CN117243886A - 一种治疗糖尿病下肢缺血病变的微针贴片及应用 - Google Patents
一种治疗糖尿病下肢缺血病变的微针贴片及应用 Download PDFInfo
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- CN117243886A CN117243886A CN202311123220.9A CN202311123220A CN117243886A CN 117243886 A CN117243886 A CN 117243886A CN 202311123220 A CN202311123220 A CN 202311123220A CN 117243886 A CN117243886 A CN 117243886A
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Abstract
一种治疗糖尿病下肢缺血病变的微针贴片,包括由肠促胰素中胰高血糖素样肽‑1短肽GLP‑1(32‑36)a混合于第一聚合物制成外壳层,由PEG‑GLP‑1(32‑36)a、光引发剂和第二聚合物制成内核层。本发明的微针贴片,具有“先暴释后缓释”的特征,不仅可以使药物快速进入体内达到有效治疗浓度,而且可以长期维持体内药物浓度在有效治疗窗内,最大发挥药物的治疗作用。
Description
技术领域
本发明涉及一种载药器件,尤其涉及一种微针贴片,将胰高血糖素样肽-1短肽(如:GLP-1(32-36)酰胺)装载于微针中,实现短肽差速释放,利于在糖尿病下肢缺血症的治疗中应用。
背景技术
随着生活方式的改变与人口老龄化的增长,糖尿病的患者人数正在逐年增长。根据全球糖尿病协会2022年的数据,全球已有超过5亿人正罹患糖尿病,还有将近一半的患者并未接受有效的治疗。一旦失去对于血糖的有效控制,糖尿病便会不断恶化发展而引起一系列包括血管、神经等部位受累在内的并发症,后期甚至会出现失明、截肢甚至死亡的结果。
糖尿病下肢缺血是糖尿病患者最为常见的慢性血管并发症,其致病原因是:高糖环境下,氧化应激信号通路被激活,产生大量氧自由基(ROS)破坏血管内皮细胞,从而造成糖尿病下肢缺血。除手术治疗之外,目前仍未发现药物能有效改善糖尿病下肢缺血。然而由于解剖因素的限制等原因,部分患者在接受外科手术治疗后其下肢缺血的症状并不能得到有效的改善,仍不可避免需要进行截肢。因此,目前亟需一种高效、低毒的药物制剂来有效治疗糖尿病下肢缺血。
胰高血糖素样肽-1短肽(GLP-1(32-36)酰胺,或GLP-1(32-36)a),作为胰高血糖素样肽-1(GLP-1)的分解产物,伴随着GLP-1葡萄糖依赖性调控血糖特性的被发现,逐渐进入大众的视野。早期的研究认为只有来自肠道L细胞的胰高血糖素样肽-1能在体内发挥血糖调节作用并改善糖尿病相关并发症的预后。而随着研究的逐渐深入,胰高血糖素样肽-1的各类分解产物的生物效应被逐渐发现。近年来,胰高血糖素样短肽被发现能抑制体外胰岛β细胞的凋亡而增加糖尿病小鼠的葡萄糖分配,同时它还对于增加基础能量消耗、抑制糖尿病小鼠的体重增加有效果。然而尚未有报道说明GLP-1(32-36)a对于治疗糖尿病下肢缺血方面的作用。本团队前期的研究发现GLP-1(32-36)a能促进内皮细胞修复,促进新生血管生成,有效改善糖尿病小鼠下肢缺血的状况。然而作为一种由5个氨基酸组成的短肽(分子量570Da),GLP-1(32-36)a在体内的半衰期极短,往往会出现未发挥治疗效果而已经被降解的情况。因此,依靠目前的皮下注射给药方式,通常需要多次注射以确保发挥多肽的生物学作用,同时下肢缺血后药物很难在肢体最远端达到有效浓度。为了减少患者因多次注射带来的痛苦,构建一种长效的局部给药缓释制剂是目前亟需解决的问题。
微针是以多个微小针体集合的阵列,针体直径一般小于300μm,针体长度一般范围为200μm至2000μm。与传统的经皮给药制剂相比,微针的优势在于可以快速递送胃肠道不易吸收的生物大分子药物,如:蛋白质、多肽、抗体、疫苗、RNA和DNA等。微针按照制备工艺和材料性质可以分为5类,即:实体微针、涂层微针、可溶性微针、水凝胶微针和空心微针。其中,水凝胶微针是目前微针给药系统的研究热点。水凝胶微针由水凝胶聚合为基质的微针,这种微针阵列在插入皮肤后迅速吸收间质液,导致水凝胶肿胀,在凝胶内产生连续畅通的孔道,药物通过组织液渗透和扩散进入皮肤组织内。其优点是可通过调节水凝胶纤维的交联密度,控制大分子药物释放速率。前期实验发现GLP-1(32-36)a分子量极小,仅靠水凝胶微针很难实现短肽的缓控释放。因此,亟需制备一种可在体内代谢的大分子前药,来辅助水凝胶微针实现长效缓释。
发明内容
本发明的一个目的在于提供一种载有胰高血糖素样肽-1短肽的微针贴片,使短肽实现差速释放,长期维持在有效治疗窗内。
本发明的另一个目的在于提供一种差速释放胰高血糖素样肽-1短肽的微针贴片在制备治疗糖尿病下肢缺血医疗器械中的应用。
本发明的再一个目的在于提供一种医疗器械,包括差速释放胰高血糖素样肽-1短肽的微针贴片。
本发明的胰高血糖素样肽-1短肽的微针贴片,包括核壳型微针,将GLP-1(32-36)a装载于微针内,使GLP-1(32-36)a具有“先暴释后缓释”的释放特征。即微针插入皮肤后,载于外壳层的GLP-1(32-36)a被快速释放,达到有效治疗浓度,载于内核层的GLP-1(32-36)缓慢释放,使药物长期维持在有效治疗窗内。
载GLP-1(32-36)a微针的外壳层由GLP-1(32-36)a混合于聚合物制成。适合的聚合物如:但不限于聚乙烯醇、聚乳酸、丝素蛋白、羧甲基纤维素钠、壳聚糖、海藻酸盐、透明质酸盐和聚乙烯吡咯烷酮等,这些聚合物单独或组合应用于本发明,其中更倾向于使用聚乙烯吡咯烷酮。
载GLP-1(32-36)a微针的内核层由PEG修饰的GLP-1(32-36)a(PEG-GLP-1(32-36)a)、光引发剂和聚合物制成。制备内核的聚合物如:但不限于聚乙烯醇、聚乳酸、丝素蛋白、羧甲基纤维素钠、壳聚糖、海藻酸盐、透明质酸盐、聚乙烯吡咯烷酮以及甲基丙烯基丝素蛋白、甲基丙烯基壳聚糖、甲基丙烯基明胶、甲基丙烯基透明质酸等,这些化合物单独或组合应用于本发明,其中,更倾向于使用甲基丙烯基明胶。
PEG分子量为1w~100w。
光引发剂如:2-羟基-2-甲基-1-苯基丙酮、1-羟基环己基苯基甲酮、2,4,6-三甲基苯甲酰基-二苯基氧化膦、2,4,6-三甲基苯甲酰基苯基膦酸乙酯、2-二甲氨基-2-苄基-1-[4-(4-吗啉基)苯基]-1-丁酮和2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮以及苯甲酰甲酸甲酯中的一种或几种。
制备PEG-GLP-1(32-36)a交联剂如:但不限于聚乙二醇-双硫键-琥珀酰亚胺碳酸酯、聚乙二醇-琥珀酰亚胺碳酸酯、聚乙二醇-双硫键-聚乙二醇-琥珀酰亚胺碳酸酯等,这些交联剂单独或组合应用于本发明,其中,更倾向于使用聚乙二醇-双硫键-琥珀酰亚胺碳酸酯和聚乙二醇-琥珀酰亚胺碳酸酯。
各个微针设置于基底层上形成微针贴片。基底层也由聚合物组成,如:但不限于聚乙烯醇、聚乳酸、丝素蛋白、羧甲基纤维素钠、壳聚糖、海藻酸盐、透明质酸盐和聚乙烯吡咯烷酮等,这些聚合物单独或组合应用于本发明,其中更倾向于使用聚乙烯吡咯烷。
多个载GLP-1(32-36)a微针按规律排列。一般来说,每个微针之间的距离相等。其中微针的形态如:但不限于圆锥形和棱形。其中,更倾向于使用圆锥形。针体长度范围100μm~1000μm。针体基底面直径范围为30μm~500μm。
一种载GLP-1(32-36)a微针贴片的实施方式,针体长度范围100μm~1000μm。
另一种载GLP-1(32-36)a微针贴片的实施方式,针体基底面直径范围为30μm~500μm。
另一种载GLP-1(32-36)a微针贴片的实施方式,在长度1mm范围内,设置1~3个微针,优先选择1~2个微针。
另一种载GLP-1(32-36)a微针贴片的实施方式,在面积为1cm2的范围内,设置10~100个微针,优先选择50~100个微针。
本发明制备微针贴片的工艺为“大小双模具灌入法”制备工艺,为便于本领域技术人员了解制取的方法,本发明示例性的,而非限制地列明如下制取工艺:
(1)PEG-GLP-1(32-36)a的制备
首先,配制GLP-1(32-36)a水溶液(pH=7.8)以及交联剂溶液(pH=7.8);之后,将交联剂溶液滴加到的GLP-1(32-36)a水溶液中进行交联反应,反应温度为4℃。采用葡聚糖凝胶柱法分离所需要的PEG-GLP-1(32-36)a溶液。最后,通过冻干技术得到PEG-GLP-1(32-36)a冻干粉。
(2)载GLP-1(32-36)a的长效微针贴片制备
配制含有PEG-GLP-1(32-36)a和含光引发剂的聚合物溶液(即工作溶液1)、用于基底层的聚合物溶液(即工作溶液2)以及含有GLP-1(32-36)a的聚合物溶液(即工作溶液3);
然后,工作溶液1加入预制的第一微针贴片模具中,通过离心或真空法,使工作溶液1完全进入第一微针模具孔中,将未进入针孔的多余液体刮掉,室温下干燥过夜;
接着,将工作溶液2加入上述第一微针贴片模具中,继续离心或真空,使所述的工作溶液2完全进入第一微针模具孔中,室温下干燥过夜后,剥离微针,紫外光固化,获得载PEG-GLP-1(32-36)a微针贴片的内核结构;然后,将所述工作溶液3加入预制的第二微针贴片模具中,通过离心或真空法,使工作溶液3完全进入微针模具孔中,再将载PEG-GLP-1(32-36)a微针贴片的内核结构压入第二微针贴片模具中,室温下干燥即得载GLP-1(32-36)a的微针贴片,具有差速释放GLP-1(32-36)a的特征。
工作溶液1中所含的PEG-GLP-1(32-36)a为1~30wt%。
工作溶液1中所含的聚合物的浓度为5~30wt%。
工作溶液2中所含的聚合物的浓度为5~30wt%,用于制成贴片的基底层。
工作溶液3中所含的GLP-1(32-36)a的浓度为1~5wt%。
工作溶液3中所含的聚合物的浓度为5~30wt%。
本发明的载GLP-1(32-36)a微针具有“先暴释后缓释”的特征,不仅可以使药物快速进入体内达到有效治疗浓度,而且可以长期维持体内药物浓度在有效治疗窗内,最大发挥药物的治疗作用。
附图说明
图1为制取PEG-GLP-1(32-36)a的路线示意图;
图2为制备GLP-1(32-36)a微针贴片的示意图;
图3为GLP-1(32-36)a微针贴片的外观形貌图;其中A为GLP-1(32-36)a微针的体式镜图,B为GLP-1(32-36)a微针的共聚焦图,图中标尺为200μm;
图4为载GLP-1(32-36)a微针的位移与承载力曲线图;
图5为载GLP-1(32-36)a微针的皮肤病理穿刺图,图中标尺为100μm;
图6为GLP-1(32-36)a微针贴片的体外释放曲线,其中,A为GLP-1(32-36)a微针贴片外壳层GLP-1(32-36)a释放曲线图;B为整个GLP-1(32-36)a微针贴片中的GLP-1(32-36)a释放曲线图;
图7为GLP-1(32-36)a制剂治疗糖尿病下肢缺血的血流图,其中,A为GLP-1(32-36)a制剂的各组动物多普勒血流图;B为GLP-1(32-36)a制剂的各组动物血流统计图;
图8为GLP-1(32-36)a制剂的药代动力学曲线,其中,A为GLP-1(32-36)a皮下注射液的药时曲线;B为载GLP-1(32-36)a微针的药时曲线。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围中。
实施例1载GLP-1(32-36)a聚乙烯醇微针贴片
首先,分别配制含2%(w/v)GLP-1(32-36)a的水溶液(pH=7.8)以及2%(w/v)PEG20K-双硫键-琥珀酰亚胺碳酸酯溶液(NHS-S-S-PEG20K);之后,将PEG20K-双硫键-琥珀酰亚胺碳酸酯溶液滴加到正在磁力搅拌的上述GLP-1(32-36)a水溶液中进行交联反应,反应温度为4℃(合成路线见图1)。进一步通过葡聚糖凝胶柱法分离所需要的GLP-1(32-36)a-PEG400溶液;最后,通过冻干技术得到GLP-1(32-36)a-PEG20K冻干粉。
配制含有3%(w/v)GLP-1(32-36)a-PEG20K、20%(w/v)聚乙烯醇1788(Polyvinylalcohol,PVA)水溶液(内核溶液)、含有0.1%(w/v)GLP-1(32-36)a和30%(w/v)聚乙烯吡咯烷酮K90(Polyvinylpyrrolidone K90,PVP)的乙醇溶液(外壳溶液)以15%(w/v)PVP乙醇溶液(基底层溶液);
之后,所述内核溶液加入100个圆锥形孔并且孔深600μm、孔最大直径250μm、针尖距离为500μm的微针模具中,通过离心或真空法,使所述的内核溶液完全进入微针模具孔中,将未进入针孔的多余液体刮掉,室温下干燥过夜,然后,将基底层溶液加入上述微针贴片模具中,继续离心或真空,使所述的基底层溶液完全进入微针模具孔中,室温下干燥过夜,剥离微针,获得载GLP-1(32-36)a-PEG20K微针贴片的内核结构(载GLP-1(32-36)a-PEG20K微针贴片);然后,将所述外壳溶液加入100个圆锥形孔并且孔深800μm,孔最大直径300μm,针尖距离为500μm的微针模具中,通过离心或真空法,使所述的外壳溶液完全进入微针模具孔中,进一步将载GLP-1(32-36)a-PEG20K微针贴片压入上述模具中,室温下干燥,即得最终的载GLP-1(32-36)a的聚乙烯醇微针贴片,具体制备路线见图2。
实施例2载GLP-1(32-36)a明胶微针贴片
首先,分别配制含2%(w/v)GLP-1(32-36)a的水溶液(pH=7.8)以及2%(w/v)PEG20K-琥珀酰亚胺碳酸酯溶液;之后,将PEG20K-琥珀酰亚胺碳酸酯溶液滴加到正在磁力搅拌的上述GLP-1(32-36)a水溶液中进行交联反应,反应温度为4℃。进一步通过葡聚糖凝胶柱法分离所需要的GLP-1(32-36)a-PEG20K溶液;最后,通过冻干技术得到GLP-1(32-36)a-PEG20K冻干粉。
配制含有3%(w/v)GLP-1(32-36)a-PEG20K、0.5%(w/v)2-羟基-2-甲基-1-苯基丙酮、5%(w/v)壳聚糖、15%(w/v)甲基丙烯基明胶的4%(v/v)醋酸水溶液(内核溶液)、含有0.1%(w/v)GLP-1(32-36)a和30%(w/v)聚乙烯吡咯烷酮K90(Polyvinylpyrrolidone K90,PVP)的乙醇溶液(外壳溶液)以15%(w/v)PVP乙醇溶液(基底层溶液);之后,所述内核溶液加入100个圆锥形孔并且孔深600μm、孔最大直径250μm、针尖距离为500μm的微针模具中,通过离心或真空法,使所述的内核溶液完全进入微针模具孔中,将未进入针孔的多余液体刮掉,室温下干燥过夜,然后,将基底层溶液加入上述微针贴片模具中,继续离心或真空,使所述的基底层溶液完全进入微针模具孔中,室温下干燥过夜,剥离微针,紫外光固化,获得载GLP-1(32-36)a-PEG20K微针贴片的内核结构(载GLP-1(32-36)a-PEG20K微针贴片);然后,将所述外壳溶液加入100个圆锥形孔并且孔深800μm、孔最大直径300μm、针尖距离为500μm的微针模具中,通过离心或真空法,使所述的外壳溶液完全进入微针模具孔中,进一步将载GLP-1(32-36)a-PEG20K微针贴片的内核结构压入上述模具中,室温下干燥即得最终的载GLP-1(32-36)a的明胶微针贴片。
实施例3载GLP-1(32-36)a透明质酸微针贴片
首先,分别配制含2%(w/v)GLP-1(32-36)a的水溶液(pH=7.8)以及2%(w/v)PEG20K-琥珀酰亚胺碳酸酯溶液;之后,将PEG20K-琥珀酰亚胺碳酸酯溶液滴加到正在磁力搅拌的上述GLP-1(32-36)a-PEG20K水溶液中进行交联反应,反应温度为4℃。采用葡聚糖凝胶柱法分离所需要的GLP-1(32-36)a-PEG20K溶液;最后,通过冻干技术得到GLP-1(32-36)a-PEG20K冻干粉。
配制含有3%(w/v)GLP-1(32-36)a-PEG20K、0.5%(w/v)2-羟基-2-甲基-1-苯基丙酮、5%(w/v)壳聚糖、5%(w/v)甲基丙烯基透明质酸钠的4%(v/v)醋酸水溶液(内核溶液)、含有0.1%(w/v)GLP-1(32-36)a和30%(w/v)聚乙烯吡咯烷酮K90(PolyvinylpyrrolidoneK90,PVP)的乙醇溶液(外壳溶液)以15%(w/v)PVP乙醇溶液(基底层溶液)。
之后,所述内核溶液加入100个圆锥形孔并且孔深600μm、孔最大直径250μm、针尖距离为500μm的微针模具中,通过离心或真空法,使所述的内核溶液完全进入微针模具孔中,将未进入针孔的多余液体刮掉,室温下干燥过夜,然后,将基底层溶液加入上述微针贴片模具中,继续离心或真空,使所述的基底层溶液完全进入微针模具孔中,室温下干燥过夜,剥离微针,紫外光固化,获得载GLP-1(32-36)a-PEG20K微针贴片的内核结构(载GLP-1(32-36)a-PEG20K微针贴片);然后,将所述外壳溶液加入100个圆锥形孔并且孔深800μm,孔最大直径300μm,针尖距离为500μm的微针模具中,通过离心或真空法,使所述的外壳溶液完全进入微针模具孔中,进一步将载GLP-1(32-36)a-PEG20K微针贴片的内核结构压入上述模具中,室温下干燥,即得最终的载GLP-1(32-36)a的透明质酸微针贴片。
2.1载GLP-1(32-36)a聚乙烯醇微针贴片的外观形貌观察
样品:按照实施例1制取的内核载有FITC-GLP-1(32-36)a-PEG20K,外壳载有罗丹明6G的微针贴片。
实验方法:
1、荧光体视镜
通过体视镜(S6D,Leica,Germany)对微针进行形貌观察。
2、共聚焦显微镜
采用共聚焦显微镜(SEM,JSM-6330F,Tokyo,Japan)对内核载有FITC-GLP-1(32-36)a-PEG20K,外壳载有罗丹明6G的微针贴片进行观察,分析微针的内部结构和药物分布。
实验结果:
图3展示了微针的形貌特点,可以观察到微针有尖锐的针尖。体视镜下(图3A),能明显观察到所制备的微针为核壳性微针,药物主要分布在针尖上。微针的内核高度为607μm,底座直径约为282μm,外壳高度约为810μm,底座直径约为325μm。外壳层与内核层有一定空隙,并可以均匀完整的将内核层包被(图3B)。
2.2微针贴片的机械性能测定
样品:按照实施例1制取的载GLP-1(32-36)a长效聚乙烯醇微针贴片和载GLP-1(32-36)a-PEG20K微针贴片。
实验方法:
用双面胶贴于上述微针的背衬层面,将其固定于压力-拉力探测器的金属台上,微针针尖面向仪器的探针,探针向微针下压,记录并绘制位移和力的曲线,通过计算该曲线斜率获得两种微针的弹性模量。
实验结果:
通过力学曲线(图4)计算得出;载GLP-1(32-36)a聚乙烯醇微针弹性模量为82Mpa,略小于载GLP-1(32-36)a-PEG20K微针(弹性模量为90Mpa)。
实施例4微针贴片的皮肤插入性能
样品:按照实施例1制取的载GLP-1(32-36)a聚乙烯醇微针贴片。
实验方法:
以Balb/C鼠(雄性,20±1g)为动物模型,使用剃须刀和脱毛膏剃除Balb/C鼠小鼠大腿外侧的毛发,用乙醇清洗暴露的皮肤表面。将载GLP-1(32-36)a聚乙烯醇微针贴片垂直插入大鼠的背部皮肤,保持5分钟后分别剥离。脱颈处死小鼠,剥离皮肤,将微针插入部位切包埋,并冷冻在液氮中。切片至5μm厚度,置于硅烷涂层玻片上。在倒置显微镜下观察皮肤切片(IX-71,奥林巴斯,东京,日本)。
实验结果:
微针的皮肤插入深度是影响药物递送和治疗效果的关键。图5显示微针插入皮肤的深度约为(210μm),证明微针可以有效插入皮肤,为微针的体外透皮释放奠定了理论基础。
实验例5微针贴片体外释放实验
样品:按照实施例1制取的载GLP-1(32-36)a聚乙烯醇微针贴片
实验方法:
1.微针针体的含药量测定
用手术刀将0.03g载GLP-1(32-36)a微针贴片的针体和基底层分离。将针体收集,置于3ml的4%(v/v)醋酸水溶液中,溶解过夜,0.22μm微孔滤膜器过滤。采用液质联用色谱法(liquid chromatography-mass spectrometry,LC-MS)测定滤液中短肽药物的含量。
色谱条件:选用Agilent C18色谱柱(100mm×2.1mm,1.7μm),流动相为0.1%(w/v)甲酸溶液和乙腈,流速为0.3ml/min,进样量为5μl。
2.微针针体的体外释放实验
将上述分离的针体置于盛有1ml生理盐水的小瓶子内,使微针完全浸没在32℃生理盐水中,以300rpm进行磁力搅拌。在预定的时间点,取出样品溶液(0.1ml),过0.22μm的滤器,补充等量等温新鲜生理盐水,采用上述液质联用色谱法测定滤液中GLP-1(32-36)a和GLP-1(32-36)a-PEG20K的量。
实验结果:
外壳中的GLP-1(32-36)a在15min内累积释放75±7.8%,60min内释放89±4.2%,预测外壳药物插入皮肤后,可以迅速溶解于皮肤内(图6A)。微针中所有药物在400h才能释放95±10.8%,证明所制备的微针具有长效缓释作用(图6B)。
实验例6载GLP-1(32-36)聚乙烯醇微针贴片的药效学研究
样品:按照实验例1制备的载GLP-1(32-36)a聚乙烯醇微针贴片。
实验方法:
选Balb/C鼠(雄性,20±1g)为模型动物,通过注射链脲佐菌素(70mg/kg)造糖尿病小鼠;之后,将上述小鼠右侧腿部主动脉血管剪断,手术线结扎,建立糖尿病小鼠下肢缺血模型。选择上述小鼠(雄性,21±1g)共18只,分为模型组、皮下注射组(每天每只小鼠皮下注射给药10μg GLP-1(32-36)a,连续给药30天)、载GLP-1(32-36)a聚乙烯醇微针组(每只小鼠微针给药350μg GLP-1(32-36)a,30天内共给药一次)三组,每组各6只。使用剃须刀剃除小鼠的背部毛发,各组小鼠利用下肢血流多普勒仪测定造模前右侧下肢血流灌注情况,模型组不加治疗,在预定的时间点内测定小鼠健侧与患侧下肢血流的灌注情况,比较不同组别小鼠下肢血流灌注的改善情况。
实验结果:
通过图7A观察到手术后,小鼠右腿处血液流动明显减少,证明糖尿病下肢缺血模型建立成功。图7A和图7B说明治疗30天后,治疗组与模型组血流灌注有明显差异,说明GLP-1(32-36)a具有显著治疗糖尿病下肢缺血的症状。微针组的治疗效果要明显优于皮下注射组,证明微针的长效性以及可以最大发挥药物的治疗作用的优势。另外,本研究制备的载GLP-1(32-36)a聚乙烯醇微针贴片,全部采用已批准的药用辅料,制备工艺简单,利于生产转化。
实验例7载GLP-1(32-36)聚乙烯醇微针贴片的药代动力学研究
样品:按照实验例1制备的载GLP-1(32-36)a聚乙烯醇微针贴片。
选Balb/C鼠(雄性,20±1g)为模型动物,通过注射链脲佐菌素(70mg/kg)造糖尿病小鼠;之后,将上述小鼠右侧腿部主动脉血管剪断,手术线结扎,建立糖尿病小鼠下肢缺血模型。选择上述糖尿病小鼠(雄性,21±1g)共12只,分为皮下注射组、载GLP-1(32-36)a聚乙烯醇微针组二组,每组各6只。使用剃须刀剃除小鼠的右侧腿部毛发,分别在此处给药。
在预定的时间点内采取血样,1500g离心10min,取上层血浆100微升,之后,加100微升乙腈,将血浆蛋白完全沉淀后,进样分析,分析方法和色谱条件采用上述实施例4所示的LC-MS方法和色谱条件,测定各组药物在小鼠体内的血药浓度,结果如图8A和图8B。
与GLP-1(32-36)a皮下注射液相比,本实施例的微针贴片给药后,不仅在血浆中达到了相当的浓度峰值,且药物代谢趋缓,更为平稳,药物在血浆中的维持时间更长。
Claims (10)
1.一种载有胰高血糖素样肽-1短肽的微针贴片,其特征在于所述的微针系核壳型,包括:
外壳层,由GLP-1(32-36)a混合于第一聚合物制成;
内核层,由PEG-GLP-1(32-36)a、光引发剂和第二聚合物制成;
所述的第一聚合物选自于聚乙烯醇、聚乳酸、丝素蛋白、羧甲基纤维素钠、壳聚糖、海藻酸盐、透明质酸盐和聚乙烯吡咯烷酮之一种或几种;
所述的第二聚合物选自于聚乙烯醇、聚乳酸、丝素蛋白、羧甲基纤维素钠、壳聚糖、海藻酸盐、透明质酸盐、聚乙烯吡咯烷酮以及甲基丙烯基丝素蛋白、甲基丙烯基壳聚糖、甲基丙烯基明胶、甲基丙烯基透明质酸之一种或几种。
2.根据权利要求1所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于所述的PEG分子量为1w~100w。
3.根据权利要求1所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于所述微针的针体长度范围100μm~1000μm。
4.根据权利要求1所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于所述微针的针体基底面直径范围为30μm~500μm。
5.根据权利要求1所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于面积为1cm2的范围内,设置10~100个所述的微针。
6.根据权利要求1所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于所述在长度1mm范围内,设置1~3个所述的微针。
7.根据权利要求1所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于按如下方法制取:
第一工作溶液加入预制的第一微针贴片模具中,通过离心或真空法,使所述第一工作溶液进入第一微针模具孔中,室温下干燥过夜;
第二工作溶液加入上述第一微针贴片模具中,离心或真空法,使所述第二工作溶液进入第一微针模具孔中,室温下干燥过夜后,剥离微针,紫外光固化,获得载PEG-GLP-1(32-36)a微针贴片的内核结构;
然后,将第三工作溶液加入预制的第二微针贴片模具中,离心或真空法,使第三工作溶液进入微针模具孔中,再将载PEG-GLP-1(32-36)a微针贴片的内核结构压入第二微针贴片模具中,室温下干燥即得载GLP-1(32-36)a的微针贴片;
所述第一工作溶液中含有PEG-GLP-1(32-36)a为1~30wt%;
所述第一工作溶液中含有第一聚合物的浓度为5~30wt%;
所述第三工作溶液中含有GLP-1(32-36)a的浓度为1~5wt%;
所述第三工作溶液中含有第二聚合物的浓度为5~30wt%。
8.根据权利要求7所述的载有胰高血糖素样肽-1短肽的微针贴片,其特征在于所述第二工作溶液中含有第三聚合物的浓度为5~30wt%。
9.根据权利要求1~8之一所述的载有胰高血糖素样肽-1短肽的微针贴片在制备治疗糖尿病下肢缺血医疗器械中的应用。
10.一种医疗器械,其特征在于包括权利要求1~8之一所述的载有胰高血糖素样肽-1短肽的微针贴片。
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