【作者】 罗军强；

【导师】 臧林泉；

【作者基本信息】 广东药学院 ， 药剂学， 2011， 硕士

【摘要】 肺癌是全球最普遍发生的恶性肿瘤之一,其发病率和死亡率居全球恶性肿瘤之首。近年来,我国肺癌的发病率呈急剧上升趋势,5年的生存率不足15 %,其主要原因是早期诊断率不高,大多数患者被确诊为肺癌时已是中晚期或出现了远端转移。目前,肺癌的治疗以手术为主,联合化疗为辅。然而,以细胞毒为主的化学治疗缺乏选择性难免产生许多不良反应和毒性,病人耐受性较差。因此,提高肺癌的早期诊断率和肺癌的靶向治疗成为国内外研究的热点。本实验研究的多肽ZS-1,是利用噬菌体随机展示肽库对非小细胞肺癌细胞筛得到的多肽。对肺癌具有较高的亲和力与靶向性,鉴于此,我们对ZS-1的靶向性作进一步系统的鉴定并研究ZS-1的药物动力学,为开发一种新型的肺癌靶向性药物载体提供理论依据。本论文首先对多肽的靶向性和特异性进行确认。从细胞水平鉴定肺癌靶向性结合多肽,本实验利用了两株人非小细胞肺癌细胞NCI-H1299和A549进行细胞免疫荧光试验,并以人胚胎肺成纤维细胞系WI-38、人正常肝细胞LO-2作为对照。结果显示,FITC标记多肽[ZS-1]FITC对人肺癌细胞有较高的特异性和亲和力,以人肺鳞癌细胞NCI-H1299的绿色荧光强度最强,而与人正常肝细胞和肺细胞的结合力极弱。并进一步从组织水平鉴定该多肽的靶向性和特异性,利用共孵育,通过多次洗涤,荧光扫描结果显示,以肺癌与对照组荧光强度比值大于2倍为阳性,肺癌的阳性的诊断率为37.3 %,本实验8例对照组织中,均没有出现假阳性现象,荧光强度均少于2倍肺对照组织。因此,假阳性的比率为0 %。实验结果提示,多肽ZS-1对人肺癌组织具有较高的特异性。在荷瘤裸鼠的动物水平,通过建立荷人肺鳞癌细胞株NCI-H1299的裸鼠模型,将[ZS-1]FITC尾静脉注射到裸鼠体内,取内脏器官并制作冰冻切片观察其荧光强度。结果显示,多肽ZS-1在肿瘤组织处分布最多,荧光最强,其次,在肝和肾主要代谢器官处也有较多ZS-1分布,而在心、脾、肺、大脑和骨髓组织处分布较少,只有较弱的荧光强度。以上体外和体内靶向性鉴定结果表明,ZS-1对肺癌具有较高和亲和力与靶向性。基于ZS-1具有较好的肺癌靶向性,然后研究ZS-1在大鼠体内的药动学以明确其在体内的稳定性与代谢情况,方法学考察结果显示,血浆中不存在内源性物质干扰ZS-1的定量,该实验色谱条件下分析ZS-1具有良好的专属性,ZS-1的保留时间是13.8 min。在0.1-50μg.mL^-1范围内,血浆中ZS-1标准曲线具有良好的线性关系,标准曲线方程是y = 19894 x -196.89,相关系数R2=1。以信号与噪音的比值等于3定义为检测限,该分析方法的检测限为0.02μg.mL^-1,准确度与精密度结果表明,以RSD表示精密度,总体的RSD都少于10 %,日内的RSD是1.5 %-8.9 %,日间的RSD是0.9 %-5.4 %。以相对回收率表示准确度,日内的回收率是95 %-102 %,日间的回收率是102 %-119.5 %。提取回收率试验结果显示,该方法学ZS-1平均提取回收率是86.1 %,以上结果表明,该方法符合药动学的分析要求。ZS-1在体外血浆中稳定性结果显示,ZS-1在大鼠血浆中的含量随时间增加而降低,放置5 min时,其含量是原来的91.2 %,RSD是2.4 %。在35 min时,回收率是12.5 %,RSD是7.8 %。在35 min后,其含量已在分析方法的定量限和检测量之下。在-20℃反复冻融3次后,回收率是86.1 %,RSD是2.4 %,分解率为13.9 %。在-20℃放置一个月后,其回收率为回收率是92.7 %,RSD是8.0 %,未见显著性的分解。-80℃放置一个月后,其回收率为回收率是71.3 %,RSD是5.3 %,分解率为28.7 %。然而,ZS-1在乙腈和水混合介质中具有良好的稳定性,ZS-1在常温放置6小时后、-20℃反复冻融3次、-20℃放置一个月和-80℃放置一个月后,回收率在96.8 % - 103.0 %,均未见分解现象,具有较好的稳定性。分别将ZS-1以20 mg.kg-1、30 mg.kg-1、45 mg.kg-1尾静脉注射到大鼠体内,然后分别在0.5,3,8,12,15,20,30,40、45 min收集血浆样品,通过反相高效液相色谱定量血浆中ZS-1含量,药动学参数3P87软件计算,结果显示,血浆中的峰浓度Cmax和药时曲线下面积AUC随给药剂量增加而增加,药时曲线符合一级房室模型,3个剂量的半衰期没有显著性差异。当给药剂量为20 mg kg-1时,药时曲线下面积（AUC）、达峰血药浓度（Cmax）、半衰期（T1/2）、表观分布容积（V）、和总清除率（CL）分别是155.5±25.7μg.min.mL^-1,17.9±1.0μg.mL^-1,7.2±1.4 min,1117.1±20.2 mL.kg -1,143.5±28.0 mL.min-1.kg-1。当给药剂量为30 mg.kg^-1时,AUC、Cmax、T1/2、V、CL分别是307.5±57.5μg.min mL^-1,31.6±2.5μg.mL^-1,8.3±1.4 min,1187.2±150.1 mL.kg -1,102.6±28.8 mL min-1.kg-1。当给药剂量为45 mg.kg-1时,AUC、C_max、T_1/2、V、CL分别是573.3±60.2μg.min.mL^-1,47.4±3.1μg.mL^-1,8.8±1.7 min,652.4±68.0 mL.kg -1,75.6±8.2mL.min-1.kg-1。最后,利用反相高效液相色谱方法研究ZS-1在荷人肺鳞癌细胞株NCI-H1299的裸鼠模型体内的组织分布,各组织在0.1-50μg.mL^-1范围内标准曲线具有良好的相关性,各组织的提取回收率在60.4 %-85.1 %,RSD是2.3 %-7.7 %。当给药剂量为30 mg.kg-1尾静脉注射到荷瘤裸鼠体内,分别在5,10,15,20,25 min后,乙醚麻醉裸鼠,把肝、肾、脾、脑、肺、心和肿瘤组织取出并匀浆,在酸性条件下提取多肽ZS-1,通过HPLC测定ZS-1的含量,结果显示,在5 min时,除大脑组织外,都可见ZS-1的分布,ZS-1主要分布在心、肝、和肾组织。在10 min时,肺组织不见ZS-1的分布,肿瘤组织ZS-1的浓度是11.3±0.31μg.mL^-1这时,肿瘤组织中ZS-1的含量最高。在15 min时,ZS-1只分布在肿瘤、肝、肾、组织,其浓度分别为4.2±0.27μg.mL^-1、1.6±0.15μg.mL^-1、3.5±0.17μg.mL^-1。此时,肿瘤组织中ZS-1的含量仍然是最高。当到了20 min时,ZS-1只分布在肿瘤和肾组织,其浓度分别为1.3±0.05μg.mL^-1、0.9±0.05μg.mL^-1。25min时,在各组织中都检测不到ZS-1信号,因此,ZS-1对肺鳞癌组织具有较高的亲和力。综上结果表明,ZS-1在体外对肺癌具有较高的结合靶向性和组织特异性,其在体内具有较好的肺癌组织靶向性分布和稳定性,为进一步的药物靶向性载体研发和分子诊断影像造影剂研制奠定了基础。更多还原

【Abstract】 Lung cancer is one of the most common malignant tumors in the worldwide. Now, the morbidity and mortality of lung cancer are the highest in the world. For the past few years, the morbidity is hoicking in China, the five-year survival rate of lung cancer is less than 15 % because of the low diagnosis, most of the patients who are defined as lung cancer are often already in advanced stage or metastasis. Now, the treatment of lung cancer is mainly operation and combination chemotherapy. Many adverse reactions are generated because of the cytotoxic chemotherapy, so it results in bad tolerance, improving the early diagnosis and targeted treatment of lung cancer are the investigation trend in the world. ZS-1 is a peptide derived from a Phage-displayed peptide library, which is specifically bound to human non-small cell lung carcinoma cell, so, to study the targeted identification and pharmacokinetics of ZS-1 in this paper to provide the theory of a new drug targeted to lung cancer.First, the targeting and the speciality of the peptide were identified in this paper . immunofluorescence assay was used to identify the speciality of ZS-1 in two non-small cell lung cancer cells NCI-H1299 and A549, human normal liver cell line LO-2 and human lung embryo fibroblasts cell line WI-38 were control cells. The result showed that the polypeptide [ZS-1]^FITC had high affinity with lung squamous carcinoma cell line NCI-H1299 and lung adenocarcinoma cell line A549, but almost no affinity with normal liver cell line and lung cell line. Human ling cancer tissues chip was used to the speciality of ZS-1, positive was defined as the fluorescence intensity of lung cancer tissues was twice than the normal control tissues, the fluorescent scanning result showed that the positive rate of [ZS-1]^FITC was 37.3 %, the fluorescence intensity of eight control tissues was twice less than the lung cancer tissues, so the false-positive rate of [ZS-1]^FITC was 0 %, the result indicated that ZS-1 had high speciality with lung cancer tissues. [ZS-1]^FITC was administrated intravenously to NCI-H1299-bearing nude mouse, tumor, heart, liver, spleen, lung, kidney, cerebrum, and myeloid were made into frozen sections to observe the fluorescence intensity. Result showed that the fluorescence intensity of the tumor was the most, ZS-1 was mainly in the tumor tissue, second, ZS-1 was distributed to the liver and kidney, the fluorescence intensity of heart, spleen, ling, cerebrum and myeloid was very low. The result indicated that ZS-1 had high affinity with lung cancer tissues.On account of good target of ZS-1, the chapter is to study the pharmacokinetics of ZS-1 in rats to learn the stability and the metabolism, the results of methodology showed that no interference from endogenous substances was observed at the retention time of ZS-1 in rat plasma. Overall chromatographic running time was established at 40 min with the retention time of ZS-1 was 13.8 min, The calibration curve was linear over a concentration range of 0.1-50μg.mL^-1. The calibration curve was y = 19894 x -196.89, the determination coefficient （R2） was 1. The LOD of the assay defined as the concentration of ZS-1 for which the signal-to-noise ratio was 3:1 was 0.02μg.mL^-1. The overall precision, expressed as RSD, was < 10 %. RSD of intra-day was ranged from 1.5 % to 8.9 % and RSD of inter-day was ranged from 0.9 % to 5.4 %. Measurement accuracy ranged between 95.0 % and 102.0 % （intra-day） and between 102.0 % and 119.5 % （inter-day）. These results indicated precision and accuracy were acceptable for pharmacokinetic analysis of ZS-1. The mean extraction recovery of ZS-1 was 86.1 %. The stability result of ZS-1 in rat plasma in vitro showed that the concentration of ZS-1 in rat plasma decreased with time increasing, at 5 min, the recovery rate was 91.2 %, and the RSD was 2.4 %. at 35 min, the recovery rate was 12.5 %, and the RSD was 7.8 %, after 35 min, the concentration of ZS-1 in rat plasma was below the limit of the determination, at -20℃three freeze-thaw cycles, the recovery rate was 86.1 %, and the RSD was 2.4 %, the degradation rate of ZS-1 was 13.9 %, at -20℃for a month, the recovery rate was 92.7 %, and the RSD was 8.0 %, the significant degradation was not observed, at -80℃for a month, the recovery rate was 71.3 %, and the RSD was 5.3 %, the degradation rate of ZS-1 was 28.7 %. However, ZS-1 had good stability in the mixture of acetonitrile and water. The results indicated there was no significant degradation of ZS-1 under room temperature （25℃） for 2, 4, 6 h, three freeze-thaw cycles, -20℃for a month, and -80℃for three months. The accuracy of ZS-1 was between 96.8 % and 103.0 %. This demonstrated that ZS-1 had a good stability under the four conditions. ZS-1 was administered at a dose of 20 mg.kg-1, 30 mg.kg-1 and 45 mg.kg-1, respectively. Blood samples were collected at 0.5, 3, 8, 12, 15, 20, 30, 40 and 45 min. ZS-1 in rat plasma was measured by RP-HPLC. Pharmacokinetic data were estimated from measured ZS-1 plasma concentrations using 3P87. The result showed that the concentration-time curves of ZS-1 fitted well to one compartment model. Area under the concentration-time curves （AUC） increased with dose. Clearance rates （CL） and elimination half-lives （T1/2） had no significant difference between different dose groups. After administration 20 mg.kg-1 of ZS-1, AUC0-45, Cmax, T1/2, V, and CL were 155.5±25.7μg.min.mL^-1, 17.9±1.0μg.mL^-1, 7.2±1.4 min, 1117.1±20.2 mL.kg -1 and 143.5±28.0 mL.min-1.kg-1. After administration of 30 mg.kg-1, the respective values were 307.5±57.5μg.min.mL^-1, 31.6±2.5μg.mL^-1, 8.3±1.4 min, 1187.2±150.1 mL.kg -1 and 102.6±28.8 mL.min-1.kg-1. After administration of 45 mg.kg-1, the respective values were 573.3±60.2μg.min.mL^-1, 47.4±3.1μg.mL^-1, 8.8±1.7 min, 652.4±68.0 mL.kg -1 and 75.6±8.2 mL.min-1.kg-1, respectively.In the end, the distribution of ZS-1 in NCI-H1299-bearing nude mouse was studied by RP-HPLC, The calibration curve of tissues was linear over a concentration range of 0.1-50μg.mL^-1. The recovery of ZS-1 in tissues was between 60.4 % and 85.1 %, and the RSD was between 2.3 % and 7.7 %. After administration of 30 mg kg-1, the nude mouse was etherized at 5, 10, 15, 20, 25 min then the tumor, heart, spleen, lung, kidney and cerebrum were homogenated, ZS-1 in tissues were measured by RP-HPLC. The result showed that ZS-1 could be determined at 5 min except for cerebrum, ZS-1 was mainly distributed in heart, liver, kidney. At 10 min, ZS-1 could not be determined in lung, the concentration of ZS-1 in tumor was 11.3±0.31μg mL^-1, it was highest in tumor. At 15 min, ZS-1 could not be determined in lung, the concentration of ZS-1 in tumor was 11.3±0.31μg.mL^-1, it was highest in tumor. ZS-1 could only be determined in tumor, liver, kidney, the corresponding concentration of ZS-1 was 4.2±0.27μg.mL^-1, 1.6±0.15μg.mL^-1、3.5±0.17μg.mL^-1, it was also highest in tumor. At 20 min, ZS-1 could only be determined in tumor and kidney, the corresponding concentration of ZS-1 was 1.3±0.05μg.mL^-1, 0.9±0.05μg.mL^-1. At 25 min, the concentration of ZS-1 in all tissues was below the limit of determination, therefor, ZS-1 had high affinity with lung squamous carcinoma tissue.The results above indicate that in vitro the peptide ZS-1 has good targeting and tussue affinity, in vivo the peptide ZS-1 has good tussue targeting and stability. Therefor, it can be a basis for developing the targeting drug delivery and diagnosis agent of lung cancer.更多还原