联苯双酯论文：联苯双酯 纳米脂质载体 组织分布 药物动力学 药效学

联苯双酯论文：联苯双酯 纳米脂质载体 组织分布 药物动力学 药效学 联苯双酯论文:联苯双酯 纳米脂质载体 组织分布 药物动力学 药效学 【中文摘要】研究制备一种新型的药物载体—纳米脂质载体(NLC),以联苯双酯为模型药物,以期提高药物的包封率、载药量,改变其体内分布。本课题采用现代靶向制剂技术,以联苯双酯(Bifendate, DDB)为模型药物,以具有良好生物相容性和生物可降解性的单硬脂酸甘油酯、硬脂酸和中链脂肪酸甘油酯混合脂质为载体材料,采用乳化蒸发—低温固化法制备联苯双酯纳米脂质载体(Bifendate-loaded nanostructured lipid carriers, DDB-NLC)。实验以包封率为主要考察指标,通过均匀法对处方工艺进行优化,确定最佳处方工艺,并系统的研究了DDB-NLC冻干粉针剂的制备、处方和工艺,考察了制剂的体外释放特点以及体内分布、药动学和体外药效学特征,以期通过肝靶向分布达到提高DDB治疗肝脏疾病的效果,降低其毒副作用的。通过均匀设计优化的最佳处方工艺为药脂比为3:20,液态脂质,固态脂质比为1:5,表面活性剂用量为450 mg,搅拌速度800 rmp,乳化温度为75?。优化工艺操作简单,所制备的DDB-NLC包封率稳定,平均包封率(91.52?1.76)%,载药量为(12.07?0.17)%。透射电镜下观察所得纳米粒为类球形,纳米粒彼此不粘连,粒径分布的范围较窄,平均粒径为277.0nm, Zeta电位为-21.91mV, pH值为5.95左右。为提高所制备DDB-NLC的储存稳定性,本文进一步对所制得的DDB-NLC 进行了冻干处理,以外观、色泽、再分散性为评价指标,将DDB-NLC混悬液制成冻干粉针剂,确定其冻干工艺为:在优化工艺下制备的联苯双酯纳米脂质载体中加入5%甘露醇作为支架剂,振摇使溶解,分装于10 ml西林瓶中,置-80?的超低温冰箱中冷冻24 h,取出,然后迅速移入冷冻干燥机中,-40?、0.10mbar条件下冻干48 h,即可得DDB-NLC的冻干品。质量评价结果表明冻干过程对药物的粒径、包封率、载药量及Zeta电位影响较小。对冻干品的DSC和X-射线衍射分析结果表明,制成纳米粒后药物已被载体包裹或吸附,不再是单纯的混合,药物不再以晶体结构存在,即DDB-NLC中形成了新的物相。用透析袋法对DDB-NLC冻干粉针剂的体外释药特性进行了研究,结果表明DDB-NLC的体外释药具有缓释制剂特征,可用一级动力学方程拟合,方程为Ln(100-Q)=-0.1951t +4.6128, r=0.9985.以DDB-NLC的外观、再分散性、含量和包封率等为指标,评价了DDB-NLC冻干针剂的初步稳定性,结果显示DDB-NLC冻干粉在室温(25?左右)、冷藏条件(4?左右)条件下放置三个月,其物理化学稳定性良好。本文采用HPLC法,分别测定了小鼠尾静脉注射DDB-Sol和DDB-NLC后在体内的组织分布情况,结果表明联苯双酯纳米脂质载体能够使DDB靶向浓集于吞噬细胞丰富的肝中,并明显延长DDB在肝中的作用时间,从而有利于提高DDB的治疗效果,具有重要临床意义。在药物动力学试验中,结果表明DDB-Sol与DDB-NLC的血药浓度-时间曲线有显著不同。以三室模型描述药物在体内的动态过程最合适,静脉注射DDB-Sol后,小鼠体内药物动力学方程为:C=0.79e-0.12t+142.6e-20.19t+0.164e-0.87t, 主要药动学参数为T1/2α=0.034h,T1/2β=0.793h,MRT=1.573h,清除 率CLs= 5.344 mg/kg/h/(μg/ml), AUC为4.678h?μg/ml,而静脉 注射DDB-NLC冻干粉针剂后,小鼠体内药物动力学有明显改变,其药 物动力学方程为: C=0.007e-0.099t+9.91e-7.66t+0.178e-0.10t,T1/2α、T1/2β均显 著延长,分别为T1/2α=0.09h, T1/2β=6.846h,MRT增加为4.114h, 清除率降低,CLS=2.836 mg/kg/h/(μg/ml), AUC则增至9.109h?μ g/ml。分析结果可知,DDB-NLC能够显著延长DDB在小鼠体内的半衰 期和体内滞留时间,AUC显著增高,清除率明显降低,说明将DDB制成 DDB-NLC后,有助于提高药物的生物利用度,并发挥长效作用。以CC14 损伤人肝细胞为模型,采用MTT法对细胞活性和增殖进行检测,研究 联苯双酯不同浓度对抗肝损伤的作用。结果发现,联苯双酯纳米脂质 载体能够增强CCl4肝损伤模型肝细胞的增殖能力。以联苯双酯为模 型药物制备纳米脂质载体的研究目前国内外尚未见报道,本文的研究 成果为难溶性药物注射剂的开发提供了思路,并对治疗肝病药物联苯 双酯注射剂的临床开发应用有着重要的意义。 【英文摘要】Bifendate is widely used in China for the treatment of chronic hepatitis by lowering alanine transaminase (ALT) in patients. Currently, there are only oral preparations on market because DDB is insoluble in water, which results in low bioavailability. However, for acute hepatitis patients and thosewith decreased liver functions after surgical operations parenteral dosages would provide the best benefit for them.In order to prepare the DDB solution for intravenous injection, several groups had tried to improve the solubility of DDB in water. In the present work, based on the solubility properties of DDB, which show high or sufficient solubility in liquid oils but poor solubility in solid lipids, DDB-loaded NLC was prepared by the emulsion-evaporation technique using glycerol monostearate (GMS) and stearic acid (SA) as solid lipids with medium chain triglycerides (MCT) as liquid lipid.As carrier material, lipids have been widely developed as the carrier of nanoparticles due to its desirable biocompatible and biodegradable properties as well as minimally toxicity. Therefore, in our investigation, bifendate was taken as the model drug, mixed lipids of glyceryl monostearate, stearic acid and medium chain triglycerides as the drug carrier, bifendate-loaded nanostructured lipid carriers (DDB-NIC) were prepared by the method of emulsion evaporation at a high temperature and solidification at a low temperature. The formula and technology for preparing DDB-NLC colloidal dispersion were optimized by the uniform design method, with the entrapment efficiency as the criterion, and its lyophilization injection was also systematically studied. The release kinetics in vitro, characteristics of distribution and pharmacokinetics in vivo were examined. DDB-NLC were expected to reach the aim of enhancing its therapeutic efficacy of hepatitis through hepatic targeting and decreasing its side effects.The optimized parameters were as follows:The ratio of drug to lipid materials was 3:20, the ratio of liquid lipid to solid lipid materials was 1:4, the amount of surfactants was 450 mg, the mount of Pluronic F68 was 3%, the stirring rate was 800rmp and emulsifying temperature was 75?.The entrapment efficiency, and actual drug loading of the nanoparticles were (91.52?1.76)% and (12.07?0.17)%, respectively. Through the observation of transmission electron microscope, we found that the nanoparticles prepared were sphere-like and regular. The size distribution of the nanoparticles was narrow, with the average particle size of 277.0 nm.The Zeta potential was-21.91 mV and the pH was 5.95.To increase the shelf life of the DDB-NLC and to further study the dissolution behavior and physical state of the formulation, the DDB-NLC dispersions were freeze-dried. Based on the appearance, color and redispersibility of the nanoparticles, we studied the major factors of the freeze-dried procedure. The final established freeze-dried method was as follows:First the DDB-NLC dispersions were poured into glass flasks and pre-frozen using an ultra-cold freezer at -80?for 24h; then the samples were freeze-dried using a lyophilizer at -40?and at 0.10 mbar of pressure for 48h to yield dry powder. The comparison between the nanoparticles before and after freeze-dried procedure showed that lyopyilization had little effects on the particle size, entrapment efficiency, drug-loading, Zeta potential and pH of the nanoparticles. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that DDB existed in the form of amorphous in the nanoparticles. The in vitro release properties of the freeze-dried DDB-NLC were evaluated by ultrafiltration-centrifugation. The results showed that the drug release pattern was in accord with two phases kinetics equation,having sustained-release character:Ln(100-Q)=-0.1951 t+4.6128, r=0.9985. The initiatory stability research showed that the freeze-dried Sily-NLC could be stored at 25?or 4?for 3 months.We utilized the HPLC method to determine and compare the content of bifendate in different tissues of mice following the tail intravenous injection of DDB-Sol and DDB-NLC. The results showed that being packed in nanoparticles, the distribution of silybin in liver were all enhanced. Moreover, the mean retention time in the tissue was also prolonged. Therefore, nanostructured lipid carriers were helpful for bifendate to improve its therapertic efficiency and achieve a long-term effect.The results of pharmaceutics showed that, the encapsulation of bifendate in nanostructured lipid carriers was remarkably effective in prolonging its blood circulation time. Pharmacokinetic parameters were obtained using the DAS 2.0. The major calculated parameters of the DDB-Sol group were as follows:T1/2z=5.92h, MRT=1.5730h, AUC=4.678 h?μg/ml, CLz=5.344 mg/kg/h/(μg/ml). Meanwhile, the major calculated parameters of the DDB-NLC group were as follows:T1/2Z=6.973 h, MRT=4.114h, AUC=9.109 h?μg/ml, CLz=2.836 mg/kg/h/(μg/ml). The results indicated that nanostructured lipid carriers could be a potential carrier for bifendate to obtain prolonged elimination half life as well as retention time.MTT results showed that DDB-NLC could effectively protect the primary cultured human hepatocytes against CCl4 induced injury as compared with DDB-Sol.It is the first report on the preparation of bifendate-loaded nanostructured lipid carriers, the results of our studies contributes to the development of injection of poorly soluble drugs, and plays a very important role in clinical application of bifendate. 【关键词】联苯双酯 纳米脂质载体 组织分布 药物动力学 药效学 【采买全文】 1.3.9.9.38.8.4.8 1.3.8.1.13.7.2.1 同时提供论文写作定制和论文发表服务.保过包发. 【说明】本文仅为中国学术文献总库合作提供，无涉版权。作者如有异议请与总库或学校联系。 【英文关键词】Bifendate Nanostructured lipid carriers Tissue distribution Pharmacokinetics Pharmacodynamics 【目录】联苯双酯纳米脂质载体给药系统的实验研究 中文摘要 13-16 ABSTRACT 16-18 符号说明 19-20 前言 20-29 第一部分 联苯双酯纳米脂质载体处方和制备工艺的研究 29-54 一、材料与仪器 29-30 1. 实验材料 29 2. 实验仪器 29-30 二、实验方法与结果 30-51 1. 联苯双酯含量测定方法的建立 30-35 1.1 标准溶液的制备 30 1.2 检测波长的确定 30-31 1.3 标准曲线的绘制 31-32 1.4 精密度试验 32-33 1.5 重复性试验 33 1.6 回收率试验 33-35 1.7 纳米脂质载体中联苯双酯的含量测定 35 2. 包封率测定方法的建立 35-37 2.1 回收率试验 35-37 2.2 包封率及载药量的测定 37 3. 联苯双酯纳米脂质载体制备工艺考察 37-51 3.1 单因素考察联苯双酯纳米脂质载体的处方与工艺 37-46 3.1.1 表面活性剂种类和用量的选择 37-39 3.1.1.1 表面活性剂种类的选择 37-38 3.1.1.2 表面活性剂用量的选择 38-39 3.1.2 有机溶剂的选择 39 3.1.3 脂质材料的选择 39-40 3.1.4 两种固态脂质比例的选择 40-41 3.1.5 药物/脂质比例的选择 41 3.1.6 液态脂质占总脂质比例的选择 41-42 3.1.7 助表面活性剂卵磷脂用量的选择 42 3.1.8 相体积比的选择 42-43 3.1.9 乳化温度的选择 43 3.1.10 乳化时间的选择 43-44 3.1.11 固化时间的选择 44 3.1.12 搅拌速度的选择 44-45 3.1.13 纳米乳液/冷却水比例的选择 45-46 3.2 均匀设计优化DDB—NLC处方与工艺 46-48 3.2.1 实验因素和水平的确定及实验表的排列 46-47 3.2.2 结果处理与评价 47 3.2.3 优化工艺 47-48 3.3 DDB-NLC胶体溶液优化处方的验证及质量评价 48-51 3.3.1 包封率和载药量 48 3.3.2 DDB-NLC的表面形态观察 48-49 3.3.3 DDB-NLC的粒径分布 49-50 3.3.4 Zeta电位的测定 50 3.3.5 pH值测定 50-51 3.3.6. DDB-NLC胶体溶液初步稳定性考察 51 三、讨论 51-54 第二部分 联苯双酯纳米脂质载体冻干粉针剂的研究 54-71 一、材料与仪器 54 1. 实验材料 54 2. 实验仪器 54 二、实验方法与结果 54-67 1. 冻干工艺的确定 54-56 1.1 冻干保护剂的评价方法 55 1.2 冻干保护剂种类的选择 55-56 1.3 冻干保护剂 用量的选择 56 1.4 冻干工艺 56 2. DDB-NLC冻干针剂的质量评价 56-61 2.1 外观及再分散性 56 2.2 表面形态观察 56-57 2.3 DDB-NLC冻干粉针剂的粒径分布 57-59 2.4 包封率与载药量测定 59 2.5 Zeta电位的测定 59 2.6 DSC分析 59-60 2.7 X-射线衍射分析 60-61 3 DDB-NLC冻干品的体外释药特性研究 61-66 3.1 色谱条件 61-62 3.2 标准曲线 62 3.3 精密度 62-63 3.4 回收率 63-64 3.5 体外释药特性研究 64-66 4 联苯双酯纳米脂质载体冻干针剂初步稳定性考察 66-67 三、讨论 67-71 第三部分 联苯双酯纳米脂质载体在小鼠体内的组织分布和药物动力学研究 71-92 一、材料与仪器 71 1. 实验材料 71 2. 实验动物 71 3. 实验仪器 71 二、实验方法与结果 71-89 1. 生物样本中联苯双酯含量测定方法的建立 71-76 1.1 样品的处理方法 71-72 1.2 色谱条件 72 1.3 方法专属性考察 72-74 1.4 DDB标准储备液的配制 74 1.5 标准曲线的建立 74-75 1.6 精密度实验 75 1.7 回收率实验 75-76 2. 动物实验 76-87 2.1 分组及给药方法 76-77 2.2 取样方法及取样点的设计 77 2.3 体内分布研究 77-87 2.3.1 组织分布测定结果 77-81 2.3.2 体内分布特点 81-85 2.3.3 靶向性评价 85-87 3. 药物动力学研究 87-89 三. 讨论 89-92 第四部分 联苯双酯纳米脂质载体体外药效学研究 92-97 一、材料与仪器 92 1. 细胞株和药物 92 2. 实验材料 92 3. 实验仪器 92 二、实验方法与结果 92-95 1. HL7702细胞的培养和冻存 92-93 2. CCl_4肝细胞损伤模型的制备 93 3. MTT比色法检测联苯双酯原料药和纳米脂质载体对CCl_4损伤HL7702细胞的保护作用 93 4. MTT法测定不同浓度CCl_4对HL7702细胞的损伤结果 93-94 5. 联苯双酯纳米脂质载体对四氯化碳损伤人肝细胞的保护作用 94-95 三、讨论 95-97 全文结论 97-99 参考文献 99-105 致谢 105-106 攻读学位期间发表论文目录 106-108 学位论文评阅及答辩情祝表 108