Background: 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 sst1-5-expressing tumors in man but, most importantly, to outline potential drawbacks and benefits associated with their use.
Methods: AT1S and AT2S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-Phe6-Phe7-Trp8/DTrp8-Lys9-Thr10-Phe11-Thr12-Ser13-Cys14-OH], respectively) were synthesized on the solid support and labeled with 111In. The sst1-5 affinity profile of AT1S/AT2S was determined by receptor autoradiography using [Leu8,dTrp22,125I-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-hsst2A+, -hsst3+ or -hsst5+ tumors.
Results: 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 hsst1-5 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 rsst2A-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-hsst2A+ (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.
Conclusions: 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.