SPECT Imaging of SST2-Expressing Tumors with (99m)Tc-Based Somatostatin Receptor Antagonists: The Role of Tetraamine, HYNIC, and Spacers

[(99m)Tc]Tc-HYNIC-TOC is the most widely used (99m)Tc-labeled somatostatin receptor (SST) agonist for the SPECT imaging of SST-expressing tumors, such as neuroendocrine tumors. Recently, radiolabeled SST antagonists have shown improved diagnostic efficacy over agonists. (99m)Tc-labeled SST antagonists are lacking in clinical practice. Surprisingly, when [(99m)Tc]Tc-HYNIC was conjugated to the SST2 antagonist SS01, SST2 imaging was not feasible. This was not the case when [(99m)Tc]Tc-N4 was conjugated to SS01. Here, we assessed the introduction of different spacers (X: β-Ala, Ahx, Aun and PEG(4)) among HYNIC and SS01 with the aim of restoring the affinity of HYNIC conjugates. In addition, we used the alternative antagonist JR11 for determining the suitability of HYNIC with (99m)Tc-labeled SST2 antagonists. We performed a head-to-head comparison of the N4 conjugates of SS01 and JR11. [(99m)Tc]Tc-HYNIC-TOC was used as a reference, and HEK-SST2 cells were used for in vitro and in vivo evaluation. EDDA was used as a co-ligand for all [(99m)Tc]Tc-HYNIC conjugates. The introduction of Ahx restored, to a great extent, the SST2-mediated cellular uptake of the [(99m)Tc]Tc-HYNIC-X conjugates (X: spacer), albeit lower than the corresponding [(99m)Tc]Tc-N4-conjugates. SPECT/CT images showed that all (99m)Tc-labeled conjugates accumulated in the tumor and kidneys with [(99m)Tc]Tc-HYNIC-PEG(4)-SS01, [(99m)Tc]Tc-N4-SS01 and [(99m)Tc]Tc-N4-JR11 having notably higher kidney uptake. Biodistribution studies showed similar or better tumor-to-non-tumor ratios for the [(99m)Tc]Tc-HYNIC-Ahx conjugates, compared to the [(99m)Tc]Tc-N4 counterparts. The [(99m)Tc]Tc-HYNIC-Ahx conjugates of SS01 and JR11 were comparable to [(99m)Tc]Tc-HYNIC-TOC as imaging agents. HYNIC is a suitable chelator for the development of (99m)Tc-labeled SST2 antagonists when a spacer of appropriate length, such as Ahx, is used.