【作者】 贾瑞；

【导师】 贺艳丽；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2013， 硕士

【摘要】 眼睛是人类的重要感觉器官,但随着人们生活习惯与环境的变化以及无纸化办公的普及,视疲劳、角膜结膜炎、干眼症等眼病患者的数量逐年增加。眼用制剂直接用于眼部治疗眼科疾病,其用药安全性一直为人们所关心。目前国内外大多数多剂量包装的眼用制剂中均含有抑菌剂(也称防腐剂),以避免微生物污染、保证药品质量。近年来,有关眼用制剂抑菌剂眼部刺激性、对角膜的毒性等不良反应的报道日益增加。研究发现,长期使用含有抑菌剂的眼用制剂患者,如白内障、青光眼和干眼症等患者,会影响其泪膜稳定性,造成角、结膜等眼组织损伤,严重者甚至失明。因此,寻找温和型抑菌剂或抑菌系统是当今眼用制剂研发的重点。聚季铵盐-1(Polyquaternium-1,以下简称PQ-1)具有抑菌作用强、眼部刺激性小的特点,已作为杀菌剂在隐形眼镜护理液中得到广泛应用,但在滴眼液等药品中的研究和应用较少。目前国内尚无厂家生产PQ-1,也没有PQ-1的相关文献报道。本课题旨在开发PQ-1在眼用制剂中的应用,对其合成工艺及产业化进行探索；建立了差示分光光度法测定PQ-1的含量；通过营养肉汤试管二倍稀释法测定PQ-1的最小抑菌浓度(Minimum inhibitory concentration,以下简称MIC)和最小杀菌浓度(Minimum bactericidal concentration,以下简称MBC),考察其抑菌稳定性并对其抑菌机理进行初步探究；研究PQ-1对人角膜上皮细胞(Human corneal epithelial cell,以下简称HCEpiC)的毒性作用；以PQ-1作为滴眼液的抑菌剂,通过抑菌效力测试,筛选PQ-1在滴眼液中的有效浓度。1聚季铵盐-1的合成、结构表征及分子量测定目的：初步确定眼用辅料PQ-1的合成工艺。方法：以N,N,N,N-四甲基-2-丁烯-1,4-二胺(DA)、反(顺)式-1,4-二氯-2-丁烯(DCB)和三乙醇胺(TEA)为原料,通过共聚合反应制备PQ-1样品；运用核磁共振仪(NMR)和傅里叶变换红外光谱仪(FT-IR)对产物结构进行表征；采用凝胶渗透色谱法(GPC法)测定产物的重均分子量(Mw)及Mw分布。结果：产物NMR图谱中各基团上的C和H的化学位移与PQ-1均一致,产物IR图谱与PQ-1对照品R图谱也一致；产物Mw为19432,Mw/Mn小于3.0。结论：所选合成工艺简单可行,制备的PQ-1样品Mw及Mw分布符合眼用级PQ-1的要求。2差示分光光度法测定眼用制剂中聚季铵盐-1的含量目的：建立简便、快捷、准确测定眼用制剂中抑菌剂PQ-1含量的方法。方法：应用PQ-1和台盼蓝之间反应发生红移现象,采用差示分光光度法,以台盼蓝溶液作为参比液,在波长681nm处,测得台盼兰在不同浓度PQ-1溶液中的差示吸光度值(△A)。结果：PQ-1浓度在5.00～15.00μg·mL-1范围内,ΔA与溶液浓度呈良好线性关系(r=0.9996),平均回收率(n=9)为97.8%,RSD=0.87%。结论：该方法操作简便、快捷,成本低,结果准确,适用于测定常规眼用制剂中PQ-1的含量。3聚季铵盐-1抑菌作用的研究目的：研究PQ-1的抑菌活性及其稳定性,并初步探究其抑菌作用机理。方法：采用营养肉汤试管二倍稀释法测定PQ-1对大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌和黑曲霉的MIC和MBC,考察紫外光照射、温度、pH和盐浓度对PQ-1稳定性的影响。以大肠杆菌和金黄色葡萄球菌作为指示菌,采用电导率法和透射电镜观察方法,考察了PQ-1对菌体细胞膜通透性的影响,并对其超微结构进行了实验室观察,同时研究了PQ-1对该两种细菌生长曲线的影响。结果：PQ-1对5种实验菌的MIC和MBC分别为：大肠杆菌MIC16μg·mL-1、 MIC16μg·mL-1金黄色葡萄球菌MIC4μg·mL-1. MBC8μg-mL-1,铜绿假单胞菌MIC16μg-mL-1MBC32μg-mL-1,白色念珠菌MIC8μg-mL-1、MBC8μg-mL-1,黑曲霉MIC64μg-mL-1、MBC128μg-mL-1;PQ-1的抑菌活性不受紫外光和pH的影响,但会随温度和盐浓度的升高而降低。菌液电导率值随着PQ-1作用时间的延长持续增加；PQ-1作用后的菌体,表面粗糙并出现许多颗粒状物,细胞壁有明显褶皱和破裂。MIC的PQ-1显著抑制细菌生长,未出现大量生长的对数期。结论：PQ-1在较低浓度具有抑制微生物生长的活性,其对紫外照射和pH较稳定,但高温处理或在高渗环境下,其抑菌活性会显著降低。PQ-1可能通过增加菌体细胞膜的通透性,促进胞内K+等电解质的渗出,逐渐破坏细胞结构而发挥抑菌作用。4聚季铵盐-1对体外培养的人角膜上皮细胞毒性作用的研究目的：考察PQ-1对体外培养的HCEpiC的毒性作用。方法：建立HCEpiC的体外培养模型,将不同浓度的PQ-1(0.01%,0.005%,0.001%,0.0005%)分别与细胞共同孵育,并于作用后10min、25min、1h、2h,利用倒置显微镜观察细胞形态学变化,并采用四唑氮蓝(MTT)分析法和细胞活性/毒性染色法评估PQ-1的细胞毒性。同时选取常用浓度的苯扎溴铵(0.01%)和羟苯乙酯(0.03%)进行比较。结果：(1)细胞形态学改变：随着作用时间延长,0.01%PQ-1、0.005%PQ-1和0.01%苯扎溴铵作用后的细胞连接逐渐松散,胞内物质逐渐泄漏,细胞变圆并逐渐裂解：0.03%羟苯乙酯对HCEpiC作用1h内细胞形态正常,但作用2h后部分细胞开始肿胀变圆,并逐渐脱落；0.001%PQ-1和0.0005%PQ-1处理组的细胞形态完整,与正常组相比无肉眼可辨的显著性差异；(2)MTT分析：随作用时间延长,0.01%PQ-1、0.005%PQ-1、0.01%苯扎溴铵和0.03%羟苯乙酯处理组的HCEpiC存活率均降低,与正常组间差异有统计学意义(P<0.05)；0.001%PQ-1和0.0005%PQ-1处理组不同作用时间的HCEpiC存活率,与正常组相比均无统计学意义(P>0.05)；(3)细胞活性／毒性检测：0.01%PQ-1和0.005%PQ-1对HCEpiC作用10min后,仍可见少数细胞存活,但作用25min、1h、2h后,细胞几乎全部死亡；0.001%PQ-1和0.0005%PQ-1对HCEpiC作用2h内的细胞活性较好,活细胞数量一直维持在较高水平；0.01%苯扎溴铵处理组各作用时问的细胞活性均较低,几乎无活细胞存在；0.03%羟苯乙酯对HCEpiC作用1h内的细胞活性较好,但作用2h后,死细胞数量明显增加。结论：高浓度PQ-1对HCEpiC有一定的毒性作用,但常用浓度的三种抑菌剂之间比较,PQ-1的细胞毒性作用最小。5聚季铵盐-1用于滴眼液防腐的可行性研究目的：考察PQ-1与常用眼用辅料的配伍稳定性,并对PQ-1用于眼用制剂防腐的可行性进行研究和探讨。方法：(1)通过配伍液的澄清度检查和透光率测定,分别考察了PQ-1与磷酸盐缓冲液、硼酸盐缓冲液、玻璃酸钠(SH)、依地酸二钠(EDTA-2Na)羟丙甲纤维素(HPMC)、聚乙烯吡咯烷酮-K30(PVP-K30)、羟苯乙酯以及Tween80的配伍稳定性；(2)考察了PQ-1与SH配伍时,SH的Mw和浓度、PQ-1浓度以及配伍液pH值对PQ-1与SH配伍液澄清度的影响；(3)选用规定范围内相对较低Mw的SH,同时加入增溶剂]ween80,将0.0001%PQ-1和0.0001%PHMB联用作为复合抑菌剂应用于牛磺酸滴眼液中,并进行了抑菌效力测试；(4)通过抑菌效力测试初步筛选了PQ-1在复方氯化钠滴眼液中的使用浓度范围。结果：(1)除SH外,PQ-1与其它常用眼用辅料均无配伍禁忌,与SH的配伍溶液有白色乳光产生；(2)PQ-1与SH的配伍溶液的澄清度与SH的Mw和浓度、PQ-1浓度均呈负相关,而受pH值(5-8)的影响较小；(3)选用0.0001%PQ-1和0.0001%PHMB作为复合抑菌剂的牛磺酸滴眼液抑菌效力符合评价标准,且性状合格；(4)复方氯化钠滴眼液中PQ-1的最低使用浓度为0.0006%时,抑菌效力才能合格,初步确定了PQ-1的使用浓度范围为0.0006%～0.0009%。结论：PQ-1与常规眼用辅料的配伍稳定性较好,适用于常规眼用制剂的防腐,但本实验制备的以PQ-1作为抑菌剂的牛磺酸滴眼液和复方氯化钠滴眼液的长期稳定性以及如何实现PQ-1与SH在眼用制剂中的共存尚需进一步实验研究。更多还原

【Abstract】 Eyes are the important sense organs for human. However, with the changes of our living habits and environment and with the popularity of paperless office, the number of patients with eye diseases such as asthenopia, keratoconjunctivitis and xerophthalmia increases every year. Then the safety of ophthalmic preparations is a question people always concern about for their direct treatment on the eyes. Most of the ophthalmic preparations in multiple dosage forms are protected against microbial contamination by means of preservatives.In recent years, there have been continuous reports about the ocular irritation and adverse toxic effects induced by preservatives. Current studies have showed that long-term use of ophthalmic preparations containing preservatives for the patients suffering from cataracts, dry eye as well as glaucoma may induce ocular surface changes such as tear film stability and cornea or conjunctiva inflammation, and even cause blindness. Developing gentle preservatives or preservative systems is therefore the key point and mainstream in the development of ophthalmic preparations.Polyquaternium-1(PQ-1) with higher antimicrobial effects and lower ocular irritation has been already used in contact lens care solutions as bactericide. However, there is little application of PQ-1in ophthalmic preparations and few relative literatures and researches in China at present. The research subject aims at studying the application of PQ-1in ophthalmic preparations. Firstly, we did the preliminary study of synthetic route for PQ-1for the purpose of commercial production in China. Secondly, the spectrophotometric method for determination of polyquaternium-1in ophthalmic preparations was established to control its quality. Thereafter, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of PQ-1through double broth dilution method. And the pilot study of the stability of PQ-1and its action mechanism was completed. Then the cytotoxicity of PQ-1to human corneal epithelial cells (HCEpiC) was performed. Finally, we prepared the eye drops preserved by PQ-1and determined its effective concentration range through preservative effectiveness tests (PE test).1Synthesis, structural characterization and molecular weight determination of polyquaternium-1Objective:To establish the synthetic route of PQ-1. Methods:PQ-1was synthesized through copolymerization using N, N, N, N-tetramethyl-2-butene-1,4-diamine (DA),1,4-dichloro-2-butene (DCB) and triethanolamine (TEA) as raw materials. The structure of target compound was characterized by nuclear magnetic resonance spectrometer (NMR) and Fourier transform infrared spectrometer (FT-IR). The molecular weight (MW) and Mv distribution were determined through gel permeation chromatography (GPC). Results:The13C-NMR,^-NMR and IR spectra showed that the structure of the synthetic was in accordance with that of the control sample. The Mw of the products was19432and the Mw/Mn was below3.0. Conclusion:The established synthetic route is simple and easy-operated, and the Mw and Mw distribution of the target compound meet the requirements of ophthalmic PQ-1.2Determination of polyquaternium-1in ophthalmic preparations by differential spectrophotometryObjective:To establish a convenient, fast and accurate method for determination of PQ-1in ophthalmic preparations. Methods:This method is based on a bathochromic shift of the trypan blue absorption which occurs when it is complexed with PQ-1. The contents of PQ-1were determinated by the differential spectrophotometry at wavelength681nm, and the reference solution was trypan blue.Results:The calibration curves showed good linearity in the range between5.00and15.00μg-mL-1(r=0.9996), and the average recovery rate was97.8%(n=9, RSD=0.87%). Conclusion:The established method is easy-operated, fast, low-cost and accurate, which can be applied for the quantitative determination of PQ-1in ophthalmic preparations.3Study on the bacteriostatic activity of polyquaternium-1Objective:To study the bacteriostatic activity and stability of PQ-1and to do preliminary research of the action mechanism. Methods:The MIC and MBC of PQ-1to E. coli, S. aureus, P. aeruginosa, C. albicans and A. niger were determined through double broth dilution method The effects of UV irradiation, temperature, pH and salt concentration on stability of PQ-1were also investigated. The influence of PQ-1on the permeability of bacterial cell membrane was studied through conductimetric method and transmission electron microscope (TEM) was used to observe the ultrastructure of the cells. Moreover, we also studied the effect of PQ-1on the growth curve of the microbes. Results:The MIC and MBC of PQ-1to the test microbes respectively were:E. coli.MLC16μg·mL-1, MBC16μg·mL-1, S. aureus MIC4μg·mL-1, MBC8μg·mL-1, P. aeruginosa MIC16μg·mL-1, MBC32μg·mL-1, C. albicans MIC8μg·mL-1, MBC8μg·mL-1,A. niger MIC64μg·mL-1, MBC128μg·mL-1.The bacteriostatic activity of PQ-1was not affected by UV irradiation and pH, but was decreased with the rise of temperature and salt concentration. The value of electric conductivity increased with the extension of the action time of PQ-1. By using the TEM, we found that after exposed to the PQ-1, the bacterial surface became rough and had some pellet substance coming out and clear folds and rupture were found on the cell walls. From the growth curve, PQ-1with MIC significantly inhibited the multiplication of the test microbes. Conclusion:PQ-1with low concentration showed strong bacteriostatic activity. It was relatively stable to UV irradiation and pH, but was heat-sensitive and easily deactivated in hyperosmotic salt solution. The action mechanism of PQ-1may be through increasing the permeability of bacterial cell which results in the leakage of electrolytes including K+in the cell.4Study on the cytotoxicity of polyquaternium-1to human corneal epithelial cells in vitroObjective:To investigate the cytotoxicity of PQ-1to cultured HCEpiC in vitro. Methods:The model of HCEpiC in vitro was established. HCEpiC were incubated with PQ-1solutions at various concentrations (0.01%,0.005%,0.001%,0.0005%) for a period of10min,25min,1h and2h respectively. Observation of cell morphology was made through inverted phase contrast microscope and the cell viability was determined by MTT assay and live/dead cell staining. The benzalkonium bromide and ethylparaben with commonly used concentration were chosen for comparison in the study. Results:(1) Morphologic changes:With the extension of exposure time, HCEpiC incubated with0.01%PQ-1,0.005%PQ-1and0.01%benzalkonium bromide became loose and round and cell breakage occurred gradually.0.03%ethylparaben didn’t affect the cell morphology within1h, but a few cells became loose and deciduous after being treated for2h. The HCEpiC cultured in0.001%PQ-1and0.0005%PQ-1were normal in morphosis.(2) MTT assay:The MTT assay showed that0.01%PQ-1,0.005%PQ-1,0.01%benzalkonium bromide and0.03%ethylparaben consistently and dramatically caused decreases in the viability of HCEpiC (P<0.05). However, there’s no statistical difference of cell viability between treated group of0.001%PQ-1or0.0005%PQ-1and the control group (P>0.05).(3) Live/Dead cell staining:Only a few live cells existed in the treated groups of0.01%PQ-1,0.005%PQ-1and0.01%benzalkonium bromide, while the number of live cells remained at a high level in the groups of0.001%PQ-1and0.0005%PQ-1.0.03%ethylparaben had no obvious effect on the cell activity within1h, but dead cells increased after being treated for2h. Conclusion:PQ-1with high concentration have some cytotoxic effect on HCEpiC, however, PQ-1with commonly used concentration is relatively less toxic than the other two preservatives, which demonstrates that PQ-1is quite an ideal preservative for ophthalmic preparations.5Feasibility research on the eye drops preserved by polyquaternium-1Objective:To investigate the compatibility between PQ-1and common ophthalmic excipients and to study the preservative application of PQ-1in eye drops. Methods:(1) The compatibility between PQ-1and common ophthalmic excipients such as phosphate buffer, borate buffer, sodium hyaluronate (SH), edetate disodium (EDTA-2Na), hypromellose (HPMC), polyvinylpyrrolidone-K30(PVP-K30), ethylparaben and Tween80, was studied through determining the clarity and light transmittance of the mixed solutions respectively.(2) The influence of the Mw and concentration of SH, the concentration of PQ-1and the pH value on the clarity of the SH-PQ-1solutions was evaluated.(3) The taurine eye drops preserved by0.0001%PQ-1and0.0001%PHMB was prepared, in which SH with relatively low Mw but up-to-standard and0.05%Tween80were added. The preservative effectiveness of the eye drops was tested.(4) The preliminary study of the applicative concentration range of PQ-1in compound sodium chloride eye drops was completed. Results:(1) PQ-1was compatible with the common ophthalmic exipients except SH. The solution became opalescent when PQ-1and SH were mixed together.(2) The clarity of the mixed solution of PQ-1and SH tended to be negtively proportional to the Mw and concentration of SH and to the concentration of PQ-1, but pH value (5-8) was demonstrated to have little influence on the clarity.(3) The preservative effectiveness of the taurine eye drops preserved by0.0001%PQ-1and0.0001%PHMB was proved to meet the evaluation standard and the character of the eye drops was qualified.(4) The preservative effectiveness of the compound sodium chloride eye drops would be up-to-standard when the concentration of PQ-1was not less than0.0006%. Then the applicable concentration range of PQ-1in the eye drops was preliminarily intended to be0.0006-0.0009%. Conclusion:PQ-1has the potential to be applied in the ophthalmic preparations as preservatives. However, the long-term stability of the prepared two kinds of eye drops and how to make PQ-1and SH compatible with each other needs further work in the future.更多还原