CN114903875B - 一种含有青藤碱的雾化药剂及其应用 - Google Patents
一种含有青藤碱的雾化药剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种含有青藤碱的雾化药剂及其应用。所述含有青藤碱的雾化药剂按质量比包括青藤碱1～20份、抛射剂80～100份、助剂0～60份；所述抛射剂为氢氟烷烃。制得的含有青藤碱的雾化药剂可应用于制备治疗银屑病样皮肤炎症的雾化吸入药物，有效避免青藤碱经胃肠道给药时出现的首过效应，同时也可以解决青藤碱经皮给药系统时药物吸收速率不稳定、药物损耗高等问题。所述含有青藤碱的雾化药剂通过呼吸道粘膜给药，青藤碱能够通过粘膜快速进入血液，可以快速起效；雾化药剂可以通过包装瓶体的阀门定量给药，不容易出现过量摄入的问题；可以实现少量多次的给药方式，解决青藤碱半衰期较短的问题，同时也能保证体内药物浓度维持在足够高的水平，以保障治疗效果。
Description
技术领域
本发明涉及医药制剂技术领域，特别涉及一种含有青藤碱的雾化药剂及其应用。
背景技术
青藤碱是从中草药植物青风藤中提取的一种纯生物碱，文献报道青藤碱通过 NF-κB/MAPK信号通路抑制LPS诱导角质形成细胞的炎症反应。另外，在胶原蛋白诱导的类风湿关节炎小鼠模型中发现青藤碱能够下调多种炎症细胞因子和单核/巨噬细胞亚群缓解类风湿关节炎的发展。同样的，在诱导的小鼠关节炎模型中青藤碱能够促进Treg细胞比例上升并降低Th17细胞比例缓解关节肿胀。
然而青藤碱口服利用度较低，因其半衰期较短，且需长时间口服给药，故有研究人员提出通过经皮给药系统(Trans dermal Therapeutic System,TTS或Trans dermal.DrugDelivery，TDD)可以绕过肝脏的首过效应及胃肠道的破坏，维持稳定、持久的血药浓度。可惜的是，经皮给药系统中的透皮吸收速率会随着药物贴片中药物浓度的降低而降低。若要维持长效、足量的药物吸收速率，则需要保证药物贴片能够长时间维持足够高的药物浓度。通常是设计经皮给药系统时预留药物贮库，将更多的有效成分封存其中，使药物贴片与用户皮肤处的药物浓度能够形成动态平衡。这样使得经皮给药系统需要贮存远超正常用量的药物，会造成浪费，更危险的是当用户遗忘按时剥离药物贴片则可能造成过量摄入药物。
发明内容
本发明目的在于提供一种含有青藤碱的雾化药剂及其应用，以解决现有技术中所存在的一个或多个技术问题，提供至少一种有益的选择或创造条件。
本发明的第一方面在于提供一种含有青藤碱的雾化药剂，所述含有青藤碱的雾化药剂按质量比包括青藤碱1～20份、抛射剂80～100份、助剂0～60份；所述抛射剂为氢氟烷烃。
优选地，所述氢氟烷烃是四氟乙烷和/或七氟丙烷。所述四氟乙烷为1,1,1,2- 四氟乙烷，所述七氟丙烷为1,1,1,2,3,3,3-七氟丙烷。
优选地，所述助剂选自助溶剂、助悬剂、潜溶剂、润湿剂、乳化剂、稳定剂和矫味剂中的一种或多种。
优选地，所述助溶剂选自聚乙烯吡咯丙酮。
优选地，所述潜溶剂选自乙醇和/或聚乙二醇。
优选地，所述矫味剂选自甜蜜素、三氯蔗糖、木糖醇和果糖中的一种或多种。由于青藤碱的酸盐具有苦味，不适合使用如柠檬酸、橙皮苷等酸味的矫味剂。
优选地，所述含有青藤碱的雾化药剂还包括抗生素、糖皮质激素、维甲酸类、免疫调节剂中的一种或多种。青藤碱联合上述化合物使用能够进一步加强其对银屑病的治疗效果。
优选地，所述抗生素选自红霉素、甲硝唑、利福平中的至少一种。由于银屑病常伴有上呼吸道感染，因此联合红霉素、甲硝唑、利福平等使用可以缩短治愈所需的周期。
优选地，所述维甲酸类选自依曲替酯、依曲替酸中的至少一种。维甲酸类能够促进上皮细胞增生、分化、角质溶解等代谢作用，与青藤碱共用能够令患处鳞屑情况更快得到抑制。
本发明的第二方面在于提供所述含有青藤碱的雾化药剂在制备治疗银屑病样皮肤炎症的雾化吸入药物中的应用。
本发明提供的含有青藤碱的雾化药剂可用于制备呼吸道的雾化吸入药物，有效避免青藤碱经胃肠道给药时出现的首过效应，同时也可以解决青藤碱经皮给药系统时药物吸收速率不稳定、药物损耗高等问题。所述含有青藤碱的雾化药剂通过呼吸道粘膜给药，青藤碱能够通过粘膜快速进入血液，可以快速起效；雾化药剂可以通过包装瓶体的阀门定量给药，不容易出现过量摄入的问题；可以实现少量多次的给药方式，解决青藤碱半衰期较短的问题，同时也能保证体内药物浓度维持在足够高的水平，以保障治疗效果。
附图说明
图1是实施例1所述小鼠皮肤差异比对图，图中A为空白对照组小鼠，B 为模型组小鼠，C为低剂量给药模型组小鼠，D为高剂量给药模型组小鼠；
图2是实施例1所述小鼠脾脏外观对比图，图中A为空白对照组小鼠，B 为模型组小鼠，C为低剂量给药模型组小鼠，D为高剂量给药模型组小鼠；
图3是实施例1所述小鼠脾脏重量对比柱状图；
图4是实施例1所述小鼠脾脏的脏器系数对比柱状图；
图5是实施例1中小鼠皮肤组织经HE染色的切片组织图，图中A为空白对照组小鼠，B为模型组小鼠，C为低剂量给药模型组小鼠，D为高剂量给药模型组小鼠；
图6是实施例1中小鼠脾脏组织免疫组化检测图，图中A为空白对照组小鼠，B为模型组小鼠，C为低剂量给药模型组小鼠，D为高剂量给药模型组小鼠；
图7是实施例1小鼠皮肤组织经过免疫荧光染色检测IL-22表达图；
图8是实施例1使用流式细胞术检测各组小鼠脾脏中Th22 (CD4+IL-17A-INF-γ-IL-22+)细胞比例结果图；
图9是实施例1通过Western blot检测IL-22蛋白表达水平的电泳图；
图10是实施例1是IL-22蛋白表达水平柱状图；
图11是实施例1是IL-22蛋白定量表达柱状图。
实施例1，青藤碱雾化药剂对银屑病样动物模型的治疗试验
采用咪喹莫特乳膏诱导小鼠构建银屑病模型，采用雾化吸入青藤碱的方式，验证其对银屑病的抑制和治疗效果。由于使用小鼠雾化给药仪(安徽耀坤生物科技有限公司，YLS-8B)进行给药，因此所使用的药剂是经溶剂稀释后的青藤碱溶液，经由小鼠雾化给药仪超声雾化。雾化给药仪每分钟输出的气体量约为2.5L，在实验中使用以下公式对雾化给药剂量进行计算：D＝C×V×DI×T。其中D为雾化给药的剂量，C为雾化装置中药物气体的饱和密度(通过对密闭容器内即时饱和气体药物进行液化收集)，V为实验动物每分钟的呼吸量(对于小鼠而言约为1.0 L·min-1·kg-1)，DI为呼吸气体在动物肺部的分布系数(对于小鼠而言约为0.3)， T为雾化吸入时间。
(1)试验小鼠的分组
将购买SPF级C57BL/6小鼠48只，体重18-20g，年龄6-8周，雄性，随机分为 4组每组12只，分别为空白对照组(Control)、模型组(Model)、低剂量给药模型组 (Model+Sino(10mg/kg))和高剂量给药模型组(Model+Sino(40mg/kg))。
饲养条件为：室内温度维持在18-26℃，相对湿度40-70％，并采用自然昼夜照明，给予小鼠以充足的水源和食物，并保持环境的稳定。小鼠采用剃毛器剃除小鼠背部2cm×2cm面积的体毛。
空白对照组：每天早上9:00，在小鼠剃毛裸露皮肤处涂抹62.5mg凡士林，晚上6:00使用小鼠雾化给药仪吸入200μL双蒸水。
模型组：每天早上9:00，在小鼠剃毛裸露皮肤处涂抹5％咪喹莫特乳膏62.5 mg，晚上6:00使用小鼠雾化给药仪吸入200μL双蒸水。
低剂量给药模型组：每天早上9:00，在小鼠剃毛裸露皮肤处涂抹5％咪喹莫特乳膏62.5mg，晚上6:00使用小鼠雾化给药仪吸入青藤碱。雾化药液浓度为100 mg·mL-1，经过公式计算，正常小鼠经过30min雾化的给药剂量约为10mg·kg-1。
高剂量给药模型组：每天早上9:00，在小鼠剃毛裸露皮肤处涂抹5％咪喹莫特乳膏62.5mg，晚上6:00使用小鼠雾化给药仪吸入青藤碱。雾化药液浓度为400 mg·mL-1，经过公式计算，正常小鼠经过30min雾化的给药剂量约为10mg·kg-1。
试验持续7天。
(2)试验样本取材
于试验开展第8天上午9：00，通过异氟烷麻醉小鼠，取各组具有代表性小鼠进行数码相机拍照对比观察小鼠剃毛裸露皮肤处的皮损情况。皮损情况如图1所示，空白对照组皮肤正常；模型组小鼠在5％咪喹莫特乳膏作用下出现明显银屑样皮损；与模型组相比较，低剂量给药模型组未能明显改善小鼠皮损；与模型组相比较，高剂量给药模型组小鼠皮肤损伤得到缓解。
观察小鼠后，通过眼眶取血收集小鼠外周血，采用高速冷冻离心机转速3000 rpm，5min，收集各组小鼠血清于-80℃超低温冰箱保存。分离各组小鼠脾脏组织进行拍照、称重之后和部分背部皮肤组织固定于4％多聚甲醛中，随后采用20％和30％蔗糖梯度脱水，采用OCT包埋，于冰冻切片机上切为厚度5μm的组织切片，存放于-80℃超低温冰箱保存。小鼠脾脏的外观对比如图2所示，各组小鼠的脾脏颜色未出现明显差异，模型组、低剂量给药模型组和高剂量给药模型组的小鼠脾脏样本均相较于空白对照组的出现了膨大，其中模型组膨大情况最为明显，其次分别是低剂量给药模型组和高剂量给药模型组。脾脏重量对比如图3所示，由于低剂量给药模型组未能明显改善小鼠皮损，所以其脾脏重量和脾脏器官系数无统计学意义。模型组小鼠脾脏与空白对照组比较，重量显著增加。高剂量给药模型组小鼠在青藤碱的作用下能够显著的降低脾脏的质量。脾脏的脏器系数对比如图 4所示，模型组小鼠脾脏脏器系数与空白对照组比较重量显著增加，高剂量给药模型组在青藤碱的作用下能够显著的降低脾脏脏器系数。以上结果表明满足一定浓度的青藤碱能够有效的抑制银屑病样小鼠脾脏肿大改善疾病症状。
(3)皮肤组织的HE染色
将存储于-80℃超低温冰箱的各组冰冻切片组织，取出放入室温平衡15 min，采用PBS泡洗5min，苏木素染色液染色5min，浸自来水中冲洗10分钟，去多余的染色液，采用PBS再冲洗一遍，伊红染色液染色1min，自来水冲洗10分钟， 70％乙醇脱水10s，80％乙醇脱水10s，90％乙醇脱水10s，无水乙醇脱水10s，二甲苯透明5min，中性树胶封片，采用显微镜拍照获取图片。结果如图5所示，空白对照组小鼠皮肤结构完整皮肤未见角化和角化不完全，皮肤颗粒层清晰，真皮周围无炎性细胞浸润。与空白对照组相比，模型组和低剂量给药模型组小鼠皮肤表层出现不同程度的角化过度，棘层肥厚伴有炎性细胞浸润，真皮层出现血管扩张及水肿。与模型组相比，高剂量给药模型组的小鼠皮肤显著改善了过度角化的问题，真皮层未出现血管扩张及水肿。说明满足一定浓度的青藤碱能够显著的降低小鼠皮肤角化。
(4)脾脏组织免疫组化检测IL-22表达
将存储于-80℃超低温冰箱的各组冰冻切片组织，置于室温5min；42℃烘烤10min；然后用PBS浸洗两遍(约5min/次)；抗原修复：将切片置于0.01M枸橼酸盐缓冲液(PH 6.0)染色缸中，98℃水浴15min；让切片在0.01M枸橼酸盐缓冲液中放置直到自然冷却至室温；PBS泡洗3次，每次5min；将切片置于0.3％ Triton 37℃破膜30min，PBS泡洗3次，每次5min；将切片置于3％的H2O2中室温孵育10min后PBS冲洗3次，每次5min，擦干周围液体；滴加5％BSA室温封闭10min；滴加抗IL-22的一抗，置于4℃湿盒内过夜，一抗孵育完成；PBS泡洗3次，每次5min；组织切片上滴加相应的二抗置于室温孵育1h；PBS洗涤3次，每次5min；DAB显色剂显色：将DAB显色剂滴加于切片，室温，镜下检测反应时间，流水冲洗10min；苏木素复染3min，自来水冲洗5min；分化液分化2s，自来水流水冲洗10min；70％乙醇脱水10s，80％乙醇脱水10s，90％乙醇脱水10s，无水乙醇脱水10s，二甲苯透明5min，中性树胶封片，采用显微镜拍照获取图片。
脾脏组织中IL-22的免疫组化间接反应脾脏中Th22细胞的分布情况如图6所示，阳性反应物经过DAB染色呈现棕色，为图中箭头指示处。空白对照组中仅有少量的IL-22阳性，模型组和低剂量给药模型组中呈现大片呈阳性的IL-22棕色颗粒密集分布，高剂量给药模型组则明显的减少。
如图7所示，皮肤组织经过固定、脱水、包埋、切片和特异性抗IL-22的荧光抗体染色各组IL-22的表达水平，结果表明在空白对照组中IL-22在组织中少量表达，在模型组和低剂量给药模型组中IL-22表达显著上升，高剂量给药模型组中发现IL-22的表达显著降低，说明满足一定浓度的青藤碱干预能够显著的降低皮肤组织中IL-22的表达。
(5)流式细胞术分析各组小鼠淋巴结中Th22细胞比例
无菌环境中分离各组小鼠腹股沟、腋下及肠系膜淋巴结，采用200目孔不锈钢筛网进行机械研磨，采用红细胞裂解液去除细胞中残留的红细胞，调整细胞密度为1×107cells/mL，进行流式抗体染色。
收集各组分离的单个淋巴细胞于流式管中，用100μL PBS重悬细胞，每管加入0.5μL FITC标记的Anti-mouse CD4单克隆抗体，4℃避光孵育30min。用 200μL PBS洗两次，加入250μL Fixation/Permeabilization溶液4℃固定20min，之后用1×BD Perm/WashTM溶液洗两次，再用50μL 1×BD Perm/WashTM溶液重悬细胞，每管加入0.5μL PE-Cy7标记的Anti-mouse IFN-γ单克隆抗体，APC标记的Anti-mouse IL-22单克隆抗体，BV421标记的Anti-mouse IL-17A单克隆抗体， 4℃避光孵育30min，用1×BD Perm/WashTM溶液洗两次，然后用PBS重悬，用流式细胞仪检测。结果如图8所示，从所有细胞中分出淋巴细胞群然后选中 CD4+T细胞群，随后选择IL-17A-INF-γ-双阴性细胞，从双阴性细胞中选择CD4+IL-22+双阳性细胞为Th22细胞。结果表明模型组相较于空白对照组，其Th22细胞比例显著上升；高剂量给药模型组相较于模型组，其显示满足一定浓度的青藤碱干预能够显著的降低Th22细胞比例，说明青藤碱的干预能够显著的降低脾脏中 Th22细胞的比例。
(6)RT-PCR和Western blot检测皮肤组织中IL-22表达水平
分离获取的各组小鼠背部皮肤组织，采用剪刀将组织剪碎，采用TRIzol法提取各样本的总RNA，紫外分光光度计测定总RNA浓度，采用逆转录试剂盒将总RNA反转录成cDNA，再以cDNA为模板进行PCR扩增反应，PCR扩增条件及引物序列为：IL-22正向5′-ATG AGT TTTTCC CTT ATG GGGAC-3′,IL-22反向 5′-GCTGGA AGT TGG ACA CCT CAA-3′,β-actin正向5′-AAC AGT CCG CCT AGA AGCAC-3′,β-actin反向5′-CGT TGA CAT CCG TAA AGA CC-3′RT-PCR 采用Step One-PlusTM Real-Time PCR System完成。RT-PCR反应条件为:95℃，5min；95℃，15s；60℃，1min，35个周期循环；72℃，30s；95℃15s；60℃， 1min；95℃；15s。β-actin为内参基因。每个样品测定重复3次，结果以2-ΔΔCt表示，结果如图9，空白对照组中IL-22mRNA表达水平较低，模型组中IL-22mRNA 相较于空白对照组显著上升，青藤碱高剂量(40mg/kg)给药模型组相较于模型组测得的IL-22mRNA表达显著降低，低剂量(10mg/kg)的无明显差异，说明高剂量的青藤碱能够抑制皮肤组织中IL-22的表达。
使用RIPA裂解液(含1％PMSF)于4℃裂解组织30min，使用高速冷冻离心机4℃16000g离心10min，收集上清液，BCA试剂盒检测总蛋白量。用10％ SDS-PAGE蛋白电泳，转膜PVDF膜上，以含5％脱脂牛奶的TBST室温下封闭1h，然后加入抗IL-22一抗4℃冰箱孵育过夜，使用TBST洗涤3次，每次5min，膜在室温下以羊抗兔/羊抗鼠IgG-HRP二抗孵育60min，使用ECL显色液曝光，通过 Quantity one进行蛋白灰度分析。图10所示，为IL-22蛋白表达水平，图11为IL-22 蛋白定量表达。空白对照组中IL-22蛋白表达水平较低，模型组中测得的IL-22相较于空白对照组显著上升，在青藤碱(40mg/kg)的干预下相较于模型组测得的 IL-22则显著降低，低剂量(10mg/kg)无明显作用，说明高剂量的青藤碱能够抑制皮肤组织中IL-22的表达。
实施例2，制备含有甲硝唑的青藤碱雾化药剂
药剂组分包括：
将青藤碱溶于无水乙醇中，再加入甲硝唑与木糖醇并搅拌均匀。在常温下真空处理，使无水乙醇挥发，获得混合物。将所述混合物与1,1,1,2-四氟乙烷加入耐压容器中，混悬均匀，而后将耐压容器中的药液在压力下经阀门充入己压好盖的气雾剂罐中，即得气雾剂；每瓶气雾剂罐装量为10mL，总掀次数100次。
实施例3，制备含有甲硝唑的青藤碱雾化药剂
药剂组分包括：
青藤碱 3.3g；
1,1,1,2,3,3,3-七氟丙烷 100mL；
依曲替酯 3.54g；
将青藤碱溶于无水乙醇中，再加入依曲替酯并搅拌均匀。在常温下真空处理，使无水乙醇挥发，获得混合物。将所述混合物与1,1,1,2,3,3,3-七氟丙烷加入耐压容器中，混悬均匀，而后将耐压容器中的药液在压力下经阀门充入己压好盖的气雾剂罐中，即得气雾剂；每瓶气雾剂罐装量为10mL，总掀次数100次。对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
SEQUENCE LISTING
<110> 南方医科大学顺德医院（佛山市顺德区第一人民医院）
<120> 一种含有青藤碱的雾化药剂及其应用
<130> 2022
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<170> PatentIn version 3.5
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Claims (8)
1.一种含有青藤碱的雾化药剂，其特征在于，按质量比由青藤碱1～20份、抛射剂80～100份、助剂0～60份组成；所述抛射剂为氢氟烷烃；所述助剂选自助溶剂、助悬剂、潜溶剂、润湿剂、乳化剂、稳定剂和矫味剂中的一种或多种，所述潜溶剂选自乙醇和/或聚乙二醇。
2.根据权利要求1所述含有青藤碱的雾化药剂，其特征在于，所述氢氟烷烃是四氟乙烷和/或七氟丙烷。
3.根据权利要求1所述含有青藤碱的雾化药剂，其特征在于，所述助溶剂选自聚乙烯吡咯丙酮。
4.根据权利要求1所述含有青藤碱的雾化药剂，其特征在于，所述矫味剂选自甜蜜素、三氯蔗糖、木糖醇和果糖中的一种或多种。
5.根据权利要求1至4任一项所述含有青藤碱的雾化药剂，其特征在于，还包括抗生素、糖皮质激素、维甲酸类中的一种或多种。
6.根据权利要求5所述含有青藤碱的雾化药剂，其特征在于，所述抗生素选自红霉素、甲硝唑、利福平中的至少一种。
7.根据权利要求5所述含有青藤碱的雾化药剂，其特征在于，所述维甲酸类选自依曲替酯、依曲替酸中的至少一种。
8.权利要求1至7任一项所述含有青藤碱的雾化药剂在制备治疗银屑病的雾化吸入药物中的应用。
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