新型靶向VPAC1高亲和力多肽的结直肠癌分子显像应用基础研究

The exact clinical stages of colorectal cancer is closely related to selection of therapeutic regimen and prognosis,which is one of the most difficult problems.At present,the detection sensitivity for early metastasis and relapse of colorectal cancers by conventional imaging has obvious limitation.Similarly, FDG PET imaging sometimes shows false positive results and radioimmunoimaging has low sensitivity and T/NT ratio. Research has shown that the expression of VPAC1 on colorectal cancers is about 22.5～57.9 times more than that in normal tissues,demonstrating that VPAC1 is a potential target for diagnosis and therapy of CRC.However,the existing VIP receptor imaging agents not only have potential risks of promoting cancers proliferation,but also lack the high selectivity toward VPAC1 receptor.And basised on the high-affinity dodecapeptide (VP2) specifically binding to VPAC1 receptor which was obtained using phage peptide display,this study program will further identify the biological activity of this peptide, label it with 99mTc with modification of G(D)AGG-Aba, study the physicochemical, receptor binding in vitro and in vivo,biology characteristic in vivo and imaging of radiopeptide in normal animals and models of inflammation and tumors.We want to explore the imaging mechanism and feasibility of 99mTc-G(D)AGG-Aba-VP2 as the novel molecular probe for colorectal cancers,to open up a new way for molecular imaging diagnosis of cancers expressing VPAC1 receptor.

结直肠癌(CRC) 准确的临床分期与治疗方案选择及预后密切相关，是临床亟待解决的难题之一。目前，常规影像学探测CRC早期转移和复发的敏感性具有明显局限性；FDG代谢显像存在假阳性；放免显像T/NT偏低，敏感性低。血管活性肠肽1型受体（VPAC1）在CRC细胞表达为正常细胞22.5～57.9倍，是一理想分子影像靶标。现有VIP受体显像剂缺乏针对VPAC1的高选择性，且VIP具有潜在促肿瘤增殖风险。本课题拟在前期利用噬菌体展示肽库技术筛选得到靶向VPAC1高亲和力十二肽（VP2）的基础上，鉴定其生物学活性；通过对其进行G(D)AGG-Aba修饰并实现99mTc间接标记，研究标记多肽理化性质、体内外受体结合特性、体内动力学与生物分布，正常、炎症模型与荷瘤模型动物显像。探索99mTc-G(D)AGG-Aba-VP2作为VPAC1受体分子显像剂的可行性及机制，拓展VPAC1阳性肿瘤分子显像新途径。

血管活性肠肽1型受体（VPAC1）在CRC细胞表达为正常细胞22.5～57.9倍，是一理想分子影像靶标。现有VIP受体显像剂缺乏针对VPAC1的高选择性，且VIP具有潜在促肿瘤增殖风险。本项目在前期利用噬菌体展示肽库技术筛选得到靶向VPAC1高亲和力十二肽（GFRFGALHEYNS，VP2）的基础上，通过对其氨基末端、羧基末端进行G(D)AGG-Aba修饰，鉴定其生物活性并实现99mTc间接标记，研究标记多肽理化性质、正常动物体内示踪动力学与生物分布，正常及炎症模型动物显像。结果：①VP2和TPN对HCT116细胞无明显促增殖及侵袭作用；②建立99mTc-TPN、99mTc-TPC最佳标记条件，标记率均＞95%；③初步制备一步法标记试剂盒，4℃保存12周，标记率仍＞90%；④99mTc-TPN、99mTc-TPC室温放置4h后的RCP分别为95.1%与92.6%，与血清蛋白的结合率分别为6.6%、5.8%，半胱氨酸置换实验未结合99mTc含量无明显增加，脂/水分配系数lgP分别为－（1.76±0.02）与－（1.82±0.02），标记溶液中标记多肽成分单一；⑤99mTc-TPN、99mTc-TPC健康家兔体内示踪动力学符合权重为1/C的二室模型，t1/2α为3.2min与4.3min，t 1/2β为61.9min与111.6min；⑥正常体内分布实验和/或SPECT显像示，血液放射性清除迅速，颈部及胃区未见异常放射性浓聚，脑部呈低放射性分布，体内放射性主要通过肾脏排泄，少量经肝胆分泌；⑦炎症模型显像示病灶放射性分布虽较正常对侧增加，但随时间延长逐渐减低，注射99mTc-TPN后0.5、1.0、3.0、5.0h，T/NT比值分别为2.19±0.12、2.17±0.11、2.02±0.15、1.68±0.21。现有研究表明：本项目制备的99mTc-TPN（TPC）方法简便，标记率高，勿需分离纯化可直接应用，易于制备一步法试剂盒，便于推广应用；体内外稳定性好，体内动力学性质优良。为后续开展VPAC1阳性肿瘤显像实验研究奠定了坚实的基础。