【作者】 王莺；

【导师】 向建南；

【作者基本信息】 湖南大学 ， 有机化学， 2009， 博士

【摘要】 抗癌药物紫杉醇其抗癌机理独特,对卵巢癌、乳腺癌、肺癌和头颈部癌均有良好的效果,是治疗晚期卵巢癌的一线药物之一。但由于其水溶性差,而其助溶剂易产生严重毒副作用,大大限制了其临床应用,为此开发具有良好水溶性的新型载药系统受到了广泛关注。近年来,生物大分子由于其良好的生物相容性、生物降解性,在医药领域应用广泛,特别是生物大分子在肿瘤组织中具有提高渗透和滞留作用(EPR)的特点,将生物大分子作为载体与抗癌药物结合,形成新的药物输送系统,可以改善药物水溶性、控制药物释放速度、延长小分子药物在体内的循环周期,提高药物利用率、增加药物稳定性和降低毒副作用等,这种药物系统成为研究热点,具有广阔的应用前景。肝素是一种天然生物大分子,广泛地存在于动物组织和器官中,具有良好的水溶性、生物相容性和生物降解性,并且具有多种生物学功能。由于以上优点,本论文选用肝素作为药物载体与抗癌药物紫杉醇结合,设计新型的药物体系。本论文进行了深入性的研究工作,首先设计、制备一种直接化学键连接的三元化肝素衍生物模拟应用于抗癌药物输送系统。同时利用该模型反应过程,分别制备出以直接化学键结合的肝素-紫杉醇药物输送系统(HD1),以及用氨基酸为连接臂的肝素-紫杉醇药物系统(HD2),并在此基础上设计出一种具有自组装性能的肝素-紫杉醇前药系统(Prodrugs)。在核磁共振(NMR)、红外光谱(FT-IR)及凝胶渗透色谱(GPC)对药物系统结构表征的基础上,开展了药物输送系统的水解动力学、药物体系的抗凝活性研究,考察了药物系统对人乳腺癌细胞MCF-7的体外抗癌抑制活性,通过应用细胞形态学和流式细胞仪分析药物系统的抗肿瘤作用机理,以及进行了体内动物实验。主要研究结果如下:1.抗癌药物模型输送系统在抗癌药物模型药物系统中分别应用乙酰化,苄氧基化和正丁胺化对肝素进行修饰,形成了一元化,二元化和三元化肝素模型药物系统。利用O-乙酰化反应对肝素进行化学修饰,得到重要活性中间体混酐,药物分子模型苄醇与肝素通过相对较易水解的酯键进行连接,靶因子模型正丁胺与肝素之间以酰胺键进行连接,形成“药物分子模型苄醇-乙酰化肝素-靶因子模型正丁胺”的三元化模型药物输送系统。研究表明活性中间体混酐对二元化肝素衍生物有较大的影响,且乙酰基取代度(重量百分数)为8.5 %,苄氧基为3.21 %,正丁胺为1.3 %,是一种理想的模型体系。引入基团重量呈下降趋势,符合实际药物体系中,要求药物分子含量大于靶因子的含量特点。抗凝活性表明模型抗癌药物系统的抗凝血活性有不同程度地降低,说明该系统能够成为一种安全,有效的药物系统,避免临床使用中的出血现象,该模型体系具有应用前景,为接下来的工作奠定了研究基础。2.直接化学键连接的肝素-紫杉醇药物输送系统(HD1)和以氨基酸连接臂连接的肝素-紫杉醇药物输送系统(HD2)应用模型反应过程,制备了两种药物输送系统:以直接化学键酯键连接的肝素-紫杉醇药物输送系统(HD1),和以缬氨酸、亮氨酸和苯丙基酸为连接臂连接的肝素-紫杉醇药物输送系统(HD2a,HD2b,HD2c)。通过测定直接化学键相连的药物体系(HD1)中,载药量为25.4 %,用氨基酸为连接臂的药物系统(HD2a,HD2b,HD2c)中,载药量分别为16.3 %,18.4 %,16.1 %。讨论了两种体系的体外药物释放作用,表明没有连接臂连接的肝素-紫杉醇药物输送系统(HD1)在中性和酸性磷酸盐缓冲液以及酶催化条件下,具有很高的稳定性很难释放出活性药物,说明该肝素-紫杉醇药物输送系统(HD1)并不适合于进一步的研究。而带有氨基酸连接臂的肝素-紫杉醇药物输送系统(HD2)具有一定的水解释放速度,随着磷酸盐缓冲液pH值下降,三种药物系统水解速度均变缓慢;而在类似体内条件酶催化下,三种体系水解速度大大增加,以缬氨酸和亮氨酸为连接臂的药物体系HD2a和HD2b能够释放出较多的活性药物,且后者快于前者;总体来说,带有氨基酸连接臂的药物系统具有较好的应用前景。抗凝活性表明两种药物体系的抗凝活性较肝素均有较大的下降,说明制备的抗癌药物系统具有较好的安全性,具有进一步应用研究的可能性。通过MTT法考察药物系统对人乳腺癌细胞MCF-7的体外抗癌抑制活性,检测表明,HD1的半数抑制浓度IC50为3.09μg/mL,高于原药紫杉醇,而以氨基酸为连接臂的肝素-紫杉醇药物输送系统的半数抑制浓度IC50分别为0.97μg/mL,0.48μg/mL,0.43μg/mL,相比较原药紫杉醇IC50低2～3倍,说明该体系能够释放出活性药物,具有良好的抑制肿瘤细胞活性。综合以上理化性质,以缬氨酸和亮氨酸为连接臂的药物体系HD2a和HD2b,具有较好的应用前景。3.自组装肝素-紫杉醇前药系统(Prodrugs)鉴于纳米尺寸的药物系统具有一定的“被动靶向性”作用,本章在前面工作基础上,设计制备具有纳米尺寸的肝素-紫杉醇前药系统。应用丁二酸酐对肝素进行化学修饰,引入更多的羧酸根,将药物分子与载体通过三种氨基酸缬氨酸、亮氨酸和苯丙氨酸为连接臂进行化学键合,形成前药体系(Prodrug1,Prodrug2,Prodrug3)。定量分析表明,前药体系中引入丁二酰基的重量百分数为8.5 %,连接紫杉醇重量百分数分别为35.4 %,37.8 %,39.1 %。该结果表明由于更多羧酸根的引入增加了连接紫杉醇的含量,较前一章的药物体系有很大的提高。经动态光散射(DLS)和扫描电镜(SEM)检测,这种前药体系能够在水溶性条件下,自组装形成载体在外而药物分子在内的稳定的不规则球形纳米颗粒,平均粒径140～180 nm之间,经测定电位在-20 mV左右。水解动力学表明前药系统在中性和酸性磷酸盐缓冲液以及血浆中均能释放出活性药物分子,并随着pH值的降低,体系释放活性药物的能力下降;而整个前药系统在血浆中具有较快的释放速度,且大鼠血浆中的稳定性低于在人血浆中的稳定性,Prodrug2对酶催化条件更为敏感,总体上来说,在两种水解条件下,Prodrug2是值得期待药物输送系统。抗凝活性表明两种药物体系的抗凝活性较载体肝素均有较大的下降,说明一定的化学修饰能够降低肝素的抗凝活性,避免用药过程中出现的出血现象,该抗癌药物系统具有较好的安全性,具有进一步应用研究的可能性。通过MTT法考察药物系统对人乳腺癌细胞MCF-7的体外抗癌抑制活性,检测表明,自组装功能的肝素-紫杉醇前药系统的半数抑制浓度IC50分别为0.10μg/mL,0.058μg/mL,0.06μg/mL,相比较原药紫杉醇IC50更低,制备的前药系统的抑癌活性均表现出高于紫杉醇的抑癌活性。建立人卵巢癌SKOV3裸鼠模型体内试验表明紫杉醇和Prodrugs单独治疗组均能抑制肿瘤生长,实验组与模型组相比差异有统计学意义( P < 0. 05),与紫杉醇组相比,模型组与Prodrugs组裸鼠体重变化较大,差异有统计学意义( P < 0. 05),而模型组与Prodrugs组裸鼠体重变化不大,差异没有统计学意义( P > 0. 05),说明相比较紫杉醇组,Prodrugs组的对裸鼠毒性不大,是一种较好的药物输送系统。综合分析,Prodrug2组对裸鼠毒性不大,能够达到类似于紫杉醇组的治疗效果,具有广泛地应用前景。更多还原

【Abstract】 Paclitaxel is a taxoid that has aroused considerable interest on account of its promising antitumor activity. It is in clinical use for treating a variety of malignancies, including ovarian, breast, and non-small-cell lung cancers. It was observed that the side effects were caused by Cremophor EL used in the paclitaxel formulation. Therefore, it is urgent to develop a novel drug delivery system. Recent years, the use of macromolecules for the targeted delivery of anticancer agents has generated considerable interest regarding their enhanced permeability and retention (EPR) effect in tumor tissues. Macromolecules with good solubility, non-toxicity, biocompatibility, biodegradability, have been employed as carriers to deliver active drugs towards intracellular compartments. It can improve water solubility, control the release hydrolysis rate and extend in vivo half life of active drugs. The novel drug delivery systems have shown a bright future for the development of anticancer therapy.Heparin is a biocompatible, biodegradable and water-soluble natural polysaccharide and is rich in animal tissues. Heparin has attracted intense attention because it demonstrates a variety of biological activities. According to this, we design and prepare a variety of drug delivery systems, in which heparin as carrier conjugates with paclitaxel.We first develop a model ternary heparin conjugate by direct covalent bond strategy applied to the drug delivery system. Based on the model procedure, we design and synthesis two drug delivery systems (HD) by the strategy, in which O-acetylated heparin as carrier conjugates with paclitaxel by direct ester bond (HD1) and by inserting amino acids as spacers (HD2). We further investigate amphiphilic heparin-paclitaxel conjugates as a type of novel prodrug, which can self-assemble to form nanoparticles due to the different hydrophilic/hydrophobic nature of the carrier and drug compound in aqueous solution. The structures of drug delivery systems have been characterized by 1H NMR, FT-IR, and GPC. The kinetics of hydrolysis for drug delivery system has been investigated under chemical and enzymatic conditions. The anticoagulant activity, in vitro cell inhibition and in vivo antitumor activity in drug delivery system are measured. In addition, we try to examine the apoptotic mechanism of these drug delivery systems by Flow Cytometry and Cytomorphology.1. A model ternary heparin conjugate by direct covalent bond strategy applied to drug delivery systemIn this study, we have developed a model ternary heparin conjugate by direct covalent bond strategy, in which modified heparin using active mix anhydride as intermediate conjugate with model drug molecule (benzyl alcohol) and model specific ligand (butylamine), respectively. Ester bonds linked model drug and carrier facilitate the release of drug from carrier. In contrast, amido bonds between model specific ligand and heparin show greater stability.It is demonstrated that the activating mixed anhydride groups largely improved the activity and selectivity toward the following esterification. It has been found that the optimal weight percentages of introduced functional groups are 8.8 % of acetyl, 3.21 % of benzyloxy and 1.3 % in ternary heparin conjugate, respectively. The decreased trend on degree of substitution (DS) is consistent with that of introduced anticancer drug and specific ligand in drug delivery system.Their anticoagulant activity has been investigated. The results show that model ternary heparin conjugate with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.2. Heparin conjugates with paclitaxel by direct ester bond and by amino acid as spacers According to the model procedure, two types of heparin-paclitaxel conjugates (HD) have been developed, in which O-acetylated heparin as carrier conjugates with paclitaxel, by direct ester bond (HD1) and by inserting amino acids with differentα-substituted group as spacers (HD2), including valine, leucine, and phenylalanine (HD2a, HD2b, and HD2c), respectively.The content of drug conjugate to O-acetylated heparin has been quantified by UV absorbance. The weight percentages of HD conjugates contained 16~25% paclitaxel. HD conjugates have been disposed to chemical and enzymatic hydrolysis to test the drug release at pH 7.4 and pH 5.0, respectively. The HD1 is highly stable under physiological and enzyme conditions. It is demonstrated that direct ester bond between carrier and drug is difficultly hydrolyzed under physiological and enzymatic condition. In the PBS, the amount of paclitaxel, which can be liberated from HD2, increased as pH increased. It is observed the hydrolysis rate of HD2a and HD2b conjugates is greatly increased under enzymatic condition.The anticoagulant activities of HD conjugates have been investigated by APTT. The anticoagulant activities of HD1 and HD2 both decreased to some extent as compared to that of heparin (150 U/mg). As a consequence, HD conjugates with reduced anticoagulant activity could be safe and effective prodrugs to further investigate.Cytotoxicity has been studied by using an MTT assay to identify cells still active in respiration. HD1 exhibites lower cytotoxicity (IC50: 3.09μg/mL) than free paclitaxel. Specifically, IC50 values of HD2a, HD2b and HD2c for MCF-7 are about 0.97μg/mL, 0.48μg/mL and 0.43μg/mL, respectively, which are about 2~3 fold lower than that of free paclitaxel. HD2 therefore demonstrated significant anticancer activity.3. Heparin-paclitaxel drug delivery system with self-assembly property In this study, we have synthesized amphiphilic heparin-paclitaxel conjugates as a type of novel prodrug, which can self-assemble to form nanoparticles due to the different hydrophilic/hydrophobic nature of the carrier and drug compound in aqueous solution. The goal to select succinylated-heparin as a carrier is to increase drug loading capacity in which hydroxyl groups of heparin react with succinic anhydride under classical acylation conditions. Amino acids are ideal linkers, which can provide a reactive carboxyl group to conjugate with paclitaxel via an ester bond and an amino group that could be easily reacted with the carboxyl groups of the carrier via N-acylation reaction.It has been found that the weight percentage of succinylated group is about 8.5 %. The content of drug conjugate to Prodrugs is 35.4 %, 37.8 % and 39.1 %, respectively. Their morphology has been investigated by SEM and DLS. The Prodrugs have a negative surface charge ( -20 mV) and their mean diameters are about 140~180 nm measured by DLS. The results demonstrate that self-assembled nanoparticles have a narrow size distribution and form an approximately spherical shape composed of a paclitaxel core and carrier shell.Prodrugs have been subjected to chemical and plasma hydrolysis to test the paclitaxel release. In the PBS, the amount of paclitaxel, which can be liberated from Prodrugs, increased as pH increased. The stability of Prodrugs in mouse plasma is lower than that in human plasma. Under physiological condition, the amount of released paclitaxel is lower than that in plasma. Apparently, Prodrug2 using leucine as spacer is appropriate to facilitate the release of pacitaxel from the carrier.The anticoagulant activity of Prodrugs has been investigated by APTT. The results show that Prodrugs with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.It shows that the Prodrugs exhibit higher cytotoxicity than free paclitaxel in MCF-7 cell after 48 h by using an MTT assay. IC50 values of Prodrug1, Prodrug2 and Prodrug3 were about 0.19μg/mL, 0.058μg/mL and 0.06μg/mL, respectively. In in vivo experiments, prodrug2 shows a similar ovarian tumor growth inhibition as the parent drug while inducing no obvious body weight loss. As a whole, Prodrug2 shows not only proper hydrolysis rate, but also better anticancer inhibition. It is demonstrate that Prodrug2 with self-assembled property offers promising potential for further clinical applications.更多还原