Ma Lixin, Yu Ping, Veerendra Bhadrasetty, Rold Tammy L, Retzloff Lauren, Prasanphanich Adam, Sieckman Gary, Hoffman Timothy J, Volkert Wynn A, Smith Charles J
Department of Radiology, The Radiopharmaceutical Sciences Institute, Columbia, MO, USA.
Mol Imaging. 2007 May-Jun;6(3):171-80.
Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.
胃泌素释放肽（GRP）受体在几种人类癌细胞上过度表达，包括乳腺癌、前列腺癌、小细胞肺癌和胰腺癌。蛙皮素（BBN）是一种14个氨基酸的肽，是人类GRP的类似物，以非常高的亲和力和特异性与GRP受体结合。本研究的目的是基于BBN开发一种新的荧光探针，该探针具有高肿瘤摄取率和最佳药代动力学，用于人类乳腺癌组织的特异性靶向和光学成像。在本研究中，采用固相肽合成法制备了具有以下通用序列的H(2)N-甘氨酰甘氨酰甘氨酸-BBN[7-14]NH(2)肽：H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2))。该缀合物通过反相高效液相色谱法纯化，并通过电喷雾电离质谱进行表征。荧光探针Alexa Fluor 680-G-G-G-BBN[7-14]NH(2)缀合物是通过在二甲基甲酰胺（DMF）中将Alexa Fluor 680琥珀酰亚胺酯与H(2)N-G-G-G-BBN[7-14]NH(2)反应制备的。以(125)I-Tyr(4)-BBN作为放射性标记金标准的体外竞争性结合试验表明，在人T-47D乳腺癌细胞中，抑制浓度50%值为7.7±1.4 nM。Alexa Fluor 680-G-G-G-BBN[7-14]NH(2)在人T-47D乳腺癌细胞中的共聚焦荧光显微镜图像表明，该荧光生物探针在体外具有特异性摄取、内化和受体阻断作用。在携带异种移植T-47D乳腺癌病变的SCID小鼠中的体内研究表明，这种新的缀合物能够以高选择性和亲和力特异性靶向肿瘤组织。
