Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors

Abstract

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH(2)-CO-c(DCys-Phe-Tyr-DAgl(8)(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst(3)-ODN-8) and a sst(2)-selective antagonist (sst(2)-ANT)], for labeling with (111/nat)In. (111/nat)In-DOTA-sst(3)-ODN-8 and (111/nat)In-DOTA-[4-NO(2)-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH(2)] ((111/nat)In-DOTA-sst(2)-ANT) showed high sst(3)- and sst(2)-binding affinity, respectively. They did not trigger sst(3) or sst(2) internalization but prevented agonist-stimulated internalization. (111)In-DOTA-sst(3)-ODN-8 and (111)In-DOTA-sst(2)-ANT were injected intravenously into mice bearing sst(3)- and sst(2)-expressing tumors, and their biodistribution was monitored. In the sst(3)-expressing tumors, strong accumulation of (111)In-DOTA-sst(3)-ODN-8 was observed, peaking at 1 h with 60% injected radioactivity per gram of tissue and remaining at a high level for >72 h. Excess of sst(3)-ODN-8 blocked uptake. As a control, the potent agonist (111)In-DOTA-[1-Nal(3)]-octreotide, with strong sst(3)-binding and internalization properties showed a much lower and shorter-lasting uptake in sst(3)-expressing tumors. Similarly, (111)In-DOTA-sst(2)-ANT was injected into mice bearing sst(2)-expressing tumors. Tumor uptake was considerably higher than with the highly potent sst(2)-selective agonist (111)In-diethylenetriaminepentaacetic acid-[Tyr(3),Thr(8)]-octreotide ((111)In-DTPA-TATE). Scatchard plots showed that antagonists labeled many more sites than agonists. Somatostatin antagonist radiotracers therefore are preferable over agonists for the in vivo targeting of sst(3)- or sst(2)-expressing tumors. Antagonist radioligands for other peptide receptors need to be evaluated in nuclear oncology as a result of this paradigm shift.

Fig. 1.

In vitro characteristics of somatostatin analogs. (A) Structure of the sst₃ antagonist DOTA-sst₃-ODN-8. (B) Effects of somatostatin analogs on forskolin-stimulated cAMP accumulation in CCL-sst₃ cells. Concentration-response curves with increasing concentrations of SS-28 (○), sst₃-ODN-8 (▴), DOTA-sst₃-ODN-8 (▾), or ^natIn-DOTA-sst₃-ODN-8 (♦), and of increasing concentrations of SS-28 in the presence of 10⁻⁶ M DOTA-sst₃-ODN-8 (□) or 10⁻⁶ M ^natIn-DOTA-sst₃-ODN-8 (▵). Data are expressed as a percentage of the 10 μM forskolin response. SS-28 inhibits forskolin-stimulated cAMP formation in CCL-sst₃ cells, whereas the sst₃-ODN-8 derivatives alone have no effect; however, they reverse the SS-28-induced effect on cAMP. (C) Effect of soma tostatin analogs on sst₃ internalization detected by immunofluorescence in HEK-sst₃ cells. Control experiment showing membrane-bound sst₃ with no peptide (a); 100 nM of the agonists SS-28 (b), or ^natIn-DOTA-NOC (c) trigger sst₃internalization. The antagonist DOTA-sst₃-ODN-8 (d) or its ^natIn-derivative (e) at 10 μM are not able to induce internalization. Internalization triggered by 100 nM of SS-28 is abolished by 10 μM ^natIn-DOTA-sst₃-ODN-8 (f). (D) Scatchard plots from saturation-binding experiments on HEK-sst₃ cells show a higher B_max for ¹¹¹In-DOTA-sst₃-ODN-8 than for ¹¹¹In-DOTA-NOC.

Fig. 2.

In vivo scans taken 30 min and 4 h after injection of ¹¹¹In-DOTA-sst₃-ODN-8 or ¹¹¹In-DOTA-NOC. Each mouse was bearing two tumors, an sst₃-expressing tumor and, as control, an sst₂-expressing tumor. Mice were placed directly on one head of a two-headed gamma-camera system (PRISM 2000, Philips, Eindhoven, The Netherlands) equipped with medium-energy collimators (acquisition time, 10 min per time point). Strong uptake is seen with the antagonist radioligand in the sst₃-expressing tumor exclusively, whereas weak uptake was found in both tumors with the sst₂/sst₃ agonist.

Fig. 3.

Effect of somatostatin analogs on sst₂ internalization detected by immunofluorescence with R2–88 in HEK-sst₂ cells. Compared with control with no peptide (a), 100 nM DTPA-TATE (b) triggers strong sst₂ internalization. ^natIn-DOTA-sst₂-ANT (10 μM) (c) does not induce sst₂ internalization but abolishes internalization induced by 100 nM DTPA-TATE (d).