δ=7.56ppm(b)为苯环上硝基间位上的H的特征峰,δ=8.29ppm(a)为苯环上硝基邻位上的H的特征峰,证明含酮缩硫醇键的交联剂合成成功。
3.交联剂的高效液相色谱表征
交联剂的高效液相色谱条件:色谱柱为InertSustain C18柱(4.6×150nm5μm),流动相为甲醇:水=80:20,检测波长为268nm,进样量为20μL,柱温为30℃,流速:1mL/min,运行时间20min。交联剂的HPLC图谱如图2所示,保留时间为10.127min,主峰处无杂峰干扰,峰形尖锐对称。由图可以看出,所得交联剂纯度高。
4.交联剂的活性氧响应性考察
分别配制浓度为50和100mM H2O2的PBS溶液,配制3份浓度为10μg/mL交联剂的DMSO溶液,取0.1mL分别加入0.2mL PBS和50、100mM H2O2 PBS溶液,涡旋,混匀,放置于37℃恒温摇床中(100rpm),于0、0.5、1、2、4、8和12小时取样,离心后进行HPLC分析,测定溶液中未被氧化的剩余交联剂含量。结果如图3所示,交联剂在PBS溶液几乎不发生断键;在50m M H2O2和100m M H2O2的PBS溶液中,6小时后,溶液中几乎无交联剂残留。说交联剂具有良好的氧化响应性能。
5.纳米粒的制备
取英夫利昔单抗的碳酸盐缓冲液0.5mL(c=0.2μmol/mL),在搅拌下加入交联剂的DMSO溶液1mL(c=2μmol/mL),室温反应过夜后,得到英夫利昔单抗的纳米复合物(IFX NP)。之后,加入0.5mL氨基化透明质酸(分子量为20kDa)(c=1μmol/mL)的碳酸盐缓冲液,调节pH值为9.6,室温搅拌反应过夜,3500Da透析袋透析5h,最后采用葡聚糖凝胶柱Sephadex G100除去杂质,即可得到载药量为39.1%,包封率为90%的透明质酸修饰的英夫利昔单抗纳米粒(IFX NP/HA),其粒径图参见图4。
6.纳米粒的表征
取制备好的纳米粒1mL,使用Zetasizer对粒径和zeta电位进行了表征。结果见图下表:
粒径(nm) | 电位(mv) | |
IFX NP | 166.4±3.1 | -13.7±1.4 |
IFX NP/HA | 182.9±3.5 | -16.2±1.2 |
IFX NP为英夫利昔单抗纳米复合物;IFX NP/HA为透明质酸修饰的英夫利昔单抗纳米粒。
7.药效学实验
使用TNBS制造肠炎小鼠模型。将肠炎小鼠随机分为6组,分别为(1)健康组;(2)结肠炎+灌胃生理盐水(对照组);(3)结肠炎+灌胃透明质酸溶液组;(4)结肠炎+尾静脉注射英夫利西单抗组(5mg/kg);(5)结肠炎+灌胃英夫利昔单抗组(5mg/kg),(6)结肠炎+灌胃英夫利昔单抗纳米粒组(5mg/kg),连续给药三天,造模后5天,处死小鼠,取出肠道组织,测量长度,结果见图5。
由图可知,口服载药的纳米粒给药组的小鼠肠道的长度,相比尾静脉给药组和口服游离药组更接近于健康组小鼠长度,说明口服载药的纳米粒可以更好的保留肠道长度,显示了优异的药效学性质。
8.TNF-α含量的测定
取出结肠组织,放入Ep管中剪碎,加入组织裂解液,用手持式高速匀浆机将组织匀浆,10s/次,共匀浆3次,冰上孵育30min后离心(10000×g,4℃,15min),取上清液备用。采用ELISA试剂盒法测定小鼠结肠粘膜组织中TNF-α含量。结果见图6。
结果发现,相比口服生理盐水组、尾静脉给药组和口服游离药组,口服纳米粒给药组的小鼠肠道组织的TNF-α含量大大降低,显示了优异的药效学性质。
实施例2:ROS响应性的英夫利昔单抗口服纳米粒制备
制备步骤:
1.交联剂的合成
同实施例1。
2.纳米粒的制备
取英夫利昔单抗的碳酸盐缓冲液0.5mL(c=0.2μmol/mL),在搅拌下加入交联剂的DMSO溶液1.2mL(c=2μmol/mL),室温反应过夜后加入0.25mL透明质酸(分子量为20kDa)(c=1μmol/mL)的碳酸盐缓冲液,调节pH值为9.6,室温搅拌反应过夜,3500Da透析袋透析5h,最后采用葡聚糖凝胶柱Sephadex G100除去杂质,即可得到载药量为52.5%,包封率为92%的英夫利昔单抗纳米粒。
实施例3:ROS响应性的英夫利昔单抗口服纳米粒制备
制备步骤:
1.交联剂的合成
同实施例1。
2.纳米粒的制备
取英夫利昔单抗的碳酸盐缓冲液0.5mL(c=0.2μmol/mL),在搅拌下加入交联剂的DMSO溶液1.5mL(c=2μmol/mL),室温反应过夜后加入0.4mL透明质酸(分子量为20kDa)(c=1μmol/mL)的碳酸盐缓冲液,调节pH值为9.6,室温搅拌反应过夜,3500Da透析袋透析5h,最后采用葡聚糖凝胶柱Sephadex G100除去杂质,即可得到载药量为43.4%,包封率为95%的英夫利昔单抗纳米粒。
以上对本发明的优选实施例及原理进行了详细说明,对本领域的普通技术人员而言,依据本发明提供的思想,在具体实施方式上会有改变之处,而这些改变也应视为本发明的保护范围。
Claims (6)
1.一种ROS响应性的单抗类药物口服纳米粒,由如下方法制备获得:以具有对活性氧响应性断裂能力的酮缩硫醇交联剂将单抗药物交联形成纳米复合物,再将透明质酸通过化学键及静电吸附作用包裹于纳米复合物表面,得到所述ROS响应性的单抗类药物口服纳米粒;所述酮缩硫醇交联剂为二(4-硝基苯基)-C,C '-二甲基硫代二碳酸二乙酯;所述单抗药物为英夫利昔单抗。
2.如权利要求1所述的单抗类药物口服纳米粒,其特征在于所述透明质酸为分子量范围为10~100kDa的氨基化透明质酸,透明质酸与单抗药物的物质的量之比为1~10:1。
3.制备权利要求1所述ROS响应性的单抗类药物口服纳米粒的方法,所述方法包括如下步骤:
(1)纳米复合物的制备:取酮缩硫醇交联剂用DMSO溶解,缓慢滴加到单抗的碳酸盐缓冲液中,室温反应过夜;
(2)纳米粒的制备:取透明质酸溶液,滴加到步骤(1)反应液中,室温反应过夜,透析除去未反应的杂质,葡聚糖凝胶柱除去未反应的单抗,即可得到所述ROS响应性的单抗类药物口服纳米粒。
4.如权利要求3所述的方法,其特征在于所述酮缩硫醇交联剂由如下方法制备获得:
(1)取β-巯基乙醇、氟化钾和冰醋酸加入到圆底烧瓶中,于80℃搅拌反应16h,反应结束后,向反应液中加入适量纯水,用适量乙酸乙酯萃取后,收集有机相,将有机相先后用饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤,取有机层用无水硫酸钠干燥,旋干,得乙酸巯基乙酯,记为产物1;
(2)称取产物1、2,2-二甲氧基丙烷和对甲苯磺酸于圆底烧瓶中,加入甲苯和5A分子筛,室温搅拌反应24h,反应结束后,除去溶剂,进行硅胶柱层析,收集洗脱液,旋干,得5,5-二甲基-4,6-二硫-壬二酸二甲酯,记为产物2;
(3)取产物2和氢氧化钾适量,加入1,4-二氧六环,室温反应,反应结束后,减压浓缩,除去1,4-二氧六环,用稀盐酸调节pH至酸性,乙酸乙酯萃取,无水硫酸钠干燥,旋干,得5,5-二甲基-4,6-二硫-壬二醇,记为产物3;
(4)取产物3和二(对硝基苯)碳酸酯,置于圆底烧瓶中,加入适量无水乙腈,搅拌溶解,再逐滴加入无水三乙胺,于室温下搅拌反应12h;反应结束后,将反应液旋转蒸发,使溶剂完全旋干,向残渣中加入适量二氯甲烷,用纯水洗涤3次,收集有机相用无水硫酸钠干燥,浓缩,过硅胶柱,收集洗脱液旋干,得到所述酮缩硫醇交联剂二(4-硝基苯基)-C,C '-二甲基硫代二碳酸二乙酯。
5.如权利要求3所述的方法,其特征在于,步骤(1)中交联剂与单抗的物质的量比为10~30:1。
6.如权利要求3所述的方法,其特征在于,步骤(2)中所述透明质酸为分子量范围为10~100kDa的氨基化透明质酸,透明质酸与单抗药物的物质的量之比为1~10:1。
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