【作者】 陈立江；

【导师】 娄红祥；

【作者基本信息】 山东大学 ， 天然药物化学， 2014， 博士

【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。更多还原

【Abstract】 Lung cancer is a common malignant tumor, which is a serious threat to human health and life. By2025, Amounts of Chinese lung cancer will be the largest in the world. Lung cancer consisits of two categories, non-small cell lung cancer (NSCLC) and small cell lung cancer. About85%of lung cancer belongs to a non-small cell carcinoma. The treatment of lung cancer and the selection of drugs are becoming increasingly diverse. The drugs which are high efficiency, low toxicity and targeting effect, have been attracted more and more attention.Bexarotene (Targretin(?)) is a synthetic retinoid analog. Its oral soft capsule and gels for external use were approved for the treatment of skin T-cell lymphoma by FDA in2000. Now, the phase Ⅲ clinical trials has been carried out in abroad, mainly used for the treatment of non-small cell lung cancer, breast cancer and psoriasis. Bexarotene showed a significant effect in the treatment of lung cancer, but it belonged to the Biopharmaceutics Classification System (BCS) Class Ⅱ compounds. Its poor solubility and low bioavailability greatly limit the clinical application. In order to solve these problems, Bexarotene was studied to provided the basis for applying Bexarotene to the treatment of lung cancer. Bexarotene, the model drug in this topic research, was made into nanosuspensions by precipitation-combined microfluidization method. The nanodrug delivery systems,method for in vitro samples and in vivo samples analysis, pharmaceutics properties, pharmacokinetics, organs distribution characters of Bexarotene nanosuspensions, and the antitumor activity in vivo and in vitro was studied.These provided theoretical and experimental basis for the development of targeted agents for lung cancer.The main contents of this study included:1. Preformulation study of Bexarotene nanosuspensionsAccording to the preformulation study, Bexarotene samples was scanned by using UV spectrophotometry, then detection wavelength and the determine method by HPLC were established. Methodological study results show that Bexarotene has a good linear relationship in the concentration range0.05～40.0μg·mL-1. And within-day precision, between-day precision, stability, reproducibility and recoveries were corresponded to technology requirement.2. Process and formulation optimization of Bexarotene nanosuspensionsBexarotene nanosuspensions formulation was optimized and screened by orthogonal design method, and zeta-potential was slected as the investigate index. The amount and type of stabilizers were investigated. The experiment revealed that, the impact of various factors on the of zeta-potential was:lecithin＞F68＞PVP K30. Nanosuspension was thermodynamically unstable systems. In order to improve the stability of nanosuspensions, nanosuspensions were prepared into lyophilized powder by using the freeze-drying method. In the lyophilization process, to ensure good physical and chemical stability of the dispersion, the freeze-drying process was investigated. By determining eutectic point and screening lyoprotectants, the freeze-drying process was identified:prepared nanosuspensions by precipitation-combined microfluidization method were added5%mannitol as lyoprotectant, and set into freeze-drying machine for24h. Then freeze-dried powder (NC-Bexarotene) was obtained.4. The study of pharmacokinetic in ratsThis study established a HPLC method for detection of Bexarotene, and the pharmacokinetics in rats was studied after oral administration and intravenous administration, and the effect of Nano-preparation in improving Pharmacokinetics was discussed.The pharmacokinetics in rats was studied for oral and intravenous administration, respectively. According to the pharmacokinetic parameter of oral administration, the control group of Bexarotene and the group of NC-Bexarotene fitted the two compartment model. The T1/2of NC-Bexarotene is significant longer than the control group, and the AUC0-∞almost2times of the control group. Drug release systems of NC-Bexarotene remained relatively durable and stable, mainly because of its good biological adhesion characteristics, that prolong the residence time of pharmaceutical in gastrointestinal tract and greatly improved the drug bioavailability. For intravenous administration, the NC-Bexarotene had a rapid dissolution in blood circulation. NC-Bexarotene using injection water as solvent, avoided the stimulation and the damage to the body from organic solvent, had a better compliance than the control group.5. The study of the tissue distribution in mice.In order to investigate the effect of nano preparation on drug distribution in the body, the tissue distribution in mice of NC-Bexarotene was studied. Compared with Bexarotene-Sol, NC-Bexarotene has significantly reduced the initial concentration of drug, and made AUC more stable and durable. MRT increased from3.82h to16.58h, AUC boosted to18.65h·μg·g-1from12.68h·μg·g-1. Compared with Bexarotene-Sol, either through oral administration or intravenous administration, NC-Bexarotene could reduce the highest drug concentration in heart and kidney, and avoid the high concentration of drug adverse effects on the heart and kidney function. Because NC-Bexarotene obviously prolonged the drugs in vivo. The MRT in heart increased from1.96h to3.15h(oral administration), from1.73h to2.90h(intravenous administration), but the percentage of distribution greatly decreased than the control group. According to the results of liver tissue distribution, in the oral administration MRT of NC-Bexarotene in liver had little increase, but the AUC and the percentage of distribution were not changed obviously. In the intravenous administration, MRT, AUC and percentage of distribution all had a little bit increased. After oral and injected with NC-Bexarotene, drug distribution in the spleen was obvious increase, and AUC and MRT was obvious increase. r e is1.50(oral administration) and1.25(intravenous administration), which indicated that the liver targeting property was obvious. In the lung tissue, the distribution of drugs in the lungs was almost similar to the distribution of spleen. AUC, AUQ and MRT all increased. In the oral administration AUC increased from48.25h·μg·g-1to79.93h·μg·g-1, MRT prolong from1.96h to3.80h. In the intravenous administration, AUC increased from32.85h·μg·g-1to54.94h·μg·g-1, MRT prolong from2.69hto6.34h. rCe=1.7(lung> spleen＞liver). And according to the relative percentage distribution figure, preparation group was significantly improved compared to the solution group. Compared with the solution group, the drug concentration of NC-Bexarotene group was obviously decreased at any time in renal. Therefore NC-Bexarotene could decrease the renal toxicity from Bexarotene.6. Pharmacodynamics study of Bexarotene nanosuspensions on the cell of Lung adenocarcinoma A549and nude mice TumorFrom the result of experiment in cell and nude mice Tumor, NC-Bexarotene and Bexarotene can inhibit the proliferation of cell of Lung adenocarcinoma A549. NC-Bexarotene can retardant G1in cell cycle and induce the cell of Lung adenocarcinoma A549death. From result of content determination of Lactate dehydrogenase (LDH) and DNA fragmentation analysis proved NS-Bexarotene has no toxic effect. During the period of drug delivery, nude Tumor mice group has a significant body weight decrease, and NC-Bexarotene group has lowest death rate. NC-Bexarotene and Bexarotene both can suppress the grow of transplantation tumor, but not much significant different from the control group; NC-Bexarotene can suppress the transplantation tumor volume after administration at13th and17th day. The investigation of the volume of transplantation tumor after autopsy showed that NC-Bexarotene has the lowest volume, But there was no significant difference between groups. From the result of study, the extension of drug administration time is needed in order to get better therapeutic effect. However, because of the high invasiveness of the cell of Lung adenocarcinoma A549, the dead rate of nude mice has increase after drug administration, especially the tumor-burdened group of nude mice model. Compared with Bexarotene, NC-Bexarotene can better inhibit Cell proliferation, induce apoptosis, and case cell cycle arrest, and tumor-burdened rat mortality is low and without obvious side effects.It was the first time to build Nano-suspension agent drug release system of Bexarotene, and it was also the first report on the pharmacokinetics and tissue distribution of Bexarotene nanosuspensions after oral and intravenous administration, and the first Pharmacodynamics study of Bexarotene nanosuspensions on the cell of Lung adenocarcinoma A549and nude mice Tumor. This paper enriched the research of passive targeting of nano-drug release system, and was helpful for the development and application of oral and intravenous formulations of nanosuspensions of Bexarotene. It has important significance on the clinical development of anti-cancer drugs of Bexarotene.更多还原