【作者】 万发里；

【导师】 邓树海；

【作者基本信息】 山东大学 ， 药剂学， 2005， 硕士

【摘要】 目的:探索白血病治疗药物骨髓靶向给药治疗的新思路、新方法和新技术。白血病是一种造血系统的恶性肿瘤,俗称“血癌”,其特征表现为正常造血细胞显著减少,而在骨髓及其他造血组织中有广泛的白血病细胞异常增生,并浸润其他组织器官,导致正常造血功能衰竭。全反式维甲酸以往主要用于治疗各种类型的皮肤病,近年来被广泛用于急性早幼粒细胞白血病(APL)的诱导分化、凋亡治疗,具有完全缓解率高(达90%以上)、弥散性血管内凝血发生率低及无骨髓抑制副作用等优点。以生物降解型高分子材料聚氰基丙烯酸正丁酯为载体,选择全反式维甲酸为模型药物,采用现代靶向制剂技术,研制骨髓靶向性全反式维甲酸聚氰基丙烯酸正丁酯毫微粒(ATRA-PBCA-NP)。对毫微粒进行外观形态学、粒径、粒度分布研究;建立质量控制方法与标准,进行毫微粒包封率、载药量、体外释放度、稳定性等实验研究;研究骨髓靶向性全反式维甲酸聚氰基丙烯酸正丁酯毫微粒动物体内药物动力学与组织分布学,进行靶向性评价;探讨骨髓靶向给药治疗机理,力求对白血病药物治疗局限的突破,为骨髓靶向给药提供科学的理论依据。 方法:采用乳化聚合法制备ATRA-PBCA-NP,根据单因素试验,采用均匀实验设计优化、筛选处方与制备工艺;采用电子透射显微镜观察毫微粒的外观形态,激光散射粒度分析仪测定毫微粒的粒径及其粒度分布,电视显微电泳仪测定毫微粒表面Zeta电位;低温超速离心法测定毫微粒的包封率、载药量;动态膜透析法考察毫微粒体外释药特性,研究体外释药机理和释药途径,并进行初步稳定性研究;以全反式维甲酸注射液为参比标准进行ATRA-PBCA-NP家兔体内药物动力学、大鼠体内组织分布学研究更多还原

【Abstract】 Objective: To develop a new idea, new method and new technique for bone marrow targeted drug delivery system (BMTDDS). Leucocythemia was a kind of serious malignant tumors of hemopoietic system, commonly called "blood cancer", the features of which were a significant reduction of normal hematopoietic cell, comprehensive of proliferation of leukemia cell in bone marrow and other hematopoiesis tissue, and infiltrating other tissues, leading to exhaustion of normal function of hematopoiesis. Utilized extensively to cure of apoptosis of APL now, ATRA (All-trans-retinoic acid), which is mainly used to cure kinds of dermatosis formerly, has the virtues as high abs remission rate (above 90 percent), low incidence rate of dispersed intravascular coagulation and having no side effect of bone marrow depression and so on. Bone marrow targeting ATRA-PBCA-NP was prepared by modern target-oriented drug delivery technique, in which the biodegradable polybutyl cyanoacrylate (PBCA) was used as carrier and ATRA was selected as a model drug. The surface morphology, diameter and size distribution of nanoparticle were studied. To establish the quality control standard for ATRA-PBCA-NP, the encapsulation efficiency, drug loading capacity, in vitro release and stability were also examined. At the same time, pharmacokinetics and tissue distribution in rabbits and mice were investigated to evaluate its virtues of bone marrow targeting. The mechanisms of BMTDDS were discussed to break through the limitation of treatments for bone marrow diseases and provide scientific theoretical basis for BMTDDS.Methods: The emulsification evaporation method was used to prepare ATRA-PBCA-NP. According to the results of the effects of different variables,the uniform experimental design was applied to optimize the technology of preparation. The properties of nanoparticle as diameter and size distribution were observed by transmission electric microscope (TEM) and laser dispersive analyzing apparatus for granularity. Micro-electrophoresis apparatus was used to measure Zeta electric potential. The encapsulation efficiency, drug loading capacity, drug concentration in blood and tissues were assayed by ultra centrifugation at low temperature. In vitro release of ATRA from nanoparticles was performed by dialysis film method. The pharmacokinetics in rabbits and tissue distribution in mice of the ATRA-PBCA-NP and ATRA injection were investigated.Results: The diameter of the nanoparticles was remarkably determined by the quantity of BCA, PluronicF-127, Dextran-70, pH and stirring duration ,et al; The optimization is as follows: BCA was 0.06ml; PluronicF-127 was 75 mg; Dextran-70 was lOOmg; pH was 1.4; stirring duration was 5h. Three batches of ATRA-PBCA-NP were prepared using the optimized formulation. From the transmission electron microscope observation, the nanoparticles were spherically shaped and ranged in size between 15 and lOOnm. The particle size distribution was 47.7nm. UV spectrophotometry method was established as the quality controlled standard, there was a good linearity of the drug concentration within the range of 0.7-6.2 u g/ml. The linear equation was y=0.1450x+0.0037 (r=0.9999) . The average recovery was 99.3% and the RSD (intra- and inter-day) were less than 2%. The drug loading and incorporationefficiency were 16.12% and 83.67% respectively.The in vitro release behavior of ATRA from nanoparticles could be described by double phase dynamic model and could be expressed by the following equation: 100-R = 42.34-e^*9’+ 58.69?e"011’(ro =0.9961, r, =0.9998). The release mechanism was diffusion. The in vitro release profiles of ATRA stuff could be described by first order dynamic model and could be expressed by the following equation: ln(l00-R) = -2.3532/ + 4.8444 (r= 0.9946) .The freeze-dry injection of ATRA-PBCA-NP was prepared. During storedat 40°C for 3 months, the properties and drug content did not alter. The stability was distinctly affected by light irradiation in 10 days. 60Co radiation had no effect on surface morphology and content of ATRA.HPLC method was established to investigate the drug content and drug distribution in rabbits and mice. The recovery of drug in blood of rabbits was more than 95% and the RSD (intra- and inter-day) were less than 2%. The recovery of drug in tissues of mice was more than 90% and the RSD (intra-and inter-day) were less than 6%. There was a good linearity within the rage of 2~1000ng/ml.The concentration-time courses of ATRA conformed to a single compartment model. The half-life was 0.916h.The concentration-time courses of ATRA-PBCA-NP conformed to a double compartment model. The half-life was 6.636h, which was prolonged remarkably compared with ATRA injection The result indicated that the ATRA-PBCA-NP had a good sustained release efficacy. Compared with ATRA injection, the relative distribution percentage of ATRA-PBCA-NP in bone marrow enhanced from 2.27% to 8.14%.Conclusion: ATRA has remarkable effects in curing of APL, but it has some disadvantages such as first pass effect, short half-life (ti/2=l.l~l .8h) , side effect and drug tolerance. Its physic-chemical characteristic was not stable in the condition of heat or light. The ATRA-PBCA-NP may increase the drug concentration in bone marrow. It also can decrease the dosage, the side effects and improve the therapeutic index (TI). The results of in vitro release and pharmacokinetics in vivo showed that the ATRA-PBCA-NP had a good sustained release efficacy and the half-life of ATRA was prolonged from 0.916h to 6.636h.The tissue distribution results of ATRA-PBCA-NP in rats indicated that the ATRA-PBCA-NP had a remarkable bone marrow targeting efficacy.更多还原