用于关节腔注射的马钱子碱微球/温敏凝胶复合系统的研究
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摘要
关节腔注射给药可以直接将药物输送至炎症部位，改变药物在体内的分布模式，避开药物在体内转运中的生理屏障，以最小的药量发挥最大的药效，然而由于一些药物的化学结构，活性物质注入关节腔后会迅速消除，在炎症部位存留时间短、药效持续时间短，需频繁给药等缺点，给病人造成了极大的痛苦，而且频繁的注射有可能导致继发性炎症反应和关节功能失调。如何延长药物在关节腔的释放，达到缓释长效的效果是关节腔给药研究的热点。本论文设计了一种新型的药物载体，即壳聚糖微球/温敏凝胶复合系统，用于控制药物的释放。以马钱子碱为模型药物。
马钱子碱是从马钱子中提取的有效成分。马钱子，又名番木鳖，始载于《本草纲目》，为历版《中国药典》所收载。是马钱科植物马钱（Strychnos nux_vomica L.）的干燥成熟种子，具通络止痛、散结消肿之功效，擅治风湿等疾病。临床常将其炮制后用于治疗风湿顽痹、麻木瘫痪、跌打损伤和痈疽肿痛等疾病，临床应用已有近千年的历史。马钱子碱为马钱子中的有效成分，具有良好的镇痛作用、抗炎免疫作用，抗肿瘤作用，对抗心律失常作用并可拮抗NO对软骨细胞增殖的抑制作用，能够有效促进软骨细胞增殖，从而对软骨降解为主要进程的骨关节炎修复起作用，其抗炎免疫作用、镇痛作用可以对自身免疫为特征的风湿性关节炎产生治疗效果，缓解患者疼痛。
本文采用喷雾干燥法制备马钱子碱壳聚糖微球，然后将微球分散于壳聚糖-甘油-硼砂温敏凝胶中，微球在凝胶中作为药物贮库，而利用温敏凝胶可阻滞微球的突释现象，形成了双重缓释系统，有效的延长了药物在关节腔的保留时间，使得马钱子碱更好的在关节腔中发挥药效。
本文建立了紫外分光光度法用于马钱子碱微球含量和释放度的测定，并进行了方法学考察。
采用瑞士BUCHI B290喷雾干燥仪制备壳聚糖微球，以收率和干燥程度为评价指标，首先确定了出口温度为120℃，鼓风流量为40m~3/h，泵速为20%·min~(-1)。之后，分别考察壳聚糖型号、壳聚糖浓度、戊二醛用量、马钱子碱与壳聚糖的质量比等处方因素对包封率和释放速率的影响。筛选对制剂性质影响较大的因素，利用正交设计优化处方：壳聚糖浓度为0.75%，戊二醛与壳聚糖的质量比为1:15，马钱子碱与壳聚糖的质量比为1:5，微球的包封率为98.61%±0.42%，载药量为15.57%±0.07%。利用SEM，FTIR，XRD等手段对微球进行了系统的表征，结果发现微球为表面光滑的球体，无粘连，粒径在0.9-4.5μm之间，平均粒径为2.45μm，呈正态分布。FTIR证明了马钱子碱与壳聚糖无新键生成，X射线衍射结果显示马钱子碱以无定形态存在于微球中。
壳聚糖温敏凝胶的处方为壳聚糖溶液、甘油、饱和硼砂溶液。以凝胶情况和凝胶时间为指标，考察壳聚糖浓度、壳聚糖与甘油的比例、pH值等因素对水凝胶理化性质的影响，最佳处方为浓度为2%的壳聚糖，甘油与壳聚糖比例为1:10，pH为6.7。采用试管法对马钱子碱壳聚糖微球（BM）、马钱子碱温敏凝胶（BH）、马钱子碱微球/温敏凝胶复合系统（BMH）进行体外释放研究，并对释放行为进行曲线拟合，探讨其释放机制，三种载体均属于双指数双相动力学释放模型。
考察了BM、BH、BMH对大鼠膝关节滑膜的生物相容性，结果显示BM、BH、BMH的生物相容性较好，可做为关节腔注射的载体。
采取活体成像技术观察了不同载体系统在关节腔内的释放行为，利用近红外染料NIRD-15为模型药物，大鼠关节腔注射后，可视化地观察载体对模型药物的控释能力，分别考察了微球和微球凝胶复合系统，结果显示，将微球分散于凝胶中，可以明显的延长在关节腔的驻留时间，缓释时间可达7天以上。
本实验对BMH进行了大鼠体内初步药代动力学研究，首先采用RP-HPLC我们建立了马钱子碱在大鼠血浆中的测定方法，研究了溶液剂（BS）、BM、BMH的大鼠药动学，运用3p97程序对三组的大鼠体内试验数据进行拟合和计算，结果表明，将微球分散于凝胶后MRT有了明显的延长，达到了制剂设计的目的。
综上所述，马钱子碱壳聚糖/温敏凝胶复合系统极大的延长了药物在关节腔的保留时间，可以将缓释作用达到的一周，减少了注射的次数，增加了患者的适应性，达到了课题的设计要求。
Intra-articular drug delivery system could directly delivery drug to an affected joint and offer thepossibility of reaching high drug concentrations at the site of action with limited systemic toxicity.However, depending on their chemical structure, some active compounds were rapidly cleared from thejoint, thus requiring numerous injections, which could cause infection or joint disability. To control therelease behavior for prolonged time periods, a novel biologically based drug delivery vehicle was designedfor intra-articular using microsphere/thermally responsive hydrogel combination system in this paper. Andthe brucine was the test drug.
Brucine is the active ingredient extracted from Strychnos nux-vomica L. Nux vomica, also known asstrychnine, first recorded in Compendium of Materia Medica, and recorded in each China Pharmacopoeia,is the dried ripe seeds of the Strychnos nux-vomica L. It has effects of activating meridians to stop pain andsubsiding swelling, especially for treating disease like RA and OA.It has been used clinical in traditionalChinese medicine for the treatment of rheumatoid paralysis, numbness, bruises and other diseases afterbeen concocted.Brucine is the active ingredient of nux_vomica, has the effect of analgesic,anti-inflammatory, anti-immune, anti-tumor, anti-arrhythmia, and antagonize the NO inhibitory effect onchondrocyte proliferation. The process of Osteoarthritis (OA) is mainly in the degration of chondrocytes;brucine can effectively promote the proliferation of chondrocytes, hence repair the damage cartilage. Oneof the pathogenesis of Rheumatoid Arthritis (RA) is self-immune, the effect of anti-immune and stop paincan alleviate distress of RA patients.
The system was constructed by disperse the brucine microspheres which was prepared by using aspray-drying method in a thermally responsive biopolymer hydrogel contained of chitosan-glycerol-borax.The hydrogel can block the burst release of microspheres, and the microspheres were incorporated inhydrogel as drug depot.The duplexing release system could effectively prolong the release behavior in thejoint cavity, increase the efficiency of brucine for a extended period of time.
The microspheres were prepared by Switzerland Buchi B290spray-drying apparatus.Firstlydetermines the craft factors of outlet temperature, Q-flow, pump speed. The outlet temperature was120℃,Q-flow was40m~3/h, Pump speed of20%·min~(-1). Then, investigate the influence of type of chitosan,chitosan concerntration, glutaral ratio, ratio of brucine to chitosan on the entrapment efficiency (EE) and in vitro drug release behavior of brucine micropheres. By single-factor test chose three factors, by orthogonaldesign optimization prescription, selected the best prescription for Chitosan0.75%, glutaral ratio of1:15,the ratio of brucine to chitosan is1:5.The Drug loading(DL%) was15.57%±0.07%and Entrapmentefficiency(EE%) was98.61%±0.42%.
The brucine microspheres were characterized by SEM, XRD, and FTIR, spherical as evidenced by thescanning electron microscopy (SEM) photographs. With an average size of2.45μm, in range of0.9–4.5μm,agree with normal distribution. Fourier transforms infrared (FT-IR) spectroscopy and X-ray diffraction(XRD) revealed the absence of drug-polymer interaction and amorphous nature of entrapped drug.
The thermosensitive converse hydrogel was constructed using chitosan-glycerol-borax as matrix,taking gelation time and gelation condition as index, investigate the influence of concerntraion of chitosan,ratio of chitosan to glycerol,pH on physical properties of hydrogel. The best prescription was CS2%, CS:glycerol10:1, pH6.7.
Taking tube method, investigated the in vitro drug release of brucine microsphere(BM), brucinehydrogel(BH),brucine microsphere hydrogel(BMH), the release behavior of each preparation was evaluatedby first-order, Higuchi, Hixon-Crowell; Peppa’s and Double exponential biphasic kinetics equation. Threepreparations were all fit Double exponential biphasic kinetics equation.
Investigated the biocompatibility of BM, BH, BMH to rats’ synovium, results showed that thepreparations appeared to be generally biocompatible with synovium and hence it might be suitable for thedevelopment of treatment strategies for arthritis diseases.
In vivo FX imaging technology was employed to determine the release behavior of preparation in vivo.Encapsulated infrared dye LF NIRD-15into microspheres instead of brucine; visually investigate theretention time of drugs in the joint cavity. The release behavior of microsphere and microsphere hydrogelwere investigated, results showed that by dispersing the microspheres into hydrogel, the burst wereobviously retarded, and could delivery drug for7days above.
The pharmacokinetics of BMH in rats were preliminarily studied.Firstly using RP-HPLC establish themethod to analyse brucine in vivo.Rats were administrated with BS, BM, BMH respectively, using3p97software calculated the pharmacokinetic parameters, results indicated that BMH has a MRT1.93times ofBM,3.38times of BS, could meet the request of design of our topic.
In conclusion, the system significantly prolonged the release of brucine in cavity for more than7days,decrease the frequency of injection and improve the compliance of patients, hence it might be suitable forthe development of treatment strategies for rheumatic diseases.
马钱子碱是从马钱子中提取的有效成分。马钱子，又名番木鳖，始载于《本草纲目》，为历版《中国药典》所收载。是马钱科植物马钱（Strychnos nux_vomica L.）的干燥成熟种子，具通络止痛、散结消肿之功效，擅治风湿等疾病。临床常将其炮制后用于治疗风湿顽痹、麻木瘫痪、跌打损伤和痈疽肿痛等疾病，临床应用已有近千年的历史。马钱子碱为马钱子中的有效成分，具有良好的镇痛作用、抗炎免疫作用，抗肿瘤作用，对抗心律失常作用并可拮抗NO对软骨细胞增殖的抑制作用，能够有效促进软骨细胞增殖，从而对软骨降解为主要进程的骨关节炎修复起作用，其抗炎免疫作用、镇痛作用可以对自身免疫为特征的风湿性关节炎产生治疗效果，缓解患者疼痛。
本文采用喷雾干燥法制备马钱子碱壳聚糖微球，然后将微球分散于壳聚糖-甘油-硼砂温敏凝胶中，微球在凝胶中作为药物贮库，而利用温敏凝胶可阻滞微球的突释现象，形成了双重缓释系统，有效的延长了药物在关节腔的保留时间，使得马钱子碱更好的在关节腔中发挥药效。
本文建立了紫外分光光度法用于马钱子碱微球含量和释放度的测定，并进行了方法学考察。
采用瑞士BUCHI B290喷雾干燥仪制备壳聚糖微球，以收率和干燥程度为评价指标，首先确定了出口温度为120℃，鼓风流量为40m~3/h，泵速为20%·min~(-1)。之后，分别考察壳聚糖型号、壳聚糖浓度、戊二醛用量、马钱子碱与壳聚糖的质量比等处方因素对包封率和释放速率的影响。筛选对制剂性质影响较大的因素，利用正交设计优化处方：壳聚糖浓度为0.75%，戊二醛与壳聚糖的质量比为1:15，马钱子碱与壳聚糖的质量比为1:5，微球的包封率为98.61%±0.42%，载药量为15.57%±0.07%。利用SEM，FTIR，XRD等手段对微球进行了系统的表征，结果发现微球为表面光滑的球体，无粘连，粒径在0.9-4.5μm之间，平均粒径为2.45μm，呈正态分布。FTIR证明了马钱子碱与壳聚糖无新键生成，X射线衍射结果显示马钱子碱以无定形态存在于微球中。
壳聚糖温敏凝胶的处方为壳聚糖溶液、甘油、饱和硼砂溶液。以凝胶情况和凝胶时间为指标，考察壳聚糖浓度、壳聚糖与甘油的比例、pH值等因素对水凝胶理化性质的影响，最佳处方为浓度为2%的壳聚糖，甘油与壳聚糖比例为1:10，pH为6.7。采用试管法对马钱子碱壳聚糖微球（BM）、马钱子碱温敏凝胶（BH）、马钱子碱微球/温敏凝胶复合系统（BMH）进行体外释放研究，并对释放行为进行曲线拟合，探讨其释放机制，三种载体均属于双指数双相动力学释放模型。
考察了BM、BH、BMH对大鼠膝关节滑膜的生物相容性，结果显示BM、BH、BMH的生物相容性较好，可做为关节腔注射的载体。
采取活体成像技术观察了不同载体系统在关节腔内的释放行为，利用近红外染料NIRD-15为模型药物，大鼠关节腔注射后，可视化地观察载体对模型药物的控释能力，分别考察了微球和微球凝胶复合系统，结果显示，将微球分散于凝胶中，可以明显的延长在关节腔的驻留时间，缓释时间可达7天以上。
本实验对BMH进行了大鼠体内初步药代动力学研究，首先采用RP-HPLC我们建立了马钱子碱在大鼠血浆中的测定方法，研究了溶液剂（BS）、BM、BMH的大鼠药动学，运用3p97程序对三组的大鼠体内试验数据进行拟合和计算，结果表明，将微球分散于凝胶后MRT有了明显的延长，达到了制剂设计的目的。
综上所述，马钱子碱壳聚糖/温敏凝胶复合系统极大的延长了药物在关节腔的保留时间，可以将缓释作用达到的一周，减少了注射的次数，增加了患者的适应性，达到了课题的设计要求。
Intra-articular drug delivery system could directly delivery drug to an affected joint and offer thepossibility of reaching high drug concentrations at the site of action with limited systemic toxicity.However, depending on their chemical structure, some active compounds were rapidly cleared from thejoint, thus requiring numerous injections, which could cause infection or joint disability. To control therelease behavior for prolonged time periods, a novel biologically based drug delivery vehicle was designedfor intra-articular using microsphere/thermally responsive hydrogel combination system in this paper. Andthe brucine was the test drug.
Brucine is the active ingredient extracted from Strychnos nux-vomica L. Nux vomica, also known asstrychnine, first recorded in Compendium of Materia Medica, and recorded in each China Pharmacopoeia,is the dried ripe seeds of the Strychnos nux-vomica L. It has effects of activating meridians to stop pain andsubsiding swelling, especially for treating disease like RA and OA.It has been used clinical in traditionalChinese medicine for the treatment of rheumatoid paralysis, numbness, bruises and other diseases afterbeen concocted.Brucine is the active ingredient of nux_vomica, has the effect of analgesic,anti-inflammatory, anti-immune, anti-tumor, anti-arrhythmia, and antagonize the NO inhibitory effect onchondrocyte proliferation. The process of Osteoarthritis (OA) is mainly in the degration of chondrocytes;brucine can effectively promote the proliferation of chondrocytes, hence repair the damage cartilage. Oneof the pathogenesis of Rheumatoid Arthritis (RA) is self-immune, the effect of anti-immune and stop paincan alleviate distress of RA patients.
The system was constructed by disperse the brucine microspheres which was prepared by using aspray-drying method in a thermally responsive biopolymer hydrogel contained of chitosan-glycerol-borax.The hydrogel can block the burst release of microspheres, and the microspheres were incorporated inhydrogel as drug depot.The duplexing release system could effectively prolong the release behavior in thejoint cavity, increase the efficiency of brucine for a extended period of time.
The microspheres were prepared by Switzerland Buchi B290spray-drying apparatus.Firstlydetermines the craft factors of outlet temperature, Q-flow, pump speed. The outlet temperature was120℃,Q-flow was40m~3/h, Pump speed of20%·min~(-1). Then, investigate the influence of type of chitosan,chitosan concerntration, glutaral ratio, ratio of brucine to chitosan on the entrapment efficiency (EE) and in vitro drug release behavior of brucine micropheres. By single-factor test chose three factors, by orthogonaldesign optimization prescription, selected the best prescription for Chitosan0.75%, glutaral ratio of1:15,the ratio of brucine to chitosan is1:5.The Drug loading(DL%) was15.57%±0.07%and Entrapmentefficiency(EE%) was98.61%±0.42%.
The brucine microspheres were characterized by SEM, XRD, and FTIR, spherical as evidenced by thescanning electron microscopy (SEM) photographs. With an average size of2.45μm, in range of0.9–4.5μm,agree with normal distribution. Fourier transforms infrared (FT-IR) spectroscopy and X-ray diffraction(XRD) revealed the absence of drug-polymer interaction and amorphous nature of entrapped drug.
The thermosensitive converse hydrogel was constructed using chitosan-glycerol-borax as matrix,taking gelation time and gelation condition as index, investigate the influence of concerntraion of chitosan,ratio of chitosan to glycerol,pH on physical properties of hydrogel. The best prescription was CS2%, CS:glycerol10:1, pH6.7.
Taking tube method, investigated the in vitro drug release of brucine microsphere(BM), brucinehydrogel(BH),brucine microsphere hydrogel(BMH), the release behavior of each preparation was evaluatedby first-order, Higuchi, Hixon-Crowell; Peppa’s and Double exponential biphasic kinetics equation. Threepreparations were all fit Double exponential biphasic kinetics equation.
Investigated the biocompatibility of BM, BH, BMH to rats’ synovium, results showed that thepreparations appeared to be generally biocompatible with synovium and hence it might be suitable for thedevelopment of treatment strategies for arthritis diseases.
In vivo FX imaging technology was employed to determine the release behavior of preparation in vivo.Encapsulated infrared dye LF NIRD-15into microspheres instead of brucine; visually investigate theretention time of drugs in the joint cavity. The release behavior of microsphere and microsphere hydrogelwere investigated, results showed that by dispersing the microspheres into hydrogel, the burst wereobviously retarded, and could delivery drug for7days above.
The pharmacokinetics of BMH in rats were preliminarily studied.Firstly using RP-HPLC establish themethod to analyse brucine in vivo.Rats were administrated with BS, BM, BMH respectively, using3p97software calculated the pharmacokinetic parameters, results indicated that BMH has a MRT1.93times ofBM,3.38times of BS, could meet the request of design of our topic.
In conclusion, the system significantly prolonged the release of brucine in cavity for more than7days,decrease the frequency of injection and improve the compliance of patients, hence it might be suitable forthe development of treatment strategies for rheumatic diseases.
引文
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[23]肖新才。正相和反相温敏型凝胶微球的制备与感温性的研究[D].
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[30] Zhang WD,The experimental research on therapeutic effect of Ostarthritis in rabbits withintra-articular injection collagenase by the compound of triptolide and hyaluronic acid[D]. Third militarymedical university(第三军医大学).2003,13
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