关节腔注射用川芎嗪缓释PLGA微球的制备、体内外评价及药效学研究
|
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。
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 microspheres
The 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 mechanism
The 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 studies
The 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 studies
The 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 study
The 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 study
Rat 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.
Conclusion
The 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.
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。
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 microspheres
The 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 mechanism
The 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 studies
The 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 studies
The 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 study
The 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 study
Rat 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.
Conclusion
The 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.
引文
[1]叶任高,陆再英.内科学[M].北京：人民卫生出版社,1999.935
[2]戴琳琅.骨关节炎发病机制及内科治疗[J].沈阳部队医药,2003,16(3)：257
[3]于顺禄,李德达,李世民等.骨关节炎研究进展[J].中国骨伤,2002,15(4)：
[4]黄荷,宋阳.骨关节炎中医药治疗的实验及,临床研究进展[J].中医正骨,2005,17(7)： 69-70
[5]黄枫,谢国平.中医药治疗膝骨关节炎的实验研究进展[J].中国中医药杂志,2005,
12(8)：109-110
[6]袁忠治,李继云,刘刚等.补肾活血中药对兔膝骨关节炎作用的组织测量学研究[J].现代中西医结合氛惠,2003,12(23)：2523-2524
[7]邱贵兴.骨关节炎流行病学和病因学进展[J].继续教育,2004,9(12)：22
[8]谈超,魏伟.骨关节炎的病因研究和治疗的进展[J].、颈腰痛杂志,2002,23(2)：166
[9]叶永平,蒋卉,曾炳芳.膝骨关节炎病因、x线片及临床表现分析[J].临床骨科杂志,2000,3(3)：169
[10]吕存贤,王维佳.膝骨关节炎病因病理研究及中医治疗概况[J].浙江中医学院学报,2005,29(1)：82
[11]娄思权.骨关节炎的病理与发病因素[J].中华骨科杂志,1996,16(1)：56-59
[12]蒋卉,曾炳芳.细胞因子与骨关节炎[J].国外医学·骨科分册,2001,22(3)：143-146
[13]肖晟,李康华,吕红斌.TMPZ对家兔软骨细胞Bcl-2蛋白表达及凋亡的影响[J].湖南医科大学学报,2003,28(3)：224-226
[14]张磊,吴林生.关节软骨损伤修复的研究进展[J].中国骨伤,1997,10(2)：60-62。
[15]杨宁.中医药治疗骨关节炎的研究进展[J].甘肃中医,2005,18(2)：7-9
[16]陈炳坤.当归四逆汤加减治疗膝关节骨关节炎85例疗效观察[J].中医骨伤,1995,7(5)：30
[17]刘金陵.补肝汤加减治疗骨关节炎76例[J].广西中医药,1995,18(2)：10
[18]吕红斌,岳珍,王嘉芙等.四种中药对体外培养兔关节软骨细胞代谢的影响[J].中国运动医学杂志,1995,14(3)：13
[19]吕红斌,徐大启,胡建中等.TMPZ关节腔内注射治疗兔骨关节炎模型的研究[J]. 中国医师杂志,2007,9(9)：1163-1165
[20]晏雪生,彭亚琴,明安萍.TMPZ注射液对体外培养软骨细胞影响的实验研究[J].中国中医骨伤科杂志,2002,10 (1)：15-17
[21]胡建中,罗承耀,康明.TMPZ关节腔内注射对膝骨关节炎的治疗作用[J].中南大学学报(医学版),2006,31(4)：591-594
[22]张建忠,王文林.推拿加正清风痛宁关节腔注射治疗增生性膝关节炎[J].陕西中医函授,2000,2：22
[23]李小军,汪巍,范雯等.甘草多糖抗骨关节炎作用及其机制研究[J].中国药理通讯[J],2009,26(2)：51
[24]鲁玉来,鲁雯,燕好军等.骨关节炎的药物治疗[J].中国矫形外科杂志,2002,10(14)：1425-1427
[25]Firestein GS. Evolving eoncep ls ofrheumatoid arthritis[J]. Nature,2003,423: 356.361.
[26]米玛顿珠,吴滨,李宁.膝关节骨关节炎的治疗概况[J].西藏医药杂志,2006,27(1)：34-36
[27]叶俊星,白书臣,吉璐宏等.骨关节炎的治疗进展[J].湖北中医杂志,2006,28(11)：55-57
[28]郭斌,邓捷,殷明等.玻璃酸钠关节腔注射治疗膝骨关节炎的疗效观察[A].见：凌沛学.玻璃酸钠及其在外科中的应用[M].北京：中国医药科技出版社,67-69
[29]Bernardeau C., Bucki B., Liote F. Acute arthritis after intra-articular hyaluronate injection:onset of effusions without crystal [J]. Ann Rheum Dis.2001,60(5):518-20
[30]王文瑞,刘宏泽,卫小春等.TMPZ防治膝关节软骨退变的实验研究[J].中国骨伤[J],2004,17(2)：80-82
[31]孙必强.正清风痛宁关节腔注射治疗骨关节炎实验研究,湖南中医药大学硕士学位论文,2007.05.
[32]杨惠琴.青藤碱关节腔注射对骨关节炎兔细胞因子及软骨结构的影响,湖北中医学院博士学位论文,2007.05：55-56.
[33]Wigginton S, Chu B, Weisman M, et al. Metholrexate pharmaeokineties after inWaartieular injeetion in patients with rheumatoid arthritis[J]. Mrthritis Rheum,1980,23: 119-122.
[34]Fernandez-Carballido A, Herrero-Vaarell It,et al. Biodergradable ibuprofen loaded PLGA microspheres for intmarticular administratiomEffect of Labrafil addition on release in vitro[J]. IntJPharm,2004,279(1-2):33-41.
[35]Bozdag S, Calis S, Kas Hs, et al. In vitro evaluation and inlra-articulat administration of biodegradable microspheres containing naproxen sodium[J]. J Microencapsul,2001,18: 443-456.
[36]JEFFREY L C, ANN D, RANDALL M. Emerging protein delivery methods[J]. Current Opinion in Biotechnology,2001,12(2):212-219.
[37]艾国,程远国.长效微球注射剂及其在糖尿病治疗领域中的应用[J].中国药剂学杂志,2008,6(4)：206-213
[38]FREIBERG S, Zhu Xiao-xin.. Polymer microspheres for controlled drug release[J]. Int J Pharm,2004,282(5):1-18.
[39]SINHA,V R, AMAN T. Biodegradable microspheres for protein delivery[J]. J Controlled Release,2003,90(3):261-280.
[40]朱盛山.药物新剂型.第1版.北京：化学工业出版社,2004：432.
[41]姚芳莲,刘畅,孟继红等.降解可控乳酸类聚合物[J].高分子通报,2003(3)：47-56.
[42]任杰,宋金星.聚乳酸及其共聚物在缓释药物中的研究及应用[J].同济大学学报,2003,31(9)：1054-1058.
[43]CANASQUILO K G, STANLEY A M, APONTE CARRO J C, et al. Non-aqueous encapsulation of excipientstabilized spray freeze-dried BSA into poly (lactide-co-glycolide) microspheres results in release of native protein[J]. J Controled Release,2001,76(3): 199-215.
[44]周定标,马郭明.PLGA-地塞米松缓释剂的合成及脑内局部应用研究[J].中国微侵袭神经外科杂志,,2006, 11(6)：324-326.
[45]何英,魏树礼等.聚乳酸微球的体外降解[J].北京大学学报(医学版),2001,33(4)：358--361.
[46]AlpaslaaC, hie K Takahashi K et al. Long-term evaluation of recombinant human bone morphogenetic protein-2 induced bone formation with a biologic and synthetic delivery system[J]. Br J oral and Maxillofac Surg,1996。 34(5):414-418.
[47]史大卓,徐浩.TMPZ临床应用和药理作用-TMPZ药理作用诌议[J].中国中西医结合杂志,2003,Vol 23(5)376-379.
[48]韩书芝,韩晓雯,霍颖芳等.TMPZ对肺心病患者急性加重期血小板功能的影响[J].中华结核和呼吸杂志,2003,26(10)：640-640.
[49]廖福龙,李斌.TMPZ抗剪刀应力诱导血小板聚集的实验研究[J].中国药理学通报,1997,13(1)：47-50.
[50]魏敏杰,王怀良,赵乃才.TMPZ对离体大鼠肺动脉作用及机理的探讨[J].中国医科大学学报,1991,20：85-88.
[51]楼正家,诸葛丽敏,郑文龙等.TMPZ对心肺复苏后脑缺血-再灌注损伤的保护作用[J].中国中西医结合急救杂志,2003,10(5)：299-301.
[52]王万铁,徐正祈,林丽娜等.TMPZ对心肌缺血再灌注损伤中一氧化氮和内皮素的干预[J].中国中医药信息杂志,2001,17(3)：230-233.
[53]徐正祈,王万铁,李东等.TMPZ对肝缺血/再灌注损伤脂质过氧化的影响[J].中国应用生理学杂志,2002,18(2)：173-177.
[54]CHANG FC.; CHEN KJ; LIN JG, e.a., Effects of tetramethylpyrazine on portal hypertensive rats Journal of Pharmacy and Pharmacoloy 1998.50(8):881-884.
[55]狄柯坪,杜军英,常立功.芎嗪在微血管运动中作用的研究进展[J].中草药,2002,33(10)：附8-附9.
[56]王锁良,王民,吴施慧。关节内注射TMPZ对兔膝关节软骨退变的影响[J].西安交通大学学报(医学版),2006,27(1)：62-65.
[57]宋桂军,柳菡,冯芳等.替米沙坦油水分配系数的测定及其意义[J].药物分析杂-志,2007,27(11)：1704-1706
[58]王雷,田青平,谢茵.两性霉素B平衡溶解度和表观油水分配系数的测定[J].山西医科大学学报,,2007,38(12)：1082-1084.
[59]Li J.K., Wang N., Wu X.S. A novel biodegradable system based on gelatin nanoparticles and poly(lactic-co-glycolic acid) microspheres for protein and peptide drug delivery [J]. J Pharm Sci.1997,86(8):891-895.
[60]吴诚,尹东锋,鲁莹等.干扰素a-2b长效注射微球的处方和制备工艺的研究[J].第二军医大学学报,2006,27(1)：76-80.
[61]Ratcliffe J.H., Hunneyball I.M., Wilson C.G., et al. Albumin microspheres for
intra-articular drug delivery:investigation of their retention in normal and arthritic knee joints of rabbits [J]. J Pharm Pharmacol.1987,39(4):290-5.
[62]Guihua Huang, Na Zhang, Xiuli Bi, Mingjin Dou. Solid lipid nanoparticles of temozolomide:Potential reduction of cardial and nephric toxicity, International Journal of Pharmaceutics, Volume 355, Issues 1-2,1 May 2008, Pages 314-320.
[63]杜彦侠,何.高效液相色谱法测定盐酸TMPZ注射液的有关物质.中华中医药杂志(原中国医药学报),2006,21(8)：p.497-499.
[64]LIU Chen;XU Hui-Nan;LI Xiao-Ling.In vitro permeation of tetramethylpyrazine across porcine buccal mucosa[J]. Acta Pharmacologica Sinica,2002,23(9):792-796.
[65]Cleland JL,Jones/US. Stable formulations of recombinant human growth hormone and interferon-7 for microcncapsulation in biodegradable microsphercs [J]. Pharm Res, 1996,13(10):1464.
[66]Cleland JL,Mac Bclyd B, el at. The stability of recombinant human growth hormone in poly(lactic-co-glycolic aeid)(PLGA)mierospheres[J]. Pharm Res,1997,14(4):420-425.
[67]BodmeierR, Mcginity JW. Solventselection in the preparation of poly(D, L-lactide)microspheres prepared by the solvent evaporation method. Int J Pharm,1988, 43:179.186
[68]石洋,李慧,徐淼,李炎,张娜,黄桂华*,正交设计优化TMPZPLGA微球处方与制备工艺,山东大学学报(医学版),2010,48(1)：154-158。
[69]Guidance for Industry:Immediate release solid oral dosage forms:Scale-up and post-approval changes(SUPACIR):Chemistry,Manufacturing and Controls, In vitro dissolution testing, and in vivo bioequivalence documentation. US Department of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research. November 1995.
[70]Shah VP,Tsong Y Sathe P, et al. In vitro dissolution profile comparison—statistics and analysis of the similarity factors, f2. Pharm Res,1998,15(6):889.
[71]Moore JW,Flanner HH. Mathematical comparison of dissolution profiles[J]. Pharmaceutical Technology,1996,20(1):64.
[72]徐昕红,于榕,贡沁燕.缓释醋酸亮丙瑞林微球对大鼠子宫异位内膜的抑制作用[J].中国临床药理学与治疗学,2004,9(8)：876-879.
[73]Zhu KJ, Jiang HL,Du XY. Preparation and characterization of hcG—loaded polylactide or poly(lactide-co—glycolide)microspheres using a modified W/O/W emulsion solvent evaporation technique. J Microencap,2001,18(2):247-260.
[74]Bao W., Zhou J., Luo J., et al. PLGA microspheres with high drug loading and high encapsulation efficiency prepared by a novel solvent evaporation technique [J]. J Microencapsul.2006,23(5):471-9.
[75]SANSDRAP P., MOES A.J. Influence of manufacturing parameters on the size characteristics and the release profiles of nifedipine from poly(DL-lactide-co-glycolide) microspheres [J]. Int J Pharm.1993,98:157-164.
[76]Bodmeier R., McGinity J.W. The preparation and evaluation of drug-containing poly(dl-lactide) microspheres formed by the solvent evaporation method [J]. Pharm Res.1987,4(6):465-71.
[77]Arica B., Kas H.S., Orman M.N., et al. Biodegradable bromocryptine mesylate microspheres prepared by a solvent evaporation technique. I:Evaluation of formulation variables on microspheres characteristics for brain delivery [J]. J Microencapsul.2002, 19(4):473-84.
[78]O'Donnell, P., J. McGinity. Preparation of microspheres by the solvent evaporation technique[J]. Advanced Drug Delivery Reviews,1997,28(1):25-42.
[79]Suhaida M.G, Yahya I.B., Darmawati M.Y. Preparation of naltrexone hydrochloride loaded poly (DL-lactide-co-glycolide) microspheres and the effect of polyvinyl alcohol (PVA) as surfactant on the characteristics of the microspheres [J]. Med J Malaysia.2004, 59 Suppl B:63-4.
[80]滕彦,梁文权,林季建等.玻璃体内注射用地塞米松微球的制备[J].中国现代应用药学杂志,2005,22(5)：393-395.
[81]Ratcliffe J.H., Hunneyball I.M., Smith A., et al. Preparation and evaluation of biodegradable polymeric systems for the intra-articular delivery of drugs [J]. J Pharm Pharmacol.1984,36(7):431-6.
[82]Yamamoto N., Fukai F., Ohshima H., et al. Dependence of the phagocytic uptake of polystyrene microspheres by differentiated HL60 upon the size and surface properties of the microspheres [J]. Colloids Surf. B.2002,25:157-162.
[83]Ahsan F., Rivas I.P., Khan M.A., et al. Targeting to macrophages:role of physicochemical properties of particulate carriers-liposomes and microspheres--on the phagocytosis by macrophages [J]. J Control Release.2002,79(1-3):29-40.
[84]梁文权.生物药剂学与药物动力学[M],第3版,北京：人民卫生出版社,2007：353.
[85]马素伟,牛国琴,陈庆华等.氟比洛芬磷脂固体分散体的制备和特性[J].中国医药工业杂志,2008,39(1)：27-29.
[86]Johansen P, Men Y, Merkle HP,et al. Revisiting PLA/PLGA microsphel-es:all analysis of thdr potential in parenteral vaccination. Eur J Pharm Biopharm,2000,50:129.
[87]中国药典,2005版,附录XIXD缓释、控释制剂指导原则。
[88]Mulye, N. V., Turco, S. J., A simple model based on first order kinetics to explain release of highly water soluble drugs from porous dicalcium phosphate dehydrate matrices, Drug Dev. Ind. Pharm.,21,943-953.
[89]Higuchi, T., Rate of release of medicaments from ointment bases containing drugs in suspensions, J. Pharm. Sci.,1961,50,874-875.
[90]John W. Skoug, Martin V. Mikelsons, Cynthia N.,et al. Qualitative evaluation of the mechanism of release of matrix sustained release dosage forms by measurement of polymer release. Journal of Controlled Release,1993(27):227-245.
[91]Yang Y.Y., Chia H.H., Chung T.S. Effect of preparation temperature on the characteristics and release profiles of PLGA microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method [J]. J Control Release.2000, 69(1):81-96.
[92]Mogi T., Ohtake N., Yoshida M., et al. Sustained release of 17β-estradiol from poly (lactide-co-glycolide) microspheres in vitro and in vivo [J]. Colloids Surf. B:Biointerfaces. 2000,17:153.
[93]L. Mua, S.S. Feng. Fabrication, characterization and in vitro release of paclitaxel (Taxol(?)) loaded poly(lactic-co-glycolic acid) microspheres prepared by spray drying technique with lipid/cholesterol emulsifiers[J]. Journal of Controlled Release,2001,76: 239-254:
[94]Huang, X., Brazel, C. On the importance and mechanisms of burst release in
matrix-controlled drug delivery systems[J]. Journal of Controlled Release 2001,73(2-3):. 121-136.
[95]Ammoury N, Fessi H, Devissaguel JP,et al. In vitro release kinetic pattern of indomethacin from poly(D, L-Lactide)nanocapsules. J Pharm Sci,1990,79(9):763.
[96]GaUagher KM, Corrigan OL Mechanistic aspects of the release of levamisole hydrochloride from biodegradable polymers. J Control Release,2000,69:261.
[97]Zhang X, Wyss UP,Pichora D, el al, A mechanistic studies of antibiotic release from biodegradablepoly(dl—lactide)cyclinders.J Control Release,1994.31:129
[98]Ogawa Y, Yamamoto M, Takada S, el al. Controlled-release of leuprolide acetate from polylacticacid or copoly(lacdc/glycolic)acid microcapsules:influence of molecularweightand copolymerratioofpolymer. ChemPharmBull,1988,36(4):1502.
[99]于芝颖,张海英,张华等.重组人生长激素聚乙交酯—丙交酯微球体外释放度研究[J].中国药学杂志,2004,39(8)：608-611.
[100]Jeyanthi R., Thanoo B.C., Metha R.C., et al. Effect of solvent removal technique on the matrix characteristics of polylactide/glycolide microspheres for peptide delivery [J]. J Control Release.1996,38(2):235-244.
[101]McGinity J.W., O'Donnell P.B. Preparation of microspheres by the solvent techniq-ue [J]. Adv Drug Deliv Rev.1997,28(1):25-42.
[102]O'Hagan D.T., JEFFERY H., DAVIS S.S. The preparation and characterization of poly (lactide-co-glycolide) microparticles. Ⅲ:Microparticle/polymer degradation rates and the in vitro release of a model protein [J]. Int J Pharm.1994,103:37-45.
[103]郑俊民主编.药用高分子材料学.北京：中国医药科技出版社,2000：49
[104]Fwulong M, Shiminng S, Y'unei L. Chintin/PLGA blend microspheres as a biodegradable drug delivery system:a new delivery system for protein [J]. Biomateriola, 2003, (1I):5023-5036.
[105]Mohr D, Wolf M,Kissel T. Gamma irradiation for terminal sterilization of 17β-estradiolloaded poly-(D, L-latide-co-glycolide)micropartides[J]. J control Release, 1999,61:203-217.
[106]林芳,沈正荣,黄诚等.60Co辐射灭菌对NAO-PLA微球理化特性及体外释放的影响[J].中国医药工业杂志,1992,23(10)：446-449.
[107]SoppimathKS, Aminabhavi TM, Kulkani AIL. Biodegradable polymeric nanoparticles as dragdelivery devices[J] J Control Release,2001,70(9):1-20.
[108]王亚敏,石庭森,蒲永林等.顺铂壳聚糖微球的制备及特性研究[J].药学学报,1996,31(4)：300-30.
[109]国家药典委员会编,中国人民共和国药典(二部)[S].2010年版.北京：化学工业出版社.
[110]Montanari L, Cilurzo F, Valvo L Gamma h'rad/ation effects oil stability of poly(lactide-co-glycolde)mierospherescontaining donazepam[J].J Control Release, 2001.75:317-330.
[111]苏德森,王思玲主编.物理药剂学.北京：化学工业出版社,2004：
[112]Zarzycki J.主编,干福熹,侯立松等译.玻璃与非晶态材料(Glasses and amorphous materials).北京：科学出版社,2001：98
[113]LINDA S. LIANG,JOHN JACKSON,WEIXIAN MI, et al. Methotrexate Loaded Poly(L-Lactic Acid) Microspheres for Intra-Articular Delivery of Methotrexate to the Joint[J], Journal of pharmaceutical sciences,2004,93(4):943-956.
[114]LINDA S. LIANG, WESLEY WONG, HELEN M. BURT. Pharmacokinetic Study of Methotrexate Following Intra-Articular Injection of Methotrexate Loaded Poly(L-Lactic Acid) Microspheres in Rabbits[J]. Journal of pharmaceutical sciences,2005,94(6):1204-1215
[115]杜彦侠,何庆国.高效液相色谱法测定盐酸TMPZ注射液的有关物质[J].中华中医药杂志(原中国医药学报),2006,21(8)：497-499.
[116]张国平,郭莹,张莉等.灌胃养阴通脑颗粒大鼠血浆中TMPZ的HPLC法测定[J].中草药,2007,38(5)：690-692
[117]Hunneyball I.M. Intra-articular administration of drugs [J]. Pharmacy Int.1986, 5:118-122
[118]Greis P.E., Georgescu H.I., Fu F.H., et al. Particle-induced synthesis of collagenase by synovial fibroblasts:an immunocytochemical study [J]. J Orthop Res.1994,12(2):286-93
[119]Simkin P.A., Nilson K.L. Trans-synovial exchange of large and small molecules [J]. Clin Rheum Dis.1981,7:99-129
[120]Nishide M., Kamei S., Takakura Y., et al. Fate of Biodegradable DL-Lactic Acid Oligomer Microspheres in the Articulus [J]. J Bioact Compat Polym.1999,14:385-398
[121]蒋学华,廖工铁.靶向微球给药系统质量评价.华西药学杂志,1993,8(2)
[122]Anderson JM, Shiver MS. Biodegradation and bioeompatibility of PL A and PLGA mierosphere[J]. AdvDrugDelivRev,1997,28(11):5-24.
[123]Anderson JM, Langone JJ. Issues and perspectives on the bioeompatibility and immunotoxidty evaluation ofimplanted controlled release systems[J]. Journal of Controlled Release,1999,57:107-113.
[124]李志平.关节腔注射用醋酸地塞米松微球的研究,中国人民解放军军事医学科学院硕士学位论文,2004,5：69
[125]Ratcliffe JH,Hunneyball M,Wilson CG etc. Albumin microspheres for intra anicular dmg deli Very.investigation of tIleir retention in nomal and arthritic knee joims of rabbits[J]. J Pharm Pharmacol,1987,39:290
[126]Roshol Hilde,Skrede Knut K,Ero Carl E.Dexamethasone and methylprednisolone affect rea peritoneal phagocyte chemiluminescence after administration in vivo. Eur J Phanllacology,1995,286:9-17
[127]JohnsonO, Ganmukhi M, Bemstein H, et al. Composition for sustained release of human growth hormone. USP 6051259.
[128]Cadee JA, Brouwer LA, Otter WD, et al. A comparative biocompatibility studies of microspheres based oll crosslinked dextran or poly(lactic-co-glycolic)acid after subcutaneous injection in rats[J]. J Miomed Mater Res,2001,56:600
[129]Anderson JM, Shive MS. Biodegradable and biocompatibility of PL A and PLGA microspheres[J]. Adv Drug Delivery Rev,1997,28:5
[130]赵锋,高永良.聚乳酸微球生物降解机制和生物相容性研究进展[J].中国新药杂志.2002.11(1)：67
[131]Pomonis JD,Boulet JM,Oottshall SL,et al.Development and pharmacological characterization of a rat model of osteoarthritis pain[J]. Pain,2005,114(3):339-346
[132]张权,黄煌渊,陈世益等.实验性膑骨关节蜕变性关节病关节软骨的病理改变[J].中华风湿病学杂志,2000,(4)：123-124
[133]LehenerKB, MarkuuT, GmeinwieserJK, etal. Structure, function, degenatlong of bovine hyaline cartilage:asses sment with MR inmging in vitro [J]. Radiology,1989(170): 495-499
[134]郭建刚,冯坤.骨关节炎软骨的生化与代谢变化[J].中国骨伤,1998,11(5)：73-75
[135]沈霖,杨家玉,高兰等.兔膝关节炎模型的复制及健骨汤对其的治疗作用[J].中医研究,1992,5(6)：18.
[136]杨峰,史宗道.用木瓜蛋白酶建立兔颞颌关节骨关节炎模型的研究[J].华西口腔医学杂志,2002,20(5)：330-332.
[137]Havdrup T, Telhag H. Papain-induced changes in the knee joints of adult rabbits[J]. Acta Orthop Scand,1997,48(2):143-149
[138]尚平,贺宪,陈孝银等.过伸位和过屈位固定复制骨关节炎动物模型的比较[J].生物骨科材料与临床研究,2006,3(1)：11.14.
[139]马建兵,沙浩渡,李向阳等.兔实验性膝骨关节炎的组织病理学及发病机理表达的影响[J].中国矫形外科杂志,2001,8(1)：45.48.
[140]McDevitt C, Gilbertson E, Muir H. An experimental model of osteoarthritis; early morphological and biochemical changes[J]. J Bone Jointt Surg Br,1977,59(1)-24-35.
Arrich, J., Piribauer, F., Mad, P., Schmid, D., Klaushofer, K., Mullner, M.,2005. Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee:systematic review and meta-analysis. Can. Med. Assoc. J.,172,1039-1043.
Ayral, X.,2001. Injections in the treatment of osteoarthritis. Bes. Pract. & Res. Clin. Rheumatol.,15,609-626.
Chen, Y., Jobanputra, P., Barton, P., Bryan, S., Fry-Smith, A., Harris, G., Taylor, R.,2008. Cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs (etodolac, meloxicam, celecoxib, rofecoxib, etoricoxib, valdecoxib and lumiracoxib) for osteoarthritis and rheumatoid arthritis:a systematic review and economic evaluation. Health. Technol. Assess. (Winchester, England),12,1-278.
Chevalier, X., Giraudeau, B., Conrozier, T., Marliere, J., Kiefer, P., Goupille, P.,2005.
Safety study of intraarticular injection of interleukin 1 receptor antagonist in patients with painful knee osteoarthritis:a multicenter study, J. Rheumatol.,32,1317-1323.
Feng, L., Xiong, Y., Cheng, F., Zhang, L., Li, S., Li, Y.,2004. Effect of ligustrazine on ischemia-reperfusion injury in murine kidney, Transplant. P.,36,1949-1951.
Foong, W. and Green, K.,1988. Retention and distribution of liposome-entrapped (super (3) H) methotrexate injected into normal or arthritic rabbit joints. J. Pharm. Pharmacol.,40, 464-468.
Gerwin, N., Hops, C., Lucke, A.,2006. Intraarticular drug delivery in osteoarthritis. Adv. Drug. Deliv. Rev.,58,226-242.
Horisawa, E., Kubota, K., Tuboi, I., Sato, K., Yamamoto, H., Takeuchi, H., Kawashima, Y., 2002. Size-dependency of DL-lactide/glycolide copolymer particulates for intra-articular delivery system on phagocytosis in rat synovium. Pharmaceut. Res.,19, 132-139.
Hunneyball, I.,1986. Intra-articular administration of drugs. Pharm. Int.,5,118-122.
Inoue, A., Takahashi, K., Arai, Y., Tonomura, H., Sakao, K., Saito, M., Fujioka, M., Fujiwara, H., Tabata, Y., Kubo, T.,2006. The therapeutic effects of basic fibroblast growth factor contained in gelatin hydrogel microspheres on experimental osteoarthritis in the rabbit knee. Arthrit. Rheum.,54,264-270.
Khanuja, H., Hungerford, M., Manjoo, A.,2003. Intraarticular injections for the treatment of osteoarthritis of the knee:basic science, results, and indications. Curr. Opin. Orthop., 14,62-72.
Kirwan, J.,2001. Is there a place for intra-articular hyaluronate in osteoarthritis of the knee? Knee,8,93-101.
Kwan, C., Gaspar, V., Shi, A., Wang, Z., Chen, M., Ohta, A., Cragoe Jr, E., Daniel, E., 1991. Vascular effects of tetramethylpyrazine:direct interaction with smooth muscle alpha-adrenoceptors. Eur. J. Pharmacol.,198,15-21.
Liang, C., Hong, C., Chen, C., Tsai, T.,1999. Measurement and pharmacokinetic study of tetramethylpyrazine in rat blood and its regional brain tissue by high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl.,724,303-309.
Liang, L., Jackson, J., Min, W., Risovic, V., Wasan, K., Burt, H.,2004. Methotrexate
loaded poly (L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint. J. Pharm. Sci.,93,943-956.
Liang, L., Wong, W., Burt, H.,2005. Pharmacokinetic study of methotrexate following intra-articular injection of methotrexate loaded poly (L-lactic acid) microspheres in rabbits. J. Pharm. Sci.,94,1204-1215.
Liggins, R., Cruz, T., Min, W., Liang, L., Hunter, W., Burt, H.,2004. Intra-articular treatment of arthritis with microsphere formulations of paclitaxel:biocompatibility and efficacy determinations in rabbits. Inflamm. Res.,53,363-372.
Lopez-Garcia, F., Vazquez-Auton, J., Gil, F., Latoore, R., Moreno, F., Villalain, J., Gomez-Fernandez, J.,1993. Intra-articular therapy of experimental arthritis with a derivative of triamcinolone acetonide incorporated in liposomes. J. Pharm. Pharmacol., 45,576-578.
Ratcliffe, J., Hunneyball, I., Wilson, C., Smith, A., Davis, S.,1987. Albumin microspheres for intra-articular drug delivery:investigation of their retention in normal and arthritic knee joints of rabbits. J. Pharm. Pharmacol.,39,290-295.
Rothenfluh, D., Bermudez, H., O'Neil, C, Hubbell, J.,2008. Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nat. Mater.,7, 248-254.
Sah, H.,1997. Microencapsulation techniques using ethyl acetate as a dispersed solvent: effects of its extraction rate on the characteristics of PLGA microspheres. J. Control Release.,47,233-245.
Sah, H.,2000. Ethyl formate-alternative dispersed solvent useful in preparing PLGA microspheres. Int. J. Pharm.,195,103-113.
Sarzi-Puttini, P., Cimmino, M. A., Scarpa, R., Caporali, R., Parazzini, F., Zaninelli, A., Atzeni, F. and Canesi, B.,2005. Osteoarthritis:An Overview of the Disease and Its Treatment Strategies. Semin. Arthritis. Rheu.,35,1-10.
Trif, M., Guillen, C., Vaughan, D., Telfer, J., Brewer, J., Roseanu, A., Brock, J. (2001). Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases. Exp. Biol. Med.,226,559-564.
Tuncay, M., ali, S., Ka, H., Ercan, M., Peksoy, I., Hincal, A.,2000. Diclofenac sodium incorporated PLGA (50:50) microspheres:formulation considerations and in vitro/in vivo evaluation. Int. J. Pharm.,195,179-188.
Wieland, H., Michaelis, M., Kirschbaum, B., Rudolphi, K.,2005. Osteoarthritis—an untreatable disease? Nat. Rev. Drug. Discov.,4,331-344.
Wu, C., Chiou, W., Yen, M.,1989. A possible mechanism of action of tetramethylpyrazine on vascular smooth muscle in rat aorta. Eur. J. Pharmacol.,169,189-195.
Xu, D. and Shen, W.,2007. Ligustrazine Reduces Cartilage Destruction in Model of Osteoarthritis in Rats. J. Sh. Sec. Med. U.,19,117-123.
Yan, X., Peng, Y., Ming, A.,2002. An experimental study of the influence of tetramethylpyrazine injection on cartilage cell of rabbit's articulation. Chin. J. Traditi. Med. Tr. Orthopedics.,10,15-17.
[2]戴琳琅.骨关节炎发病机制及内科治疗[J].沈阳部队医药,2003,16(3)：257
[3]于顺禄,李德达,李世民等.骨关节炎研究进展[J].中国骨伤,2002,15(4)：
[4]黄荷,宋阳.骨关节炎中医药治疗的实验及,临床研究进展[J].中医正骨,2005,17(7)： 69-70
[5]黄枫,谢国平.中医药治疗膝骨关节炎的实验研究进展[J].中国中医药杂志,2005,
12(8)：109-110
[6]袁忠治,李继云,刘刚等.补肾活血中药对兔膝骨关节炎作用的组织测量学研究[J].现代中西医结合氛惠,2003,12(23)：2523-2524
[7]邱贵兴.骨关节炎流行病学和病因学进展[J].继续教育,2004,9(12)：22
[8]谈超,魏伟.骨关节炎的病因研究和治疗的进展[J].、颈腰痛杂志,2002,23(2)：166
[9]叶永平,蒋卉,曾炳芳.膝骨关节炎病因、x线片及临床表现分析[J].临床骨科杂志,2000,3(3)：169
[10]吕存贤,王维佳.膝骨关节炎病因病理研究及中医治疗概况[J].浙江中医学院学报,2005,29(1)：82
[11]娄思权.骨关节炎的病理与发病因素[J].中华骨科杂志,1996,16(1)：56-59
[12]蒋卉,曾炳芳.细胞因子与骨关节炎[J].国外医学·骨科分册,2001,22(3)：143-146
[13]肖晟,李康华,吕红斌.TMPZ对家兔软骨细胞Bcl-2蛋白表达及凋亡的影响[J].湖南医科大学学报,2003,28(3)：224-226
[14]张磊,吴林生.关节软骨损伤修复的研究进展[J].中国骨伤,1997,10(2)：60-62。
[15]杨宁.中医药治疗骨关节炎的研究进展[J].甘肃中医,2005,18(2)：7-9
[16]陈炳坤.当归四逆汤加减治疗膝关节骨关节炎85例疗效观察[J].中医骨伤,1995,7(5)：30
[17]刘金陵.补肝汤加减治疗骨关节炎76例[J].广西中医药,1995,18(2)：10
[18]吕红斌,岳珍,王嘉芙等.四种中药对体外培养兔关节软骨细胞代谢的影响[J].中国运动医学杂志,1995,14(3)：13
[19]吕红斌,徐大启,胡建中等.TMPZ关节腔内注射治疗兔骨关节炎模型的研究[J]. 中国医师杂志,2007,9(9)：1163-1165
[20]晏雪生,彭亚琴,明安萍.TMPZ注射液对体外培养软骨细胞影响的实验研究[J].中国中医骨伤科杂志,2002,10 (1)：15-17
[21]胡建中,罗承耀,康明.TMPZ关节腔内注射对膝骨关节炎的治疗作用[J].中南大学学报(医学版),2006,31(4)：591-594
[22]张建忠,王文林.推拿加正清风痛宁关节腔注射治疗增生性膝关节炎[J].陕西中医函授,2000,2：22
[23]李小军,汪巍,范雯等.甘草多糖抗骨关节炎作用及其机制研究[J].中国药理通讯[J],2009,26(2)：51
[24]鲁玉来,鲁雯,燕好军等.骨关节炎的药物治疗[J].中国矫形外科杂志,2002,10(14)：1425-1427
[25]Firestein GS. Evolving eoncep ls ofrheumatoid arthritis[J]. Nature,2003,423: 356.361.
[26]米玛顿珠,吴滨,李宁.膝关节骨关节炎的治疗概况[J].西藏医药杂志,2006,27(1)：34-36
[27]叶俊星,白书臣,吉璐宏等.骨关节炎的治疗进展[J].湖北中医杂志,2006,28(11)：55-57
[28]郭斌,邓捷,殷明等.玻璃酸钠关节腔注射治疗膝骨关节炎的疗效观察[A].见：凌沛学.玻璃酸钠及其在外科中的应用[M].北京：中国医药科技出版社,67-69
[29]Bernardeau C., Bucki B., Liote F. Acute arthritis after intra-articular hyaluronate injection:onset of effusions without crystal [J]. Ann Rheum Dis.2001,60(5):518-20
[30]王文瑞,刘宏泽,卫小春等.TMPZ防治膝关节软骨退变的实验研究[J].中国骨伤[J],2004,17(2)：80-82
[31]孙必强.正清风痛宁关节腔注射治疗骨关节炎实验研究,湖南中医药大学硕士学位论文,2007.05.
[32]杨惠琴.青藤碱关节腔注射对骨关节炎兔细胞因子及软骨结构的影响,湖北中医学院博士学位论文,2007.05：55-56.
[33]Wigginton S, Chu B, Weisman M, et al. Metholrexate pharmaeokineties after inWaartieular injeetion in patients with rheumatoid arthritis[J]. Mrthritis Rheum,1980,23: 119-122.
[34]Fernandez-Carballido A, Herrero-Vaarell It,et al. Biodergradable ibuprofen loaded PLGA microspheres for intmarticular administratiomEffect of Labrafil addition on release in vitro[J]. IntJPharm,2004,279(1-2):33-41.
[35]Bozdag S, Calis S, Kas Hs, et al. In vitro evaluation and inlra-articulat administration of biodegradable microspheres containing naproxen sodium[J]. J Microencapsul,2001,18: 443-456.
[36]JEFFREY L C, ANN D, RANDALL M. Emerging protein delivery methods[J]. Current Opinion in Biotechnology,2001,12(2):212-219.
[37]艾国,程远国.长效微球注射剂及其在糖尿病治疗领域中的应用[J].中国药剂学杂志,2008,6(4)：206-213
[38]FREIBERG S, Zhu Xiao-xin.. Polymer microspheres for controlled drug release[J]. Int J Pharm,2004,282(5):1-18.
[39]SINHA,V R, AMAN T. Biodegradable microspheres for protein delivery[J]. J Controlled Release,2003,90(3):261-280.
[40]朱盛山.药物新剂型.第1版.北京：化学工业出版社,2004：432.
[41]姚芳莲,刘畅,孟继红等.降解可控乳酸类聚合物[J].高分子通报,2003(3)：47-56.
[42]任杰,宋金星.聚乳酸及其共聚物在缓释药物中的研究及应用[J].同济大学学报,2003,31(9)：1054-1058.
[43]CANASQUILO K G, STANLEY A M, APONTE CARRO J C, et al. Non-aqueous encapsulation of excipientstabilized spray freeze-dried BSA into poly (lactide-co-glycolide) microspheres results in release of native protein[J]. J Controled Release,2001,76(3): 199-215.
[44]周定标,马郭明.PLGA-地塞米松缓释剂的合成及脑内局部应用研究[J].中国微侵袭神经外科杂志,,2006, 11(6)：324-326.
[45]何英,魏树礼等.聚乳酸微球的体外降解[J].北京大学学报(医学版),2001,33(4)：358--361.
[46]AlpaslaaC, hie K Takahashi K et al. Long-term evaluation of recombinant human bone morphogenetic protein-2 induced bone formation with a biologic and synthetic delivery system[J]. Br J oral and Maxillofac Surg,1996。 34(5):414-418.
[47]史大卓,徐浩.TMPZ临床应用和药理作用-TMPZ药理作用诌议[J].中国中西医结合杂志,2003,Vol 23(5)376-379.
[48]韩书芝,韩晓雯,霍颖芳等.TMPZ对肺心病患者急性加重期血小板功能的影响[J].中华结核和呼吸杂志,2003,26(10)：640-640.
[49]廖福龙,李斌.TMPZ抗剪刀应力诱导血小板聚集的实验研究[J].中国药理学通报,1997,13(1)：47-50.
[50]魏敏杰,王怀良,赵乃才.TMPZ对离体大鼠肺动脉作用及机理的探讨[J].中国医科大学学报,1991,20：85-88.
[51]楼正家,诸葛丽敏,郑文龙等.TMPZ对心肺复苏后脑缺血-再灌注损伤的保护作用[J].中国中西医结合急救杂志,2003,10(5)：299-301.
[52]王万铁,徐正祈,林丽娜等.TMPZ对心肌缺血再灌注损伤中一氧化氮和内皮素的干预[J].中国中医药信息杂志,2001,17(3)：230-233.
[53]徐正祈,王万铁,李东等.TMPZ对肝缺血/再灌注损伤脂质过氧化的影响[J].中国应用生理学杂志,2002,18(2)：173-177.
[54]CHANG FC.; CHEN KJ; LIN JG, e.a., Effects of tetramethylpyrazine on portal hypertensive rats Journal of Pharmacy and Pharmacoloy 1998.50(8):881-884.
[55]狄柯坪,杜军英,常立功.芎嗪在微血管运动中作用的研究进展[J].中草药,2002,33(10)：附8-附9.
[56]王锁良,王民,吴施慧。关节内注射TMPZ对兔膝关节软骨退变的影响[J].西安交通大学学报(医学版),2006,27(1)：62-65.
[57]宋桂军,柳菡,冯芳等.替米沙坦油水分配系数的测定及其意义[J].药物分析杂-志,2007,27(11)：1704-1706
[58]王雷,田青平,谢茵.两性霉素B平衡溶解度和表观油水分配系数的测定[J].山西医科大学学报,,2007,38(12)：1082-1084.
[59]Li J.K., Wang N., Wu X.S. A novel biodegradable system based on gelatin nanoparticles and poly(lactic-co-glycolic acid) microspheres for protein and peptide drug delivery [J]. J Pharm Sci.1997,86(8):891-895.
[60]吴诚,尹东锋,鲁莹等.干扰素a-2b长效注射微球的处方和制备工艺的研究[J].第二军医大学学报,2006,27(1)：76-80.
[61]Ratcliffe J.H., Hunneyball I.M., Wilson C.G., et al. Albumin microspheres for
intra-articular drug delivery:investigation of their retention in normal and arthritic knee joints of rabbits [J]. J Pharm Pharmacol.1987,39(4):290-5.
[62]Guihua Huang, Na Zhang, Xiuli Bi, Mingjin Dou. Solid lipid nanoparticles of temozolomide:Potential reduction of cardial and nephric toxicity, International Journal of Pharmaceutics, Volume 355, Issues 1-2,1 May 2008, Pages 314-320.
[63]杜彦侠,何.高效液相色谱法测定盐酸TMPZ注射液的有关物质.中华中医药杂志(原中国医药学报),2006,21(8)：p.497-499.
[64]LIU Chen;XU Hui-Nan;LI Xiao-Ling.In vitro permeation of tetramethylpyrazine across porcine buccal mucosa[J]. Acta Pharmacologica Sinica,2002,23(9):792-796.
[65]Cleland JL,Jones/US. Stable formulations of recombinant human growth hormone and interferon-7 for microcncapsulation in biodegradable microsphercs [J]. Pharm Res, 1996,13(10):1464.
[66]Cleland JL,Mac Bclyd B, el at. The stability of recombinant human growth hormone in poly(lactic-co-glycolic aeid)(PLGA)mierospheres[J]. Pharm Res,1997,14(4):420-425.
[67]BodmeierR, Mcginity JW. Solventselection in the preparation of poly(D, L-lactide)microspheres prepared by the solvent evaporation method. Int J Pharm,1988, 43:179.186
[68]石洋,李慧,徐淼,李炎,张娜,黄桂华*,正交设计优化TMPZPLGA微球处方与制备工艺,山东大学学报(医学版),2010,48(1)：154-158。
[69]Guidance for Industry:Immediate release solid oral dosage forms:Scale-up and post-approval changes(SUPACIR):Chemistry,Manufacturing and Controls, In vitro dissolution testing, and in vivo bioequivalence documentation. US Department of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research. November 1995.
[70]Shah VP,Tsong Y Sathe P, et al. In vitro dissolution profile comparison—statistics and analysis of the similarity factors, f2. Pharm Res,1998,15(6):889.
[71]Moore JW,Flanner HH. Mathematical comparison of dissolution profiles[J]. Pharmaceutical Technology,1996,20(1):64.
[72]徐昕红,于榕,贡沁燕.缓释醋酸亮丙瑞林微球对大鼠子宫异位内膜的抑制作用[J].中国临床药理学与治疗学,2004,9(8)：876-879.
[73]Zhu KJ, Jiang HL,Du XY. Preparation and characterization of hcG—loaded polylactide or poly(lactide-co—glycolide)microspheres using a modified W/O/W emulsion solvent evaporation technique. J Microencap,2001,18(2):247-260.
[74]Bao W., Zhou J., Luo J., et al. PLGA microspheres with high drug loading and high encapsulation efficiency prepared by a novel solvent evaporation technique [J]. J Microencapsul.2006,23(5):471-9.
[75]SANSDRAP P., MOES A.J. Influence of manufacturing parameters on the size characteristics and the release profiles of nifedipine from poly(DL-lactide-co-glycolide) microspheres [J]. Int J Pharm.1993,98:157-164.
[76]Bodmeier R., McGinity J.W. The preparation and evaluation of drug-containing poly(dl-lactide) microspheres formed by the solvent evaporation method [J]. Pharm Res.1987,4(6):465-71.
[77]Arica B., Kas H.S., Orman M.N., et al. Biodegradable bromocryptine mesylate microspheres prepared by a solvent evaporation technique. I:Evaluation of formulation variables on microspheres characteristics for brain delivery [J]. J Microencapsul.2002, 19(4):473-84.
[78]O'Donnell, P., J. McGinity. Preparation of microspheres by the solvent evaporation technique[J]. Advanced Drug Delivery Reviews,1997,28(1):25-42.
[79]Suhaida M.G, Yahya I.B., Darmawati M.Y. Preparation of naltrexone hydrochloride loaded poly (DL-lactide-co-glycolide) microspheres and the effect of polyvinyl alcohol (PVA) as surfactant on the characteristics of the microspheres [J]. Med J Malaysia.2004, 59 Suppl B:63-4.
[80]滕彦,梁文权,林季建等.玻璃体内注射用地塞米松微球的制备[J].中国现代应用药学杂志,2005,22(5)：393-395.
[81]Ratcliffe J.H., Hunneyball I.M., Smith A., et al. Preparation and evaluation of biodegradable polymeric systems for the intra-articular delivery of drugs [J]. J Pharm Pharmacol.1984,36(7):431-6.
[82]Yamamoto N., Fukai F., Ohshima H., et al. Dependence of the phagocytic uptake of polystyrene microspheres by differentiated HL60 upon the size and surface properties of the microspheres [J]. Colloids Surf. B.2002,25:157-162.
[83]Ahsan F., Rivas I.P., Khan M.A., et al. Targeting to macrophages:role of physicochemical properties of particulate carriers-liposomes and microspheres--on the phagocytosis by macrophages [J]. J Control Release.2002,79(1-3):29-40.
[84]梁文权.生物药剂学与药物动力学[M],第3版,北京：人民卫生出版社,2007：353.
[85]马素伟,牛国琴,陈庆华等.氟比洛芬磷脂固体分散体的制备和特性[J].中国医药工业杂志,2008,39(1)：27-29.
[86]Johansen P, Men Y, Merkle HP,et al. Revisiting PLA/PLGA microsphel-es:all analysis of thdr potential in parenteral vaccination. Eur J Pharm Biopharm,2000,50:129.
[87]中国药典,2005版,附录XIXD缓释、控释制剂指导原则。
[88]Mulye, N. V., Turco, S. J., A simple model based on first order kinetics to explain release of highly water soluble drugs from porous dicalcium phosphate dehydrate matrices, Drug Dev. Ind. Pharm.,21,943-953.
[89]Higuchi, T., Rate of release of medicaments from ointment bases containing drugs in suspensions, J. Pharm. Sci.,1961,50,874-875.
[90]John W. Skoug, Martin V. Mikelsons, Cynthia N.,et al. Qualitative evaluation of the mechanism of release of matrix sustained release dosage forms by measurement of polymer release. Journal of Controlled Release,1993(27):227-245.
[91]Yang Y.Y., Chia H.H., Chung T.S. Effect of preparation temperature on the characteristics and release profiles of PLGA microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method [J]. J Control Release.2000, 69(1):81-96.
[92]Mogi T., Ohtake N., Yoshida M., et al. Sustained release of 17β-estradiol from poly (lactide-co-glycolide) microspheres in vitro and in vivo [J]. Colloids Surf. B:Biointerfaces. 2000,17:153.
[93]L. Mua, S.S. Feng. Fabrication, characterization and in vitro release of paclitaxel (Taxol(?)) loaded poly(lactic-co-glycolic acid) microspheres prepared by spray drying technique with lipid/cholesterol emulsifiers[J]. Journal of Controlled Release,2001,76: 239-254:
[94]Huang, X., Brazel, C. On the importance and mechanisms of burst release in
matrix-controlled drug delivery systems[J]. Journal of Controlled Release 2001,73(2-3):. 121-136.
[95]Ammoury N, Fessi H, Devissaguel JP,et al. In vitro release kinetic pattern of indomethacin from poly(D, L-Lactide)nanocapsules. J Pharm Sci,1990,79(9):763.
[96]GaUagher KM, Corrigan OL Mechanistic aspects of the release of levamisole hydrochloride from biodegradable polymers. J Control Release,2000,69:261.
[97]Zhang X, Wyss UP,Pichora D, el al, A mechanistic studies of antibiotic release from biodegradablepoly(dl—lactide)cyclinders.J Control Release,1994.31:129
[98]Ogawa Y, Yamamoto M, Takada S, el al. Controlled-release of leuprolide acetate from polylacticacid or copoly(lacdc/glycolic)acid microcapsules:influence of molecularweightand copolymerratioofpolymer. ChemPharmBull,1988,36(4):1502.
[99]于芝颖,张海英,张华等.重组人生长激素聚乙交酯—丙交酯微球体外释放度研究[J].中国药学杂志,2004,39(8)：608-611.
[100]Jeyanthi R., Thanoo B.C., Metha R.C., et al. Effect of solvent removal technique on the matrix characteristics of polylactide/glycolide microspheres for peptide delivery [J]. J Control Release.1996,38(2):235-244.
[101]McGinity J.W., O'Donnell P.B. Preparation of microspheres by the solvent techniq-ue [J]. Adv Drug Deliv Rev.1997,28(1):25-42.
[102]O'Hagan D.T., JEFFERY H., DAVIS S.S. The preparation and characterization of poly (lactide-co-glycolide) microparticles. Ⅲ:Microparticle/polymer degradation rates and the in vitro release of a model protein [J]. Int J Pharm.1994,103:37-45.
[103]郑俊民主编.药用高分子材料学.北京：中国医药科技出版社,2000：49
[104]Fwulong M, Shiminng S, Y'unei L. Chintin/PLGA blend microspheres as a biodegradable drug delivery system:a new delivery system for protein [J]. Biomateriola, 2003, (1I):5023-5036.
[105]Mohr D, Wolf M,Kissel T. Gamma irradiation for terminal sterilization of 17β-estradiolloaded poly-(D, L-latide-co-glycolide)micropartides[J]. J control Release, 1999,61:203-217.
[106]林芳,沈正荣,黄诚等.60Co辐射灭菌对NAO-PLA微球理化特性及体外释放的影响[J].中国医药工业杂志,1992,23(10)：446-449.
[107]SoppimathKS, Aminabhavi TM, Kulkani AIL. Biodegradable polymeric nanoparticles as dragdelivery devices[J] J Control Release,2001,70(9):1-20.
[108]王亚敏,石庭森,蒲永林等.顺铂壳聚糖微球的制备及特性研究[J].药学学报,1996,31(4)：300-30.
[109]国家药典委员会编,中国人民共和国药典(二部)[S].2010年版.北京：化学工业出版社.
[110]Montanari L, Cilurzo F, Valvo L Gamma h'rad/ation effects oil stability of poly(lactide-co-glycolde)mierospherescontaining donazepam[J].J Control Release, 2001.75:317-330.
[111]苏德森,王思玲主编.物理药剂学.北京：化学工业出版社,2004：
[112]Zarzycki J.主编,干福熹,侯立松等译.玻璃与非晶态材料(Glasses and amorphous materials).北京：科学出版社,2001：98
[113]LINDA S. LIANG,JOHN JACKSON,WEIXIAN MI, et al. Methotrexate Loaded Poly(L-Lactic Acid) Microspheres for Intra-Articular Delivery of Methotrexate to the Joint[J], Journal of pharmaceutical sciences,2004,93(4):943-956.
[114]LINDA S. LIANG, WESLEY WONG, HELEN M. BURT. Pharmacokinetic Study of Methotrexate Following Intra-Articular Injection of Methotrexate Loaded Poly(L-Lactic Acid) Microspheres in Rabbits[J]. Journal of pharmaceutical sciences,2005,94(6):1204-1215
[115]杜彦侠,何庆国.高效液相色谱法测定盐酸TMPZ注射液的有关物质[J].中华中医药杂志(原中国医药学报),2006,21(8)：497-499.
[116]张国平,郭莹,张莉等.灌胃养阴通脑颗粒大鼠血浆中TMPZ的HPLC法测定[J].中草药,2007,38(5)：690-692
[117]Hunneyball I.M. Intra-articular administration of drugs [J]. Pharmacy Int.1986, 5:118-122
[118]Greis P.E., Georgescu H.I., Fu F.H., et al. Particle-induced synthesis of collagenase by synovial fibroblasts:an immunocytochemical study [J]. J Orthop Res.1994,12(2):286-93
[119]Simkin P.A., Nilson K.L. Trans-synovial exchange of large and small molecules [J]. Clin Rheum Dis.1981,7:99-129
[120]Nishide M., Kamei S., Takakura Y., et al. Fate of Biodegradable DL-Lactic Acid Oligomer Microspheres in the Articulus [J]. J Bioact Compat Polym.1999,14:385-398
[121]蒋学华,廖工铁.靶向微球给药系统质量评价.华西药学杂志,1993,8(2)
[122]Anderson JM, Shiver MS. Biodegradation and bioeompatibility of PL A and PLGA mierosphere[J]. AdvDrugDelivRev,1997,28(11):5-24.
[123]Anderson JM, Langone JJ. Issues and perspectives on the bioeompatibility and immunotoxidty evaluation ofimplanted controlled release systems[J]. Journal of Controlled Release,1999,57:107-113.
[124]李志平.关节腔注射用醋酸地塞米松微球的研究,中国人民解放军军事医学科学院硕士学位论文,2004,5：69
[125]Ratcliffe JH,Hunneyball M,Wilson CG etc. Albumin microspheres for intra anicular dmg deli Very.investigation of tIleir retention in nomal and arthritic knee joims of rabbits[J]. J Pharm Pharmacol,1987,39:290
[126]Roshol Hilde,Skrede Knut K,Ero Carl E.Dexamethasone and methylprednisolone affect rea peritoneal phagocyte chemiluminescence after administration in vivo. Eur J Phanllacology,1995,286:9-17
[127]JohnsonO, Ganmukhi M, Bemstein H, et al. Composition for sustained release of human growth hormone. USP 6051259.
[128]Cadee JA, Brouwer LA, Otter WD, et al. A comparative biocompatibility studies of microspheres based oll crosslinked dextran or poly(lactic-co-glycolic)acid after subcutaneous injection in rats[J]. J Miomed Mater Res,2001,56:600
[129]Anderson JM, Shive MS. Biodegradable and biocompatibility of PL A and PLGA microspheres[J]. Adv Drug Delivery Rev,1997,28:5
[130]赵锋,高永良.聚乳酸微球生物降解机制和生物相容性研究进展[J].中国新药杂志.2002.11(1)：67
[131]Pomonis JD,Boulet JM,Oottshall SL,et al.Development and pharmacological characterization of a rat model of osteoarthritis pain[J]. Pain,2005,114(3):339-346
[132]张权,黄煌渊,陈世益等.实验性膑骨关节蜕变性关节病关节软骨的病理改变[J].中华风湿病学杂志,2000,(4)：123-124
[133]LehenerKB, MarkuuT, GmeinwieserJK, etal. Structure, function, degenatlong of bovine hyaline cartilage:asses sment with MR inmging in vitro [J]. Radiology,1989(170): 495-499
[134]郭建刚,冯坤.骨关节炎软骨的生化与代谢变化[J].中国骨伤,1998,11(5)：73-75
[135]沈霖,杨家玉,高兰等.兔膝关节炎模型的复制及健骨汤对其的治疗作用[J].中医研究,1992,5(6)：18.
[136]杨峰,史宗道.用木瓜蛋白酶建立兔颞颌关节骨关节炎模型的研究[J].华西口腔医学杂志,2002,20(5)：330-332.
[137]Havdrup T, Telhag H. Papain-induced changes in the knee joints of adult rabbits[J]. Acta Orthop Scand,1997,48(2):143-149
[138]尚平,贺宪,陈孝银等.过伸位和过屈位固定复制骨关节炎动物模型的比较[J].生物骨科材料与临床研究,2006,3(1)：11.14.
[139]马建兵,沙浩渡,李向阳等.兔实验性膝骨关节炎的组织病理学及发病机理表达的影响[J].中国矫形外科杂志,2001,8(1)：45.48.
[140]McDevitt C, Gilbertson E, Muir H. An experimental model of osteoarthritis; early morphological and biochemical changes[J]. J Bone Jointt Surg Br,1977,59(1)-24-35.
Arrich, J., Piribauer, F., Mad, P., Schmid, D., Klaushofer, K., Mullner, M.,2005. Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee:systematic review and meta-analysis. Can. Med. Assoc. J.,172,1039-1043.
Ayral, X.,2001. Injections in the treatment of osteoarthritis. Bes. Pract. & Res. Clin. Rheumatol.,15,609-626.
Chen, Y., Jobanputra, P., Barton, P., Bryan, S., Fry-Smith, A., Harris, G., Taylor, R.,2008. Cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs (etodolac, meloxicam, celecoxib, rofecoxib, etoricoxib, valdecoxib and lumiracoxib) for osteoarthritis and rheumatoid arthritis:a systematic review and economic evaluation. Health. Technol. Assess. (Winchester, England),12,1-278.
Chevalier, X., Giraudeau, B., Conrozier, T., Marliere, J., Kiefer, P., Goupille, P.,2005.
Safety study of intraarticular injection of interleukin 1 receptor antagonist in patients with painful knee osteoarthritis:a multicenter study, J. Rheumatol.,32,1317-1323.
Feng, L., Xiong, Y., Cheng, F., Zhang, L., Li, S., Li, Y.,2004. Effect of ligustrazine on ischemia-reperfusion injury in murine kidney, Transplant. P.,36,1949-1951.
Foong, W. and Green, K.,1988. Retention and distribution of liposome-entrapped (super (3) H) methotrexate injected into normal or arthritic rabbit joints. J. Pharm. Pharmacol.,40, 464-468.
Gerwin, N., Hops, C., Lucke, A.,2006. Intraarticular drug delivery in osteoarthritis. Adv. Drug. Deliv. Rev.,58,226-242.
Horisawa, E., Kubota, K., Tuboi, I., Sato, K., Yamamoto, H., Takeuchi, H., Kawashima, Y., 2002. Size-dependency of DL-lactide/glycolide copolymer particulates for intra-articular delivery system on phagocytosis in rat synovium. Pharmaceut. Res.,19, 132-139.
Hunneyball, I.,1986. Intra-articular administration of drugs. Pharm. Int.,5,118-122.
Inoue, A., Takahashi, K., Arai, Y., Tonomura, H., Sakao, K., Saito, M., Fujioka, M., Fujiwara, H., Tabata, Y., Kubo, T.,2006. The therapeutic effects of basic fibroblast growth factor contained in gelatin hydrogel microspheres on experimental osteoarthritis in the rabbit knee. Arthrit. Rheum.,54,264-270.
Khanuja, H., Hungerford, M., Manjoo, A.,2003. Intraarticular injections for the treatment of osteoarthritis of the knee:basic science, results, and indications. Curr. Opin. Orthop., 14,62-72.
Kirwan, J.,2001. Is there a place for intra-articular hyaluronate in osteoarthritis of the knee? Knee,8,93-101.
Kwan, C., Gaspar, V., Shi, A., Wang, Z., Chen, M., Ohta, A., Cragoe Jr, E., Daniel, E., 1991. Vascular effects of tetramethylpyrazine:direct interaction with smooth muscle alpha-adrenoceptors. Eur. J. Pharmacol.,198,15-21.
Liang, C., Hong, C., Chen, C., Tsai, T.,1999. Measurement and pharmacokinetic study of tetramethylpyrazine in rat blood and its regional brain tissue by high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl.,724,303-309.
Liang, L., Jackson, J., Min, W., Risovic, V., Wasan, K., Burt, H.,2004. Methotrexate
loaded poly (L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint. J. Pharm. Sci.,93,943-956.
Liang, L., Wong, W., Burt, H.,2005. Pharmacokinetic study of methotrexate following intra-articular injection of methotrexate loaded poly (L-lactic acid) microspheres in rabbits. J. Pharm. Sci.,94,1204-1215.
Liggins, R., Cruz, T., Min, W., Liang, L., Hunter, W., Burt, H.,2004. Intra-articular treatment of arthritis with microsphere formulations of paclitaxel:biocompatibility and efficacy determinations in rabbits. Inflamm. Res.,53,363-372.
Lopez-Garcia, F., Vazquez-Auton, J., Gil, F., Latoore, R., Moreno, F., Villalain, J., Gomez-Fernandez, J.,1993. Intra-articular therapy of experimental arthritis with a derivative of triamcinolone acetonide incorporated in liposomes. J. Pharm. Pharmacol., 45,576-578.
Ratcliffe, J., Hunneyball, I., Wilson, C., Smith, A., Davis, S.,1987. Albumin microspheres for intra-articular drug delivery:investigation of their retention in normal and arthritic knee joints of rabbits. J. Pharm. Pharmacol.,39,290-295.
Rothenfluh, D., Bermudez, H., O'Neil, C, Hubbell, J.,2008. Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nat. Mater.,7, 248-254.
Sah, H.,1997. Microencapsulation techniques using ethyl acetate as a dispersed solvent: effects of its extraction rate on the characteristics of PLGA microspheres. J. Control Release.,47,233-245.
Sah, H.,2000. Ethyl formate-alternative dispersed solvent useful in preparing PLGA microspheres. Int. J. Pharm.,195,103-113.
Sarzi-Puttini, P., Cimmino, M. A., Scarpa, R., Caporali, R., Parazzini, F., Zaninelli, A., Atzeni, F. and Canesi, B.,2005. Osteoarthritis:An Overview of the Disease and Its Treatment Strategies. Semin. Arthritis. Rheu.,35,1-10.
Trif, M., Guillen, C., Vaughan, D., Telfer, J., Brewer, J., Roseanu, A., Brock, J. (2001). Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases. Exp. Biol. Med.,226,559-564.
Tuncay, M., ali, S., Ka, H., Ercan, M., Peksoy, I., Hincal, A.,2000. Diclofenac sodium incorporated PLGA (50:50) microspheres:formulation considerations and in vitro/in vivo evaluation. Int. J. Pharm.,195,179-188.
Wieland, H., Michaelis, M., Kirschbaum, B., Rudolphi, K.,2005. Osteoarthritis—an untreatable disease? Nat. Rev. Drug. Discov.,4,331-344.
Wu, C., Chiou, W., Yen, M.,1989. A possible mechanism of action of tetramethylpyrazine on vascular smooth muscle in rat aorta. Eur. J. Pharmacol.,169,189-195.
Xu, D. and Shen, W.,2007. Ligustrazine Reduces Cartilage Destruction in Model of Osteoarthritis in Rats. J. Sh. Sec. Med. U.,19,117-123.
Yan, X., Peng, Y., Ming, A.,2002. An experimental study of the influence of tetramethylpyrazine injection on cartilage cell of rabbit's articulation. Chin. J. Traditi. Med. Tr. Orthopedics.,10,15-17.