蓓萨罗丁纳米混悬剂释药系统的构建与评价

发布时间：2018-09-12 14:28

【摘要】：肺癌是导致人类疾病的主要癌症之一,严重威胁人类健康和生命,到2025年,中国将成为世界第一肺癌大国。肺癌有非小细胞肺癌(NSCLC)和小细胞肺癌两大类,而肺癌中大约有85%为非小细胞癌。肺癌的治疗方式和药物选择正日趋多样化。其中具有高效、低毒、且具有一定靶向性的药物,越来越成为研究热点。蓓萨罗丁(Bexarotene)为新型维甲酸类似物,其口服软胶囊和外用凝胶剂获FDA批准于2000年在美国上市,用于治疗皮肤T-细胞淋巴瘤。目前,国外正在开展蓓萨罗丁三期临床试验,主要用于治疗非小细胞肺癌、乳腺癌及牛皮癣。化合物蓓萨罗丁对肺癌有显著的治疗效果,但是其属于生物药剂学分类系统(BCS) Class Ⅱ类化合物,溶解性差,生物利用度低,以及给药靶向性不好等诸多因素严重的限制了其临床的应用。本课题主要以药物蓓萨罗丁为研究重点,以期解决上述难题,并为蓓萨罗丁开发为治疗肺癌新的剂型提供基础。本课题研究以蓓萨罗丁为模型药物,采用沉淀法与高压均质法结合(Precipitation-Microfluidisation)技术将蓓萨罗丁制备成纳米制剂,构建了药物纳米混悬剂给药系统,建立了蓓萨罗丁药物体外以及其生物样品的分析方法,考察其制剂学特征、大鼠体内药物动力学以及小鼠体内组织分布特征。并对纳米制剂的体内外抗肿瘤活性进行考察,为肺癌靶向制剂的开发提供理论和实验依据。本研究的主要内容包括： 1、蓓萨罗丁纳米混悬剂处方前研究根据处方前研究,利用紫外分光光度法,对蓓萨罗丁样品进行紫外扫描,确定了蓓萨罗丁药物最适宜的检测波长,建立了药物的高效液相测定方法,方法学考察结果表明,蓓萨罗丁在浓度范围0.05～40.0μg·mL-1线性关系良好含量测定方法的日内、间精密度、稳定性、重现性以及回收率均符合方法学考察的要求。 2、蓓萨罗丁纳米混悬剂的工艺研究与处方优化采用正交设计法蓓萨罗丁纳米混悬剂进行处方优化筛选,以辅料的用量为考察因素,以不同稳定剂对纳米混悬剂Zeta-电位的影响作为筛选指标。实验结果显示,各因素对Zeta-电位的影响大小顺序依次为：卵磷脂Pluronic F68＞PVPK30。制备得到纳米混悬剂属于热力学不稳定系统,为提高纳米混悬剂的稳定性,采用冷冻干燥法将其制备成了冻干粉。在冻干过程中,为保证蓓萨罗丁纳米混悬剂良好的分散性和物理化学稳定性,对其冻干工艺进行考察。通过对蓓萨罗丁纳米混悬剂共晶点的测定和冻干保护剂的筛选,确定冷冻干燥工艺为：采用沉淀法进行预处理,同时结合高压均质技术制备蓓萨罗丁纳米混悬剂,加入5%甘露醇作为冻干保护剂,置冷冻干燥机中,冻干24h,得到纳米混悬剂冻干粉(NC-Bexarotene)。 3、蓓萨罗丁纳米混悬剂制剂学性质的研究经过冻干工艺制备得到蓓萨罗丁纳米混悬剂之后,对其进行制剂学性质的考察。采用DelsaTM Nano C Particle Analyzer测得NC-Bexarotene的粒径及Zeta-电位；对蓓萨罗纳米混悬剂进行扫描电子显微镜(SEM)观察,从纳米混悬剂NC-Bexarotene放大倍数为5,000和10,000的图像中可以看出,纳米混悬剂粒度分布较均匀,且外观呈不规则的颗粒状；透射电镜(TEM)观察结果表明蓓萨罗丁纳米混悬剂基本上呈完整球形外观,且粒度分布均一；从NC-Bexarotene原子力显微镜(AFM)分析的3D表面形态图像中可以看出,纳米制剂在水中复溶后并没有出现聚集的现象,说明制剂冻干之后是稳定的。将药物与辅料处方比例的物理混合物,NC-Bexarotene样品和三个月后的NC-Bexarotene进行差示扫描量热分析(DSC), X-射线衍射(?)XRPD)测定和共聚焦显微拉曼光谱检测。DSC曲线中可以看到各样品中的蓓萨罗丁有相同的吸热峰；蓓萨罗丁的特征峰均可以在X-射线衍射(XRPD)的衍射图谱中找到；在拉曼谱图中,我们可以看到1607cm-1的峰显示的是蓓萨罗丁的C=C。根据DSC,XRPD和拉曼谱图,可以充分的表明在以沉淀法-高压均质法制备制剂以及冷冻干燥的过程中蓓萨罗丁的晶型并没有发生改变,体现出了良好的物理化学稳定性。用恒温电磁搅拌法对NC-Bexarotene的饱和溶解度进行考察,实验结果表明其PBS (pH=7.4)中溶解度显著提高。体外溶出采用桨法进行测定,结果表明与原料药相比,纳米制剂很大程度的改善了蓓萨罗丁的溶出速率。实验中为了对照纳米制剂和已上市软胶囊药物(Soft Gelatin Capsule, SGC)的体外释放情况,采用透析的方法进行比较,纳米制剂和已上市软胶囊药物的体外释放实验结果表明,纳米制剂的释放速度比SGC有明显的优越性。NS-Bexarotene和NC-Bexarotene的初步稳定考察以外观,粒径以及分布为指标,结果证明了将NS-Bexarotene制备成NC-Bexarotene显著的增加了制剂的稳定性。 4、蓓萨罗丁纳米混悬剂在大鼠体内的药代动力学研究本文建立了蓓萨罗丁生物样品的HPLC检测方法,考察了NC-Bexarotene分别经口服给药和静脉注射给药后在大鼠体内的药物动力学过程,探讨了纳米制剂在改善药物动力学特征方面所起到的作用。分别研究了经灌胃给药以及静脉内给药后药物在大鼠体内的药代动力学行为,根据口服药动学参数具体分析,蓓萨罗丁原料药对照组和纳米混悬剂NC-Bexarotene组口服给药均符合二室模型。根据药动学参数分析,NC-Bexarotene制剂组的半衰期T1/2明显长于溶液组；而NC-Bexarotene混悬剂组AUC0-∞为接近溶液组的2倍,纳米释药系统维持相对持久且平稳的药-时曲线,主要由于其具有良好的生物黏附性的特点,延长了药物在胃肠道的滞留时间,很大程度提高了药物生物利用度。而经静脉注射后,药物能够迅速进入到血液循环,NC-Bexarotene以注射用水为溶剂,避免了有机溶剂对机体的刺激和损害,比起蓓萨罗丁溶液组有更好的顺应性。 5、纳米混悬剂在小鼠体内组织分布的研究为了考察纳米制剂对药物体内分布特征的改变,该实验研究了NC-蓓萨罗丁制剂在小鼠体内组织分布的特征。对口服后药物在血浆的动力学过程进行分析,与Bexarotene-Sol相比,NC-Bexarotene明显的降低了药物的初始浓度,并且使药-时曲线更加平稳且持久。MRT从3.82h增加到了16.58h, AUC从17.02h·μg·g-1增加至103.2h·μg·g-1;而注射组的MRT变化却不大,但AUC由12.68h·μg·g-1提高到了18.65h·μg·g-1。与Bexarotene-Sol相比,不论通过口服给药还是经静脉注射给药,NC-Bexarotene均可降低心脏以及肾脏中最高药物浓度,可以避免过高的药物浓度对心脏以及肾脏机能产生不良影响。由于NC-Bexarotene明显延长了药物在体内的滞留时间,Bexarotene在心脏中的MRT也由1.96h增加到3.15h(口服),1.73h增加到2.90h(注射),但在心脏中的分布百分率与溶液相比却大大降低。根据肝脏组织分布结果,口服过程中,NC-Bexarotene在肝脏中的MRT虽也有所延长,但药物AUC变化并不明显,而且分布百分比也并不突出；注射组中,MRT略有延长,AUC相对于Bex-Bexarotene有一定的提高,分布百分率也增加。口服以及注射NC-Bexarotene之后,药物在脾中的分布均较溶液组有明显的提高,药物在脾中的AUC、AUQ值明显增大,MRT值也明显的延长,rCe为1.50(口服组)和1.25(注射组)均较肝脏的靶向性明显,但从药物的分布百分比图中可看出肾脏中药物百分比明显低于其他脏器。在肺组织分布中,药物在肺中的分布情况几乎与脾中的分布情况相似。不论是AUC、AUQ还是MRT均得到增加,口服中AUC由48.25增加到79.93,MRT由1.96延长到3.80；注射组中,AUC由32.85增加到54.94,MRT由2.69延长至6.34。rCe为1.7(肺脾肝)。而且根据相对分布百分比率图比较,相比于溶液组有明显的提高。与溶液组相比,NC-Bexarotene几乎在各时间点的肾脏浓度均有明显的降低,因此将蓓萨罗丁制备成NC-Bexarotene后有利于降低Bexarotene可能带来的肾脏毒性。 6、蓓萨罗丁纳米混悬剂对人肺腺癌细胞A549及其裸鼠移植瘤的药效学考察从细胞学实验和在体裸鼠移植瘤实验结果表明,蓓萨罗丁和蓓萨罗丁纳米混悬剂可抑制人体肺腺癌上皮A549细胞系增殖,蓓萨罗丁纳米混悬剂作用相对较强；蓓萨罗丁纳米混悬剂显著诱导人肺腺癌上皮A549细胞的凋亡,细胞周期的G1期阻滞；通过细胞渗漏乳酸脱氢酶(LDH)含量测定证实蓓萨罗丁和蓓萨罗丁纳米混悬剂对人体肺腺癌上皮A549细胞没有明显的毒性作用；在给药期间,荷瘤模型组裸鼠的体重下降更明显,蓓萨罗丁纳米混悬剂组裸鼠死亡率最低；在给药期间,蓓萨罗丁和蓓萨罗丁纳米混悬剂可以抑制裸鼠移植瘤瘤体积的增长,但与荷瘤模型组比较作用不显著；给药第13天和第17天之后,蓓萨罗丁纳米混悬剂组可明显抑制裸鼠移植瘤相对体积RTV；解剖后离体的裸鼠移植瘤体积和重量,蓓萨罗丁纳米混悬剂组最低,但各组间无显著性差异。从在体实验结果看,要达到更有效的治疗效果,还需要延长给药时间,但可能由于人肺腺癌上皮A549细胞的侵袭性比较强,给药后期裸鼠死亡的比例加大,裸鼠的体重偏低,特别是荷瘤模型组的裸鼠。相对蓓萨罗丁,蓓萨罗丁纳米混悬剂有更好的抑制人体肺腺癌上皮A549细胞增殖、诱导凋亡、引起细胞周期阻滞的作用,荷瘤鼠死亡率低,无明显的毒副作用。本课题首次构建了蓓萨罗丁纳米混悬剂释药系统,首次对蓓萨罗丁纳米混悬剂的口服以及注射制剂在动物体内的药物动力学,组织分布特征进行了评价,并首次对蓓萨罗丁纳米混悬剂对人肺腺癌细胞A549及其裸鼠移植瘤的药效学进行考察,本文丰富了纳米释药系统被动靶向的研究内容,对蓓萨罗丁口服纳米制剂以及注射制剂的开发和应用提供了一定实验参考,对抗癌药物蓓萨罗丁的临床开发有着重要的意义。
[Abstract]:Lung cancer is one of the main cancers causing human diseases, which seriously threatens human health and life. By 2025, China will become the world's largest lung cancer country. Lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer, and about 85% of lung cancer is non-small cell cancer. Drugs with high efficiency, low toxicity and certain targeting properties have increasingly become a research hotspot.
Bexarotene is a new retinoic acid analogue. Its oral soft capsules and topical gels were approved by FDA to be marketed in the United States in 2000 for the treatment of cutaneous T-cell lymphoma. It has a significant therapeutic effect on lung cancer, but it belongs to Class II compounds of Biopharmaceutics Classification System (BCS). Its poor solubility, low bioavailability, and poor drug targeting seriously limit its clinical application. Beza Rodin provides a basis for the development of new dosage forms for lung cancer.
In this study, Besarodine was used as a model drug to prepare nanoparticles by precipitation and high pressure homogenization (Precipitation-Microfluidisation) technology. The drug nanosuspension delivery system was constructed, and the analytical method of Besarodine in vitro and its biological samples was established. Pharmacokinetics in vivo and tissue distribution in mice were studied. The antitumor activity of nanoparticles in vitro and in vivo was investigated to provide theoretical and experimental basis for the development of lung cancer targeting agents.
1, pre formulation study of Bei sardin nanosulfan.
According to the pre-prescription study, the Besarodine samples were scanned by ultraviolet spectrophotometry, and the optimum detection wavelength was determined. A high performance liquid chromatographic method for the determination of Besarodine was established. The results of methodological investigation showed that Besarodine had a good linear relationship in the concentration range of 0.05-40.0 ug.mL-1. Intraday density, stability, reproducibility and recovery were in line with methodological requirements.
2, the technology research and Prescription Optimization of Bei sardin nano suspensions.
The orthogonal design method was used to optimize the formulation of Bexarodin nanosuspension. The influence of different stabilizers on Zeta-potential of the nanosuspension was taken as the screening index. The results showed that the order of influence of various factors on Zeta-potential was lecithin Pluronic F68 > PVPK30. In order to improve the stability of nano-suspension, freeze-drying method was used to prepare freeze-dried powder. In order to ensure good dispersion and physicochemical stability of nano-suspension, the freeze-drying process was investigated. The determination of eutectic point and the selection of freeze-drying protectant were carried out. The freeze-drying process was determined as follows: pretreatment by precipitation method, preparation of Bexarotine nanosuspension by high-pressure homogenization technology, adding 5% mannitol as freeze-drying protectant, freeze-drying machine, and freeze-drying for 24 hours to obtain NC-Bexarotene nanosuspension powder.
3, the study of the properties of Bei sardin nano suspensions.
Bexarotine nanosuspension was prepared by freeze-drying process and its pharmacological properties were investigated. The particle size and Zeta-potential of NC-Bexarotene were measured by DelsaTM Nano-C Particle Analyzer, and the magnification of NC-Bexarotene nanosuspension was observed by scanning electron microscopy (SEM). The results of transmission electron microscopy (TEM) showed that the particle size distribution of the nanosuspension was uniform and the appearance was irregular. The results of TEM showed that the nanosuspension was basically spherical and uniform in size distribution. The surface morphology of the nanosuspension was analyzed by NC-Bexarotene atomic force microscopy (AFM). It can be seen that there is no aggregation of nanoparticles in water after re-dissolution, indicating that the preparation is stable after lyophilization.
Differential scanning calorimetry (DSC), X-ray diffraction (?) XRPD and confocal microscopic Raman spectroscopy were used to determine the ratio of drug to excipient, NC-Bexarotene samples and NC-Bexarotene samples three months later. The peaks can be found in the X-ray diffraction (XRPD) spectra. In the Raman spectra, we can see that the peak of 1607 cm-1 shows C=C of Besarodine. According to DSC, XRPD and Raman spectra, it can be fully shown that the crystalline form of Besarodine prepared by precipitation-high pressure homogenization method and freeze-drying process is not found. The change shows good physical and chemical stability.
The saturated solubility of NC-Bexarotene was investigated by electromagnetic stirring at constant temperature. The results showed that the solubility of NC-Bexarotene in PBS (pH=7.4) was significantly improved. The dissolution of NC-Bexarotene in vitro was determined by paddle method. The results showed that the dissolution rate of NC-Bexarotene was greatly improved by nano-preparation compared with the raw material. In vitro release of marketed soft gelatin capsule (SGC) was compared by dialysis. The results of in vitro release experiments of nano-preparations and marketed soft capsules showed that the release rate of nano-preparations was obviously superior to that of SGC. The results showed that the preparation of NC-Bexarotene from NS-Bexarotene significantly increased the stability of the preparation.
4, pharmacokinetics of Beza Rodin nano suspensions in rats
A method for the determination of Bexarotine in biological samples by HPLC was developed. The pharmacokinetic process of NC-Bexarotene in rats after oral administration and intravenous administration was investigated. The role of nanoparticles in improving the pharmacokinetic characteristics was discussed.
The pharmacokinetic behavior of the drug in rats after intragastric administration and intravenous administration was studied. According to the specific analysis of oral pharmacokinetic parameters, the two compartment model was established in the control group of Bexarotine and the NC-Bexarotene group. Phase T1/2 was significantly longer than that of the solution group, while AUC0-uuuuuuuuuuuuuuuuuuuuuuuuuuu After intravenous injection, the drug can quickly enter the blood circulation, NC-Bexarotene injection of water as a solvent, to avoid organic solvent stimulation and damage to the body, compared with the Bexarotine solution group has better compliance.
5, the tissue distribution of nanossuspensions in mice.
To investigate the effect of nanoparticles on the distribution of NC-Bexarotine in mice, the tissue distribution of NC-Bexarotine in mice was studied. MRT increased from 3.82 hours to 16.58 hours, AUC increased from 17.02 hours to 103.2 hours. MRT did not change much in the injection group, but AUC increased from 12.68 hours to 18.65 hours. Because NC-Bexarotene significantly prolonged the drug retention time in the body, the MRT of Bexarotene in the heart increased from 1.96 h to 3.15 h (oral) and from 1.73 h to 2.90 h (injection), but the percentage of distribution in the heart increased from 1.96 h to 2.90 h (injection). According to the results of liver tissue distribution, the MRT of NC-Bexarotene in the liver was prolonged, but the change of AUC was not obvious and the percentage of distribution was not prominent. In the injection group, the MRT was slightly prolonged, the percentage of distribution of AUC was increased compared with that of Bex-Bexarotene. After taking NC-Bexarotene and injecting NC-Bexarotene, the distribution of the drug in spleen was significantly higher than that of the solution group, the AUC and AUQ values in spleen were significantly increased, and the MRT values were significantly prolonged. Both rCe 1.50 (oral group) and 1.25 (injection group) were more targeted to the liver, but the percentage distribution of the drug in kidney could be seen from the drug percentage map. The distribution of drugs in lung tissue was similar to that in spleen. Both AUC, AUQ and MRT increased, AUC increased from 48.25 to 79.93, MRT from 1.96 to 3.80 in oral administration, AUC increased from 32.85 to 54.94 in injection group, and MRT from 2.69 to 6.34.rCe to 1.7 (lung, spleen and liver). Compared with the solution group, the concentration of NC-Bexarotene in the kidney decreased significantly at almost every time point. Therefore, the preparation of Bexarotene into NC-Bexarotene can reduce the possible renal toxicity of Bexarotene.
6, the pharmacodynamics of Bei sardin nano suspension on human lung adenocarcinoma cell A549 and nude mice transplanted tumor.
Cytological and xenograft tumors in nude mice showed that besarotine and besarotine nanosuspensions could inhibit the proliferation of human lung adenocarcinoma epithelial A549 cell line, and the effect of besarotine nanosuspension was relatively strong. The results showed that Besarodine and Besarodine nanosuspensions had no obvious toxic effect on human lung adenocarcinoma epithelial A549 cells. During the administration period, the weight of nude mice in tumor-bearing model group decreased more significantly, and the mortality of nude mice in Besarodine nanosuspension group was the lowest. Besarodine and Besarodine nanosuspensions inhibited the growth of tumor volume in nude mice, but had no significant effect compared with the tumor-bearing model group; Besarodine nanosuspension group significantly inhibited the relative volume of tumor RTV in nude mice after 13 and 17 days of administration; Anatomically isolated tumor volume and weight in nude mice Salodine nanosuspension group was the lowest, but there was no significant difference between the groups. From the in vivo experimental results, to achieve more effective treatment, we need to extend the administration time, but it may be due to the strong invasiveness of human lung adenocarcinoma epithelial A549 cells, increase the proportion of death in nude mice, nude mice with low body weight, especially tumor-bearing model. Compared with Besarodine, Besarodine nanosuspension could inhibit the proliferation of human lung adenocarcinoma epithelial A549 cells, induce apoptosis, and cause cell cycle arrest. The mortality of tumor-bearing mice was low without obvious side effects.
The release system of Besarodine nanosuspension was constructed for the first time. The pharmacokinetics and tissue distribution of Besarodine nanosuspension were evaluated in vivo for the first time. The pharmacodynamics of Besarodine nanosuspension on human lung adenocarcinoma cell line A549 and its transplanted tumor in nude mice were studied for the first time. This study enriched the research contents of passive targeting of nanodrug delivery system, oral nanopreparations and injection preparation of Besarodine.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R734.2;R943

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