【作者】 张维芬；

【导师】 陈西广；

【作者基本信息】 中国海洋大学 ， 生态学， 2007， 博士

【摘要】 支气管哮喘是一种呼吸系统严重危害人民健康的常见病,在过去的20年中,其发病率和病死率在世界范围内呈上升趋势,这不仅引起了呼吸学界的普遍关注,也得到了WHO和许多国家政府的关注,如何有效控制哮喘成为各国的公共卫生问题。氨茶碱是治疗支气管哮喘的常用药物,具有松弛呼吸道平滑肌的作用,尚有一定的抗炎和免疫调节作用。由于治疗指数小(10～20μg/ml),普通片的半衰期短(约6 h),需频繁给药才能达到治疗浓度。口服给药后容易出现血药浓度峰谷现象及引起对胃肠道和心脏的不良反应。制成缓控释制剂虽然可以提供较平稳的血药浓度,但不能避免长期口服的副作用。肺部吸入缓释制剂药物可维持局部治疗浓度,减少全身副作用,延长药物作用时间,提高患者的顺应性。因此,运用生物降解材料及控释技术与呼吸临床治疗技术的紧密结合,探索一种新的控释技术和剂型,成为目前研究的热点。壳聚糖是一种聚阳离子多糖,因具有良好的物理化学特性、生物相容性和生物可降解性,来源丰富,无毒,成为药剂学研究的热点而被广泛应用于药物控释体系中。壳聚糖作为药物载体可以控制药物释放,提高难溶性药物的溶出。已有大量文献报道用壳聚糖作为缓释药物载体如珠、膜、海绵、凝胶、微球等递药系统。壳聚糖阳离子聚合物的性质能与粘膜的负电荷相互作用,提高粘附性和降低清除率。最近研究显示,壳聚糖能打开细胞连接,提高生物利用度5～10倍,而且对粘膜功能的影响是可逆的,尤其适用于肺部给药系统。本文采用壳聚糖和β-环糊精为原料,以氨茶碱为模型药物,采用喷雾干燥法制备不同药物/载体比例的茶碱/壳聚糖/β-环糊精微球,旨在研究其理化性质及粉末特性,生物相容性、降解性,并对其药效进行评价。利用喷雾干燥法成功地制备了三种不同药物/载体比例的茶碱/壳聚糖/β-环糊精微球,通过扫描电镜、激光粒度测定仪、红外光谱仪、紫外分光光度计对微球的物理化学特性进行观察。结果微球外观圆整,表面光滑或有皱折,粒径分布范围窄,符合肺部粒子吸入的要求。微球具有较高的产率、载药量和包封率;红外光谱结果显示茶碱与壳聚糖和β-环糊精高分子间发生氢键反应;体外释放结果证实茶碱/壳聚糖/β-环糊精微球具有缓释作用,并且与释放介质、载药量、溶胀性及药物性质有关,茶碱/壳聚糖/β-环糊精微球B具有良好的缓释效果。稳定性实验表明,在实验条件下茶碱/壳聚糖/β-环糊精微球载药量和形态无明显的变化,药物较易透过粘膜。茶碱/壳聚糖/β-环糊精微球B吸湿速率为0.3071 mg/h,固密度为0.44 g/cm3。为克服壳聚糖酸溶性的不足,利用喷雾干燥法、以壳聚糖衍生物羧甲基壳聚糖为主要载体,成功地制备了三种不同药物/载体比例的茶碱/羧甲基壳聚糖/β-环糊精微球,并对其物理化学特性进行了研究。结果得到的微球外观圆整,表面光滑或有皱折,理论空气动力学直径小5μm。茶碱/羧甲基壳聚糖/β-环糊精微球微球具有高的较高的产率、载药量和包封率;红外光谱结果显示茶碱与羧甲基壳聚糖高分子间发生了氢键反应。茶碱/羧甲基壳聚糖/β-环糊精微球在实验条件下保存稳定,其载药量和形态无明显的变化。微球具有较高的药物透过粘膜透过率(>14.7%),较低的固密度(<0.44 g/cm3),吸湿速率在0.0538～0.1864 mg/h之间,因而具有良好的粉末特性;体外释放结果证实茶碱/羧甲基壳聚糖/β-环糊精微球在pH6.8介质中的释放快于pH1.2,并且与载药量有关,微球F(pH1.2)具有良好的缓释效果。通过细胞毒性实验、溶血实验、微核实验、肺泡灌洗液乳酸脱氢酶及肌肉植入实验等对茶碱/壳聚糖微球、茶碱/壳聚糖/β-环糊精及茶碱/羧甲基壳聚糖/β-环糊精微球等进行了一系列生物学性能的表征。结果表明,茶碱/壳聚糖/β-环糊精及茶碱/羧甲基壳聚糖/β-环糊精微球无毒,不引起溶血,试验材料组微核率为0.99‰,小于3‰,骨髓微核实验呈阴性。茶碱/壳聚糖、茶碱/壳聚糖/β-环糊精微球及茶碱/羧甲基壳聚糖/β-环糊精微球的纤毛持续运动时间分别为493.00、512.33和514.33 min,纤毛运动频率为92.4%、96.0%和96.4%,对蟾蜍口腔上腭纤毛无明显毒性;气管给药后总蛋白含量增加,LDH活性与正常组无差异显著性;肌肉埋植实验证明,肌肉植入后各个时期伤口无红肿、化脓、坏死等现象,对造血组织和血红蛋白的合成没有影响,对肝肾功能无损伤;病理结果显示埋植组织的反应与缝合丝线相比无差别,具有良好生物相容性,能够作为缓释药物的安全载体用于肺部给药。微球的体外降解结果表明,微球具有良好的降解性,体外酶解快于体外水解。微球A的降解快于微球B、C、和空白微球;电镜结果表明体外酶解和水解至12周,微球形貌完全改变,表面不再光滑且粘连成一体,有微孔出现;体内降解至8周,茶碱/壳聚糖/β-环糊精微球多数已降解成为不规则、片状、多孔结构,直径小于5μm,茶碱/壳聚糖/β-环糊精微球和壳聚糖/β-环糊精微球体内降解无明显差异。通过建立大鼠哮喘病模型、高血压及哮喘合并高血压大鼠模型,开展茶碱/壳聚糖/β-环糊精微球疗效的实验研究,并与茶碱溶液雾化的疗效进行比较。通过全自动生化分析仪测定BALF中LDH、ALT、AST和Cr,计数BALF中白细胞计数及分类、全自动血液分析仪测定血液中白细胞计数及分类,并HE染色观察肺组织水肿及Eos浸润情况,探讨茶碱/壳聚糖/β-环糊精微球对气道炎症、LDH及血压、肝肾功能的影响。结果显示,哮喘模型组与正常对照组相比,BALF中白细胞总数、Eos计数均明显增加(p < 0.05),血液中白细胞总数、Eos计数也具有显著差异(p < 0.05);地塞米松治疗组与哮喘模型组相比,具有显著差异(p < 0.05),BALF和血液中白细胞总数、中性粒细胞、Eos均明显下降;茶碱溶液雾化治疗组、茶碱/壳聚糖/β-环糊精微球肺部给药治疗组、茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘口服茶碱/壳聚糖/β-环糊精微球口服治疗组经治疗后BALF和血液中白细胞总数、Eos数与哮喘模型组相比明显下降(p <0.05);而茶碱/壳聚糖/β-环糊精微球肺部给药治疗组、茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘茶碱/壳聚糖/β-环糊精微球口服治疗组相互比较,差异无显著性(p﹥0.05)。肺组织病理可见哮喘组大鼠气道粘膜、粘膜下组织大量Eos浸润,粘膜水肿,腔内可见脱落的上皮细胞、渗出的炎性细胞,而正常组大鼠无上述改变;经地塞米松治疗后病理可见粘膜水肿减轻,炎性细胞浸润明显减少,肺组织结构基本正常;茶碱溶液雾化治疗组病理可见肺泡间隔断裂,水肿,细支气管周围有少量炎性细胞浸润,肺组织结构基本正常,证明茶碱对中支气管哮喘有明显的治疗作用;茶碱/壳聚糖/β-环糊精微球肺部给药治疗组有少量炎性细胞浸润,可见Eos,肺组织结构基本正常;茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘口服茶碱/壳聚糖/β-环糊精微球口服治疗组病理表现与哮喘组比较炎症明显减轻,细支气管周围有少量炎性细胞浸润,粘膜断裂,肺间质水肿,肺组织结构基本正常。茶碱/壳聚糖/β-环糊精微球肺部给药对BALF中LDH、蛋白含量及肝、肾功能皆无影响,口服也具有同样的规律;正常大鼠诱发哮喘后没有引起血压升高,高血压大鼠诱发哮喘亦未致血压升高;肺部和口服给药后对血压皆亦没有影响,提示肺部给药有可能在支气管哮喘的治疗中发挥作用。更多还原

【Abstract】 Asthma is a serious commonly chronic airway disease which do harm to health of people, in the past two decades, the prevalence and the mortality rates of asthma are increasing in all countries throughout the world, which has been paid much more attention not only by the specialist of respiratory, but also by the WHO and the government of many countries, so how to manage asthma effectively has become a public health problem. Theophylline (TH) is one of the most important drugs for treatment of asthma and recent studies indicate that it has anti-inflammatory effects. Thus, great attentions have been shown due to the new effect on treatment of asthma. Unfortunately, since its short half-life (6 h), conventional dosage forms have to be administered 3-4 times a day in order to avoid large fluctuations in plasma concentrations, which lead to poor patient compliance. The large fluctuations of plasma TH concentration lead to gastrointestinal and cardiovascular adverse effects. Moreover, its therapeutic index is narrow (10-20μg/ml). The therapeutic effects of TH require plasma concentration of TH at least 5-10μg/ml and toxic effects are frequent above 20μg/ml. Sustained release dosage forms can overcome these drawbacks, but long-term oral therapy can’t avoid the side effects of gastrointestinal, cardiovascular and central nervous system’s disturbances because its elimination half-life varies widely between patients. Hence a long-term TH therapy also leads to serious management problems in asthmatics. Despite the occurrence of numerous new sustained release products of TH, an optimal therapeutic use continues to evolve. Inhalation of aerosolized sustaining release of drugs in the lung can prolong drug action, reduce side effects and improve patient compliance. Therefore new sustained microparticles have been paid much attention by applying biomaterial and the techniques of pharmaceutics and clinical respiratory. Chitosan (CTS) is a cationic natural copolymer of glucosamine, which is abundant biodegradable biomaterial. It has been widely used in several pharmaceutical formulations as sustained release carrier systems such as beads, gels, films, sponges and microspheres for its many unique properties such as low toxicity, biocompatibility, biodegradability and good physicochemical properties. Furthermore, CTS has been used as a drug carrier to attain the desirable drug release profile and enhance dissolution rate of low water soluble drugs. Recently, CTS has become a good candidate for using in pulmonary delivery because its effect on the lung epithelium is reversable. It can bind with mucosal surfaces due to its cationic nature, which leads to bioadhesion and a reduced mucociliary clearance. In addition, CTS has another dramatic effect in terms of improving drug absorption (5～10 fold) by opening the intercellular tight junctions of the lung epithelium. In this paper, TH is used as model drug, while CTS andβ-cyclodextrin (β-CD) are used as drug carriers. The main aim is to study the characterization of TH/CTS/β-CD micropheres with different formulations made by spray drying as pulmonary sustained drug delivery systems. The microspheres are characterized by a series of physicochemical and powder properties, biocompability, degradability and pharmacodynamic effect.The TH/CTS/β-CD microspheres are prepared by spray drying method. The characteristics of TH/CTS/β-CD microspheres are carried out by Scanning Electron Microscopy (SEM), Laser diffraction, Fourier Transform Infrared Spectroscope (FT-IR) and UV-Vis Spectrophotometer. The result of SEM shows spherical microspheres with smooth or slightly wrinkled surfaces. The microspheres B have a narrower particle size distribution with the diameter between l and 10μm. FT-IR spectroscopy reveals that hydrogen bonds are formed between TH and CTS orβ-CD. The drug entrapments significantly increase from 13.33% to 35.70% with an increase of the ratio of drug/polymer. The encapsulation efficiencies are from 85.16% to 91.40%. The in vitro release of TH from microspheres is slower at pH 6.8 than that at pH 1.2 and also relate to the swelling ability, especially to the ratio of drug/polymer. The microspheres B have a prolonged release pattern with the release rate of 60.20% (pH 6.8) within 8 h. The results of permeation test reveale that the microspheres are easy to penetrate the membrane.The microspheres are stability under the experimental condition. The microspheres B have the low rate of moisture uptake (0.3071 mg/h) and low tap density (0.44 g/cm3).In order to overcome the disadvantage of acid soluble of chitosan, three kinds of carboxymethyl chitosan(CMCS)/β-CD microspheres loaded TH are successfully made by spray drying method and the characteristics are investigated. The microspheres obtained after spray drying are found to be spherical shaped with smooth or wrinkled surface. The mean particle size is between 3.39 and 6.06μm. The micropheres demonstrate high product yield (43.7-50.2%), high drug loading (13.7-38.1%), and high encapsulation efficiency (86.9-92.8%). The results of FT-IR indicate that there are interactions of TH with CMCS matrix. The TH/CMCS/β-CD microspheres are stability under the experimental condition which have the low rate of moisture uptake (0.0538～0.1864 mg/h) and tap density (<0.44 g/cm3). The in vitro release of TH from microspheres is slower at pH 1.2 than that at pH 6.8 and also related to the ratio of drug/polymer. The microsphere F has a prolonged release pattern (pH1.2).To study the biocompability of the microspheres, the tests such as the cell toxicity experiment, hemolytic test, acute cell toxicity experiment (LDH and protein in BALF), micronucleus test and muscle implantation test have been investigated. The results demonstrate that the microspheres have no toxicity and don’t cause hemolysis. The micronucleus ratio of the microspheres is 0.99‰, being less than 3‰. The result of medullary micronucleus test is reported negative. The ciliary movement time of TH/CTS microspheres, TH/CTS/β-CD microspheres, and TH/CMCS/β-CD microspheres are 493.00、512.33 and 514.33 min,and the ciliary beat frequencies are 92.4%、96.0% and 96.4%.This result shows that the microspheres have no significant toxicity to palate mucosa. This result suggests that the microspheres can effectively reduce the ciliotoxicity and possess better adaptability. The total contents of protein and LDH in BALF of pulmonary delivery group are not significantly different from that of normal group. The wounds are free from suppuration and necrosis after muscle implantation in all periods. The results of implantation show that the microspheres have no effect on the hematopoisis, hemoglobin and have no toxicity in the liver and kidney. The inflammations of muscle tissue are not significantly different from that of operative suture, therefore, the TH/CTS/β-CD microspheres possess good biocompability and can be applied as pulmonary sustained release systems.In vitro degradation was performed in PBS buffer solution and with enzyme solution. Microspheres are found to be degraded by enzymatic hydrolysis and buffer solution, which indicate that enzymatic hydrolysis is faster than that in buffer solution. The degradation rate of MS A is faster than that of other microspheres (MS B、MS C and blank microspheres). Morphology of SEM of microparticles with PBS and enzymic solution develope rough surfaces and become irregularly shaped and porous after 12 weeks. In addition, microparticle aggregation is also observed both in PBS and enzyme solution. Microparticles degrade faster in vivo and show similar morphology after 8 weeks, which the TH/CTS/β-CD microspheres also degrade into irregular, sheet, porous shape, and the diameters are smaller than 5μm. The degradation rate of TH/CTS/β-CD microspheres has no different from that of the TH/CTS microspheres.The objective of this experiment is to study the influence of TH/CTS/β-CD microspheres on the allergen induced asthmatic rat model and the asthma-hypertension rat model, which also compare with the effect of TH solution. The quantity of WBC and its classification in blood is detected by automatic blood analyzer and in BALF is smeared sedimentation of the cell before stained with Wright-Giemsa and count differential leukocyte number. The LDH in BALF, ALT、AST and Cr in blood is detected by automatic clinical analyzer. The contents of protein in BALF are measured by protein quantification kit-rapid. To investigate the change of inflammation in the lung tissue and the blood pressure in asthma–hypertension rat model, the middle lobe of right lung is fixed in 10% formalin, then stained with hematoxylin-eosin and the blood pressure is measured. The total of WBC and Eos in blood and BALF of asthmatic group are higher than that of normal group (p < 0.05). Compared with the asthmatic group, the total of WBC and Eos in blood and BALF from DXM group is significantly different from asthmatic group (p < 0.05), the decrease tendency is found. The total of WBC and Eos in blood and BALF results of TH solution group, the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po) are all significantly different from asthmatic group (p < 0.05), which are lower than that of asthmatic group, furthermore, there are no significant difference between these groups (p>0.05). The result of histological changes in asthmatic model reveal obvious allergic inflammation such as dissepiments of bronchioles and small vessels becoming thicker, epithelial damage and the tissue edema compared to normal group. The lung tissue of DXM group seem normal except a few inflammatory cell infiltration. The lung tissue of TH solution group seem normal too except a few inflammatory cell and Eos infiltration. In the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po), the lung tissue also seem normal, but still a few inflammatory cell infiltration and tissue edema, which are signicantly different from asthmatic group. The routes of pulmonary delivery and po have no damages to the liver and kindney, and also have no effect on the blood pressure. There is no effect on the LDH and contents of protein in BALF. In short, the TH/CTS/β-CD microspheres can significantly reduce the quantity of Eos and the total of inflammatory cell in blood and in BALF and can inhibit airway inflammation, whose efficiency is similar to dexamethasone. This study indicates that the TH/CTS/β-CD theophylline/chitosan/β-cyclodextrin microspheres applied as pulmonary delivery is feasible.更多还原