锝-99m标记硝基咪唑-氨基酸配合物的构建及肿瘤双重靶向显像研究

Hypoxia is present in the vast majority of solid tumors and has been associated with an increased recurrence rate, tumor aggressiveness, metastatic spread. Thus cancer cells can become resistant to radiation treatment and chemotherapy. Non-invasive detection of tumor hypoxia is very important to determine diagnosis, prognosis and treatment options for tumors. Among the image-based modalities for hypoxia assessment, positron emission tomography (PET) radionuclide imaging is one of the most extensively investigated based on the various advantages it offers. But common hypoxia imaging agents such as 18F-MISO exist the following limitations: Lack of correlation in all tumors , low tumor-to-background ratio,slow plasma clearance, variable reproducibility. 64Cu-ATSM is evidence more limited compared to 18F-MISO.Less clear mechanism of uptake in tumor hypoxia compared to nitroimidazole compounds. Moreover, they are very expensive. This project is based on the growth of tumor cells can not synthesize asparagine and need glutamine to apply the ATP.We will use a high hypoxic selective 5-nitroimidazole as a precursor connected with asparagine or glutamine to form 5- nitroimidazole-amino acids complexes , and then labeled by 99mTc to obtain novel hypoxia molecular probes.To explore radionuclide labeling methods, biological identification, study the mechanism of cellular uptake, biodistribution properties of mice and hypoxia positioning performance, in order to get a relatively inexpensive, high tumor uptake and tumor/non-target tissue ratios of novel tumor dual targeted imaging agent for SPECT/CT.

乏氧是实体肿瘤的重要特征，是导致肿瘤复发、侵袭、转移的重要原因，也导致肿瘤对放化疗产生抵抗，因此，肿瘤乏氧区的无创性检测对其诊断、预后和治疗方案确定十分重要。PET核素显像是当前研究最为广泛的乏氧影像技术，但常用的乏氧显像剂如18F-MISO等存在着肿瘤/本底比值偏低、血浆清除慢、显像间隔时间长、价格贵等缺点。本项目基于肿瘤细胞不能自己合成生长必需的天冬酰胺以及需要谷氨酰胺供能，以有较高乏氧选择性的2-甲基-5-硝基咪唑为前体连接上述氨基酸，并进行结构修饰并用99mTc标记获得新型分子探针，探讨核素标记方法、进行生物学鉴定，研究其乏氧细胞选择性、生物分布性能、小鼠肿瘤乏氧定位性能，以期得到相对便宜、用于SPECT/CT、具有高肿瘤摄取和肿瘤/非靶组织比值的新型肿瘤双重靶向显像剂。

随着SPECT/CT的进展，研发99mTc标记的乏氧放射性药物很有必要。本课题成功设计一种新型5-硝基咪唑-天冬氨酸酰胺衍生物，同时自主合成了未在国内研究过的2-硝基咪唑代表BRU59-21进行对比研究。结果显示：1.两种配体化合物的NMR与MS分析显示与设计的结构一致，新型硝基咪唑天冬酰胺衍生物（代号NASn）采用99m[Tc(v)N]2+核标记，优化了NASn的放射性标记条件。获得两种理化性质稳定、制备相对容易的标记配合物。2.通过CCK-8、Wetern Blot和RT-PCR实验确定了A549肺腺癌细胞培养最佳的乏氧时间；通过体外细胞摄取实验，表明99mTc-NASn及99mTc-BRU59-21在乏氧细胞中摄取良好，约为常氧状态下的5倍以上。3.通过荷A549腺癌裸鼠生物分布实验表明99mTc-NASn及99mTc-BRU59-21亲脂性相近，通过肝肠排泄，虽然血液清除速率较慢，但肿瘤摄取及滞留时间较好，摄取高峰时99mTc-NASn瘤与肌肉摄取比值（T/M）为6.01，高于99mTc-BRU59-21的3.5。4.荷瘤裸鼠SPECT/CT显像可见肿瘤清晰显影，肿瘤与非靶比值（T/N）与生物分布的大致相符；标记物结构中含有两个乏氧还原基团以及适中的血液清除速度是决定肿瘤摄取和滞留的关键因素。5.病理免疫组化PIMO和HIF1α染色证实了构建的肿瘤模型的乏氧，通过相同肿瘤切片的放射自显影和免疫化学分析显示肿瘤内放射性摄取和PIMO染色阳性区域一致，证实了两种标记物均有良好的乏氧选择性，99mTc-NASn与PIMO染色的相关性更好。6.99mTc-NASn作为肿瘤乏氧放射性药物的候选较99mTc-BRU59-21具有更大的潜力，值得进一步研究。