CN100348198C - 单唾液酸四已糖神经节苷脂脂质体复合物制剂 - Google Patents
单唾液酸四已糖神经节苷脂脂质体复合物制剂 Download PDFInfo
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Abstract
本专利涉及单唾液酸四己糖神经节苷脂脂质体复合物制剂的组份、制备工艺和应用。经实验证明脂质体形式的单唾液酸四己糖神经节苷脂能显著提高体内生物利用度，能快速地进入脑组织中，更有效地治疗急慢性中枢神经损伤。
Description
单唾液酸四己糖神经节苷脂(GM1)属于含唾液酸的酸性糖脂，由含有亲脂的神经酰胺和一个亲水的唾液酸寡糖基团组成，分子量为1574道尔顿，主要分布于中枢神经组织中。具有促进神经元增生，神经营养作用和修复功能，抗兴奋性神经性作用等多重功效。单唾液酸四己糖神经节苷脂主要从牛猪的脑组织提取，临床上用于治疗中枢神经的损伤，如急性脊髓损伤，脑卒中和帕金森氏症。已证明在血脑屏障中存在GM1受体，能快速主动进入脑组织。GM1在血中的有效浓度与进入脑组织的量有密切关系。GM1进入血液后主要由肝脏和肾脏代谢和排泄。
脂质体是由带极性和非极性的脂类物形成的单层或多层的囊泡，一般直径为100-1000nm。由于脂质体在体内具有逐渐释放被包裹药物的作用，能极大地提高药物的体内生物利用度，减少药物的毒性作用，增强体内的药物治疗效果。脂质体作为一种药物载体制剂，已应用于临床多年。由于GM1属于酸性糖脂，同样被作为脂质体的膜成份用于形成脂质体。与其它磷脂相比，有一定的优势如物理化学稳定性增强和体内清除减慢。常用于形成脂质体的脂类物质主要由两大类组成：胆固醇和磷脂，其中磷脂包括中性和酸性磷脂。根据不同脂质体的要求选择磷脂的种类，目前多用的天然磷脂是大豆卵磷脂和蛋黄卵磷脂，合成磷脂是由含C14-18的饱和和不饱和脂肪链的磷脂，有磷脂酰胆碱(PC)，磷脂酰丝氨酸(PS)，磷脂酰乙醇氨(PE)，磷脂酰甘油(PG)和磷脂酸(PA)。如二棕榈酰、二亚油酰、二硬脂酰形成的各种磷脂。按照脂质体双层膜中磷脂的特征，常用的合成磷脂有DPPC、DPPS、DSPG、DOPC等。脂质体中的胆固醇与磷脂按比例的要求配方，结合被包裹物质的特性可形成单层或多层脂质体，形成不同粒径大小的脂质体。
通过将被包裹药物与脂类物质按比例进行脂质体的制备方法有多种，目前比较常用的方法有超声法、均质法和蒸发法、离心法，薄膜法和前脂质体等方法。根据不同的制备工艺可形成不同粒径大小的脂质体。在进行药物脂质体包封中，首先要考虑被包裹药物的包封率，包封后的渗漏率，形成的脂质体的粒径大小，以及包被膜上磷脂的稳定性。脂质体一般是悬液，为了提高其稳定性如减少渗漏、防止氧化、减少脂质体凝聚和融合等，目前经常将脂质体制成冻干粉。通过加入一定比例的糖类保护物质，经冷冻干燥后形成。常用的糖类物质包括单糖和多糖如甘露醇、苷蔗糖、山梨糖、蔗糖、麦芽糖、海藻糖、乳糖、淀粉等。
GM1作为一种有效的治疗中枢神经损伤的药物，临床目前为注射水针剂型。虽然GM1注射后可进入血脑屏障，但在血中的最大有效浓度时间短，临床用量大(20-100mg/60kg/日)，病人需每日注射给药1-2次。本发明在于提供一种将GM1制成脂质体，以提高在血液中的生物利用度，提高脑组织中GM1的含量。进而达到减少给药的剂量，给药周期或给药途径。对于神经损伤特别中枢神经损伤的治疗更有效。本发明属于单唾液酸四己糖神经节苷脂的脂质体剂型，它是将GM1作为一种被包裹的药物，与胆固醇和磷脂共同形成一定粒径的脂质体复合物。GM1脂质体复合物中包含了部分游离的GM1，部分作为脂质体双层膜中GM1，部分被包裹在脂质体内囊中的GM1。根据不同工艺和制备方法，使GM1脂质体复合物中未包裹的游离GM1的含量低于30％以下。使GM1脂质体复合物中的脂质体的粒径为50-300nm之间。
本发明提供了将单唾液酸四己糖神经节苷脂脂质体复合物制成冻干粉的工艺和配方。一定粒径的单层GM1脂质体复合物悬液中加入糖类保护剂后，经冷冻干燥可形成稳定性更好的GM1脂质体干粉。虽然常用的糖类保护剂有单糖和多糖，本发明中GM1脂质体干粉中的糖类保护剂为多糖如淀粉、海藻糖、麦芽糖、蔗糖。为了使糖类物质加入后达到有效的保护作用，加入比例是GM1脂质体悬液中按体积重量比的5-20％。制备而成的GM1脂质体干粉可进一步添加各种辅助剂制成非注射的口服给药剂型。在实例中提供了GM1脂质体干粉的口服胶囊经胃肠吸收进入脑组织的结果。
实例一：单唾液酸四己糖神经节苷脂脂质体的制备
1.薄膜超声法
精密称定一定量的DSPG 2.0克和胆固醇1.0g(DERMOPRO.U.S.A，)，GM1500mg，氯仿溶解，于40℃水浴中减压旋转蒸发除去有机溶剂，使脂质在瓶内壁均匀成膜，抽干。向其中加入100ml用氮气饱和的注射用水充分和，瓶口膜封，旋涡混合5min，使脂质膜全部从瓶壁上转移入水相，磁力搅拌15min，充分溶胀、水合，再于水浴式超声仪中超声30min，旋风式匀浆器处理30min，即得采用薄膜超声法制备GM1脂质体。
2.改良均质法
精密称定一定量的DSPG 2.0g和胆固醇1.0g(DERMOPRO，U.S.A，)，GM1500mg，加热至85℃左右，然后喷射加入100ml用N2饱和的注射用水和、不断搅拌，再于旋风式匀浆器(Lipex)处理30min，用100nm稳定过滤支持剂透析和粒径成型器处理样品，样品充氮气密封，即得采用改良均质法制备的GM1脂质体。
实例二：单唾液酸四己糖神经节苷脂脂质体包封率和粒径测定
1.激光散射学研究
分别取少量的薄膜超声法制备GM1脂质体和改良均质法GM1脂质体适量，用缓冲液稀释后用激光粒度分析仪进行测定，经动态光散射处理软件处理。测定结果表明，采用薄膜超声法制备GM1脂质体，平均粒径为245nm。改良质体制剂的粒径分布范围比较前者窄，粒径分布比较均匀，平均粒径为110nm。
2.包封率的测定
精密量取改良均质法制备的GM1脂质体制剂1ml置12ml容量超高速冷冻离心管中，用缓冲溶液稀释配平，100000g离心2小时，吸取上清，补足缓冲液体，再次超高速冷冻离心100000分种。收取离心上清，采用建立的二阶倒数紫外分光光度检测GM1含量。并按下式计算制备包封率(EN％)。
EN％＝(Wt-Ws/Wt)×100％
其中Ws，Wt分别表示上清液体药物量和总投药量，测定结果见表1。
| 批号 | 主药含量(mg/ml) | 包封率(％) |
| 020102020203 | 4.904.874.99 | 87.7786.7989.10 |
由结果可见，制备的GM1脂质体的包封率高于85％，试验结果的重现性好。
实例三：单唾液酸四己糖神经节苷脂脂质体渗漏测定和体外稳定性
1.制剂外观评价和初步稳定性预测
制备的薄膜超声法制备GM1脂质体为乳白色胶体溶液。改良均质法制备的GM1脂质体为乳白色溶液。常温高速离心3000rpm、10min，5000rpm、10min，10000rpm、10min，15000rpm、10min，20000rpm、10min，无沉淀和分层现象发生。样品孵浴40℃后，高速离心3000rpm、10min，5000rpm、10min，10000rpm、10min，15000rpm、10min，20000rpm、10min，无沉淀和分层现象发生。样品4℃冷藏3个月后，高速离心3000rpm、10min，5000rpm、10min，10000rpm、10min，15000rpm、10min，20000rpm、10min，无沉淀和分层现象发生。
2.形态学研究
分别取少量的薄膜超声法制备GM1脂质体和改良均质法GM1脂质体，稀释到恰当浓度，滴至载玻片上，用1％磷钨酸进生负染，再滴至专用铜网上，自然挥干，使粒子在铜网上浓缩沉积，用透射电子显微镜观察并摄制照片。电镜照片显示制备的GM1脂质体，形态结构清晰，呈单层状，磷脂双分子膜特异性结构清晰。分别观察4℃放置4周后，未有形态学改变发现。
3.体外释放率的测定
精密量取系列改良均质法制备的GM1脂质体制剂1ml，置12ml容量超高速冷冻离心管中中，分别用PBS缓冲溶液稀释配平，37℃水浴保存，不同时相100,000g离心2小时，吸取上清，补足缓冲液体，再次超高速冷冻离心100,000分钟。收取离心上清，采用建立的二阶倒数紫外分光光度器检测GM1含量。以此获得制备的改良均质法制备的GM1脂质体制剂体外释放曲线。制备的GM1脂质体制剂呈程序释放，符合缓释模型。
实例四：单唾液酸四己糖神经节苷脂脂质体复合物的冻干
取改良均质法制备的GM1脂质体复合物500ml，加入50克蔗糖，在饱和N2下搅拌混匀后置冻干机中-45℃预冻6小时。设定程序20小时，最后冻干温度低于20℃，冻干时间20小时。冻干的GM1脂质体显白色疏松状，加入注射用水后快速溶解显乳白色。按实例二测定，10,000rpm离心10min后无沉淀和分层，粒径为110nm，分布均匀无凝集和融合，冻干溶解后漏渗率低于5％。
实例五：单唾液酸四己糖神经节苷脂脂质体复合物的口服制剂
精确称取冻干的GM1脂质体干粉1克，加入医用淀粉1克，彻底混匀过筛后，装入肠溶胶囊，每颗含GM1 5mg，置室温干燥封闭保存。
实例六：单唾液酸四己糖神经节苷脂脂质体复合物的血中药代动力学
取家兔耳静脉注射GM1脂质体复合物悬浮液(1mg/kg)，口服GM1脂质体复合物胶囊(5mg/kg)，对照用GM1注射液(1mg/kg)。于给药后48小时内不同时间点取血或脑组织。血和脑中GM1含量测定用ELISA的方法。脑组织样品称量后加入适量PBS均浆，血液用肝素抗凝后取血浆。脑组织匀浆液和血浆用4倍四氢呋喃抽提GM1二次，再加入丙酮分层，取水相冻干后用水溶解。不同倍数稀释后样品测定样品中GM1含量与标准GM1曲线比较，计算血中Cmax，AUC和T1/2，以及脑组织中GM1含量。
| 血 | 脑GM1含量(ng/克) | ||||||
| 剂量(mg/kg) | Cmax(ng/ml) | T1/2(hr) | AUC(ng/ml.min) | 2hr | 8hr | 24hr | |
| GM1注射液(静注)GM1脂质体(静注)GM1脂质体(口服) | 115 | 42514252 | 8.42.6- | 1189045203280 | 18922151 | 592242189 | 292138113 |
表中显示GM1脂质体在血和脑中的生物利用度和含量均高出GM1注射液3-4倍。口服GM1脂质体的生物利用度约25％。
Claims (5)
1.一种含有单唾液酸四己糖神经节苷脂脂质体复合物，其特征在于其中含有胆固醇、DSPG和单唾液酸四己糖神经节苷脂。
2.如利要求1中所述的脂质体复合物，其特征在于其中DSPG和胆固醇之间的摩尔比为1∶1～1∶2。
3.如权利要求1所述的脂质体复合物，其特征在于可在复合物中加入多糖冻干保护剂制成干粉。
4.如权利要求3所述的脂质体复合物，其特征在于其中所述的多糖冻干保护剂选自蔗糖、麦芽糖、海藻糖、淀粉或山梨醇。
5.如权利要求3所述的脂质体复合物，其特征在于加入的多糖冻干保护剂的比例为5-20％。
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| CN100411610C (zh) * | 2005-12-29 | 2008-08-20 | 武汉海特生物制药股份有限公司 | 一种可透血脑屏障的脂质体和以该脂质体为载体的药物组合物及其制备方法 |
| CN101700230B (zh) * | 2009-11-06 | 2012-02-01 | 山东大学 | 一种单唾液酸四己糖神经节苷酯微球及其制备方法 |
| CN102366408B (zh) * | 2011-09-14 | 2013-03-20 | 海南灵康制药有限公司 | 单唾液酸四己糖神经节苷脂钠脂质体注射剂 |
| CN102988284B (zh) * | 2012-12-13 | 2014-04-02 | 哈药集团技术中心 | 一种单唾液酸四己糖神经节苷脂钠注射液的制备方法 |
| CN106361718B (zh) * | 2016-10-14 | 2019-01-25 | 珠海赛隆药业股份有限公司 | 单唾液酸四己糖神经节苷脂钠的结肠靶向生物粘附片 |
| CN106619545A (zh) * | 2016-10-14 | 2017-05-10 | 珠海赛隆药业股份有限公司 | 在胃肠道稳定的单唾液酸四己糖神经节苷脂钠口服制剂及制备方法 |
| CN108404139A (zh) * | 2018-01-29 | 2018-08-17 | 南昌大学 | 低浓度单唾液酸四己糖神经节苷脂修饰的重组脂蛋白的制备方法及其应用 |
| CN110755405A (zh) * | 2019-10-25 | 2020-02-07 | 南昌大学 | 具有靶向性的神经节苷脂gm3在制备治疗动脉粥样硬化疾病药物中的应用 |
| CN113304276B (zh) * | 2021-06-04 | 2022-11-15 | 沈阳药科大学 | 用单唾液酸四己糖神经节苷脂修饰的脂质体及其制法和冻干应用 |
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