新型c-MET靶向小分子喹啉类探针18F-AZC PET/CT监测非小细胞肺癌C-MET TKI分子治疗敏感性的实验研究

Mesenchymal epithelial transition (c-MET) factor is a very important molecular target for therapeutic intervention. An abnormally activated c-Met promotes cell proliferation, survival, motility and invasion via ErbB3/PI3K/AKT signaling pathway. Amplification of c-Met is also a secondary resistance mechanism against EGFR TKI therapy. Until now, many molecular targeted agents for c-MET have been developed and evaluated in the clinical phase II-III trials. However, there is no technique available for noninvasive detecting c-MET accurately in vivo. In our pilot experiments, we designed and synthesized a series of small molecule c-MET inhibitors. Among them, 3,5-disubstituted and 3,5,7-trisubstituted quinolines are promising small molecule scaffolds to develop a molecular imaging probe with high binding affinity and selectivity to c–MET. In this proposal, on the basis of our pilot experiments, we aim to develop an 18F labeled positron emission tomography (PET) probe, 18F-AZC, with high specificity and affinity to c-MET by a one-step nucleophilic substitution reaction. Then we will use 18F-AZC/PET to further detect c-MET expression in vivo. Moreover, we will evaluate the efficacy of 18F-AZC/PET for monitoring monitoring the sensitivity to c-MET TKI therapy in NSCLC. In summary, the c-MET targeted PET probe is expected to be a valuable tool for management of c-MET targeted treatment of cancer patients, which will promote the rational use of the molecular targeted agents. Thus the imaging strategy developed in this proposal will have major implications for the improvement of the efficacy of c-MET targeted treatment.

肝细胞生长因子受体(HGF-R/c-MET)能激活ErbB3/PI3K/AKT信号途径，参与肿瘤增殖、侵袭和转移全过程，并引发对EGFR激酶抑制剂的耐药，是肺癌分子靶向治疗的关键靶点，相关研究已进入II/III期临床阶段。但目前尚缺乏一种在活体状态下，无创、直观、实时、准确检测c-MET表达的方法。本课题预实验成功设计合成了一类具有高靶向亲和力的小分子喹啉类ATP竞争性c-MET抑制剂，其特征性的3，5-双取代和3，5，7-叁取代喹啉骨架结构是理想的用于开发PET探针的前体化合物。本项目将在此基础上，通过氟化亲核取代一步法，构建18F标记的PET成像探针18F-AZC，建立活体检测肺癌c-MET表达的PET分子成像方法，并进一步明确其在监测非小细胞肺癌c-MET TKI分子治疗敏感性中的应用价值。该方法可指导个体化治疗方案的选择和实施，对切实提高肺癌分子靶向治疗疗效具有重大意义。

上皮间质转化因子（c-Met）在许多肿瘤表达，研发肿瘤成像的c-Met靶向PET探针，为肿瘤c-Met靶向治疗提供无创可视化工具。方法：本项目合成了具有高靶向的小分子喹啉类ATP竞争性c-MET抑制剂并命名为18F-AZC。选择不同的肺癌细胞（NCI-H1993，HCC-827和A549）和无c-Met表达的LNCaP肿瘤做阴性对照，体外细胞实验包括18F-AZC与c-Met在有/无抑制剂阻断的情况下的特异性结合和竞争实验。对c-Met阳性或阴性肺癌皮下移植瘤鼠进行microPET/CT成像和生物分布研究。此外，还进一步评估了18F-AZC在不同级别原位胶质瘤模型鼠的PET/CT成像、MRI显像和放射自显影，并进行相应的离体组织免疫荧光染色和H&E染色，以定性评估肿瘤组织中c-Met的表达。结果：探针18F-AZC合成简单，放射化学特征表征结果显示合成产率大于20%，比活度为52.9-75.8 GBq/µmol，放射化学纯度大于98%，且稳定好。体内细胞实验1h NCI-H1993、HCC-827和A549对18F-AZC细胞摄取率分别为10.19±1.25%、9.15±0.43%和5.23±0.81%。竞争抑制实验显示，18F-AZC的结合被过量的19F-AZC和c-Met抑制剂Crizonitib或Cabozantinib取代，并且IC50值为98.7±7.61 nM；18F-AZC在NCI-H1993皮下移植瘤中快速聚集，并具有良好的肿瘤/肌肉比（3.24±0.13），并且明显高于对照组LNCaP（1. 09 ± 0.11 %ID/g）（P = 0.006）和阻断组（19F-AZC、Crizonitib或Cabozantinib分别为1.06 ± 0.16 %ID/g，1.00 ± 0.08 %ID/g或0.99 ± 0.08 %ID/g）。此外，18F-AZC对不同等级、不同c-Met表达水平的原位胶质瘤显示出良好的识别能力，其在U87MG、U251MG和Hs683肿瘤组织中的积累量分别为4.52±1.00、4.47±1.05和3.17±0.46 ID%/g，高于周围脑组织（2.19±0.23、1.79±0.44和2.06±0.11 ID%/g）（p<0.05）。结论 18F-AZC可指导个体化治疗方案的选择和实施，对提高肿瘤分子靶向治疗具有重要意义。