AF Medicina Nucleare, Istituto Nazionale Tumori, Fondazione G. Pascale, Via M. Semmola, 80131 Naples, Italy.
Eur J Nucl Med Mol Imaging. 2011 Aug;38(8):1417-25. doi: 10.1007/s00259-011-1816-y. Epub 2011 Apr 20.
Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COST Action BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the present study, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project.
Determination of IC(50) values was performed using autoradiography, with DOTA-peptides displacing (125)I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using (111)In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (K(d)) and apparent number of binding sites (B(max)) were determined. Internalization was determined in receptor-expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface-bound peptide was then stripped either by acid wash or subsequent incubation with 1 μM unlabelled peptide at 4°C.
All peptides showed high receptor affinity with IC(50) values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with K(d) values in the 10(-9)-10(-8) M range. B(max) values estimated in A431-CCK2R cells ranged from 0.6 to 2.2 × 10(6) per cell. All peptides showed high levels of internalization when incubated at 37°C.
All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue.
胆囊收缩素 2（CCK2）/胃泌素受体在几种神经内分泌来源的肿瘤中特异性过表达。在这些癌症类型中，有些类型，如甲状腺髓样癌（MTC），仍然缺乏敏感的诊断方法，并且需要为不可手术的病变提供治疗选择。肽受体放射性核素治疗（PRRT）可能是这些患者治疗管理的可行策略。近年来已经描述了几种 CCK2R 靶向放射性药物。作为欧盟 COST 行动 BM0607 的一部分，我们研究了 12 个 1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）-缀合的 CCK2R 结合肽的体外和体内特性。在本研究中，我们分析了结合和内化特性。通过项目中涉及的其他中心同时进行了稳定性、生物分布和成像研究。
使用放射性自显影术测定 IC（50）值，用 DOTA-肽置换组织切片上的（125）I-CCK 与来自人类肿瘤的受体。使用（111）In 标记的肽进行饱和结合和内化实验。使用大鼠 AR42J 细胞系和转染人 A431-CCK2R 的细胞系进行体外实验；测定解离常数（K（d））和表观结合位点数（B（max））。通过在 37°C 和 4°C 下孵育不同时间（最长 120 分钟），用示踪量的肽孵育来确定受体表达细胞中的内化。然后通过酸洗涤或随后在 4°C 下用 1 μM 未标记的肽孵育来除去表面结合的肽。
所有肽均表现出高受体亲和力，IC（50）值在 0.2 至 3.4 nM 范围内。饱和实验也显示出 K（d）值在 10（-9）-10（-8）M 范围内的高亲和力。在 A431-CCK2R 细胞中估计的 B（max）值范围为每个细胞 0.6 至 2.2×10（6）。当在 37°C 下孵育时，所有肽均表现出高水平的内化。
正如本期其他文章所述，所有 DOTA 缀合的肽均表现出高受体结合和内化特性，似乎适合进一步表征。
