Novel nastorazepide-based radioligands demonstrate effective cholecystokinin-2 receptor targeting with potential for theranostic applications.

UNLABELLED: Radiolabeled gastrin antagonists are emerging as a new tool for the diagnosis and treatment of cholecystokinin-2 receptor (CCK2R) expressing tumors such as medullary thyroid carcinoma and small cell lung cancer, overcoming limitations of agonist-based ligands. In this study, the CCK2R antagonist nastorazepide (Z-360) was used as a targeting scaffold in combination with either a peptidic (NPP) or a hydrophilic polysaccharide (NPS) linker and the chelators DOTA and DOTAGA, yielding four novel radioligands. These were evaluated alongside the reference minigastrin analogs DOTA-PP-F11 and DOTA-MGS5. METHODS: All ligands were synthetized via solid-phase peptide synthesis and labeled with In-111 and Lu-177. In vitro characterization included receptor affinity, hydrophilicity and cellular uptake in A431-CCK2R. In vivo biodistribution and imaging were performed in A431-CCK2R xenografts. [177Lu]Lu-DOTAGA-NPP was further assessed in a proof-of-concept therapy study and metabolic stability assays. RESULTS: 111In-labeled antagonists showed 3-4-fold higher site recognition than [111In]In-DOTA-PP-F11 and nanomolar affinities comparable to the reference ligand. Among the tested ligands, [111In]In-DOTAGA-NPP demonstrated the highest cellular uptake (22.0 ± 0.2%), approximately twice that of the other ligands and moderately higher than [111In]In-DOTA-PP-F11 (16.2 ± 1.8%) after 4 h at 37 °C. In vivo, all 177Lu-labeled antagonists demonstrated high tumor uptake (range: 22.98 ± 7.04 - 29.00 ± 3.30% I.A./g tissue) with [177Lu]Lu-DOTAGA-NPP exhibiting the most favorable tumor-to-tissue ratios. Compared with [177Lu]Lu-DOTA-MGS5, [177Lu]Lu-DOTAGA-NPP showed rapid tumor accumulation with faster washout but reduced stomach exposure and high in vivo stability. CONCLUSION: [¹⁷⁷Lu]Lu-DOTAGA-NPP emerged as the most promising lead compound, combining high tumor uptake, favorable tumor-to-organ ratios, and excellent in vivo stability. Although no significant therapeutic efficacy was observed in the tested xenograft model, the results support [177Lu]Lu-DOTAGA-NPP as a promising lead for optimization. Overall, these findings highlight the potential of Z-360-based CCK2R antagonists as scaffolds for next-generation theranostic radioligands and support their development as alternatives to minigastrin-based tracers.