托吡卡胺眼用温度敏感原位凝胶的研制

发布时间：2018-08-26 09:24

【摘要】：目的制备符合眼部应用要求的托吡卡胺（TR）温敏凝胶。运用流变学方法评价并筛选处方，对优化处方所制备凝胶的质量、凝胶溶蚀、药物释放、眼部滞留时间和刺激性进行全面考察。方法 1.星点设计-效应面法优化TR眼用温敏凝胶处方通过单因素试验，以P407和P188作为温敏材料基质，获取了对相变温度影响较大的因素；采用星点设计-效应面法设计并进行处方筛选试验，星点设计试验结果以SPSS13.0统计学软件进行方程拟合，建立了模拟泪液稀释前、后凝胶的相变温度（T1、T2）对P407、P188质量分数的线性模型；运用Matlab2011b统计学软件绘制以上两方程的三维效应面、二维等高线图，由两等高线的叠加获得两温敏材料质量分数的优化区域；代表性处方从上述优化区域中选出后，对所拟合方程进行预测效果的验证。根据2010版《中国药典》的要求考察抑菌剂、渗透压调节剂和（或）粘附性材料对T1、T2的影响，根据结果调整温敏材料的含量即获得符合眼部应用要求的处方。 2. TR眼用温敏凝胶流变学性质的考察采用Kinexus Pro旋转流变仪，在动态振荡模式下进行处方的流变学评价试验。分别应用应力扫描选取处方的线性粘弹区，温度扫描获得相变温度，频率扫描考察粘度随频率的变化情况，时间扫描对TR温敏凝胶由液态转变为凝胶状态的时间进行考察。 3.质量控制方法考察根据2010版《中国药典》二部对眼用制剂的相关规定，对TR温敏凝胶的含量测定方法、制剂的含量、性状、pH、初步稳定性、无菌检查等进行全面考察，建立TR温敏凝胶的质量控制标准。 4. TR眼用温敏凝胶的凝胶溶蚀、药物释放行为的考察根据TR眼用温敏凝胶的含量测定方法，运用无膜溶出法考察TR温敏凝胶的凝胶溶蚀和药物的释放行为。 5. TR温敏凝胶兔眼滞留时间和刺激性考察以荧光素钠标记已制备好的TR温敏凝胶和普通滴眼液制剂，交叉给药，用紫外灯观察荧光消失时间即代表各制剂的滞留时间。采用自身对照法、Draize评分法对TR温敏凝胶单次给药、多次给药的兔眼局部刺激性进行评价。结果 1.由单因素试验结果可知两温敏材料的质量分数均显著影响处方的相变温度，且P407质量分数与相变温度呈负相关，P188质量分数与相变温度呈正相关；星点设计试验结果以SPSS13.0拟合的方程为：T1=74.270-2.578X+0.934Y (r=0.9187，P 0.01)；T2=89.264-3.018X+1.191Y (r=0.8899，P 0.01)。Matlab2011b绘出优化的温敏材料质量分数区域并从该区域中选取了4个代表性处方，对拟合方程进行预测能力的验证，结果可知T1、T2偏差均5%，表明其预测能力良好。在上述代表处方中加入渗透压调节剂、抑菌剂和（或）HPMC K4M或HPMC E50后，发现处方的相变温度降低；由相变温度的改变调整P407和P188的质量分数，获得了相变温度适宜的符合眼部应用的A~G七个处方，分别为A：20%P407-4%P188；B：20%P407-5%P188-0.5%HPMCK4M；C：21%P407-8%P188-0.5%HPMC K4M；D：22%P407-10%P188；E：20%P407-5%P188-1%HPMC E50；F：21%P407-6%P188；G：21%P407-9%P188-1%HPMCE50，各处方均含有0.25%TR、0.03%尼泊金乙酯和0.9%氯化钠（至全量）。 2.应力扫描结果显示模拟泪液稀释前、后的线性粘弹区范围分别为5Pa~13Pa、1Pa~13Pa，因此剪切应力分别选择为10Pa、5Pa。TR眼用温敏凝胶的弹性模量（G’）和粘性模量（G”）随温度升高呈S形趋势变化；温度低于相变温度时呈自由流动的液体状态，达相变温度时发生相变，转变为半固态；同时，模拟泪液稀释后温敏凝胶的G’和G”相比之前均有所下降，，说明其凝胶强度降低；随着温度的升高，相角δ逐渐降低。同样地，随着剪切频率的升高，粘度η’也有所降低；且模拟泪液稀释后仍呈现较好的粘度；TR温敏凝胶不同处方的相变时间随着温敏材料含量的增加而缩短。通过对比处方A~G各流变学参数，处方D：0.25%TR-22%P407-10%P188-0.03%尼泊金乙酯-0.9%氯化钠（至全量）相变温度适宜，粘度随剪切频率升高下降最为缓慢，而且具有最短的相变时间，因此确定其为最优处方。 3. HPLC测定结果显示，TR的保留时间为7min；辅料对其的含量测定无干扰；峰面积（Y）对TR浓度（C）进行线性回归并绘制标准曲线，得标准曲线为，Y=16.163C+52.731(R2=0.9997)，表明TR浓度在19.9~199μg/mL范围内与峰面积呈现良好的线性关系；24h溶液稳定性试验和精密度试验中，峰面积的RSD分别为0.35%(n=6)、0.19%(n=6)；低、中、高浓度的平均回收率分别为99.01%、98.94%、100.21%，对应的RSD分别为0.97%、0.14%、0.12%(n=6)；质量研究显示TR温敏凝胶低温时呈透明、均匀的可流动液体，温度高于相变温度即变为透明均匀的凝胶；各处方pH值均位于6.6~6.7之间，符合眼部用药的要求；无菌检查结果符合要求；初步稳定性结果显示制剂6个月的稳定性良好。 4. TR温敏凝胶的凝胶溶蚀和药物释放试验显示TR呈缓慢释放；凝胶溶蚀量与TR释放量呈正比，说明药物释放是由凝胶溶蚀控制的释放行为。 5.测得荧光标记的TR温敏凝胶与市售TR滴眼液的眼部滞留时间分别为20min、5min，温敏凝胶滞留时间更长；且其单次给药、多次给药对兔眼均无刺激性。结论本课题采用星点设计-效应面法优化了TR眼用温敏凝胶的处方，并运用旋转流变仪对处方的流变学进行评价，表征了其相变过程，获得了符合眼部应用要求的最佳处方；采用HPLC法建立了TR温敏凝胶的含量测定方法；考察了其质量控制方法、凝胶溶蚀和TR的释放；考察了TR温敏凝胶的兔眼滞留时间和局部刺激性。所得制剂不仅给药剂量准确、减少患者用药次数，提高患者的顺应性，而且与普通滴眼液相比延长了药物在眼内的滞留时间，提高药物局部作用效果，具有良好的临床应用前景。
[Abstract]:objective
To prepare topicamide (TR) thermosensitive gel which meets the requirements of ophthalmic application, rheological method was used to evaluate and screen the prescription, and the gel quality, gel dissolution, drug release, eye retention time and irritation were investigated.
Method
Optimization of TR ophthalmic thermosensitive gel formulation by central composite design response surface methodology 1.
Through single factor experiment, using P407 and P188 as the matrix of temperature-sensitive materials, the factors which have great influence on the phase change temperature were obtained; Star design-response surface methodology was used to design and carry out the prescription screening test; Star design test results were fitted by SPSS 13.0 statistical software, and the phase change temperature of the gel before and after the dilution of the simulated tear was established. T1, T2) Linear model of mass fraction of P407 and P188; 3-D effect surface and 2-D contour plot of the above two equations were drawn by using MATLAB 2011B statistical software, and the optimal region of mass fraction of two thermosensitive materials was obtained by the superposition of two contours; representative prescriptions were selected from the above optimized region, and the predicted results of the fitting equation were obtained. The effects of antimicrobial agents, osmotic pressure regulators and/or adhesive materials on T1 and T2 were investigated according to the requirements of the Chinese Pharmacopoeia (2010 edition).
Investigation on rheological properties of 2. TR ophthalmic thermosensitive gel
The Kinexus Pro rotary rheometer was used to evaluate the rheology of the prescription under dynamic oscillation mode. The linear viscoelastic region of the prescription was selected by stress scanning, and the phase transition temperature was obtained by temperature scanning. The change of viscosity with frequency was investigated by frequency scanning, and the time of time scanning for the transition of TR thermosensitive gel from liquid to gel state was investigated by time scanning. Investigate.
3. quality control method investigation
According to the related regulations of ophthalmic preparations in the second part of Chinese Pharmacopoeia 2010 edition, the content determination method, properties, pH, initial stability and sterility test of TR thermosensitive gel were investigated comprehensively, and the quality control standard of TR thermosensitive gel was established.
Investigation of gel dissolution and drug release behavior of 4. TR ophthalmic thermosensitive gel
According to the content determination method of TR ophthalmic thermosensitive gel, the gel dissolution and drug release behavior of TR thermosensitive gel were investigated by non-membrane dissolution method.
Retention time and irritation of 5. TR thermosensitive gel in rabbit eyes
The prepared TR thermosensitive gel and common eye drops were labeled with sodium fluorescein, and the fluorescence disappearance time was observed by ultraviolet lamp.
The local irritation of TR thermosensitive gel was evaluated by Draize score and self-control method.
Result
1. The results of single factor experiment showed that the mass fraction of the two thermosensitive materials significantly affected the phase transition temperature of the prescription, and the mass fraction of P407 was negatively correlated with the phase transition temperature, and the mass fraction of P188 was positively correlated with the phase transition temperature. The equation fitted by SPSS 13.0 was T1 = 74.270-2.578X + 0.934Y (r = 0.9187, P 0.01); T2 = 89.2. 64-3.018X+1.191Y (r=0.8899, P 0.01). The optimized mass fraction region of temperature-sensitive materials was plotted by MATLAB 2011b. Four representative prescriptions were selected from the region. The predictive ability of the fitting equation was verified. The results showed that the deviations of T1 and T2 were all 5%, indicating good predictive ability. (or) After HPMC K4M or HPMC E50, it was found that the phase transition temperature of the prescription decreased; P407 and P188 mass fractions were adjusted by the change of phase transition temperature, and seven prescriptions of A~G with suitable phase transition temperature were obtained, which were A: 20% P407-4% P188; B: 20% P407-5% P188-0.5% HPMCK4M; C: 21% P407-8% P188-0.5% HPMCK4M; D: 22% P407-10% P188-0.5% HPMCK4M; D: 22% P407-10% P407-10% P188-0.5% MCK % P188; E: 20% P407-5% P188-1% HPMC E50; F: 21% P407-6% P188; G: 21% P407-9% P188-1% HPMC E 50. Each prescription contains 0.25% TR, 0.03% ethyl Nipagin ester and 0.9% sodium chloride (to the full amount).
2. The results of stress scanning show that the linear viscoelastic region of the simulated tear before and after dilution is 5Pa~13Pa and 1Pa~13Pa respectively, so the shear stress is 10Pa and 5Pa. At the same time, the G'and G'of the thermosensitive gel decreased after dilution, indicating that the gel strength decreased; with the increase of temperature, the phase angle delta decreased gradually. Similarly, with the increase of shear frequency, the viscosity_'also decreased; and the simulated tear dilution decreased. The phase transition time of TR thermosensitive gel was shortened with the increase of the content of thermosensitive materials.
By comparing the rheological parameters of prescription A~G, prescription D: 0.25% TR-22% P407-10% P188-0.03% ethyl Nipagin ester-0.9% sodium chloride (to the full amount) has the best phase transition temperature, viscosity decreases most slowly with the increase of shear frequency and has the shortest phase transition time, so it is the best prescription.
3. The results of HPLC showed that the retention time of TR was 7 minutes; the excipients had no interference in the determination of TR content; the peak area (Y) was linearly regressive to the concentration of TR (C) and the standard curve was drawn. The standard curve was Y=16.163 C+52.731 (R2=0.9997), indicating that the TR concentration had a good linear relationship with the peak area in the range of 19.9-199 ug/mL; the solution was stable in 24 hours. In qualitative test and precision test, the RSD of peak area was 0.35% (n=6) and 0.19% (n=6), respectively; the average recoveries of low, medium and high concentrations were 99.01%, 98.94%, 100.21%, and the corresponding RSDs were 0.97%, 0.14%, 0.12% (n=6), respectively; the quality study showed that TR thermosensitive gel was a transparent and uniform flowable liquid at low temperature, and the temperature was higher than the phase transition temperature. The pH value of each prescription ranged from 6.6 to 6.7, which met the requirements of ophthalmic medication. The results of sterility test met the requirements. The preliminary stability results showed that the preparation had good stability for 6 months.
4. The gel dissolution and drug release test of TR thermosensitive gel showed that TR was released slowly, and the gel dissolution was proportional to the TR release, indicating that the drug release was controlled by the gel dissolution.
5. The retention time of fluorescence-labeled TR thermosensitive gel and commercial TR eye drops were 20 minutes and 5 minutes respectively, and the retention time of thermosensitive gel was longer than that of the commercial TR eye drops.
conclusion
In this study, the prescription of TR ophthalmic thermosensitive gel was optimized by star design-response surface methodology, and the rheology of the prescription was evaluated by rotational rheometer, and the phase transformation process was characterized, and the best prescription was obtained, and the content determination method of TR ophthalmic thermosensitive gel was established by HPLC. Methods: The gel dissolution and release of TR were studied. The retention time and local irritation of TR thermosensitive gel in rabbit eyes were investigated. The prospect of clinical application.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

【参考文献】