Spacer effects on in vivo properties of DOTA-conjugated dimeric [Tyr3]octreotate peptides, synthesized via a 'Cu(I)-Click' and 'sulfo-Click' ligation method

Objectives: The somatostatin analog [Tyr3]octreotate, has a high binding affinity for somatostatin receptor (SSTR) expressing tumor cells and is used for the diagnosis and treatment of a variety of neuroendocrine tumors. Preclinical studies with RGD peptides have shown that dimeric analogs have an increased tumor uptake compared to the monomeric analog. We prepared a series of four dimeric [Tyr3]octreotate analogs, with different spacer lengths (9-, 19-, 41-, and 57-atoms) between the peptides, determined their SSTR-binding affinity and evaluated their in vivo SSTR targeting properties. Methods: The DOTA-conjugated mono- and dimeric [Tyr3]octreotate peptides were synthesized via a two-step “click” ligation method, using the facile Cu(I)-catalyzed 1,3-dipolar cycloaddition ('Click' reaction) and metal-free thio acid/sulfonyl azide amidation ('sulfo-Click' reaction). Subsequently, these compounds were radiolabeled with 111In. SSTR-binding characteristics were determined in a competitive binding assay using AR42J cells and the biodistribution of the 111In-labeled compounds was determined in BALB/c nude mice with s.c. AR42J tumors at 1, 4 and 24 h p.i. Results: The two-step “click” ligation procedure yielded mono- and dimeric [Tyr3]octreotate analogs conjugated with DOTA via a triazole-sulfonamide linker. The IC50 values of these DOTA-conjugated [Tyr3]octreotate analogs were comparable, and internalization studies showed that 111In-DOTA-labeled [Tyr3]octreotate (9-atoms) dimer had rapid and high receptor binding. Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the 111In-labeled [Tyr3]octreotate (9- atoms) dimer had a high tumor uptake at 1 h p.i. (38.8 ± 8.3 %ID/g), and excellent tumor retention at 4 h p.i. (40.9 ± 2.5 %ID/g). However, the introduction of the extended hydrophilic 57-atoms spacer led to a rapid clearance from the circulation, which limited tumor accumulation of the radiotracer (21.4 ± 4.9 %ID/g at 1 h p.i.). Conclusions: We have developed a versatile synthesis and systemically investigated the binding properties of a series of dimeric [Tyr3]octreotate analogs and evaluated the effects of different spacers between two cyclic [Tyr3]octreotate sequences on in vitro characteristics. In vivo study showed that the 111In-labeled dimeric [Tyr3]octreotate analog with 9-atom spacer had a high tumor uptake (38.8 ± 8.3 %ID/g) at 1 h p.i., which was maintained at 4 p.i. (40.9 ± 2.5 %ID/g). However, the introduction of a long hydrophilic 57-atom spacer in our dimeric [Tyr3]octreotate design resulted in higher kidney uptake and faster clearance from the circulation, with concomitant lower tumor accumulation (21.36 ± 4.87 %ID/g at 1 h p.i.). These findings provide important information about spacer length effects of dimeric [Tyr3]octreotate peptides on tumor targeting and pharmacokinetics, which can be translated into future research towards applications of [Tyr3]octreotate-based conjugates.