Unnatural Amino Acid Substitutions to Improve In Vivo GRPR-Targeting Capability of [68Ga]Ga-SP01011 for Cancer Imaging

Gastrin-releasing peptide receptor (GRPR) shows elevated expression in several malignancies and represents an attractive target in cancer imaging and radioligand therapy. However, improving in vivo stability without compromising high tumor uptake and low pancreatic uptake remains a key challenge for the development of GRPR-targeted radiopharmaceuticals. In the present study, we investigated whether incorporating unnatural amino acid substitutions at potential cleavage sites of the previously reported GRPR-targeted agonist [68Ga]Ga-SP01011 (Ga-DOTA-Pip-d-Phe-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Ac6c14-NH2; Ac6c: 1-amino-1-cyclohexanecarboxylic acid) could enhance in vivo stability without compromising tumor-targeting capability. SP01012, SP01013, and SP01019 incorporating NMe-His12, Ala(tBu)13 and Tle10/NMe-His12 substitutions, respectively, were synthesized by Fmoc solid-phase peptide synthesis and obtained in 22-60% yield. Their corresponding nonradioactive Ga complexes were obtained in 58-89% yield by reacting the DOTA-conjugated precursors with excess GaCl3 in acetate buffer, and the products were subsequently purified by HPLC. In vitro competition binding assays demonstrated that the Ki values of Ga-SP01012, Ga-SP01013 and Ga-SP01019 were 1.36 ± 0.12, 11.5 ± 0.89 and 2.29 ± 0.77 nM, respectively. All three ligands retained agonistic characteristics as confirmed by the calcium release assay. Their 68Ga-labeled analogs were obtained by reacting the precursors with [68Ga]GaCl3 in HEPES buffer with microwave heating (100 °C, 1 min). Following HPLC purification, 68Ga-labeled tracers were obtained in 33-47% decay-corrected radiochemical yield with radiochemical purities >95%. PET/CT imaging together with ex vivo biodistribution analyses conducted at 1 h postinjection in PC-3 tumor-bearing mice demonstrated clear tumor visualization and predominantly renal excretion for all three tracers. Among them, [68Ga]Ga-SP01012 containing the NMe-His12 substitution alone, had the most favorable profile, with the highest tumor uptake (15.8 ± 0.65% ID/g), low pancreas uptake (1.27 ± 0.32% ID/g), and improved in vivo plasma stability (78.1 ± 0.83% intact tracer at 15 min postinjection compared to 63.5 ± 6.24% for [68Ga]Ga-SP01011). In contrast, [68Ga]Ga-SP01019, containing both Tle10 and NMe-His12 substitutions, showed the highest plasma stability (88.3 ± 2.90% intact tracer at 15 min postinjection), but this was accompanied by lower tumor uptake (12.4 ± 1.54% ID/g) and markedly increased pancreas uptake (23.7 ± 3.47% ID/g). [68Ga]Ga-SP01013, containing the Ala(tBu)13 substitution, showed reduced GRPR binding affinity and lower tumor uptake (8.33 ± 1.41% ID/g). Our findings indicate that [68Ga]Ga-SP01012 is a potential PET tracer for visualizing GRPR-expressing tumors, and the NMe-His12 substitution is a favorable modification for the design of GRPR-targeted agonist-based radiopharmaceuticals.