胸腺五肽长效注射微球突释控制技术的研究
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摘要
作为双相免疫调节剂，胸腺五肽(TP-5)临床上主要用于免疫功能低下的治疗以及多种重症疾病的辅助用药。由于其肽类结构特点，口服易被胃肠道酶降解，目前临床使用制剂为冻干粉针，规格1mg，一次1支，每日1-2次，皮下注射或静脉滴注，用药时间1～24个月不等。长时间频繁注射患者依从性差，影响治疗效果。为提高用药顺应性，减少注射次数，改善治疗效果，本课题组前期对胸腺五肽长效微球进行了研究，以不同规格的乳酸—羟基乙酸共聚物(PLGA)为材料，可获得释药周期15-60天的载药微球。但由于胸腺五肽极性强，水溶性大，其长效微球存在明显的突释效应，24小时突释量高达40％左右，使其长效微球注射剂的发展受到限制。
目的：突破多肽蛋白类药物长效微球制剂发展的关键技术，降低胸腺五肽微球突释，制备突释量低、粒径分布均匀、释药性能稳定、质量可控的胸腺五肽微球，为长效注射剂的研究与发展奠定基础。
方法：建立微球形态、分散性、粒径及分布、含量、包封率及载药量、释放度等微球质量控制方法。以包封率和24小时突释量为主要指标，单因素考察多种添加剂方案对微球制备结果的影响，并对添加剂降低突释的机理进行探讨。在添加剂筛选基础上，对PLGA材料的规格及来源、粒径控制工艺、微球干燥方式及灭菌条件等进行考察。采用正交试验设计，以突释量、包封率及粒径为指标，优化微球处方和制备工艺。通过影响因素及室温留样试验，评价胸腺五肽微球质量稳定性。建立切实可行的微球残余药量测定方法，并进行系统方法学考察。大鼠皮下注射胸腺五肽微球，于不同时间取注射部位皮下组织，依法测定微球残余药量，绘制体内释药曲线，考察体内释药行为。通过体内外结果的比较，评价微球体内外释药相关性。
结果：[1]突释控制技术在内水相加入Tween-20、葡萄糖，油相加入碳酸锌、甘油或乙酸乙酯，外水相加入渗透压物质氯化钠，均可降低微球突释，其中外水相加入氯化钠降低突释的作用最为显著，制得的微球流动性最好。突释降低与氯化钠产生的渗透压有关，氯化钠浓度越大，外水相渗透压越高，内外水相渗透压差越小，微球表面形成的孔隙越少，骨架密度越大，突释量越低。氯化钠浓度在10～15mg／ml，微球突释可控制在13％以下；选择聚合比为50∶50、内在黏度为0.55～0.75dl／g的PLGA，两种来源(美国伯明翰聚合物公司和中国科学院成都高分子材料研究所)的材料制备的微球均具有较高的包封率和较低的突释量；加大高剪切乳化分散机的功率、提高次级乳化速度、延长乳化时间或调节乳化剂PVA的浓度，均有利于微球粒径分布的均一性；冷冻干燥是首选的微球干燥方式，γ射线灭菌对胸腺五肽的化学稳定性有影响，建议采用无菌操作工艺。正交试验优化结果表明，PLGA浓度对微球突释和包封率的影响最为显著，其次是氯化钠浓度，次级乳化速度和PVA浓度主要影响微球的粒径分布。最优组合为PLGA浓度250mg／ml，氯化钠浓度10或15mg／ml，次级乳化速度13000r·min~(-1)，PVA浓度0.5％。[2]稳定性微球在高温、高湿和强光条件下，不同程度地结块，分散性明显变差；室温放置两个月，微球粒径、含量及突释量没有明显变化，质量稳定。[3]药代动力学研究采用HPLC法测定微球残余药量，加入甲醇破坏皮下组织中的生物酶活性，可确保测定过程中药物的稳定。在选定的测定条件下，内源性物质无干扰，最低检测限1ng，最低定量限3ng；在0.97μg·ml~(-1)～97μg·ml~(-1)范围内线性关系良好，日内精密度1.80％，日间精密度9.16％，皮下组织取样及测定的总回收率为59.94％。中科院来源的PLGA材料制备的微球，体内释药周期为1个月，但释药速率明显快于体外，无明显平台期，24小时突释分别为12.03％和3.70％，释药模式基本符合零级方程。选用pH7.4释放介质，可获得良好的体内外相关性。
结论：通过突释控制技术及系统处方和工艺优化，制备的微球粒径成正态分布，平均粒径为30～65μm，包封率在85％以上，突释量在5％以下，室温贮存两个月质量稳定。胸腺五肽微球体内1个月累积释放度达93.46％，释药速率比较平稳。课题研究实现了预期目标，为开发1个月给药一次的胸腺五肽长效微球注射剂奠定了基础。
Thymopentin was a synthet(?)c penta-peptide corresponding to the active site of thymopoietin, a 49 amino acid hormone of the thymus. Its biological acitivity was similar to that of thymopoietin, and it had been used clinically as a modulator of the immune response or a co-adjuvant for serious illnesses. So far, the pharmaceutical products of TP-5 were injections, with the specification of 1mg. Once or twice per day by s.c. or i.v., and the course of treatment lasted from one month to 24 months. Frequent injections for long time impared the compliance of the patients and therapeutical efficacy. To overcome the shortcomings referred to, our research team prepared PLGA based micro-spheres with a water-oil-water emulsion solvent evaporation technique for 15～60 days controlled release. For the high solubility of TP-5 in water, significant burst effect(＞40%) happened in this sustained-release inject-able micro-spheres, which was an obstacle to the development of sustained-release inject-able micro-spheres.
Objective: Prepare tp-5-loaded microspheres having the characteristics of low burst effect, well-distributed particle size, controlled release and good quality to break through the bottle-neck of sustained-release inject-able micro-spheres.
Method: to establish the quality control method for items including the appearance、dispersibility、particle size and distribution、drug content、encapsulation efficiency、drug loading and release profile. Set the encapsulation efficiency and the burst effect as the main indexes, to investigate the influence of kinds of addictives on the characteristics of the microspheres. Further study was conducted on the mechanism about the burst effect reduced by addictive. Furthermore, different specifications and manufacturers of the PLGA, the production process for controlling particle size, drying process andγirradiation process were investigated. An orthogonal experiment was undertaken to optimize the formulation and production process. Evaluate the stability of TP-5 microspheres in influential factors experiment and long-term storage experiment. The drug release kinetics of TP-5/PLGA microspheres was studied by determining the residue drug in the injection site.
Results: [1] burst effect controlling strategies add Tween-20、glucose to the inner water phase, or zinc carbonate、glycerol、acetoacetate to the oil phase, or sodium chloride to the outer water phase all could reduce the burst effect. Interestingly, the sodium chloride has better results than other addictives. The extent of reducing the burst effect was correlated with the osmotic pressure. When the concentration of sodium chloride in the external water phase was adjusted within 10～15mg/ml, low initial release (＜13%) was achieved. PLGA(50:50,0.55～0.75dl/g) provided by BPI or COCC could prepare high encapsulation and low initial release microspheres. Increasing the power of the homogenizers、the second emulsifying speed、extending the emulsifying time or adjusting the PVA concentration were benefit for the homogenicity of the particle size distribution. Freezing-drying was the best drying process, and theγirradiation would impact the stability, so sterile operation was suggested as the sterilization process. The results from the orthogonal experiment showed that the PLGA concentration was the most striking influential factor for the burst effect and the encapsulation efficiency, the secondly influential factor was the sodium chloride concentration. The optimization combination was comprised by the PLGA 250mg/ml, sodium chloride 10 or 15mg/ml, the second emulsifying speed 13000r·min~(-1), PVA 0.5%. [2] Stability Under the conditions of high temperature, high humidity and highlight, the microspheres became to agglomerate to different extent, and the dispersibility turned bad. But in the long-term storage experiment, no change on the particle size, the drug content or the burst effect was found. [3] The study of pharmacokinetics Determining the residual drug in the injection site by HPLC, with the addition of methanol to destroy the enzyme in the subcutaneous tissue. The PLGA (provided by the COCC) based microspheres, could controlled release for one month, and the initial release in vitro and in vivo were 12.03% and 3.70% respectively. The release profile in vivo meted the zero order equation. Good relativity was detected between the in vivo drug release and in vitro drug release.
Conclusions: The normal distribution of particle size, D(4,3) within 30～65μm, high encapsulation(＞85%), low initial release (＜5%) and controlled release for one month are achieved by the optimization of the formulation and production process. This study have obtained the proposed objects.
目的：突破多肽蛋白类药物长效微球制剂发展的关键技术，降低胸腺五肽微球突释，制备突释量低、粒径分布均匀、释药性能稳定、质量可控的胸腺五肽微球，为长效注射剂的研究与发展奠定基础。
方法：建立微球形态、分散性、粒径及分布、含量、包封率及载药量、释放度等微球质量控制方法。以包封率和24小时突释量为主要指标，单因素考察多种添加剂方案对微球制备结果的影响，并对添加剂降低突释的机理进行探讨。在添加剂筛选基础上，对PLGA材料的规格及来源、粒径控制工艺、微球干燥方式及灭菌条件等进行考察。采用正交试验设计，以突释量、包封率及粒径为指标，优化微球处方和制备工艺。通过影响因素及室温留样试验，评价胸腺五肽微球质量稳定性。建立切实可行的微球残余药量测定方法，并进行系统方法学考察。大鼠皮下注射胸腺五肽微球，于不同时间取注射部位皮下组织，依法测定微球残余药量，绘制体内释药曲线，考察体内释药行为。通过体内外结果的比较，评价微球体内外释药相关性。
结果：[1]突释控制技术在内水相加入Tween-20、葡萄糖，油相加入碳酸锌、甘油或乙酸乙酯，外水相加入渗透压物质氯化钠，均可降低微球突释，其中外水相加入氯化钠降低突释的作用最为显著，制得的微球流动性最好。突释降低与氯化钠产生的渗透压有关，氯化钠浓度越大，外水相渗透压越高，内外水相渗透压差越小，微球表面形成的孔隙越少，骨架密度越大，突释量越低。氯化钠浓度在10～15mg／ml，微球突释可控制在13％以下；选择聚合比为50∶50、内在黏度为0.55～0.75dl／g的PLGA，两种来源(美国伯明翰聚合物公司和中国科学院成都高分子材料研究所)的材料制备的微球均具有较高的包封率和较低的突释量；加大高剪切乳化分散机的功率、提高次级乳化速度、延长乳化时间或调节乳化剂PVA的浓度，均有利于微球粒径分布的均一性；冷冻干燥是首选的微球干燥方式，γ射线灭菌对胸腺五肽的化学稳定性有影响，建议采用无菌操作工艺。正交试验优化结果表明，PLGA浓度对微球突释和包封率的影响最为显著，其次是氯化钠浓度，次级乳化速度和PVA浓度主要影响微球的粒径分布。最优组合为PLGA浓度250mg／ml，氯化钠浓度10或15mg／ml，次级乳化速度13000r·min~(-1)，PVA浓度0.5％。[2]稳定性微球在高温、高湿和强光条件下，不同程度地结块，分散性明显变差；室温放置两个月，微球粒径、含量及突释量没有明显变化，质量稳定。[3]药代动力学研究采用HPLC法测定微球残余药量，加入甲醇破坏皮下组织中的生物酶活性，可确保测定过程中药物的稳定。在选定的测定条件下，内源性物质无干扰，最低检测限1ng，最低定量限3ng；在0.97μg·ml~(-1)～97μg·ml~(-1)范围内线性关系良好，日内精密度1.80％，日间精密度9.16％，皮下组织取样及测定的总回收率为59.94％。中科院来源的PLGA材料制备的微球，体内释药周期为1个月，但释药速率明显快于体外，无明显平台期，24小时突释分别为12.03％和3.70％，释药模式基本符合零级方程。选用pH7.4释放介质，可获得良好的体内外相关性。
结论：通过突释控制技术及系统处方和工艺优化，制备的微球粒径成正态分布，平均粒径为30～65μm，包封率在85％以上，突释量在5％以下，室温贮存两个月质量稳定。胸腺五肽微球体内1个月累积释放度达93.46％，释药速率比较平稳。课题研究实现了预期目标，为开发1个月给药一次的胸腺五肽长效微球注射剂奠定了基础。
Thymopentin was a synthet(?)c penta-peptide corresponding to the active site of thymopoietin, a 49 amino acid hormone of the thymus. Its biological acitivity was similar to that of thymopoietin, and it had been used clinically as a modulator of the immune response or a co-adjuvant for serious illnesses. So far, the pharmaceutical products of TP-5 were injections, with the specification of 1mg. Once or twice per day by s.c. or i.v., and the course of treatment lasted from one month to 24 months. Frequent injections for long time impared the compliance of the patients and therapeutical efficacy. To overcome the shortcomings referred to, our research team prepared PLGA based micro-spheres with a water-oil-water emulsion solvent evaporation technique for 15～60 days controlled release. For the high solubility of TP-5 in water, significant burst effect(＞40%) happened in this sustained-release inject-able micro-spheres, which was an obstacle to the development of sustained-release inject-able micro-spheres.
Objective: Prepare tp-5-loaded microspheres having the characteristics of low burst effect, well-distributed particle size, controlled release and good quality to break through the bottle-neck of sustained-release inject-able micro-spheres.
Method: to establish the quality control method for items including the appearance、dispersibility、particle size and distribution、drug content、encapsulation efficiency、drug loading and release profile. Set the encapsulation efficiency and the burst effect as the main indexes, to investigate the influence of kinds of addictives on the characteristics of the microspheres. Further study was conducted on the mechanism about the burst effect reduced by addictive. Furthermore, different specifications and manufacturers of the PLGA, the production process for controlling particle size, drying process andγirradiation process were investigated. An orthogonal experiment was undertaken to optimize the formulation and production process. Evaluate the stability of TP-5 microspheres in influential factors experiment and long-term storage experiment. The drug release kinetics of TP-5/PLGA microspheres was studied by determining the residue drug in the injection site.
Results: [1] burst effect controlling strategies add Tween-20、glucose to the inner water phase, or zinc carbonate、glycerol、acetoacetate to the oil phase, or sodium chloride to the outer water phase all could reduce the burst effect. Interestingly, the sodium chloride has better results than other addictives. The extent of reducing the burst effect was correlated with the osmotic pressure. When the concentration of sodium chloride in the external water phase was adjusted within 10～15mg/ml, low initial release (＜13%) was achieved. PLGA(50:50,0.55～0.75dl/g) provided by BPI or COCC could prepare high encapsulation and low initial release microspheres. Increasing the power of the homogenizers、the second emulsifying speed、extending the emulsifying time or adjusting the PVA concentration were benefit for the homogenicity of the particle size distribution. Freezing-drying was the best drying process, and theγirradiation would impact the stability, so sterile operation was suggested as the sterilization process. The results from the orthogonal experiment showed that the PLGA concentration was the most striking influential factor for the burst effect and the encapsulation efficiency, the secondly influential factor was the sodium chloride concentration. The optimization combination was comprised by the PLGA 250mg/ml, sodium chloride 10 or 15mg/ml, the second emulsifying speed 13000r·min~(-1), PVA 0.5%. [2] Stability Under the conditions of high temperature, high humidity and highlight, the microspheres became to agglomerate to different extent, and the dispersibility turned bad. But in the long-term storage experiment, no change on the particle size, the drug content or the burst effect was found. [3] The study of pharmacokinetics Determining the residual drug in the injection site by HPLC, with the addition of methanol to destroy the enzyme in the subcutaneous tissue. The PLGA (provided by the COCC) based microspheres, could controlled release for one month, and the initial release in vitro and in vivo were 12.03% and 3.70% respectively. The release profile in vivo meted the zero order equation. Good relativity was detected between the in vivo drug release and in vitro drug release.
Conclusions: The normal distribution of particle size, D(4,3) within 30～65μm, high encapsulation(＞85%), low initial release (＜5%) and controlled release for one month are achieved by the optimization of the formulation and production process. This study have obtained the proposed objects.
引文
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