CN104013537B - 填充剂的效能维持用组合物 - Google Patents
填充剂的效能维持用组合物 Download PDFInfo
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- CN104013537B CN104013537B CN201410073360.4A CN201410073360A CN104013537B CN 104013537 B CN104013537 B CN 104013537B CN 201410073360 A CN201410073360 A CN 201410073360A CN 104013537 B CN104013537 B CN 104013537B
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Abstract
本发明涉及包含胶原蛋白肽作为有效成份而使填充剂的效能长时间维持的组合物。本发明的目的在于提供一种在透明质酸填充剂手术后能够使其效能长时间维持的组合物。为达成该目的，本发明提供一种包含胶原蛋白作为有效成份的透明质酸填充剂效能维持用组合物及包含胶原蛋白作为有效成份的透明质酸合成促进用组合物。本发明的组合物能够促进透明质酸的合成，抑制透明质酸酶的活性，能够使透明质酸填充剂效能维持更长时间，因而有用。
Description
技术领域
本发明涉及包含胶原蛋白作为有效成份而使填充剂的效能长时间维持的组合物。
背景技术
移植到生物组织的物质应无毒性，在完成所希望的功能后，应借助于生物体内代谢活动而分解及排出。最近，向皮肤组织内移植特定物质的美容目的的手术正在迅速发展。已知透明质酸作为在这种手术主要使用的物质，在插入或移植到人体内后，与相应物质的浓度或包含相应物质的组合物的种类无关，自数日后起，实现相应物质的代谢及排出。手术后为了长时间维持所希望的美容效果，要求能够克服透明质酸的较短的人体内代谢时间的方法，对具有数个月以上的人体内维持时间的填充剂材料的开发等的要求正在增加。
因此，本发明人确认了当胶原蛋白肽给药与透明质酸填充剂手术并行时，胶原蛋白肽能够使注入皮肤内的透明质酸填充剂的生物体内维持时间延长，从而完成了本发明。
发明内容
本发明的目的在于提供一种在透明质酸填充剂手术后能够使其效能长时间维持的组合物。
为达成所述目的，本发明提供一种包含胶原蛋白作为有效成份的透明质酸填充剂效能维持用组合物及包含胶原蛋白作为有效成份的透明质酸合成促进用组合物。
本发明的组合物能够促进透明质酸的合成，抑制透明质酸酶（hyaluronidase）的活性，能够使透明质酸填充剂效能维持更长时间，因而有用。因此，具有即使以更少次数进行填充剂手术，其效果也能够维持，减小皮肤刺激的优点。
附图说明
图1是表示通过胶原蛋白肽的处理而透明质酸合成量增加的图表。
图2是目视观察填充剂注入后，随着处理胶原蛋白肽而实验对象的体积变化的照片。
图3以图表表示填充剂注入后，随着处理胶原蛋白肽而实验对象的体积变化的程度。
具体实施方式
本发明的一个方式涉及包含胶原蛋白作为有效成份的透明质酸填充剂效能维持用组合物。
胶原蛋白是硬蛋白质之一，含有少量的糖。胶原蛋白是结缔组织基质的主成份，就哺乳动物而言，占总蛋白质的约30重量%。在动物的真皮、软骨等中以纤维状存在，聚集成胶原纤维。构成纤维的基本单位是分子量约30万、长280nm、粗1.5nm的原胶原蛋白。
作为本发明的一个方式的组合物在注入透明质酸填充剂后，能够使填充剂在皮肤中更自然地维持更长时间。
本发明的另一个方式涉及包含胶原蛋白作为有效成份的透明质酸合成促进用组合物。
本发明的胶原蛋白肽能够通过它的给药而促进透明质酸的合成，能够增进与透明质酸的合成相关的之前步骤的酶或蛋白质的活性或量。
在本说明书中，“透明质酸填充剂”可以意味着将透明质酸（hyaluronic acid,HA）作为主成份的填充剂。所述透明质酸作为由氨基酸与糖醛酸构成的复杂的多糖之一，是由N-乙酰氨基葡萄糖与葡糖醛酸构成的高分子化合物。所述填充剂（filler）可以意味着为填平皱纹或凹陷的疤痕等而向皮肤细胞直接注入或插入的补充材料。所述填充剂能够以缓解皮肤凹凸补充水份感等多种目的来使用，没有限制。所述填充剂可以在身体所有部位进行手术，具体可以包括脸、颈等。
在本说明书中，“维持填充剂效能”包括使注入的填充剂更缓慢分解或促进生物体内透明质酸的合成，使作为填充剂主成份的透明质酸的含量增加。
本发明的组合物能够使通过填充剂而缓解皮肤凹凸、补充水份感等非限定的效能长时间持续，因而能够有用地使用。具体而言，能够延长填充剂的注入周期，不仅使皮肤刺激减小，还具有经济上的优点。
本发明的一个方式的组合物能够促进透明质酸的合成，能够促进制备透明质酸的之前步骤（up-stream）的酶、蛋白质等的活性，或增加其量。
在本发明的一个方式的组合物中，所述胶原蛋白包括胶原蛋白肽。
在本说明书中，所述胶原蛋白肽作为分子量为500～1000Da的肽，只要是氨基酸通过肽键而结合在一起，均可无限制地应用。具体而言，所述胶原蛋白肽可以包括Gly-X-Y的胶原三肽，所述X及Y可以包括通常在自然中存在的所有种类的氨基酸。而且，所述X及Y可以是相同或互不相同种类的氨基酸。
在本发明的一个方式的组合物中，所述组合物可以相对于组合物全部重量包括0.01～50重量%的胶原蛋白肽。在所述范围内，相应组合物可以促进透明质酸的合成，使透明质酸填充剂的效能长时间维持。从如上观点而言，本发明的一个方式的组合物可以包括相对于组合物全部重量为0.05～48重量%、相对于组合物全部重量为0.1～46重量%、相对于组合物全部重量为0.5～44重量%、相对于组合物全部重量为1～42重量%，或相对于组合物全部重量为5～40重量%的胶原蛋白肽。
在本发明的一个方式的组合物中，所述胶原蛋白肽可以包括Gly-X-Y的胶原三肽。
在本发明的一个方式的组合物中，所述X及Y可以包括相同或互不相同的氨基酸的情形，所述氨基酸可以选自甘氨酸（Glycine,Gly）、丙氨酸（Alanine,Ala）、缬氨酸（Valine,Val）、亮氨酸（Leucine,Leu）、异亮氨酸（Isoleucine,Ile）、苏氨酸（Threonine,Thr）、丝氨酸（Serine,Ser）、半胱氨酸（Cysteine,Cys）、蛋氨酸（Methionine,Met）、天冬氨酸（Aspartic acid,Asp）、天冬酰胺（Asparagine,Asn）、谷氨酸（Glutamic acid,Glu）、谷氨酰胺（Glutamine,Gln）、赖氨酸（Lysine,Lys）、精氨酸（Arginine,Arg）、组氨酸（Histidine,His）、苯丙氨酸（Phenylalanine,Phe）、酪氨酸（Tyrosine,Tyr）、色氨酸（Tryptophan,Trp）及脯氨酸（Proline,Pro）中。
具体而言，所述胶原三肽可以包括甘氨酸-脯氨酸-羟基脯氨酸，但并不限定于此。
在本发明的一个方式的组合物中，所述组合物可以相对于组合物总重量包括胶原蛋白或胶原蛋白肽1～80重量%。胶原蛋白或胶原蛋白肽的含量如果不足1重量%，则难以获得所需的效果，如果超过80重量%，则在制成剂型方面存在困难。从上述观点考虑，本发明的组合物可以相对于组合物总重量，含有1～80重量%、3～78重量%、5～76重量%、7～74重量%、9～72重量%、11～70重量%、13～68重量%、15～66重量%、17～64重量%或19～62重量%的胶原蛋白或胶原蛋白肽。
在本发明的一个方式的组合物中，所述胶原蛋白或胶原蛋白肽可以抑制透明质酸酶（hyaluronidase）的活性。所述胶原蛋白或胶原蛋白肽抑制透明质酸酶的活性或使量减少，防止透明质酸的分解，因而能够促进透明质酸的合成，能够使填充剂的效能长时间维持。
在本发明的一个方式的组合物中，所述组合物可以是药物组合物。
在将本发明的组合物应用于医药品的情况下，可以以所述组合物为有效成份，加入常用的无机或有机载体，以固体、半固体或液态的形态，制成口服给药剂。
作为用于所述口服给药的剂型，可举出片剂、丸剂、颗粒剂、胶囊剂、散剂、微粒剂、粉剂、乳浊剂、糖浆剂、微丸剂等。为了对本发明的有效成份进行制剂，按照通常的方法实施便可以容易地制剂，可以适当使用表面活性剂、赋形剂、着色料、香料、防腐剂、稳定剂、缓冲剂、悬浊剂、其它常用辅助剂。
本发明的所述药物组合物可以经由口服给药。
另外，所述活性成份的给药量因接受治疗的对象的年龄、性别、体重和将治疗的特定疾病或病理状态、疾病或病理状态的严重度、给药途径及开处方者的判断而异。基于这种因素决定给药量，在本领域技术人员的水平之内。普通的给药量为0.001mg/kg/日～2000mg/kg/日，更具体而言，为0.5mg/kg/日～1500mg/kg/日。
就本发明的一个方式的组合物而言，所述组合物可以是健康食品组合物。
在本发明的一方面，所述健康食品组合物例如可以举出粉末、颗粒、片剂、胶囊剂及口服液等，但并不限定于此，可以以多种形态进行制剂。
另外，所述健康食品组合物根据需要，可以包括以下添加剂中的1种或2种以上。作为所述添加剂，例如可以是葡萄、苹果、橙子、柠檬、菠萝、香蕉、梨等的浓缩果汁或粉末果汁，视黄醇棕榈酸酯、核黄素、吡哆醇、氰钴胺素（cyanocobalamine）、抗坏血酸、烟酰胺、泛酸钙、叶酸、生物素、胆钙化醇（cholecalciferol）、重酒石酸胆碱、生育酚、β-胡萝卜素等水溶性或脂溶性维生素类，柠檬香精、橙子香精、草莓香精、葡萄香精、香草精等香味剂，谷氨酸、谷氨酸钠、甘氨酸、丙氨酸、天冬氨酸、天冬氨酸钠或肌苷酸等氨基酸、核酸或其盐类，聚葡萄糖、果胶、黄原胶、葡甘聚糖、褐藻酸等植物纤维，或氯化钠、硫酸镁、氯化钾、氯化镁、碳酸镁、氯化钙、磷酸氢二钾、磷酸二氢钠、甘油磷酸钙、枸橼酸亚铁钠、枸橼酸铁铵、枸橼酸铁、硫酸锰、硫酸铜、碘化钠、山梨酸钾、锌、锰、铜、碘或钴等矿物质类。
本发明的另一个方式可以是包括透明质酸填充剂组合物以及技术方案1或2的组合物的填充剂手术用试剂盒。
就本发明的一个方式的试剂盒而言，所述试剂盒还包括说明书 在所述说明书中可以公开所述透明质酸经皮给药，本发明
的另一个方式的组合物口服给药。
所述填充剂可以涂布于皮肤表面，使其吸收到皮肤中，可以使其插入皮肤内。
本发明的所述试剂盒在填充剂手术时使用，能够使填充剂的效能持续时间延长。
以下通过实施例，进一步详细说明本发明。这些实施例只用于举例说明本发明，不得解释为本发明的范围限定于这些实施例，这是本领域的技术人员清楚的。
[制备例1]胶原蛋白肽的准备
在本发明中用作活性成份的胶原蛋白肽购自杰乐公司（HATC,Jellice Co.,JAPAN），是含有如甘氨酸-脯氨酸-羟基脯氨酸形态的三肽（tripeptide）15%以上的复合物。
[实验例1]角质形成细胞中的透明质酸（HA）合成促进能力
将人源的角质形成细胞株HaCaT细胞（Dr N.E.Fusenig，德国癌症研究中心，德国海德堡）在包含10%FBS的DMEM培养基中，在37℃、5%CO2的条件下进行培养。在96孔板中培养细胞，当长到80%以上时，更换为不含FBS的培养基，培养24小时。然后，将制备例1的胶原蛋白肽按200倍浓度溶解于PBS，处理成最终浓度分别为100ppm及200ppm，培养24小时。24小时后，回收培养基，用于透明质酸测定（HR assay），利用剩余的细胞，进行细胞生存率定量实验（CCK8assay）。
CCK8测定（Dojindo公司）和透明质酸测定（Echelon公司）按照试剂盒提供厂商的协议进行了实验，将定量的透明质酸值除以细胞生存率值并进行补正。
结果，如图1所示，进行胶原蛋白肽处理的组与不进行处理的对照组相比，可以确认透明质酸合成量增加30%～44%以上，这种合成促进能力与进行胶原蛋白肽处理的浓度成比例。因此可以确认，胶原蛋白肽能够在皮肤细胞中促进透明质酸的合成。
[实验例2]透明质酸酶（hyaluronidase）的活性抑制能力
为了了解能否抑制作为填充剂主要成份的透明质酸的水解，观察了透明质酸酶的活性抑制效能。
在0.1M的透明质酸酶液（7900unit/mL）50μL中，每次添加胶原蛋白肽20μL，使得最终浓度达到0.2、0.4、0.6、0.8、1.0mg/mL，为了激活酶，在混合12.5mM的CaCl2200μL后，在37℃的水溶液上培养20分钟。对照组放入蒸馏水取代胶原蛋白肽，在水溶液上培养20分钟。在以Ca2+激活的透明质酸酶酶溶液中添加0.1M透明质酸液（12mg/5mL）250μL，再次在水溶液上培养40分钟。培养后，将0.4N的NaOH溶液100μL和0.4M四硼酸钾（potassium tetraborate）100μL添加到反应混合物，在沸水槽中培养3分钟后冷却。在冷却的反应物中添加二甲氨基苯甲醛溶液（对二甲氨基苯甲醛4g、100%乙酸350mL及10N的HCl50mL的混合液）3.28mL后，在37度的水浴上培养20分钟，在585nm下测定了吸光度。
结果，如表1所示，可知胶原蛋白肽抑制透明质酸酶活性的效能优秀。而且，确认了所述效能依赖于胶原蛋白肽的浓度。
【表1】
| 胶原蛋白肽浓度(mg/ml) | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 |
| 透明质酸酶活性抑制率(％) | 30.4 | 32.8 | 48.6 | 75.2 | 82.4 |
[实验例3]透明质酸填充剂的生物组织内维持能力
使用作为市销的透明质酸填充剂的葛莉斯黛II（GLYTONE II）（太平洋制药，HA20mg/g），注入无毛鼠的背部后，观察体积维持时间。
15～17周龄的无毛鼠分成两组，一组维持正常食物（对照组），其余一组将胶原蛋白肽以500mg/kg浓度口服给药3周（试验组）。根据填充剂的注入量，如以下表2所示，将对照组和试验组分成亚组进行实施。
【表2】
| 类别 | 填充剂注入量 |
| 对照组1 | 0.02ml |
| 对照组2 | 0.04ml |
| 试验组1 | 0.02ml |
| 试验组2 | 0.04ml |
注入填充剂后，对照组及试验组均维持正常食物，只对试验组维持每周胶原蛋白肽口服给药。对所述对照组及试验组的体积变化进行了4周时间观察。结果，如图2及图3所示，可以确认，注入对照组背部的填充剂随着时间而减少，对照组的体积在更短时间内持续减少，相反，胶原蛋白肽口服给药的试验组与对照组相比，体积维持效果优秀。
以下说明本发明的组合物的剂型例，但药物组合物及化妆品组合物可以以多种剂型应用，这并非要限定本发明，仅用于具体说明。
[制剂例1]软质胶囊剂
混合制备例1的胶原蛋白肽150mg、棕榈油2mg、氢化棕榈油8mg、黄蜡4mg及卵磷脂6mg，根据通常的方法，每个胶囊填充400mg，制备了软质胶囊。
[制剂例2]片剂
混合制备例1的胶原蛋白肽150mg、葡萄糖100mg、红参提取物50mg、淀粉96mg及硬脂酸镁4mg，添加30%乙醇40mg，形成颗粒后，在60℃下干燥，利用压片机压成片剂。
[制剂例3]颗粒剂
混合制备例1的胶原蛋白肽150mg、葡萄糖100mg、红参提取物50mg及淀粉600mg，添加30%乙醇100mg，形成颗粒后，在60℃下干燥，形成颗粒后装袋。内容物的最终重量为1g。
[制剂例4]口服液
混合制备例1的胶原蛋白肽150mg、葡萄糖10g、红参提取物50mg、柠檬酸2g及纯净水187.8g并装瓶。内容物的最终容量为200ml。
以上详细记述了本发明内容的特定部分，这种具体记述只是优选实施方式，本发明的范围并非限定于此，这是本领域技术人员清楚的。因此，本发明的实质范围由权利要求及其等价物来确定。
Claims (7)
1.一种胶原蛋白在制备透明质酸填充剂效能维持用组合物中的用途，
其中，在将透明质酸填充剂组合物给药至对象后将包含所述胶原蛋白的组合物口服给药至所述对象，
其中，所述胶原蛋白包括胶原蛋白肽，所述胶原蛋白肽包括甘氨酸-脯氨酸-羟基脯氨酸的胶原三肽，并且
所述胶原蛋白或胶原蛋白肽抑制透明质酸酶的活性。
2.根据权利要求1所述的用途，其中，所述组合物相对于组合物全部重量包含0.01～50重量％的胶原蛋白肽。
3.根据权利要求1所述的用途，其中，所述组合物相对于组合物总重量包含1～80重量％的胶原蛋白或胶原蛋白肽。
4.根据权利要求1所述的用途，其中，所述组合物为药物组合物。
5.根据权利要求1所述的用途，其中，所述组合物为健康食品组合物。
6.一种胶原蛋白在制备填充剂手术用试剂盒中的用途，其中，所述试剂盒包括：
透明质酸填充剂组合物，和
透明质酸填充剂效能维持用组合物，并且
其中，所述透明质酸填充剂效能维持用组合物包含胶原蛋白，并且
其中，所述胶原蛋白包括胶原蛋白肽，所述胶原蛋白肽包括甘氨酸-脯氨酸-羟基脯氨酸的胶原三肽，并且
所述胶原蛋白或胶原蛋白肽抑制透明质酸酶的活性，并且
将包含所述胶原蛋白的组合物口服给药至对象。
7.根据权利要求6所述的用途，其中，所述试剂盒还指示：所述透明质酸经皮给药，
所述透明质酸填充剂效能维持用组合物口服给药。
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