【作者】 王秀敏；

【导师】 邓英杰； 赵春杰；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2005， 博士

【摘要】 β-榄香烯是从中药温莪术（Curcuma wenyujin Chen et C. Ling）的干燥根茎中提取分离得到的倍半萜烯类化合物。β-榄香烯是具有特异性细胞毒性的广谱抗癌药物，具有抗肿瘤、抗菌、抗炎、抗病毒、提高机体免疫力、抑制血小板凝集、改善微循环等作用。其现有制剂，乳注射剂、口服乳等，在临床上主要用于治疗癌性胸腹水、肝癌、肺癌、脑瘤等，疗效确切。近几年来，β-榄香烯已经成为抗癌药物的一个研究热点，也是中药西制的一个典范。β-榄香烯不溶于水，口服生物利用度极低，腹腔注射生物利用度也仅有8.49％，静脉注射可迅速被机体消除。β-榄香烯制剂在临床应用中的一个严重的副作用是血管刺激性较大，静脉炎发生率达30％。基于以上原因，本论文的主要研究目的就是通过脂质体新剂型解决β-榄香烯血管刺激性、提高其生物利用度、延长其静脉注射给药时的体内半衰期，进而靶向肿瘤，提高抗肿瘤疗效。为此我们设计了三种不同类型的脂质体，即β-榄香烯普通脂质体（CLE）、长循环脂质体（PLE）和热敏长循环脂质体（TLLE）。本论文主要包括以下内容：1．β-榄香烯的分离和理化性质测定。温莪术挥发油中分离提取得到了β-榄香烯，并对其理化性质，如油水分配系数、折光率、旋光度、在不同溶剂中的溶解度进行了测定，同时考察了β-榄香烯在乙醇溶液中的稳定性。结果，β-榄香烯油水分配系数为199.5，折光率为1.4932，比旋度为-16.359°，在各种有机溶剂中溶解度均较大。在40℃、60℃加热的条件下，β-榄香烯含量均有所下降。2．CLE、PLE、TLLE的制备及制剂性质考察。乙醇注入法为CLE、PLE的制备方法，在单因素考察和正交试验基础上优化CLE处方。结果表明，脂药比、磷脂浓度、水相介质种类对CLE包封率影响较大，其他因素对包封率影响较小。最后确定CLE的最优处方，得到了包封率较高（92.3％）的CLE；通过考察DSPE-PEG₂₀₀₀的用量，在CLE最优处方的基础上，确定了PLE的处方（包封率95.2％）；以相变温度和包封率为指标，确定了薄膜分散法制备TLLE的处方，该处方相变温度为41.8℃，符合临床热疗的要求，包封率为87.9％；对CLE、PLE和TLLE的理化性质及制剂稳定性进行了考察。结果，CLE、PLE和TLLE为小单室脂质体，CLE、PLE较TLLE稳定，4℃、25℃下12个月内稳定性较好。3．CLE、PLE和TLLE的药物动力学和组织分布的研究。建立了快速、准确、简单的生物样品中p-榄香烯气相色谱分析方法。大鼠尾静脉注射50 mg／kg以上三种制剂后，与市售制剂EE相比，TLLE的t_1/2、AUC_（0→t）、MRT_（0→t）分别为EE的3.2、4.2、2.3倍，统计学分析有显著性差异（P＜0.001）；PLE的t_1/2、AUC_（0→t）、MRT_0→t)分别为EE的2.7、3.9、2.3倍（P＜0.001）；而与EE相比，CLE只使AUC_（0→t）提高了1.5倍（P＞0.05），二者其他药动学参数间无显著性差异。药动学结果表明，PLE、TLLE均可使β-榄香烯体内循环时间延长，有长循环性；TLLE、PLE、CLE均可使β-榄香烯生物利用度得到显著提高。荷瘤小鼠尾静脉注射60mg／kg各种制剂，不同方案的组织分布结果表明，各治疗组肿瘤中药物分布由大到小的顺序为：TLLE+HT＞PLE+HT＞PLE＞CLE＞EE+HT＞EE。在热疗条件下，以EE+HT为对照，TLLE+HT有最大的肿瘤靶向性，靶向参数为2.57。在生理温度下，PLE、CLE较EE有更好的肿瘤靶向性（靶向参数分别为2.17、1.33）。TLLE、PLE在肝脾中药物分布量均降低，证明了TLLE、PLE可降低肝脾的摄取，更多地靶向肿瘤组织。尤其是TLLE有在加热部位快速释药的热敏性，使其对肿瘤组织的靶向性大大增加。另外，TLLE、PLE、CLE在脂肪中的分布均减少，有望可以克服EE在脂肪中的蓄积现象。4．CLE、PLE和TLLE药效学研究。在体外抗肿瘤试验中，以EE作为阳性对照药，用MTT试验测定了三种制剂对6种人癌细胞的半数抑制浓度。结果，细胞毒活性为PLE＞CLE≈TLLE＞EE，透射电镜对癌细胞结构的观察结果与MTT试验结果一致。体内药效学试验中，以荷瘤小鼠为动物模型，考察了不同给药方案荷瘤鼠的抑瘤率和生命延长率，结果显示，在40mg／kg剂量下，各组抑瘤效果由强至弱依次为：TLLE+HT（69％）＞PLE+HT（62％）＞CLE+HT（58％）=TLLE（58％）＞PLE（56％）＞EE+HT（51％）＞CLE（48％）＞EE（40％）＞HT（11％）。TLLE+HT组的抑瘤率高达69％。在生理体温（无热疗）条件下，各组比较时，PLE的抑瘤率较高，为56％。生命延长率实验中，生命延长百分率由大到小的顺序为PLE＞CLE＞TLLE。PLE、CLE可使荷瘤鼠存活时间延长约1倍；TLLE使荷瘤鼠存活时间延长约25.7％；EE无延长存活时间的作用。各组荷瘤鼠体重变化的结果表明，在抑瘤实验过程中荷瘤鼠的体重变化未超过正常范围，说明给药剂量比较适合，各种制剂毒副作用均较小。总之，CLE、PLE和TLLE体内外抗肿瘤效果均优于EE。5．CLE、PLE和TLLE的制剂安全性研究。CLE、PLE、TLLE均为静脉注射剂，本文对制剂的安全性从以下几个方面进行了评价。急性毒性实验考察小鼠最大耐受剂量；家兔溶血性试验考察制剂的渗透压是否合格，能否引起溶血；豚鼠的过敏试验考察制剂能否引起过敏反应；家兔的耳缘静脉刺激性试验考察制剂的血管刺激性。结果，三种制剂在制剂最大浓度和小鼠最大耐受剂量下均有良好的耐受性，无溶血现象、无过敏反应发生、对血管无刺激性，静脉给药是安全可行的。更多还原

【Abstract】 β-elemene is a sesquiterpene ingredient extracted from one of the traditional Chinese herbal medicine Curcuma wenyujin Chen et C. Ling. Recent studies have shown thatβ-elemene possesses specific cytotoxicity. As a broad-spectrum anti-tumor agent,β-elemene exhibits pharmacological effects including anti-tumor, antibacterial, antivirus, improving immunity, inhibiting platelet aggregation and improving microcirculation, etc. The preparations ofβ-elemene, such as oral emulsion, injection, are extensively used in clinic to treat ascites resulting from cancer, lung cancer, hepatoma, brain cancer, etc. in China. Due to the potent efficacy ofβ-elemene in clinic to treat these diseases, the research ofβ-elemene has become a hot topic in recent years in China andβ-elemene has also become a typical example of TCM made by Hesperian and modern preparation technology.It has been reported that the oral bioavailability ofβ-elemene is very low due to its insolubility in water. Theβ-elemene is rapidly eliminated by injection and the bioavailability ofβ-elemene by i.p. is 8.49%. One of the adverse effects ofβ-elemene formulation used in clinic is the irritability to blood vessel, and the incidence rate of phlebitis is about 30%. Therefore, the purposes of this paper are to reduce irritability to blood vessel, to increase its bioavailability and to prolong its duration in the circulation after i.v. and to target to tumor and to improve efficacy. Three kinds of liposomes, including conventional liposome, PEGylated liposome, thermosensitive long-circulating liposome, were used as the means of fulfilling these purposes.The following parts are included in this paper:1.β-elemene was isolated from Rhizoma Curcumae. The physicochemical properties ofβ-elemene, including n-octanol/water partition coefficient, the solubilities in various solvent, refractive index, specific optical rotation, stability, were determined. The results indicate that:β-elemene is insoluble in water or PBS and soluble in organic solvents. Its n-octanol/water partition coefficient is 199.5, refractive index is 1.4932, specific optical rotation is -16.359°. The concentration ofβ-elemene in ethanol aqueous decreased at 40℃or 60℃for 6 or 12 months.2. The CLE、PLE were prepared by ethanol injection method. Single factor experiment and orthogonal experimental design were used to optimize the formulation of CLE. The effects of various factors on the encapsulation efficiency （EE％） were investigated. The results indicated that the ratio of PC toβ-elemene, different aqueous media and concentration of PC had larger effect on EE％than the other factors. The optimized formulation of CLE was 5％PC, 0.83％CH, 0.5％β-elemene, pH7.4 PBS （33.23mmol/L）. The EE％of optimized CLE formulation was above 92％. The quantity of DSPE-PEG₂₀₀₀ in formulation was investigated and the formulation of PLE was optimized on the basis of CLE formulation. The EE％of PLE was 95.2％. The T_m and EE％were used to evaluate the formulation of TLLE prepared by film hydration method. The T_m of TLLE formulation was 41.8℃, which accord with the requirement of the clinical heat treatment. The EE％of TLLE formulation was 87.9％. The characteristics of CLE、PLE、TLLE were investigated. The results indicated that the liposomes were mainly unilamellar and the stability of CLE, PLE and TLLE were investigated. The results of stability indicated that CLE, PLE were stable at 4℃or 25℃for 12 months. But the stability of TLLE was not good.3. The pharmacokinetics and tissue distribution of CLE, PLE and TLLE were investigated. A simple, accurate, rapid GC method for the determination of 13-elemene in biological sample was developed. The pharmacokinetic behavior in rats via i.v. administration showed that the mean t_1/2, AUC_（0→t） and MRT_（0→t） of PLE was 2.7 fold, 3.9 fold, 2.3 fold higher than those of EE. There was significant difference （P＜0.001）. The mean t_1/2, AUC_（0→t） and MRT_（0→t） of TLLE was 3.2, 4.3, 2.3 times higher than those of EE. Meanwhile, AUG_（0→t） showed a significant difference （P＜0.05） between CLE and EE. Other PK parameters showed no significant difference （P＞0.05） between CLE and EE. The results obtained from pharmacokinetics study showed that the circulation time in vivo ofβ-elemene in TLLE and PLE was significantly prolonged （P＜0.001） and the bioavailability of TLLE, PLE, CLE was significantly improved （P＜0.001）. In in vivo tissue distribution study, the dosage was 60mg/kg and the biodistribution behavior ofβ-elemene in the H₂₂ bearing mice via i.v. was changed by encapsulated in conventional liposome, PEGylated liposome and thermosensitive long-circulating liposome. The results of biodistribution showed that: the quantity ofβ-elemene in tumor in different groups was in order that, TLLE+HT＞PLE+HT＞PLE＞CLE＞EE+HT＞EE. The ability to target to tumor of TLLE+HT was maximal and TI of it was 2.57 when it compared with EE+HT. At 37℃, PLE and CLE had greater tumor target than EE. The TI of PLE and CLE was 2.17, 1.33, respectively. The accumulation ofβ-elemene in liver and spleen in TLLE+HT group and PLE group decreased, which indicated that TLLE and PLE can decrease uptake of liver and spleen and can target to tumor. Especially, TLLE had the thermosensitive property and the ability to target to tumor rapidly increased with HT. Moreover, the accumulation ofβ-elemene encapsulated into TLLE, PLE, CLE in fat drastically decreased, which can be used to resolve the accumulation of EE in fat in clinic.4. In pharmacodynamics, we studied the anti-tumor effect of PLE, CLE, TLLE and EE for six cancer cell lines in vitro by MTr assay. The cytotoxicity of different formulations was in order that: PLE＞CLE≈TLLE＞EE. Morphologic change of CoCl cell observed by transsion electron micrograph was consistent with the result of MTT assay. In in vivo anti-tumor experiment, H₂₂ beating mice was used as animal model and IRT and lifespan were used to evaluate the efficacy following intravenous administration at a dose of 40mg/kg. The IRT of different groups was in order that: TLLE+HT（69％）＞PLE+HT （62％）＞CLE+HT（58％）=TLLE （58％）＞PLE（56％）＞EE+HT（51％）＞CLE （48％）＞EE（40％）＞HT（11％）。The IRT of TLLE combining with HT was 69％. PLE had high IRT（56％） at normal animal heat. The survival time of different groups was in order that: PLE≈CLE＞TLLE. The survival time of PLE and CLE group increased 100％. The percent weight loss was used to evaluate the toxicity of different formulations. The percent weight loss was not above 15％, which indicated that the 40mg/kg dosage is proper and the toxicity of CLE, PLE, TLLE and EE was low. In conclusion, the anti-tumor activities of CLE, PLE and TLLE in vivo and in vitro were superior to EE.5. We also studied the pharmaceutical safety of CLE, PLE, TLLE to determine if these formulations were safe via iv administration. The results indicated that （1） no hemolysis, no irritability and no hypersensitivity were found in three formulations, （2） These formulation were tolerated for mice at maximal concentration and maximal injection volume （150mg/kg）, which showed the safety of these formulations.更多还原