CN104399084B - Peg化维甲酸及其自组装胶束在药物传递中的应用 - Google Patents
Peg化维甲酸及其自组装胶束在药物传递中的应用 Download PDFInfo
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Abstract
本发明涉及一种两亲性PEG化维甲酸前药及其自组装胶束在药物传递中应用。所述的两亲性前药嵌段以聚乙二醇为亲水端，通过酯键与一分子疏水性的维甲酸结合，得到了AB型两亲性前药嵌段。该聚合物具有潜在治疗白血病，延长药物半衰期作用，同时具有较高的载药量，有效的增加胃肠道粘膜透过而增加口服生物利用度的优势。该PEG化前药在水性介质中自组装形成胶束，可作为难溶性药物维甲酸的储库，缓慢释放维甲酸。该胶束安全性好，可用于口服给药，具有较大的市场应用前景。
Description
技术领域
本发明属于药物制剂新辅料和新剂型领域，涉及PEG化维甲酸及其自组装胶束在药物传递中应用，具体涉及不同链长聚乙二醇维甲酸两亲性前药作为药物载体在药物传递中的应用。
背景技术
纳米载体由于能够提高抗癌药物的疗效并且减少药物副作用而被广泛用于靶向药物传递系统，PEG化的纳米载体更能显示出一些独特的优越性，例如延长纳米粒体内循环时间；通过EPR效应提高药物在肿瘤的聚集量；提高药物的耐受性等等。在众多载体中，PEG化纳米胶束凭借可修饰的化学结构和水溶液中自组装成核壳结构而受到更多的关注。然而，研究发现胶束表面的PEG水化层会阻碍药物从胶束中释放出来，从而降低了药物的疗效，限制了临床应用。
与静脉注射，口服吸收纳米粒为更加复杂的过程,涉及黏液层截留和渗透、肠道细胞吸收和肝脏首过效应。PEG化的胶束吸收入血后，PEG作为亲水基，能形成空间立体屏障，从而能避免网状内皮系统(RES)的摄取，延长循环时间，进而通过EPR效应使更多纳米粒聚集在肿瘤部位。然而,关于PEG链长的水化层对口服纳米粒的吸收影响几乎没有文献报道。在此，我们旨在研究不同PEG链长对胶束渗透效率和口服生物利用度的的影响。本课题制备了PEG化维甲酸，其本身既可作为前药胶束的口服给药，具有潜在治疗白血病，延长药物半衰期作用，同时具有较高的载药量，有效的增加胃肠道粘膜透过而增加口服生物利用度的优势，也可作为载体应用于药物传递系统，将具有很大的应用前景。
发明内容
本发明旨在提供一种具有治疗急性粒细胞白血病、延长药物半衰期、具有较高的载药量的两亲性PEG化维甲酸的自组装前药分子。
本发明第二个目的旨在提供上述不同PEG链长的维甲酸前药胶束的制备方法。
本发明的第三个目的旨在提供两亲性前药嵌段胶束在药物传递中的应用。
本发明通过以下技术方案实现上述目的：
该PEG化维甲酸以聚乙二醇为亲水端，以酯键链接一分子疏水性的维甲酸而形成两亲性前药，是一种稳定性好、功能性强的PEG化前药。
所述的PEG化前药(Ⅰ)的结构式通式如下：
所述R为C1-C4烷基。
所述的PEG化前药，其特征在于所用的聚乙二醇的分子量范围500-5000,n为其相应的聚合度，为11到112；优选500-1000，n的值为11到22。
所述的聚乙二醇：优选单甲氧基聚乙二醇(mPEG)。
以单甲氧基聚乙二醇(mPEG)为例，本发明的PEG化维甲酸是通过如下方法制备的：全反式维甲酸(ATRA)溶于少量二氯甲烷中，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和4-二甲氨基吡啶(DMAP)催化作用下，避光冰浴0.5-2h，然后与不同链长聚乙二醇(PEG)在30-40℃N2保护下反应10-12h，经分离纯化得到黄色油状的聚乙二醇化维甲酸两亲性前药。反应式如下：
步骤中n为11到112。
所述的两亲性不同PEG链长的PEG化前药具有治疗急性粒细胞白血病、延长药物半衰期作用，具有较高的载药量。
所述的PEG化维甲酸可采用超声法或乳化溶剂挥发法制备聚合物胶束，其特征在于采用下述步骤：是将上述的PEG化维甲酸完全溶解于少量的二氯甲烷中，再转移到适量的蒸馏水中，探头超声并避光搅拌12h后，过0.22微米滤膜过滤，得到前药胶束溶液。
载药胶束制备：将PEG化维甲酸和抗肿瘤药物(阿霉素)完全溶解于少量的二氯甲烷中，再转移到适量的蒸馏水中，探头超声并避光搅拌12h后，13000rpm离心10min，取上清液即得到载药胶束溶液。
所述的药物选自：常用的抗肿瘤药物如阿霉素等。
本发明具有以下有益效果：制备的PEG化维甲酸前药嵌段，载体制备过程温和，易操作。所制备的前药聚合物胶束，制备简便，粒径较小且均一，临界胶束浓度低，稳定性好，可作为难溶性药物、蛋白质或基因药物的储库，且具有潜在治疗白血病，延长药物半衰期作用，同时具有较高的载药量，有效的增加胃肠道粘膜透过而增加口服生物利用度。
附图说明
图1为本发明单甲氧基聚乙二醇维甲酸(ATRA-PEGs)前药嵌段结构的合成过程示意图。
图2为本发明实施例2的单甲氧基聚乙二醇500维甲酸(ATRA-PEG500)前药嵌段结构的1HNMR谱图。
图3为本发明实施例2的单甲氧基聚乙二醇1000维甲酸(ATRA-PEG1000)前药嵌段结构的1HNMR谱图。
图4为本发明实施例2的单甲氧基聚乙二醇2000维甲酸(ATRA-PEG2000)前药嵌段结构的1HNMR谱图。
图5为本发明实施例2的单甲氧基聚乙二醇5000维甲酸(ATRA-PEG5000)前药嵌段结构的1HNMR谱图。
图6为本发明实施例2的单甲氧基聚乙二醇500维甲酸(ATRA-PEG500)前药嵌段结构的IR谱图。
图7为本发明实施例2的单甲氧基聚乙二醇1000维甲酸(ATRA-PEG1000)前药嵌段结构的IR谱图。
图8为本发明实施例2的单甲氧基聚乙二醇2000维甲酸(ATRA-PEG2000)前药嵌段结构的IR谱图。
图9为本发明实施例2的单甲氧基聚乙二醇5000维甲酸(ATRA-PEG5000)前药嵌段结构的IR谱图。
图10为本发明实施例2的单甲氧基聚乙二醇2000维甲酸(ATRA-PEG2000s)前药嵌段结构的UV谱图。
图11为本发明实施例4的前药聚合物胶束(ATRA-PEGs)的动态光散射测定胶束粒径图。
图12为本发明实施例4的前药聚合物胶束(ATRA-PEGs)的透视电镜图。
图13为实施例5中单甲氧基聚乙二醇维甲酸前药嵌段荧光激发光强度比值I383/I377与浓度对数关系图。
图14为本发明实施例7中单甲氧基聚乙二醇维甲酸(ATRA-PEGs)前药胶束的在体原位肠灌流的(Papp)柱状图。
图15为本发明实施例7中单甲氧基聚乙二醇维甲酸(ATRA-PEGs)前药胶束的在体原位肠灌流的(Ka)柱状图。
图16为本发明单甲氧基聚乙二醇维甲酸(ATRA-PEG1000)前药胶束肠道摄取机制图。
图17为本发明实施例10中单甲氧基聚乙二醇维甲酸(ATRA-PEGs)前药胶束口服生物利用度图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将发明限制在所述的实施例范围之中。
实施例1
不同PEG链长的维甲酸前药嵌段的制备。
全反式维甲酸(ATRA)溶于少量二氯甲烷中，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和4-二甲氨基吡啶(DMAP)催化作用下，避光冰浴0.5-2h，然后与不同链长聚乙二醇(PEG)在30-40℃N2保护下反应10-12h，经分离纯化得到黄色油状的聚乙二醇化维甲酸两亲性前药。反应式如下：
实施例2
反应式中的mPEG可以为mPEG500，mPEG1000，mPEG2000和mPEG5000即所选用的聚乙二醇分子量为500,1000,2000或者5000，本发明的聚乙二醇是一端甲基化修饰的聚乙二醇，但是并不局限于以上四种物质。
采用核磁共振测定1HNMR氢谱来确定嵌段共聚物的结构，选用的溶剂为CHCl3，结果如图2到图5。ATRA-PEG500的质谱图，质子峰从1.1到2.3ppm，5.8到7.1ppm是为维甲酸的特征峰，3.52-3.75ppm间的质子峰为PEG中的H，3.37-3.38ppm的质子峰为PEG端头的甲基(-CH3)特征峰，即从3.3到3.8ppm为mPEG质子峰，而新出现的峰值(a)约为4.2ppm是属于亚甲基(图2)附近的酯键。
实施例3
采用红外图谱来确定实施例2中嵌段共聚物的结构，选用KBr为空白辅料，结果如图6到图9。ATRA-PEG500，在3444cm-1羟基的宽吸收峰消失和新的峰值约1707cm-1出现，新出现的峰位是由于ATRA的羧羟基转变成ATRA-PEG500中的酯键，这些结果表明PEG化维甲酸的形成。
实施例4
乳化探头超声制备载维甲酸前药胶束
乳化探头超声法：称取适量的上述的前药嵌段，乳化探头超声法是将上述的PEG化维甲酸完全溶解于少量的二氯甲烷中，再转移到适量的蒸馏水中，探头超声并避光搅拌12h后，过0.22微米滤膜过滤，得到前药胶束溶液。
将实施例2中制备的两种载药胶束通过动态光散射和透视电镜测定胶束的粒径大小和形态，结果如图5、图6。动态光散射测定四种不同PEG链长的前药胶束的粒径为13-20nm；粒径分布较窄；透视电镜图表明载药胶束为粒径均一的球形，透射电镜的粒径比动态光散射测定的结果略小。
实施例5
两亲性ATRA-PEG聚合物临界胶束浓度的测定。
临界胶束浓度的测定广泛采用芘荧光探针法。芘是一种脂溶性的荧光探针，在极性环境中荧光较弱，而在非极性环境中荧光较强。当极性溶剂中有胶团或疏水区域时，芘由极性环境会自发向非极性环境转移，引起荧光加强。通常芘的这一特性通过第一激发峰和第三激发峰强度的比值来反应，当这一比值显著增大时，为芘由极性环境向非极性环境迁移，即为胶束或疏水区域产生。
将5×10-6mol/L芘的无水乙醇溶液加入到10mL的具塞烧瓶中，氮气流吹干除去无水乙醇。精密称定10mg的实施例2制备的聚合物单甲氧基聚乙二醇维甲酸前药嵌段分别溶解得到溶液浓度为1mg/mL。再将聚合物溶液稀释成系列浓度(10-3，10-4，10-5，10-6，10-7，10-8，10-9mol/L)各5mL，分别加入到含有芘的烧瓶中，使芘的终浓度为5×10-7mol/L，避光超声4h，避光放置过夜。酶标仪测定荧光强度，激发波长为336nm，发射波长分别为I1＝383nm，I2＝377nm。
以I383/I377的荧光强度比值和聚合物胶束的浓度对数作图如图13，曲线的拐点即为聚合物的临界胶束浓度(CMC)，测定结果为四种不同单甲氧基聚乙二醇维甲酸前药嵌段的CMC值分别为14.3μg/mL,3.30μg/mL,3.45μg/mL和30.57μg/mL。
图13结果证明本发明的聚合物载体临界胶束浓度较低，易于自组装形成胶束，且形成的胶束具有较好的稀释稳定性。
实施例6
大鼠体外稳定性试验：大鼠禁食麻醉后，眼眶取血后，打开腹腔，取出肝脏，十二指肠，制备出血浆，肝匀浆，肠匀浆。同时配置PH1.2和PH6.8的缓冲液进行稳定性试验。
稳定性实验结果(如下表)表明：ATRA-PEG2000具有较高的稳定性，其次ATRA-PEG1000，ATRA-PEG500和ATRA-PEG5000较差；但四种前药胶束制剂都容易在肝匀浆中代谢。
实施例7
大鼠在体肠灌流：实验前用供试液将蠕动泵管路冲洗至出口处药物溶液浓度与供试液浓度相同，大鼠禁食不禁水12h，腹腔注射麻醉后，将大鼠固定，打开腹腔，分离出十二指肠、空肠、回肠、结肠段进行肠灌流试验。
试验结果如图14和图15，与原料药溶液剂相比，ATRA-PEG1000胶束制剂在小肠中具有较高的粘膜透过性。
实施例8
大鼠肠道染色试验：大鼠禁食不禁水12h，灌胃给予香豆素-6溶液剂或载香豆素-6的胶束制剂。45min后，腹腔注射麻醉，分别取出一小段十二指肠、空肠，回肠和结肠，再冷冻、切片、固定、组织透化、染色后，共聚焦显影。
共聚焦结果表明：ATRA-PEG前药胶束在整个肠段均有分布，沿着肠道顺序依次减少。
实施例9
大鼠外翻肠环摄取机制研究：大鼠禁食麻醉后，打开腹腔，取出十二指肠段，清洗、外翻、切成小段，加入了内吞抑制剂(氯丙嗪为网格蛋白抑制剂，吲哚美辛为小窝蛋白抑制剂，秋水仙碱为巨胞饮抑制剂，槲皮素为非网格蛋白非小窝蛋白抑制剂，叠氮钠为能量抑制剂)进行摄取抑制试验。同时进行37℃不加抑制剂的肠段吸收和4℃不加抑制剂的肠段吸收情况考察。
实验结果表明：ATRA-PEG1000胶束制剂的摄取为小窝蛋白和网格蛋白介导，能量依赖性的内吞过程。
实施例10
不同PEG链长的维甲酸前药嵌段胶束的药代动力学研究
取35只健康、雄性大鼠，体重200g左右，随机分为5组，给药前禁食12h，自由饮水。一组静脉口服维甲酸溶液(33.4mg/Kg)作为对照，另一组分别口服实施例1到实施例4制备的前药胶束，剂量为16.7mg/kg的维甲酸，眼眶取血，测定维甲酸浓度，药时曲线见图17。
由图17可知，给予维甲酸前药胶束后，与溶液组相比，维甲酸胶束消除半衰期显著延长，具有显著的长循环作用。同时，ATRA-PEG1000药时曲线下面积明显增加，维甲酸的生物利用度提高了2倍，ATRA-PEG500提高了1.2倍，明显提高了药物的生物利用度。
Claims (5)
1.聚乙二醇化维甲酸，其特征在于，以聚乙二醇为亲水端，以酯键链接一分子维甲酸为疏水端，结构式如下所示：
R为C1-C4烷基，所述聚乙二醇分子量为1000。
2.根据权利要求1所述的聚乙二醇化维甲酸，其特征在于，所述的聚乙二醇为单甲氧基聚乙二醇。
3.权利要求1或2所述的聚乙二醇化维甲酸在制备提高药物生物利用度的药物中的应用。
4.一种载药胶束，其特征在于，通过如下方法制备：将权利要求1或2所述的PEG化维甲酸和抗肿瘤药物完全溶解于少量的二氯甲烷中，再转移到适量的蒸馏水中，探头超声并避光搅拌后，离心，取上清液即得到载药胶束溶液。
5.如权利要求4所述的载药胶束，其特征在于，所述的药物为阿霉素。
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