【作者】 郭盛磊；

【导师】 阎秀峰；

【作者基本信息】 东北林业大学 ， 细胞生物学， 2012， 博士

【摘要】 随着社会的发展,现代人的生活方式和环境污染导致癌症患者剧增。癌症的治疗是世界性的难题,人类至今无法攻克。为了治疗癌症,科学家们花费了大量的精力用于抗癌药物研究与开发。喜树碱(Camptothecin, CPT)是从我国特有树种喜树(Camptotheca acuminata)中提取出的一种拓扑异构酶I (Topo I)抑制剂,具广谱抗肿瘤活性。喜树碱类药物因其独特的抗肿瘤机制已被美国国家癌症研究所(NCI)所列为重点研究的抗肿癌药物之一。但是由于喜树碱的活性形式(内酯形式)在人体内正常生理环境下(pH=7.4)不稳定、水溶性差、毒性大等问题限制了它在临床上的应用。CZ48(camptothecin-C20-propionate),喜树碱丙酸酯,是喜树碱前体药物,CZ48通过在体内水解酯键释放活性药物CPT实现抗肿瘤作用。与CPT相比,CZ48在体内血浆中可保持CPT内酯环的稳定性,从而具有极高的抗肿瘤活性和极低的毒性,在美国已进入Ⅰ期临床研究,是极具发展前景的喜树碱类抗肿瘤药物。然而,水溶性差,口服生物利用度低等不足仍是阻碍CZ48临床应用的主要问题。本研究以生物相容性好且可生物降解高分子材料聚乳酸(PLA)为载体材料,研究制备了载CZ48聚乳酸纳米粒和载CZ48微泡超声造影剂两种载药系统,并对它们的形态、包封率、载药量、体外释放性能和体内药动学进行了研究,目的是利用药物载体系统良好的水分散性、缓控释性,延长药物在体内滞留时间,以解决CZ48在水溶性差、生物利用率低等方面的不足。本研究主要有如下结果：在载CZ48聚乳酸纳米粒载药系统研究中,(1)采用乳化溶剂挥发法成功地制备了载有抗肿瘤药物CZ48的聚乳酸纳米粒-——CZ48-PLA纳米粒,其包封率和载药量分别为88.7%和11.8%,平均粒径为260.6nm；(2)建立了在体外用荧光分光光度计测定磷酸缓冲液(PBS, pH=7.4)中CZ48浓度的方法。体外释放实验结果表明CZ48-PLA纳米粒具良好的缓释效果；(3)采用液-液萃取法(LLE)处理生物样品,联合HPLC分析、建立了在生物样品中同时测定CZ48和其主要代谢产物CPT药物浓度的方法,并分别对所建方法进行了全面的方法学论证。所建立的方法简单易行,选择性特异性强,灵敏度高,方法的准确率高,重现性好；(4)用wistar大鼠进行的体内药动学实验表明CZ48-PLA纳米粒未表现出所期待的持续释药效果,这可能与其在体内被巨噬细胞大量吞噬有关。在载CZ48微泡超声造影剂载药系统研究中,(1)采用双乳溶剂挥发法和冷冻干燥技术相结合的方法成功地制备了载CZ48的聚乳酸微泡超声造影剂——CZ48-Loaded微泡。制备材料为25mg PLA、5mL二氯甲烷和80mg CZ48时,CZ48-Loaded微泡具有最大包封率和载药量,分别为85.73%和26.07%,粒径分布范围为0.5～6.7μm,98%的粒径小于7μm,能够保证微泡注射进入人体后通过肺滤而实现全身循环,满足临床使用的基本要求。(2)体外释药研究发现,CZ48-Loaded微泡超声造影剂具非常好的药物缓释效果。超声可促进其中药物的释放,超声时间越长,促进效果越明显。(3)体内外超声显影结果表明,CZ48-Loaded微泡超声造影剂具有良好的回声特性和较强的体内稳定性。(4)大鼠体内药代动力学进行研究结果表明,通过CZ48-loaded微泡的形式静脉注射给药,延迟了药物达峰时间(Tmax)、T1/2和MRT,提高了AUC0-∞,从而提高CZ48的生物利用度和抑制肿瘤效果。本研究工作的意义在于：通过构建载CZ48聚乳酸纳米粒和载CZ48微泡超声造影剂两种载药系统,较全面的研究了它们的体内外释药行为,为进一步研究这两种载药系统乃至研发CZ48的新型药物载体系统奠定了基础；本研究中建立的在生物样本中同时测定CZ48及其活性代谢产物CPT浓度的方法,将为CZ48的其它载药系统研究中体内药物含量测定方法提供借鉴。更多还原

【Abstract】 With the development of society, modern lifestyles and environmental pollution result in dramatic increase in cancer patients. Treatment of cancer is a worldwide problem that humanity has not yet overcome. In order to treat cancer, scientists have spent a lot of energy for the anti-cancer drug research and development. Camptothecin (CPT) is a well-established topoisomerase I inhibitor against a broad spectrum of cancers. Because of its unique anti-tumor mechanism, CPT has been listed as one of the key research of anticancer drugs by the U.S. National Cancer Institute (NCI). However, poor aqueous solubility, instability of the lactone ring in vivo under physiological conditions at pH7.4, and toxic effects to normal tissues have limited CPT clinical development. CZ48(camptothecin-C20-propionate), a C20-propionate ester of camptothecin, was synthesized as a derivative resistant to lactone hydrolysis. CZ48has demonstrated strong anticancer activity with an exceptional lack of toxicity. CZ48acts as a prodrug and exerts its anticancer activity by carboxylesterases mediated hydrolysis to the active metabolite CPT in vivo. CZ48has entered phase I clinical studies in the United States, which of great prospects anticancer drugs development. However, poor aqueous solubility and low degree of bioavailability by oral is still the main problems that hinder the CZ48clinical applications. In this study, two drug carrier systems were fabricated, i.e., a biocompatible and biodegradable polymer material polylactic acid (PLA) as the carrier material, contained CZ48polylactic acid nanoparticles and contained CZ48microbubble ultrasound contrast agent, whose morphology, encapsulation efficiency, drug loading efficiency and release performance in vitro and pharmacokinetic in vivo were studied, The purpose is to extend the residence time of drugs in the body with the aid of good water dispersion and sustained controlled drug release of the drug delivery system. so as to make up for the poor aqueous solubility and low bioavailability of CZ48. The study results are as follows:in the study of CZ48loaded PLA nanoparticles drug carrier systems (1) emulsion solvent evaporation method was used to prepare polylactic acid nanoparticles containing anticancer drugs CZ48nanoparticles, whose encapsulation efficiency and drug loading efficiency were88.7%and11.8%respectively, The average particle size being260.6nm;(2) a determination method of using the fluorescence spectrophotometer determinate concentration of CZ48in phosphate buffered saline (PBS,pH=7.4) was created. Experimental results of drug release in vitro show that CZ48-PLA nanoparticles have a good release effect;(3) using liquid-liquid extraction (LLE) to deal with biological samples, plus HPLC analysis, the methods of simultaneous determination of CZ48content and its major metabolite CPT in biological samples was established, and the comprehensive methodology demonstration were carried out. The advantages of this method lie in the simplicity and practicability, selective specificity, high sensitivity, high accuracy and good reproducibility;(4) pharmacokinetic experiments demonstrated that CZ48-PLA nanoparticles did not show the expected sustained release effect, which may be related to the large number of nanoparticles that were phagocytosed by macrophage in body. In the study of CZ48-loaded microbubbles ultrasound contrast agent drug carrier systems,(1) CZ48-loaded microbubbles ultrasound contrast agent was successfully prepared using double emulsion solvent evaporation and lyophilization methods, a biocompatible, safe and biodegradable high molecular polymer being employed as the shell material. The morphology of the polymeric microbubbles observed with scanning electron microscope appeared regular spherical with hollow structure and a tight size distribution. When preparation of materials contain25mg of PLA,5mL dichloromethane and80mg CZ48, CZ48-loaded microbubbles have the largest encapsulation efficiency and drug loading efficiency of85.73%and26.07%, respectively. The particle size distribution ranges from0.5to6.7urn,98%of which is less than7μm, which meet the requirement of the size for ultrasound contrast agent.(2) The in vitro release studies have found that CZ48-loaded microbubble ultrasound contrast agent had a very good drug release effect. Ultrasound can promote the release of drugs and the longer ultrasound is the promote effects were more obvious.(3) In vitro and in vivo ultrasound contrast effects indicated that CZ48-loaded microbubbles showed high echogenicity and strong stability in vivo.(4) Pharmacokinetics results show that CZ48-loaded microbubbles have delayed time to peak (Tmax), T1/2and MRT, and have increased the AUC0-∞thereby enhancing the CZ48bioavailability and inhibit tumor effect. The significance of this research is:Through fabricating the two drug delivery systems of CZ48contained PLA nanoparticles and carrier the CZ48microbubble ultrasound contrast agent, their drug release behavior in vivo and in vitro was comprehensively studied, which can build the basis for further study of these two systems and even for the research and development CZ48new drug delivery system; the method of simultaneous determination of CZ48and CPT content in biological samples in this study may provide some reference for determinating durg content in other drug delivery system research for CZ48.更多还原