【作者】 黄桂华；

【导师】 王凤山；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2010， 博士

【摘要】 骨关节炎(Osteoarthritis, OA)是关节炎中最常见的疾病,发病率高,治疗周期长。关节腔注射给药是目前OA常用的治疗方法,但常规注射剂关节腔注射给药,大量药物渗漏进入体循环,导致局部药物浓度低,治疗效果差,频繁用药,副作用大,病人的顺应性差。本文创新性提出了川芎嗪缓释微球用于关节腔注射,通过局部给药达到治疗OA的目的。微球制剂作为能够缓释药物的剂型已被广泛研究,这种剂型通过其特殊骨架材料—乳酸-羟基乙酸共聚物(PLGA)的降解溶蚀达到缓释药物的目的,该骨架材料最终降解为水和CO2,对人体无毒无害,已经被FDA批准为药用辅料。因此,将药物制成缓释微球注射剂,关节腔注射给药治疗OA,可有效提高关节腔局部药物浓度,降低药物毒性、延长药物作用时间,延长用药间隔,减少病人用药次数,提高病人的顺应性和用药水平。本课题以活血化瘀中药TMPZ为模型药物,以PLGA为骨架材料,采用改良的O/W型乳化—溶剂挥发法制备了关节腔注射用TMPZ缓释微球,研究内容主要包括TMPZ缓释微球处方前研究、处方设计和工艺研究、质量表征与体外释药机理研究、灭菌方法与稳定性研究、体内渗漏和关节腔局部组织药物动力学研究、初步安全性评价和药效学研究。1.关节腔注射用TMPZ缓释微球的处方前研究考察了TMPZ的溶解特性和油水分配系数,建立了TMPZ缓释微球质量评价的主要指标及测定方法,结果表明,TMPZ在二氯甲烷、乙酸乙酯等有机溶剂中溶解性较好,在正辛醇-水中的油水分配系数为12.33,通过方法学考察,建立了紫外分光光度法测定TMPZ缓释微球的载药量和包封率,HPLC法测定有关物质；选择含1%十二烷基硫酸钠、0.02%叠氮钠的pH7.4磷酸盐缓冲溶液为释放介质,建立了动态膜透析法测定体外释药百分率,为TMPZ缓释微球处方和工艺优化提供依据。2.关节腔注射用TMPZ缓释微球的处方设计、工艺优化与理化性质表征针对药物的性质和微球临床应用的要求,对经典O/W型乳化-溶剂挥发法进行改良,以改良的O/W型乳化-溶剂挥发法制备关节腔注射TMPZ缓释微球。以微球粒径、载药量和包封率、突释程度等作为质量评价指标,通过单因素考察和正交试验优化确定最佳处方和工艺；选择低温冷冻干燥法制备干燥微球。结果表明,改良的O/W型乳化-溶剂挥发法,操作简便,条件温和可控,制备工艺稳定,重现性良好,是一种可以广泛应用的载药微球制备方法。该法制备的TMPZ缓释微球平均粒径为(10.0±2.11)μm；包封率为(81.36±1.15)%；载药量为(8.2±0.25)%。24小时累积释放(16.57±2.94)%,32天时累积释放达(82.24±0.51)%。结果表明,TMPZ缓释微球包封率达到了现行版药典规定要求,药物体外释放指标达到了预定设计要求。3.关节腔注射用TMPZ缓释微球体外释药方法的建立与释药机理研究采用DSC法验证了TMPZ缓释微球的形成；对微球体外释药条件(包括释放介质和抑菌剂)进行了考察,同时建立了加速释药方法以缩短评价周期,对常规释放和加速释放试验数据进行点对点相关性拟合,结果表明,二者相关性较好(r=0.99),可用体外短期加速释放实验预测常规释放的数据。通过失重法和对体外释放不同时间点的电镜照片分析,评价了TMPZ缓释微球的体外降解行为。结果显示,体外释放20天,微球粘连、融合,无明显界线,微球表面孔隙增多,但聚合物并没有明显降解,微球形状变化不明显。体外释放30天微球表面呈蜂窝状空洞,外观形态发生彻底改变,基本不见微球存在,聚合物明显降解。说明TMPZ缓释微球的释药过程并非主要依靠聚合物的降解。通过测定不同时间微球在释放介质中的粒径变化评价微球的溶胀程度。采用Higuchi、零级、一级方程和Ritger-Peppas指数模型对释药曲线进行拟合,结果表明TMPZ缓释微球的释药曲线符合Higuchi方程。方程拟合结果与体外降解结果皆说明在微球释药过程中,药物的自身扩散起到了较为重要的作用。4.关节腔注射用TMPZ缓释微球的灭菌与稳定性研究通过对微球的外观形态、药物含量、体外释放行为的考察,表明TMPZ缓释微球在高温、高湿条件下性质均不稳定,应低温、干燥保存。分别采用10kGy和15kGy两个剂量对微球进行60Co辐射灭菌,经微生物检查,结果表明,10kGy60Co辐射灭菌对该微球制剂无明显影响,灭菌后的微球微生物检查合格。5.关节腔注射用TMPZ缓释微球体内渗漏和局部组织药物动力学研究以大鼠为模型动物,以TMPZ注射液为对照制剂,关节腔注射TMPZ缓释微球,通过测定不同时间点大鼠体内血药浓度变化和大鼠注射部位(关节组织和滑液)残留的药物浓度的变化,比较两种制剂渗漏进入体循环的药物量和关节腔局部的药物量,通过药动学参数评价TMPZ缓释微球的缓释长效作用,探讨TMPZ缓释微球局部用药的优势。结果表明,关节腔注射给药后,血浆中的A UC0-∞注射液组是微球组的5.02倍；关节组织和滑液总药物浓度与时间的关系采用统计矩法求得药物动力学参数,微球组和溶液组半衰期(t1/2)分别为130.75h和16.93h,延长了7.72倍；平均滞留时间(MRT0-∞)分别为223.37h和26.09h,微球组较溶液组延长了8.56倍；血药浓度-时间曲线下面积(AUC0-∞)分别为52586.0mg/L*h和3644.5mg/L*h,微球组是溶液组的14.4倍。药物被包封于微球后,渗漏进入体循环的药量减少,关节腔局部药物浓度明显增加,药物在关节腔的作用时间明显延长。6.关节腔注射用TMPZ缓释微球的初步安全性评价以大鼠为模型动物,以生理盐水为对照,关节腔注射空白PLGA微球和TMPZ缓释微球,对不同时间点大鼠膝关节组织进行病理切片观察,评价空白微球和载药微球对局部组织的损伤程度。结果显示,微球仅引起轻微的炎症反应,没有组织渗透液蓄积,没有观察到明显血管与纤维的增生,说明空白微球和载药微球关节腔注射给药生物相容性良好。7.关节腔注射用TMPZ缓释微球的体内药效学研究以大鼠为模型动物,采用关节腔注射木瓜蛋白酶法建立大鼠膝关节OA模型,造模后膝关节腔分别注射TMPZ注射液(每周一次,2.1mg/只,连续给药6周)和TMPZ缓释微球混悬液(给药一次,4.2mg/只)。给药6周后处死大鼠,分别采用①参照Freemont分度法,光学显微镜下观察关节软骨的退变情况；②参照Mankin骨关节病组织学分类法,对软骨和滑膜评价积分；③造模前、后及给药后不同时间膝关节的直径和活动度；④检测关节滑液中细胞因子IL-p和PGE2的含量等,探讨和比较TMPZ注射液与TMPZ缓释微球对大鼠OA的治疗作用。结果表明,关节腔注射TMPZ缓释微球减少了给药次数(由6次变为1次),降低了给药剂量(总剂量由12.6 mg/只变为4.2mg/只),对关节组织的炎症治疗微球组优于注射液组,对关节软骨的修复治疗,微球组与注射液组治疗效果基本相同。充分证明了TMPZ缓释微球的有效性和长效作用。结论本文制备的关节腔注射用TMPZ缓释微球有效克服了常规注射剂治疗时药物全身吸收导致的毒副作用大、用药频繁等不足,可减小用药剂量,减少用药次数,延长给药间隔,减轻病人痛苦,提高病人用药的顺应性和用药水平,具有良好的社会和经济效益。同时本文建立了改良的O/W型乳化-溶剂挥发法制备载药微球,在提高包封率的同时降低了药物突释,为同类药物的微球制剂研究提供了重要参考价值；本文建立了体外短期加速释放试验法用以考察长效制剂的体外释放情况,结果表明,加速释药方法可以很好的预测长效制剂的体外释药行为,且可缩短评价周期。综上所述,本文通过关节腔注射TMPZ缓释微球的制备、体内外评价和药效学研究,为关节腔注射给药治疗局部疾病探索了新的思路和手段,丰富了中药制剂和长效制剂的研究内容,具有较高的科学技术价值和临床应用前景。更多还原

【Abstract】 Osteoarthritis (OA) is one of the common arthritis with high attack rate and it usually needs multiple treatment cycles to cure it. Intra-articular injection is one of the most common treatment methods for OA. However, drug solution injection always leaks into the systemic circulation after the intra-articular injection. Thus, the local drug concentration falls quickly and the therapy effect will be low. The other problems meet with this treatment including frequent administration frequency, serious side effects and relative low patient compliance. This article innovationally designed sustained-release microspheres encapsulating tetramethylpyrazine (ligustrazine, TMPZ) for intra-articular injection with the purpose of improving the therapeutic effects and avoiding the existing problems of the intra-articular injection of drug solution mentioned above.As one of the widely accepted sustained release drug delivery systems, microspheres have been deeply investigated. It exerts sustained-release effects through the specific skeleton material, poly(lactic-co-glycolic acid, PLGA), which is biodegradable with harmless products H2O and CO2. PLGA was used as adjuvant which had been approved by the Food and Drug Administration (FDA), USA. The intra-articular injectable sustained-release microspheres preparation to heal OA can reduce administration frequency, extent therapy duration, reduce the patient pain and enhance the patient compliance.TMPZ was selected as the model drug and PLGA used as the carrier material in this experiment. The TMPZ loaded sustained-release microspheres were prepared by O/W emulsion solvent evaporation method. This article was composed of the following seven parts:the preformulation study, the optimization of the formulation and technological parameters, the characteristics of the preparations and in vitro drug release study, the sterilization and stability study, the leakage and local tissue distribution study in vivo, and the in vivo biocompatibility and pharmacodynamic study.1. The preformulation study The equilibrium solubility and oil-water partition coefficient of TMPZ were investigated firstly, in order to determine the evaluating indexes and the preparation method of the TMPZ loaded sustained-release microspheres. The results indicated that TMPZ was well dissolved in some organic solvents such as methylene chloride and ethyl acetate, and the coefficient of distribution in propyl butyl-carbinol-water was 12.33. Encapsulation efficiency and loading efficiency were evaluated by ultraviolet-visible spectroscopy and related material was determined by high performance liquid chromatography (HPLC) according to the results of the methodology investigation. The in vitro release of the TMPZ from the sustained-release microspheres were evaluated by dynamic membrane dialysis method using pH 7.4 phosphate buffer solution (with 1% lauryl sodium sulfate and 0.02% nitrine sodium) as release media.2. The optimization of the formulation and the technological parameters of the TMPZ loaded sustained-release microspheresThe TMPZ loaded sustained-release microspheres were prepared using modified O/W emulsion solvent evaporation method under the consideration of the drug properties and the requirements of the clinical application of the intra-articular injectable sustained-Release microspheres. The evaluating indexes included diameter, encapsulation efficiency, drug loading and initial burst release. The formulation and technological parameters were optimized based on the results of the single factor study and the orthogonal design. The obtained microspheres were dried using freeze-drying method. The results showed that the modified O/W emulsion solvent evaporation method was suitable for the preparation of the TMPZ loaded sustained-release microspheres with the advantages of easy to operation, the temperate preparation conditions and perfect reproducibility. The mean diameters of the 3 batch TMPZ loaded sustained-release microspheres prepared under the optimized formulation and the technological parameters was (10.0±2.11)μm, the encapsulation efficiency was (81.36±1.15)% and the drug loading was (8.2±0.25)%.The results of the in vitro drug release study showed that (16.57±2.94)% of total loaded drug released from the sustained-release microspheres in 24 hours and (82.24±0.51)% released within 32 days. These results indicated that the encapsulation efficiency achieved the requirements of pharmacopoeia and in vitro drug release met the predetermined requirements of the project.3. The establishment of in vitro drug release method and the research on drug release mechanismThe formation of the TMPZ loaded sustained-release microspheres was validated by different scanning calorimetry (DSC). The different in vitro drug released conditions (including the release medium and the bacteriostatic agent) was compared; meanwhile, an accelerated release method was established to shorten the experimental time of in vitro release study. The linear relationship between the data obtained from the conventional release method and the data from the novel accelerated release method was confirmed by point-to-point regression equation and the result showed that it’s available to anticipate the conventional release property by the data from the novel accelerated release method. Thus, the novel accelerated release method is expected to be popularized and applicated widely.The in vitro drug release mechanism of the TMPZ loaded sustained-release microspheres was analyzed by determining the weight loss and by comparing the scanning electron microscope (SEM) photographs. The results indicated that after releasing for 20 days, the microspheres were adhered and fused; demarcation line disappeared and pores of the microspheres increased. However, no obvious shape changes were observed. After releasing about 30 days, the microspheres were porous and the shape changed thoroughly. These results suggested that the release process of the TMPZ loaded sustained-release microspheres was not mainly depended on the polymer degradation. Swelling degree was evaluated by determination of the microspheres particle size in release medium and calculation of the size changes. These results showed that the in vitro releases curve of the TMPZ loaded sustained-release microspheres was depicted by Higuchi equation. The results of the equation fitting and the drug release mechanism research showed that the in vitro release of the drug from the TMPZ loaded sustained-release microspheres was mainly by diffusion mechanism.4. The sterilization and stability studiesThe stability of the TMPZ loaded sustained-release microspheres was evaluated through changes including appearance, drug contents and in vitro release behavior. The results showed that high temperature and high moisture would affect the properties of the TMPZ loaded sustained-release microspheres. The TMPZ loaded sustained-release microspheres were sterilized under lOkGy and 15kGy of 60Co exposure. lOkGy of 60Co exposure was selected to sterilize the TMPZ loaded sustained-release microspheres and the result of the microorganism test indicated that treatment with lOkGy of 60Co was acceptable.5. In vivo leakage and local tissue distribution studiesThe in vivo leakage and the local tissue distribution of the TMPZ loaded sustained-release microspheres after intra-articular injection in rats were investigated and TMPZ injection was used as control. After intra-articular injection of the TMPZ loaded sustained-release microspheres, the plasma drug concentration and the local drug concentration of the joint cavity (joint tissue and synovial fluid) of two kind of preparation were compared. The results indicated that the AUC0-∞of TMPZ injection group was 5.02 times more than that of the TMPZ loaded sustained-release microspheres group. Pharmacokinetic parameters were calculated by statistical moment method. The t1/2 of microspheres group and injection group are 130.75h and 16.93h, respectively, showing of 7.72 folds increasing. MRT0-∞were 26.09h and 223.37h, respectively, showing of 8.56 folds enhancement. AUC0-∞of the TMPZ loaded sustained-release microspheres group was 14.4 times more than that of TMPZ injection group (52586.0 mg/L*h and 3644.5 mg/L*h, respectively). After encapsulated in the microspheres, the drug leakage into the body circulation reduced and the local drug concentration increased obviously, thus the therapy effect was expected to be enhanced obviously.6, The biocompatibility studyThe biocompatibility of the TMPZ loaded sustained-release microspheres and the blank PLGA microspheres in rats was evaluated by pathological examination using normal saline as control. The results showed the TMPZ loaded sustained-release microspheres induced slight inflammation response, but infiltration liquid accumulation, blood vessels and fibrous proliferation were not detected. Therefore, the results showed good compatibility of both the TMPZ loaded sustained-release microspheres and the blank PLGA microspheres.7. The pharmacodynamic studyRat OA model was induced by intra-articular injection of papain and aminothiopropionic acid. The successful modeled rats were injected with TMPZ injection (2.1mg/0.1ml, once a week,6 times in total) and the TMPZ loaded sustained-release microspheres suspensions (4.2mg/0.1ml, injected once), respectively. The rats were sacrificed after injection of different preparation for 6 weeks. Therapy effect was evaluated via (1) the Freemont indexing under the optical microscope for the examination of the articular cartilage changes; (2) the Mankin arthrosis histology classification for the characterization of the cartilage and synovial membrane; (3) knee joint diameter and the knee joint activity; (4) examination of joint synovial fluid cell factor IL-βand PGE2 content for the discussion and comparison of the therapy effect of TMPZ injection and the TMPZ loaded sustained-release microspheres. The results indicated that the administration frequency and total dose can be reduced by TMPZ loaded sustained-release microspheres (from 6 times to 1 time and 12.6 mg to 4.2 mg, respectively). Furthermore, the effect of inflammation treatment of-the TMPZ loaded sustained-release microspheres was better than that of the TMPZ injection, the cartilage repair of the TMPZ loaded sustained-release microspheres was equal with that of the TMPZ injection. The results proved the better therapy effect and the sustained activity of the TMPZ loaded sustained-release microspheres.ConclusionThe TMPZ loaded sustained-release microspheres overcame the disadvantages of the TMPZ injection, including decreasing drug leakage and the frequent administration. The application of the novel dosage form is expected to extend the therapeutic duration, reduce the pain of patients, enhance the patient compliance and access to good social and economic benefits. The TMPZ loaded sustained-release microspheres was prepared using modified emulsion solvent evaporation method with reduced burst release property and increased encapsulation efficiency compared with the conventional preparation method. The accelerated release method was established to shorten the experiment time. It was available to expect the conventional in vitro release characteristics using the novel accelerated release method.In conclusion, the preparation, in vitro and in vivo property studies and pharmacodynamic study of the TMPZ loaded sustained-release microspheres was completed. A novel idea and method were explored for the treatment of joint diseases. This project possessed scientific and technological value and clinical prospect, and the research area of traditional Chinese medicine and long-acting preparations were enriched by this study.更多还原