Giblin Michael F, Sieckman Gary L, Shelton Tiffani D, Hoffman Timothy J, Forte Leonard R, Volkert Wynn A
Research Service, Harry S. Truman Memorial Veterans Administration Hospital, Columbia, MO 65201, USA.
Nucl Med Biol. 2006 May;33(4):481-8. doi: 10.1016/j.nucmedbio.2006.01.009. Epub 2006 May 2.
The human E. coli heat-stable enterotoxin (ST(h), amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. Analogs of ST(h) are currently being used as vectors targeting human colon cancers. Previous studies in our laboratory have focused on development of 111Indium-labeled ST(h) analogs for in vivo imaging applications. Here, we extend the scope of this work to include targeting of the therapeutic radionuclides 90Y and 177Lu. The peptide DOTA-F19-ST(h)(1-19) was synthesized using conventional Fmoc-based solid-phase techniques and refolded in dilute aqueous solution. The peptide was purified by RP-HPLC and characterized by MALDI-TOF MS and in vitro receptor binding assay. The DOTA-conjugate was metallated with nonradioactive Lu(III)Cl3 and Y(III)Cl3, and IC50 values of 2.6+/-0.1 and 4.2+/-0.9 nM were determined for the Lu- and Y-labeled peptides, respectively. 177Lu(III)Cl3 and 90Y(III)Cl3 labeling yielded tracer preparations that were inseparable by C18 RP-HPLC, indicating that putative differences between Lu-, Y- and In coordination spheres are not observed in the context of labeled ST(h) peptides. In vivo biodistribution studies of the 177Lu-labeled peptide in severe combined immunodeficient (SCID) mice bearing T-84 human cancer tumor xenografts showed rapid clearance from the bloodstream, with >90 %ID in the urine at 1 h pi. Localization of the tracer within tumor xenografts was 1.86+/-0.91 %ID/g at 1 h pi, a value higher than for all other tissues with the exception of kidney (2.74+/-0.24 %ID/g). At 24 h pi, >98 %ID was excreted into the urine, and 0.35+/-0.23 %ID/g remained in tumor, again higher than in all other tissues except kidney (0.91+/-0.46 %ID/g). Biodistribution results at 24 h pi for the 90Y-labeled peptide mirrored those for the 177Lu analog, in agreement with the identical behavior of the labeled analogs by C18 RP-HPLC. These results demonstrate the ability of 177Lu- and 90Y-labeled ST(h) molecules to specifically target GC-C receptors expressed on T-84 human colon cancer cells.
人源大肠杆菌热稳定肠毒素（ST(h)，氨基酸序列为N1SSNYCCELCCNPACTGCY19）特异性结合鸟苷酸环化酶C（GC-C）受体，该受体在正常肠上皮细胞的顶端表面以及人结肠癌细胞表面高密度存在。ST(h)类似物目前正被用作靶向人结肠癌的载体。我们实验室之前的研究集中于开发用于体内成像应用的111铟标记的ST(h)类似物。在此，我们将这项工作的范围扩展至包括靶向治疗性放射性核素90钇和177镥。使用基于Fmoc的传统固相技术合成肽DOTA-F19-ST(h)(1-19)，并在稀水溶液中重折叠。该肽通过反相高效液相色谱（RP-HPLC）纯化，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和体外受体结合试验进行表征。用非放射性的Lu(III)Cl3和Y(III)Cl3对DOTA缀合物进行金属化，分别测定Lu标记肽和Y标记肽的半数抑制浓度（IC50）值为2.6±0.1 nM和4.2±0.9 nM。177Lu(III)Cl3和90Y(III)Cl3标记产生的示踪剂制剂通过C18 RP-HPLC无法分离，表明在标记的ST(h)肽的情况下未观察到Lu、Y和In配位球之间的假定差异。在携带T-84人癌肿瘤异种移植物的严重联合免疫缺陷（SCID）小鼠中对177Lu标记肽进行的体内生物分布研究表明，其从血液中快速清除，在注射后1小时尿液中放射性占注入剂量的百分比（%ID）>90%。在注射后1小时，示踪剂在肿瘤异种移植物中的定位为1.86±0.91 %ID/g，该值高于除肾脏（2.74±0.24 %ID/g）外的所有其他组织。在注射后24小时，>98 %ID排泄到尿液中，肿瘤中残留0.35±0.23 %ID/g，再次高于除肾脏（0.91±0.46 %ID/g）外的所有其他组织。90Y标记肽在注射后24小时的生物分布结果与177Lu类似物的结果相似，这与通过C18 RP-HPLC显示的标记类似物的相同行为一致。这些结果证明了177Lu和90Y标记的ST(h)分子能够特异性靶向T-84人结肠癌细胞上表达的GC-C受体。
