CN101961486A - 一种滴眼液及其制备方法 - Google Patents
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Abstract
一种滴眼液及其制备方法,涉及一种滴眼液。提供一种与现有的滴眼液相比,作用时间更短、价格较低、效果相对较好、可同时起到抗角膜新生血管及快速修复上皮的滴眼液及其制备方法。所述滴眼液的组成及其按体积百分比的含量为:牛血清0.1%~5%,增稠剂5%~15%,酸碱调节液1%~5%,渗透压缓冲剂0.1%~2%,抗生素0.5%~2%,SA3K原溶液5%~30%,余为平衡盐溶液。按滴眼液的配方,将增稠剂、牛血清、抗生素和SA3K原溶液加到平衡盐溶液中混合均匀,用酸碱调节剂调pH值为7.2~7.4,用渗透压缓冲剂调渗透压为350~380mOsm/L,经膜过滤除菌,即得SA3K滴眼液。
Description
技术领域
本发明涉及一种滴眼液,尤其是涉及一种治疗炎症性角膜新生血管及角膜上皮缺损性疾病的滴眼液及其制备方法。
背景技术
角膜病是常见的致盲性眼病之一,临床上各种因素所致的角膜炎症和角膜损伤均可致角膜缺损、糜烂和溃疡,若得不到有效治疗,则将严重损伤患者的视功能和生活质量。据世界卫生组织报告,目前,角膜病是引起视力丧失的第二位主要病因,仅次于白内障,每年因角膜溃疡、眼外伤等造成的新增角膜盲为(150~200)万,严重威胁着患者的健康([1]赵家良.深入开展防盲治盲是我国眼科医师的社会责任.中华眼科杂志,2005,41:3-5)。而在角膜病中,最主要的致盲原因是的角膜血管化和瘢痕化,由于参与角膜新生血管及角膜炎症的因素较多,发病原因复杂,文献报道较少,同时缺乏系统研究,有关其发生机制、病理过程等面的问题还尚未完全阐明,寻找新的药物治疗方向也已经成为当前眼科界的一个研究热点。角膜新生血管(corneal neovascularization,CNV)可以继发于多种角膜炎症性疾病(角膜炎、溃疡等)及各类角膜损伤(角膜外伤、烧伤等),是角膜对缺氧、炎症等多种刺激因素综合的病理反应,可能导致角膜移植时的排斥反应和炎症反应,严重影响着患者视力恢复。然而对于角膜新生血管、淋巴管及角膜炎症的药物研究,有望解决角膜创口愈合困难和术后免疫排斥反应二大关键问题,是目前治疗角膜盲的新途径。
SA3K,(SERPINA3K,亦称为KBP kallikrein-binding protein)是新近发现的一种内源性激肽释放酶结合蛋白,其基因序列也已发现。SA3K主要产生于肝脏及眼部组织,具有抑制激肽释放酶作用([2]Chai KX,Ma J-X,Murray SR,Chao J,Chao L:Molecular cloningand analysis of the rat kallikrein-binding protein gene.J BiolChem 1991;266:16029-16036)。SA3K属丝氨酸蛋白酶抑制剂家族(serine proteinase inhibitor,SERPIN),享有重要的同源序列([3]Tombran-Tink J,Mazuruk K,Rodriguez IR,Chung D,Linker T,Englander E,Chader GJ:Organization,evolutionary conservation,expression and unusual Alu density ofthehuman gene for pigment epithelium-derived factor,a unique neurotropHic serpin.Mol Vis 1996;2:11)。最近的研究发现,SA3K在视网膜有抗新生血管、抗炎、抗氧化、抗纤维化活性([4]Yamagishi S,Inagaki Y,Nakamura K,Abe R,Shimizu T,Yoshimura A,Imaizumi T:Pigment epithelium-derived factor inhibits TNF-alpHa-induced interleukin-6expression inendothelial cells by suppressing NADpH oxidase-mediated reactive oxygen species generation.JMol Cell Cardiol 2004;37:497-506;[5]Wang JJ,Zhang SX,Mott R,Chen Y,Knapp RR,Cao W,Ma JX:Anti-inflammatory effects of pigment epithelium-derived factor in diabeticnepHropathy.Am J pHysiol Renal pHysiol 2008;294:F1166-1173;[6]Wang JJ,Zhang SX,MottR,Knapp RR,Cao W,Lau K,Ma JX:Salutary effect of pigment epithelium-derived factorin diabetic nepHropathy:evidence for antifibrogenic activities.Diabetes 2006;55:1678-1685;[7]Zhang SX,Wang JJ,Dashti A,Wilson K,Zou MH,Szweda L,Ma JX,Lyons TJ:Pigmentepithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed tooxidized low-density lipoprotein.J Mol Endocrinol 2008;41:135-143;[8]Zhang SX,Wang JJ,Gao G,Shao C,Mott R,Ma JX:Pigment epithelium-derived factor(PEDF)is an endogenousantiinflammatory factor.Faseb J 2006;20:323-325)。最新的研究还提示SA3K在视网膜的抗新生血管、抗炎、抗氧化、抗纤维化活性与Wnt信号通路有关,SA3K通过抑制、调控Wnt通路而起作用([9]Zhang B,Abreu JG,Zhou K,Chen Y,Hu Y,Zhou T,He X,MaJX.Blocking the Wnt pathway,a unifying mechanism for an angiogenic inhibitor in the serineproteinase inhibitor family.Proc Natl Acad Sci U S A.2010;107(15):6900-6905)
Wnt信号转导通路是细胞重要的信号转导通路之一,在细胞的分化、迁移和增殖中起到重要作用,是当前国际生物及医学界的前缘、热点研究领域之一([10]Huang H and He X.Wnt/b-catenin signaling:new(and old)players and new insights.Current Opinion in Cell Biology2008,20:119-125;[11]Gordon MD,Nusse R.Wnt signaling:multiple pathways,multiplereceptors,and multiple transcription factors.J Biol Chem.2006;281(32):22429-33;[12]CleversH.Wnt/beta-catenin signaling in development and disease.Cell.2006;127(3):469-80)。目前,研究主要集中在肿瘤、神经发育、干细胞、视网膜等领域([13]13.Luo J,Chen J,Deng ZL,Luo X,Song WX,Sharff KA,Tang N,Haydon RC,Luu HH,He TC.Wnt signaling andhuman diseases:what are the therapeutic implications?Lab Invest.2007;87(2):97-103;[14]Takahashi-Yanaga F,Sasaguri T.The Wnt/β-Catenin Signaling Pathway as a Target in DrugDiscovery.J pHarmacol Sci.2007;104,293-302;[15]Katohl M,Katoh M.WNT SignalingPathway and Stem Cell Signaling Network.Clin Cancer Res 2007;13(14):4042-4045)。近年来,研究者发现Wnt通路在视网膜新生血管形成中起到重要作用([16]Lad N,Cheshier S,KalaniY.Wnt Signaling in Retinal Development and Disease.Stem Cells Dev 2009;18(1):7-16;[17]ChenY,Hu Y,Lu K,Flannery JG,Ma JX:Very low density lipoprotein receptor,a negativeregulator of the wnt signaling pathway and choroidal neovascularization.J Biol Chem 2007;282:34420-34428),然而对其在眼表组织中作用的研究较少([18]Lyu J,Joo CK.Expressionof Wnt and MMP in epithelial cells during corneal wound healing.Cornea 2006;25(10):S24-28;[19]Lyu J,Joo CK.Wnt-7a Up-regulates Matrix Metalloproteinase-12Expression and PromotesCell Proliferation in Corneal Epithelial Cells during Wound Healing.J Biol Chem 2005;280(22):21653-21660)。对它们在正常角膜组织中的功能、与角膜组织炎症以及新生血管的形成机理、与角膜的损伤和修复过程以及角膜其它疾病的关系还不清楚,作为Wnt信号转导通路抑制剂-全新的LRP抑制剂SA3K则受到了越来越多眼科学者的重视([9]Zhang B,Abreu JG,Zhou K,Chen Y,Hu Y,Zhou T,He X,Ma JX.Blocking the Wnt pathway,a unifyingmechanism for an angiogenic inhibitor in the serine proteinase inhibitor family.Proc Natl Acad SciU S A.2010;107(15):6900-5)。
目前,市面上尚无抗炎、修复上皮及抗新生血管的滴眼液产品,临床上应用的大多数眼科药物具有局限的治疗功能,而且各种药品成分中可能存在相互影响和制约的因素,加上多种联合应用眼药中的毒性成分及防腐剂会给眼表微环境造成破坏,给本身就存在的眼表疾患雪上加霜,对多种角膜病的治疗带来一定的困惑;此外,大多数抗炎产品多含有激素类成分,长期使用可严重影响患者的眼表微环境,部分抗炎药物还有相关的并发症,如氯霉素滴眼液大剂量用药可致眼睑、角膜水肿,眼球运动受限及视盘萎缩,长期应用可致再生障碍性贫血等;抗新生血管药物,如青蒿琥酯([20]陈欢欢,周慧君.青蒿琥酯的抗血管生成作用.药学学报2004;39(1):29-33)滴眼液,成分比较复杂,效果尚不够肯定,且没有抗炎抗淋巴管作用;强力霉素滴眼剂是目前治疗炎症的有效药物,但目前我国尚无正式商品化的制剂。
发明内容
本发明的目的是提供一种与现有的滴眼液相比,作用时间更短、价格较低、效果相对较好、可同时起到抗角膜新生血管及快速修复上皮的滴眼液及其制备方法。
本发明所述滴眼液(以下称为SA3K滴眼液)的组成及其按体积百分比的含量为:牛血清0.1%~5%,增稠剂5%~15%、酸碱调节液1%~5%,渗透压缓冲剂0.1%~2%,抗生素0.5%~2%,SA3K原溶液5%~30%,余为平衡盐溶液。
本发明所述滴眼液的组成及其按体积百分比的含量最好为:牛血清0.5%~2.5%,增稠剂9.5%~11.5%、酸碱调节液2%~3%,渗透压缓冲剂0.1%~2%,抗生素1%~1.5%,SA3K原溶液10%~20%,余为平衡盐溶液。
所述牛血清最好采用胎牛血清,尤其是特级胎牛血清,特级胎牛血清是在无菌条件下通过心脏穿刺的方式采血取到,经过40nm正压气流过滤的胎牛血清,具有极佳的促细胞生长作用及产品稳定性,内毒素含量≤10EU/ml,血红蛋白含量≤10mg/dl,按体积百分比,所述牛血清的浓度为1%~15%,最好为10%。
所述增稠剂可选自硫酸软骨素、右旋糖酐、透明质酸钠、羧丙基甲基纤维素、羧甲基纤维素、聚乙烯吡咯烷酮、聚丙烯醇、卡波姆、壳聚糖等中的至少一种。
所述酸碱调节剂可选自碳酸钠-碳酸氢钠缓冲对、磷酸氢钠-磷酸二氢钠缓冲对、羟乙基哌嗪乙硫磺酸(英文名为2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid,简写为HEPES,分子式为C8H18N2O4S)、硼酸-硼砂缓冲对等中的一种,最好选用作用较强的羟乙基哌嗪乙硫磺酸。
所述渗透压缓冲剂可选自氯化钠、磷酸氢二钠、磷酸二氢钠、硼酸、硼砂、甘油、葡萄糖等中的一种;所述渗透压缓冲剂的用量参照眼科制剂标准。
所述抗生素可选自庆大霉素、链霉素、妥布霉素等中的一种,最好采用妥布霉素。
所述SA3K原溶液的制备可参照上述文献[9],其具体制备方法如:首先将SA3K纯化,即将含SA3K重组DNA的质粒在BL21株的大肠杆菌(E.coli)中扩增,后挑取大肠杆菌单克隆接入后含KANA的LB培养基中,加入0.5IPTG过夜诱导,后用镍柱分离SA3K。
所述平衡盐溶液可采用PBS溶液。PBS溶液是最常用的平衡盐溶液,也是抗炎药物的基础溶液,其成分包含了部分离子成分,可以满足用药期间阴阳离子平衡和渗透压的需要。
所述SA3K滴眼液,以10ml计,一般含有SA3K蛋白2.5mg,pH值为7.2~7.4。
本发明所述滴眼液的制备方法的具体步骤如下:
按滴眼液的配方,将增稠剂、牛血清、抗生素和SA3K原溶液加到平衡盐溶液中混合均匀,用酸碱调节剂调pH值为7.2~7.4,用渗透压缓冲剂调渗透压为350~380mOsm/L,经膜过滤除菌,即得SA3K滴眼液。
与现有的滴眼液相比,本发明具有以下优点:
1)由于滴眼液成分简单,因此成本相对较低,配制方便;
2)由于没有添加激素类成分,因此可以长期使用,大大减少用药期间并发症和后遗症的发生;
3)由于添加抗生素后无污染,因此病原菌培养观察发现此滴眼液能较长时间保存(3个月),应用于大鼠角膜碱烧伤模型后发现,不仅可以预防和治疗角膜新生血管,促进上皮修复的发生,而且对控制角膜炎症也发挥了一定的作用;
4)由于加入牛血清,无病原微生物,低内毒素,低补体,因此可以显著减少使用其他血清的不良反应;
5)由于加入一定剂量的增稠剂,因此可明显提高药物的舒适度;
6)可方便临床医生工作,为术前控制和稳定角膜新生血管及角膜炎症性疾病患者的病情提供更合理的手术时机;
7)不仅可以用于抗角膜新生血管角膜炎症性疾病,预防和治疗角膜新生血管的发生,而且能控制角膜炎症,将来可能会用于各种炎症或相关新生血管的药物治疗,并能被广大患者接受而应用于临床,同时将为其作用机制的进一步研究提供重要的线索和技术支持;
8)制作流程简单、可靠,且易于实施。
附图说明
图1为经过本发明所述不同浓度的SA3K滴眼液(20nM,40nM,80nM,160nM,320nM)分别作用于HUVEC(人脐静脉血管内皮细胞)和HCE(人角膜上皮细胞系)两种细胞72小时后细胞存活率的比较情况。在图1中,横坐标为SK3K滴眼液浓度(nM),纵坐标为细胞成活率(%)。
图2为大鼠角膜碱烧伤后,经过PBS滴眼液连续治疗8天角膜上皮缺损染色的裂隙灯照片。
图3为大鼠角膜碱烧伤后,经过本发明所述SA3K滴眼液连续治疗8天角膜上皮缺损染色的裂隙灯照片。
图4为大鼠角膜碱烧伤后,经过本发明所述SA3K滴眼液,PBS滴眼液和BSA滴眼液连续治疗8天内的角膜上皮缺损面积比较情况和统计分析。在图4中,横坐标为时间(d),纵坐标为荧光计数;
图5为大鼠角膜碱烧伤后,经过PBS滴眼液连续治疗8天新生血管面积的裂隙灯照片。
图6为大鼠角膜碱烧伤后,经过本发明所述SA3K滴眼液连续治疗8天新生血管面积的裂隙灯照片。
图7为大鼠角膜碱烧伤后,经过本发明所述SA3K滴眼液,PBS滴眼液和含BSA滴眼液连续治疗8天内的新生血管面积变化情况和统计分析。在图7中,横坐标为时间(d),纵坐标为新生血管面积(mm2)。
图8为大鼠角膜碱烧伤后,经过本发明所述SA3K滴眼液,PBS滴眼液和含BSA滴眼液连续治疗8天内的炎症指数变化情况和统计分析。在图8中,横坐标为时间(d),纵坐标为炎症指数。
图9为大鼠角膜碱烧伤后,经过PBS滴眼液连续治疗8天中央角膜HE染色照片。
图10为大鼠角膜碱烧伤后,经过本发明所述SA3K滴眼液连续治疗8天中央角膜HE染色照片。
图11为经过本发明所述SA3K滴眼液及PBS滴眼液,BSA滴眼液连续治疗8天的大鼠角膜碱烧伤的VEGF和PEDF的Western blot图片。
图12为经过本发明所述SA3K滴眼液及PBS滴眼液,BSA滴眼液连续治疗8天的大鼠角膜碱烧伤的TNF-α的Western blot图片。
具体实施方式
以下给出一种优化的SA3K滴眼液制作方法
实施例1
以10ml滴眼液计,以7.73ml PBS为基础溶液,含有胎牛血清200μl、低分子右旋糖酐100μl、HEPES200μl,妥布霉素100μl,SA3K原溶液1.67ml,pH值为7.2~7.4,渗透压为350~380mOsm/L。
制备方法:将1.67ml SA3K原溶液和100μg低分子右旋糖酐溶解于2ml PBS溶液中,然后和200μl胎牛血清,1MHERES 200μl及10%妥布霉素100μl混合均匀,补加PBS溶液于10ml,调节pH值为7.2~7.4,渗透压为350~380mOsm/L,经0.2μm膜过滤除菌,即得SA3K滴眼液。
实施例2
以10ml滴眼液计,以7.73ml HBSS为基础溶液,含有胎牛血清200μl、低分子右旋糖酐100μl、HERES 200μl,妥布霉素100μl,SA3K原溶液1.67ml,pH值为7.2~7.4,渗透压为350~380mOsm/L。
制备方法:将1.67ml SA3K原溶液和100μg低分子右旋糖酐溶解于2ml HBSS溶液中,然后和200μl胎牛血清,1M HERES 200μl及10%妥布霉素100μl混合均匀,补加HBSS溶液于10ml,调节pH值为7.2~7.4,渗透压为350~380mOsm/L,经0.2μm膜过滤除菌,即得SA3K滴眼液。
实施例3
以10ml滴眼液计,以7.73ml生理盐水为基础溶液,含有胎牛血清200ul、低分子右旋糖酐100μl、HERES 200μl,妥布霉素100μl,SA3K原溶液1.67ml,pH值为7.2~7.4,渗透压为350~380mOsm/L。
制备方法:将1.67ml SA3K原溶液和100μg低分子右旋糖酐溶解于2ml生理盐水溶液中,然后和200μl胎牛血清,1M HERES 200μl及10%妥布霉素100μl混合均匀,补加生理盐水溶液于10ml,调节pH值为7.2~7.4,渗透压为350~380mOsm/L,经0.2μm膜过滤除菌,即得SA3K滴眼液。
实施例4
以10ml滴眼液计,以7.73ml林格氏液为基础溶液,含有胎牛血清200μl、低分子右旋糖酐100μl、HEPES 200μl,妥布霉素100μl,SA3K原溶液1.67ml,pH值为7.2~7.4,渗透压为350~380mOsm/L。
制备方法:将1.67ml SA3K原溶液和100μg低分子右旋糖酐溶解于2ml林格氏液溶液中,然后和200μl胎牛血清,1M HEPES 200μl及10%妥布霉素100μl混合均匀,补加林格氏液溶液于10ml,调节pH值为7.2~7.4,渗透压为350~380mOsm/L,经0.2μm膜过滤除菌,即得SA3K滴眼液。
实施例5
以10ml滴眼液计,以7.73ml Harks液为基础溶液,含有胎牛血清200μl、低分子右旋糖酐100μl、HEPES 200μl,妥布霉素100μl,SA3K原溶液1.67ml,pH值为7.2~7.4,渗透压为350~380mOsm/L。
制备方法:将1.67ml SA3K原溶液和100μg低分子右旋糖酐溶解于2ml Harks液溶液中,然后和200μl胎牛血清,1M HEPES 200μl及10%妥布霉素100μl混合均匀,补加Harks液溶液于10ml,调节pH值为7.2~7.4,渗透压为350~380mOsm/L,经0.2μm膜过滤除菌,即得SA3K滴眼液。
实施例6
以下给出一种优化的SA3K滴眼液在体外影响HUVEC(人脐静脉血管内皮细胞)和HCE(人角膜上皮细胞系)细胞生长的初步实验研究:
目的:了解不同浓度SA3K滴眼液在体外对HUVEC和HCE细胞的增殖的影响。
方法:采用原代HUVEC细胞株进行细胞培养,传至第3代时,将3×10-4个/cm3细胞培养于96孔板中,待细胞贴壁后,在培养液中分别加入含不同浓度SA3K(20nM,40nM,80nM,160nM,320nM)的相应培养液(HUVEC培养液为M-200,HCE培养液为DMEM)培养,利用MTT实验检测培养72h后两组细胞增殖情况。
结果:MTT实验结果显示72h后SA3K组的HUVEC细胞的生长明显受到抑制,成活细胞随浓度逐渐减少,呈剂量效应关系;而对HCE细胞增殖则没有明显抑制作用。
结论:不同浓度SA3K组在体外对HUVEC的增殖有抑制作用,而对HCE细胞则没有明显影响,提示SA3K的抗新生血管的作用可能与其抑制血管内皮细胞有关。
参见图1,HUVEC(人脐静脉血管内皮细胞)和HCE(人角膜上皮细胞系)细胞在相同密度接种后,待细胞完全贴壁后给予含分别不同浓度SA3K的培养基连续处理72小时的变化情况。结果显示在浓度为40,80,160及320nM SA3K培养基培养下,HUVEC细胞(a)的生长明显受到抑制,成活细胞随浓度逐渐减少,呈剂量效应关系;而在相应浓度(40,80,160及320nM)的SA3K培养基培养下,HCE细胞(b)的生长没有受到抑制,说明上述浓度SA3K培养基对HUVEC细胞增殖有明显抑制作用,而对HCE细胞增殖无抑制影响,提示SA3K的抗新生血管的作用可能与其抑制血管内皮细胞有关。
实施例7
以下给出一种优化的SA3K滴眼液促进大鼠碱烧伤角膜的角膜上皮生长初步实验研究
目的:了解SA3K滴眼液对大鼠角膜碱烧伤模型中角膜上皮生长的影响。
方法:先将24只Wistar大鼠用1%NaOH制作角膜碱烧伤模型,分为4组,第一组为未烧伤对照组,第二组给予PBS滴眼液,(每次滴眼容量为10μl,每天滴眼4次,下同),第三组给予BSA滴眼液(每次滴眼容量为10μl,浓度为1.5mg/ml,每天滴眼4次,下同)和第四组给予SA3K滴眼液(剂量为20μg/天,每次滴眼容量为10μl,浓度为500μg/ml,每天滴眼4次,下同)点眼1,2,5,8天后,观察大鼠角膜上皮修复情况。
结果:SA3K组角膜上皮缺损面积明显少于PBS滴眼液及BSA滴眼液组,且在8天后存在有统计学意义(P值<0.05),说明SA3K滴眼液能有效促进上皮修复.
结论:SA3K滴眼液能显著促进大鼠碱烧伤角膜的角膜上皮生长。
参见图2~4,大鼠角膜碱烧伤模型制作后,给予PBS滴眼液(a)(图2)、BSA滴眼液(b)和SA3K滴眼液(c)(图3)连续点眼8天,观察大鼠角膜上皮细胞修复改变情况。图4为PBS滴眼液(a)、BSA滴眼液(b)和SA3K滴眼液(c)在碱烧伤后1,2,5,8天角膜上皮细胞修复的变化情况和统计分析,结果显示SA3K组角膜上皮缺损面积明显少于PBS滴眼液组及BSA滴眼液组,且在8天后存在有统计学意义(P值<0.05),说明SA3K滴眼液能有效促进上皮修复.
实施例8
以下给出一种优化的SA3K滴眼液抑制大鼠角膜新生血管和炎症的初步实验研究
目的:了解SA3K滴眼液对大鼠角膜碱烧伤模型中新生血管和炎症反应的影响。
方法:将24只Wistar大鼠用1%NaOH制作角膜碱烧伤模型,分为4组,第一组为未烧伤对照组,第二组给予PBS滴眼液,(每次滴眼容量为10μl,每天滴眼4次,下同),第三组给予BSA滴眼液(每次滴眼容量为10μl,浓度为1.5mg/ml,每天滴眼4次,下同)和第四组给予SA3K滴眼液(剂量为20μg/天,每次滴眼容量为10μl,浓度为500μg/ml,每天滴眼4次,下同)点眼1,2,5,8天后,观察两组大鼠角膜新生血管生长和炎症指数情况。取点药8天后角膜,采用Western blot技术对比两组TNF-α、VEGF以及PEDF表达情况。
结果:在连续用药8天后,SA3K组的角膜炎症指数和新生血管面积明显小于PBS滴眼液组和BSA滴眼液。Western blot结果显示,与PBS滴眼液组相比较,VEGF在SA3K组明显下调而PEDF则明显上调;Western blot结果还显示TNF-α在SA3K组明显下调。
结论:SA3K滴眼液能显著抑制大鼠碱烧伤角膜的新生血管形成和炎症反应发生。
参见图5~10,大鼠角膜碱烧伤模型制作后,给予PBS滴眼液(a)(图5)、BSA滴眼液(b)和SA3K滴眼液(c)(图6)连续点眼8天,大鼠角膜新生血管面积和炎症改变情况。图7为浓度为PBS滴眼液(a)、BSA滴眼液(b)和SA3K滴眼液(c)在碱烧伤后1,2,5,8天角膜新生血管面积变化情况和统计分析,结果显示SA3K组角膜新生血管面积明显少于PBS滴眼液及BSA滴眼液组,且在第5,8天时间点存在有统计学意义(P值均<0.05),说明SA3K滴眼液能有效抑制大鼠碱烧伤角膜的新生血管形成.图8为浓度为PBS(a)、BSA(b)和SA3K滴眼液(c)在碱烧伤后1,2,5,8天角膜炎症变化情况统计分析,结果显示,SA3K组角膜炎症指数明显小于PBS及BSA滴眼液组,且在第5,8天时间点存在有统计学意义(P值均<0.05),说明SA3K滴眼液能有效控制大鼠碱烧伤角膜的炎症。图9~10,大鼠角膜碱烧伤模型制作后,给予PBS滴眼液(图9)和SA3K滴眼液(图10)连续点眼8天,大鼠中央角膜的HE照片情况,结果显示SA3K滴眼液组中央角膜上的炎症细胞明显少于PBS滴眼液组,进一步说明了SA3K滴眼液能显著控制大鼠碱烧伤角膜的炎症浸润。
图11~12为在碱烧伤后给予PBS滴眼液、BSA滴眼液和SA3K连续点眼8天,角膜VEGF、PEDF和TNF-α的Western Blot变化情况。图11结果显示,SA3K组较PBS滴眼液组和BSA滴眼液组VEGF水平明显下调而PEDF水平明显上调,说明了SA3K滴眼液是通过使VEGF表达下降,PEDF表达升高而抑制大鼠碱烧伤角膜新生血管形成的。图12结果显示,SA3K组较PBS滴眼液组和BSA滴眼液组TNF-α水平明显下调,说明了SA3K滴眼液也通过使TNF-α表达下降而抑制大鼠碱烧伤角膜新生血管形成以及抑制炎症发生的。
Claims (10)
1.一种滴眼液,其特征在于其组成及其按体积百分比的含量为:牛血清0.1%~5%,增稠剂5%~15%、酸碱调节液1%~5%,渗透压缓冲剂0.1%~2%,抗生素0.5%~2%,SA3K原溶液5%~30%,余为平衡盐溶液。
2.如权利要求1所述的一种滴眼液,其特征在于其组成及其按体积百分比的含量为:牛血清0.5%~2.5%,增稠剂9.5%~11.5%、酸碱调节液2%~3%,渗透压缓冲剂0.1%~2%,抗生素1%~1.5%,SA3K原溶液10%~20%,余为平衡盐溶液。
3.如权利要求1所述的一种滴眼液,其特征在于所述牛血清为胎牛血清,最好是特级胎牛血清。
4.如权利要求1所述的一种滴眼液,其特征在于按体积百分比,所述牛血清的浓度为1%~15%,最好为10%。
5.如权利要求1所述的一种滴眼液,其特征在于所述增稠剂选自硫酸软骨素、右旋糖酐、透明质酸钠、羧丙基甲基纤维素、羧甲基纤维素、聚乙烯吡咯烷酮、聚丙烯醇、卡波姆、壳聚糖中的至少一种。
6.如权利要求1所述的一种滴眼液,其特征在于所述酸碱调节剂选自碳酸钠-碳酸氢钠缓冲对、磷酸氢钠-磷酸二氢钠缓冲对、羟乙基哌嗪乙硫磺酸、硼酸-硼砂缓冲对中的一种,最好为羟乙基哌嗪乙硫磺酸。
7.如权利要求1所述的一种滴眼液,其特征在于所述渗透压缓冲剂选自氯化钠、磷酸氢二钠、磷酸二氢钠、硼酸、硼砂、甘油、葡萄糖中的一种。
8.如权利要求1所述的一种滴眼液,其特征在于所述抗生素选自庆大霉素、链霉素、妥布霉素中的一种,最好为妥布霉素。
9.如权利要求1所述的一种滴眼液,其特征在于所述平衡盐溶液为PBS溶液。
10.如权利要求1所述的一种滴眼液的制备方法,其特征在于其具体步骤如下:
按滴眼液的配方,将增稠剂、牛血清、抗生素和SA3K原溶液加到平衡盐溶液中混合均匀,用酸碱调节剂调pH值为7.2~7.4,用渗透压缓冲剂调渗透压为350~380mOsm/L,经膜过滤除菌,即得SA3K滴眼液。
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CN102614120A (zh) * | 2012-03-30 | 2012-08-01 | 湖南正清制药集团股份有限公司 | 一种制备盐酸青藤碱滴眼剂的方法 |
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CN104147592A (zh) * | 2014-09-10 | 2014-11-19 | 厦门大学 | 一种滴眼液及其制备方法 |
WO2016083767A3 (en) * | 2014-11-21 | 2016-10-27 | Anant Sharma | Epithelial treatment |
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