[¹¹¹In-DOTA]Somatostatin-14 analogs as potential pansomatostatin-like radiotracers - first results of a preclinical study

In this study, we report on the synthesis, radiolabeling, and biological evaluation of two new somatostatin-14 (SS14) analogs, modified with the universal chelator DOTA. We were interested to investigate if and to what extent such radiotracer prototypes may be useful for targeting sst_1-5-expressing tumors in man but, most importantly, to outline potential drawbacks and benefits associated with their use.

AT1S and AT2S (DOTA-Ala¹-Gly²-c[Cys³-Lys⁴-Asn⁵-Phe⁶-Phe⁷-Trp⁸/DTrp ⁸ -Lys⁹-Thr¹⁰-Phe¹¹-Thr¹²-Ser¹³-Cys¹⁴-OH], respectively) were synthesized on the solid support and labeled with ¹¹¹In. The sst_1-5 affinity profile of AT1S/AT2S was determined by receptor autoradiography using [Leu⁸,d Trp²²,¹²⁵I-Tyr²⁵]SS28 as radioligand. The ability of AT2S to stimulate sst₂ or sst₃ internalization was qualitatively analyzed by an immunofluorescence-based internalization assay using hsst₂- or hsst₃-expressing HEK293 cells. Furthermore, the internalization of the radioligands [¹¹¹In]AT1S and [¹¹¹In]AT2S was studied at 37 °C in AR4-2J cells endogenously expressing sst₂. The in vivo stability of [¹¹¹In]AT1S and [¹¹¹In]AT2S was tested by high-performance liquid chromatography analysis of mouse blood collected 5 min after radioligand injection, and biodistribution was studied in normal mice. Selectively for [¹¹¹In]AT2S, biodistribution was further studied in SCID mice bearing AR4-2J, HEK293-hsst_2A ⁺, -hsst₃ ⁺ or -hsst₅ ⁺ tumors.

The new SS14-derived analogs were obtained by solid phase peptide synthesis and were easily labeled with ¹¹¹In. Both SS14 conjugates, AT1S, and its DTrp⁸ counterpart, AT2S, showed a pansomatostatin affinity profile with the respective hsst_1-5 IC₅₀ values in the lower nanomolar range. In addition, AT2S behaved as an agonist for sst₂ and sst₃ since it stimulated receptor internalization. The ¹¹¹In radioligands effectively and specifically internalized into rsst_2A-expressing AR4-2J cells with [¹¹¹In]AT2S internalizing faster than [¹¹¹In]AT1S. Ex vivo mouse blood analysis revealed a rapid degradation of both radiopeptides in the bloodstream with the DTrp⁸ analog showing higher stability. Biodistribution results in healthy mice were consistent with these findings with only [¹¹¹In]AT2S showing specific uptake in the sst₂-rich pancreas. Biodistribution of [¹¹¹In]AT2S in tumor-bearing mice revealed receptor-mediated uptake in the AR4-2J (1.82 ± 0.36 %ID/g - block 0.21 ± 0.17 %ID/g at 4 h post injection (pi)), the HEK293-hsst_2A ⁺ (1.49 ± 0.2 %ID/g - block 0.27 ± 0.20 %ID/g at 4 h pi), the HEK293-hsst₃ ⁺ (1.24 ± 0.27 %ID/g - block 0.32 ± 0.06 %ID/g at 4 h pi), and the HEK293-hsst₅ ⁺ tumors (0.41 ± 0.12 %ID/g - block 0.22 ± 0.006 %ID/g at 4 h pi). Radioactivity washed out from blood and background tissues via the kidneys.

This study has revealed that the native SS14 structure can indeed serve as a motif for the development of promising pansomatostatin-like radiotracers. Further peptide stabilization is required to increase in vivo stability and, consequently, to enhance in vivo delivery and tumor targeting.