CN105534882B - 一种青霉胺鼻凝胶剂及其制备方法 - Google Patents
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Abstract
一种青霉胺鼻凝胶剂及其制备方法，所述鼻凝胶由如下重量百分比的原料制成：青霉胺0.1~1%，壳聚糖1~5%，β‑甘油磷酸钠2~8%，酸溶液0.05~0.5%，去离子水85~98%。制备方法为（1）按照所述质量百分比，室温下将D‑青霉胺溶解于酸溶液中，搅拌至完全溶解；（2）加入壳聚糖搅拌溶解，控制搅拌速度为200~1000r/min；（3）控制温度0~4°C，保温搅拌30~60min；（4）加入β‑甘油磷酸钠，0~4°C搅拌10~40min，控制搅拌速度为100~800r/min，得到青霉胺鼻凝胶剂。本发明的青霉胺鼻凝胶剂在动物模型试验中表现具有减少老年斑和治疗阿尔茨海默病的疗效。
Description
技术领域
本发明涉及一种鼻凝胶的制备，具体涉及一种青霉胺鼻凝胶剂及其制备方法。
背景技术
阿尔茨海默病(Alzheimer disease, AD)是一种中枢神经系统变性病，起病隐袭，病程呈慢性进行性，是老年期痴呆最常见的一种类型。主要表现为渐进性记忆障碍、认知功能障碍、人格改变及语言障碍等神经精神症状，严重影响社交、职业与生活功能。随着人口的老龄化，AD的发病率逐年上升，患病率研究显示，年龄每增加5岁，阿尔茨海默病病人的百分数将上升2倍[1]。AD晚期患者生活不能自理，给患者、家庭和社会均带来沉重的痛苦和负担。目前，AD的病因及发病机制尚未阐明，深入研究AD发病机理与防治策略具有重要的理论意义和现实意义。
AD特征性病理改变为β淀粉样蛋白(β-amyloid peptide，Aβ)沉积形成的细胞外老年斑和细胞内tau蛋白的过度磷酸化形成神经原纤维缠结(neurofibrillary tangles，NFTs)。其中Aβ的细胞毒性及其脑内沉积是导致AD脑内神经元死亡的重要原因[2, 3]。Aβ由跨膜蛋白β-淀粉样蛋白前体蛋白 (amyloid-β precursor protein, APP)的淀粉样代谢途径，即经过 β-分泌酶 (BACE1) 和γ-分泌酶依次酶切产生。首先，APP膜外区被BACE1 剪切，产生可溶性APPβ (sAPPβ) 并分泌到细胞外，然后剩余在膜上的C99的跨膜区域进一步被γ-分泌酶剪切，由此产生Aβ且也被分泌到细胞外。在AD的发病过程中，APP淀粉样代谢途径被激活，产生的大量Aβ以可溶性Aβ或以寡聚肽Aβ形式存在于细胞外，具有极强的神经毒性，能导致神经元死亡以及相关的神经功能障碍或丧失，随着AD病程的进展，Aβ在脑内逐渐沉积并最终聚集成老年斑。Aβ的产生和沉积导致脑内胆碱能神经元死亡和神经胶质细胞激活等，引起学习记忆能力减退直至痴呆等进行性神经退行性临床症状[4]。因此，减少Aβ的形成，抑制Aβ的沉积，是预防和治疗AD的根本途径。
研究表明，Aβ是一个拥有多金属键合位点的金属蛋白，其N端的6，13，14位组氨酸残基是金属离子键合的关键位点[1, 4]。Aβ并不是自发地聚集，而是与脑中的过量金属(如Cu2+，Zn2+)发生了一种年龄依赖性的作用，它使Aβ沉淀为富含金属的斑块。青霉胺，英文名称为D(-)-Penicillamine，中文别名为D-青霉胺；二甲基半胱胺酸，CAS号为52-67-5，分子式为C5H11NO2S，在临床上主要用于Wilson疾病及类风湿性关节炎的治疗，还用于重金属中毒的解毒治疗。最近的研究表明，D-青霉胺能够减弱阿尔茨海默病人血清中的氧化应激反应[5, 6]，减少血红蛋白中铜锌超氧化物歧化酶的活性[5, 7]。提示D-青霉胺可能是防治AD的药物之一。而揭示D-青霉胺预防和治疗AD的机理，对寻找AD新的治疗靶点具有重要的意义。D-青霉胺具有高度的亲水性，但不易于通过血脑屏障[8]。因此，亟需一种D-青霉胺新剂型作为药物传递系统，将亲水性青霉胺有效的转运至脑组织内为基础，来验证D-青霉胺影响Aβ产生和老年斑形成的机理。
近年来，药物制剂领域内研究者们致力于通过鼻粘膜给药使药物发挥全身治疗作用，鼻腔给药的优点主要体现在以下几个方面：1、药物无首过效应，生物利用度高；2、鼻粘膜下血管丰富，药物可迅速吸收；3、胃肠道中容易破坏和难吸收的药物，鼻粘膜都能很好吸收；4、用药方便，适用于各类患者。目前临床应用的滴鼻剂多为液体制剂，使用时易从鼻腔流入口腔，往往给患者带来不必要的困扰，并且降低了药物的吸收率。凝胶剂是药物与适宜的辅料制成的均一、混悬或乳剂型的乳胶稠厚液体或半固体制剂，具有良好的亲和性、涂展性和粘附性，还具有良好的生物相容性。
发明内容
针对现有靶向性青霉胺新剂型用于治疗阿尔茨海默病的研究空白，并结合鼻腔给药和凝胶剂的一系列优点，本发明提供一种青霉胺鼻凝胶剂及其制备方法，将青霉胺开发成鼻凝胶剂，通过动物模型的鼻饲给药方式探讨青霉胺鼻凝胶剂预防和治疗AD的机理，为研究治疗阿尔茨海默病的方法提供新思路。
本发明以壳聚糖与甘油磷酸钠体系为载体，壳聚糖是自然界唯一大量存在的碱性多糖，生物毒性小，具有良好的组织相容性，生物可降解性和粘附性，在医学，生物学领域得到了深入的研究和广泛的应用，是一种理想的医用植入材料，并且壳聚糖与甘油磷酸钠体系是研究最多的给药缓释载体。
本发明的技术方案为：
一种青霉胺鼻凝胶剂，由如下重量百分比的原料制成：青霉胺0.1~1%，壳聚糖1~5%，β-甘油磷酸钠2~8%，酸溶液0.05~0.5%，去离子水85~98%。
所述青霉胺鼻凝胶剂为无色到白色透明凝胶，pH值为7.0~7.4。
所述酸溶液为醋酸和盐酸的混合溶液，两者在混合溶液中的浓度分别为17.5mol/L和12mol/L。
所述青霉胺鼻凝胶剂的制备方法，按照以下工艺步骤进行：
（1）按照所述质量百分比，室温下将D-青霉胺溶解于酸溶液中，搅拌至完全溶解；
（2）加入壳聚糖搅拌溶解，控制搅拌速度为200~1000r/min；
（3）控制温度0~4°C，保温搅拌30~60min；
（4）加入β-甘油磷酸钠，0~4°C搅拌10~40min，控制搅拌速度为100~800r/min，得到青霉胺鼻凝胶剂。
本发明的青霉胺鼻凝胶剂在动物模型试验中表现具有减少老年斑和治疗阿尔茨海默病的疗效。
本发明以青霉胺为主药，以壳聚糖为载体，β-甘油磷酸钠为pH调节剂，酸溶液为溶剂，制成pH为7.4的青霉胺鼻凝胶剂，在20~30°C为无色透明溶液，35~45°C为白色透明凝胶，具有良好的温度敏感性和生物粘附性。
本发明的有益效果如下：
1、本发明的青霉胺鼻凝胶剂以壳聚糖与甘油磷酸钠体系为载体，为一种水性凝胶剂，pH在7.0~7.4，对皮肤无刺激，并且具有良好的亲和性、涂展性和粘附性，还具有良好的温度敏感性和生物相容性，给药方便，适合各类患者使用。
2、本发明的青霉胺鼻凝胶剂在动物实验中显示出对减少老年斑和治疗阿尔茨海默病具有疗效，并对氧化损伤具有保护作用。
3、本发明方法简单易行，制备条件温和，不使用任何有机化学试剂及其他有害物质，适合药物释放和组织工程领域的应用。
附图说明
图1为动物模型试验中4个给药组小鼠大脑的金属自显影染色对比图，4个给药组分别为本发明的青霉胺鼻凝胶剂组、对照组、青霉胺水溶液组和壳聚糖组。
图2为动物模型试验中4个给药组小鼠大脑的Aβ免疫组织化学染色图，4个给药组分别为本发明的青霉胺鼻凝胶剂组、对照组、青霉胺水溶液组和壳聚糖组。
图3为动物模型试验中4个给药组小鼠大脑的蛋白质印迹法检测对比图，其中1为对照组，2为本发明的青霉胺鼻凝胶剂组，3为青霉胺水溶液组，4为壳聚糖组。
具体实施方式
本发明实施采用的青霉胺为市售纯度在98%以上的原料药，使用前经过纯化处理，待液相归一化纯度达到99.9%以上进行本发明的青霉胺鼻凝胶剂的制备。
本发明实施采用的壳聚糖、β-甘油磷酸钠为市售医药级试剂。
实施例1
一种青霉胺鼻凝胶剂，由如下重量百分比的原料制成：青霉胺0.2%，壳聚糖3%，β-甘油磷酸钠4%，酸溶液0.5%，去离子水92.3%。所述青霉胺鼻凝胶剂的pH值为7.4，无色到白色透明凝胶。
所述酸溶液为醋酸和盐酸的混合溶液，两者在混合溶液中的浓度分别为17.5mol/L和12mol/L。
所述青霉胺鼻凝胶剂的制备方法，按照以下工艺步骤进行：
（1）按照所述质量百分比，室温下将D-青霉胺溶解于酸溶液中，搅拌至完全溶解；
（2）加入壳聚糖搅拌溶解，控制搅拌速度为1000r/min；
（3）控制温度0~4°C，保温搅拌60min；
（4）加入β-甘油磷酸钠，0~4°C搅拌20min，控制搅拌速度为800r/min，得到青霉胺鼻凝胶剂。
实施例2
一种青霉胺鼻凝胶剂，由如下重量百分比的原料制成：青霉胺0.5%，壳聚糖2%，β-甘油磷酸钠6%，酸溶液0.5%，去离子水91%。所述青霉胺鼻凝胶剂的pH值为7.4，无色到白色透明凝胶。
所述酸溶液为醋酸和盐酸的混合溶液，两者在混合溶液中的浓度分别为17.5mol/L和12mol/L。
所述青霉胺鼻凝胶剂的制备方法，按照以下工艺步骤进行：
（1）按照所述质量百分比，室温下将D-青霉胺溶解于酸溶液中，搅拌至完全溶解；
（2）加入壳聚糖搅拌溶解，控制搅拌速度为500r/min；
（3）控制温度0~4°C，保温搅拌40min；
（4）加入β-甘油磷酸钠，0~4°C搅拌30min，控制搅拌速度为700r/min，得到青霉胺鼻凝胶剂。
实施例3
一种青霉胺鼻凝胶剂，由如下重量百分比的原料制成：青霉胺0.8%，壳聚糖5%，β-甘油磷酸钠8%，酸溶液0.2%，去离子水86%。所述青霉胺鼻凝胶剂的pH值为7.4，无色到白色透明凝胶。
所述酸溶液为醋酸和盐酸的混合溶液，两者在混合溶液中的浓度分别为17.5mol/L和12mol/L。
所述青霉胺鼻凝胶剂的制备方法，按照以下工艺步骤进行：
（1）按照所述质量百分比，室温下将D-青霉胺溶解于酸溶液中，搅拌至完全溶解；
（2）加入壳聚糖搅拌溶解，控制搅拌速度为600r/min；
（3）控制温度0~4°C，保温搅拌30min；
（4）加入β-甘油磷酸钠，0~4°C搅拌20min，控制搅拌速度为800r/min，得到青霉胺鼻凝胶剂。
本发明的青霉胺鼻凝胶剂的药理学试验
1、金属自显影染色
金属自显影染色是特异性针对游离锌离子的一种染色方法。首先将待测组织在0.1%的硫化钠溶液中浸泡3天，再做冰冻切片，乳酸银染色后老年斑中的锌离子可以清晰表达。
以20只5月龄的雄性APP/PS1转基因小鼠为动物模型，将它们随机分为四组：1）对照组，2）本发明的青霉胺鼻凝胶剂组，3）青霉胺水溶液组，4）壳聚糖组。给药方式采取隔日鼻饲给药，剂量为青霉胺2mg/kg， 持续3个月后动物处死，取小鼠大脑进行金属自显影染色，结果如图1所示，大量的锌离子沉积在小鼠大脑皮层老年斑周围，呈深褐色或深棕色颗粒，大脑皮层各层也有锌离子的沉积。与对照组相比，本发明的青霉胺鼻凝胶剂组给药的小鼠大脑皮层锌离子斑块数量减少，青霉胺水溶液鼻饲组、壳聚糖组以及对照组之间进行比较，锌离子斑块数量并无明显差异，各组大脑皮层中锌离子含量并未发生明显改变。
以上实验结果表明，本发明的青霉胺鼻凝胶剂能够有效减少APP/PS1小鼠大脑皮层中老年斑的数量。
2、Aβ免疫组织化学染色
以20只5月龄的雄性APP/PS1转基因小鼠为研究对象，将它们随机分为四组：1）对照组，2）本发明的青霉胺鼻凝胶剂组，3）青霉胺水溶液组，4）壳聚糖组。给药方式采取隔日鼻饲给药，剂量为青霉胺2mg/kg， 持续3个月后动物处死，取小鼠大脑进行Aβ免疫组织化学染色，考察青霉胺鼻凝胶剂的药理作用。Aβ免疫组织化学染色结果如图2所示，Aβ阳性反应在小鼠大脑皮层中呈棕黄色斑块状，分布于细胞外，斑块大小与形态不均，高倍镜下呈圆形或不规则形。与对照组相比，本发明的青霉胺鼻凝胶剂给药的小鼠大脑皮层Aβ阳性斑块的数目减少，斑块变小。青霉胺水溶液鼻饲组与对照组相比Aβ阳性斑块的数目也有所减少，斑块形状呈圆形或不规则形。壳聚糖鼻饲组与对照组相比Aβ阳性斑块数目和形状无明显差异。以上结果表明，本发明的青霉胺鼻凝胶剂能够有效减少Aβ的沉积，具有较大潜力开发成为阿尔茨海默症的治疗制剂。
3、蛋白质印迹法检测
细胞色素C氧化酶调节亚基IV ( Cytochrome c oxidase IV, COX-IV ) 作为线粒体呼吸链中的关键组成蛋白，其活性需要铜离子来维持；COX-IV蛋白水平表达的变化与线粒体氧化损伤密切关联，而线粒体的氧化损伤作用与阿尔茨海默病退行性神经疾病发生相关。
以12只5月龄的雄性APP/PS1转基因小鼠为研究对象，将它们随机分为四组：1）对照组，2）本发明的青霉胺鼻凝胶剂组，3）青霉胺水溶液组，4）壳聚糖组。给药方式采取隔日鼻饲给药，剂量为青霉胺2mg/kg， 持续3个月后动物处死，取小鼠大脑采用蛋白质印迹法检测，检测各给药组COX-IV蛋白水平表达的变化情况，来确定D-青霉胺对铜离子的螯合能力。检测结果为：本发明的青霉胺鼻凝胶剂组、青霉胺水溶液组、壳聚糖组和对照组的COX-IV蛋白水平分别为0.45±0.02、0.72±0.058、0.79±0.14、0.77±0.07。本发明的青霉胺鼻凝胶剂组与其它三组相比，COX-IV蛋白水平表达明显降低（P < 0.05）。该结果提供给我们一种启示，即本发明的青霉胺鼻凝胶剂组对APP/PS1小鼠大脑皮层中的铜离子具有显著下调作用，并对氧化损伤也具有明显保护作用。
背景技术部分的参考文献：
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Claims (4)
1.一种青霉胺鼻凝胶剂，其特征在于由如下重量百分比的原料制成：青霉胺0.1～1％，壳聚糖1～5％，β-甘油磷酸钠2～8％，酸溶液0.05～0.5％，去离子水85～98％，所述青霉胺鼻凝胶剂通过鼻饲给药方式用于预防和治疗阿尔茨海默病。
2.根据权利要求1所述的一种青霉胺鼻凝胶剂，其特征在于所述青霉胺鼻凝胶剂为无色到白色透明凝胶，pH值为7.0～7.4。
3.根据权利要求1所述的一种青霉胺鼻凝胶剂，其特征在于所述酸溶液为醋酸和盐酸的混合溶液，两者在混合溶液中的浓度分别为17.5mol/L和12mol/L。
4.权利要求1所述的一种青霉胺鼻凝胶剂的制备方法，其特征在于按照以下工艺步骤进行：
(1)按照所述质量百分比，室温下将D-青霉胺溶解于酸溶液中，搅拌至完全溶解；
(2)加入壳聚糖搅拌溶解，控制搅拌速度为200～1000r/min；
(3)控制温度0～4℃，保温搅拌30～60min；
(4)加入β-甘油磷酸钠，0～4℃搅拌10～40min，控制搅拌速度为100～800r/min，得到青霉胺鼻凝胶剂。
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| CN101053550A (zh) * | 2007-04-25 | 2007-10-17 | 昆明制药集团股份有限公司 | 天麻素鼻腔凝胶剂 |
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| Development, characterization and application of in situ gel systems for intranasal delivery of tacrine;Shuai Qian等;《International Journal of Pharmaceutics》;20140405;第468卷;272–282 * |
| Huperzine A Activates Wnt/b-Catenin Signaling and Enhances the Nonamyloidogenic Pathway in an Alzheimer Transgenic Mouse Model;Chun-Yan Wang等;《Neuropsychopharmacology》;20110202;第36卷;1073–1089 * |
| Intranasal therapeutic strategies for management of Alzheimer’s disease;Sumeet Sood等;《J Drug Target》;20140109;279–294 * |
| Novel D-penicillamine carrying nanoparticles for metal chelation therapy in Alzheimer’s and other CNS diseases;Zhengrong Cui;《European Journal of Pharmaceutics and Biopharmaceutics》;20041002;第59卷;263–272 * |
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