The overexpression of cholecystokinin-2/gastrin receptor (CCK2R) on the cell surface of certain tumour types makes CCK2R a promising target for the development of radiolabelled ligands, which is in the last years still mainly focused on the optimization and development of radiolabelled agonists based on minigastrin (MG). The use of MG analogues is limited by the low tumour uptake and/or high kidney retention, and also the possible adverse side effects caused by the agonistic properties. The latter could be bypassed by the use of radiolaelled CCK2R antagonists; therefore, the main aim of the doctoral thesis was to design, synthesiszed and evaluate novel radiolabelled CCK2R antagonists. Additionally, by comparising such compounds with the radiolabelled agonists currently under clinical investigation (PP11), we sought to answer whether radiolabelled CCK2R antagonists represent suitable option for further development of radiolabelled CCK2R ligands. Novel radiolabelled CCK2R antagonists were designed based on the small-molecule CCK2R antagonist % nastorazepide/Z360, which was further functionalized with spacer and bifunctional chelator DOTA to ensure the theranostic approach. After the determination of the minimal length of the spacer determined by using in silico homology modelling and docking the literature reference compound was chosen. To optimize the reference compounds three approaches were undertaken: a) the extension of spacer by the introduction of polyethylene glycol chains of variable length, b) the identification of crucial aminoacid residues in the spacer region, and c) replacement of bifunctional chelator. For all synthesized and analytically evaluated (by RP-HPLC, ESI-MS, HRMS) compounds the antagonistic properties were determined in the functional cell-based test on the A431-CCK2R+ cell line. Afterwards, the compounds were successfully radiolabelled with different radionuclides e.g. gallium-68, indium-111 and lutetium-177. For radiolabelled compounds high stability in human serum and reasonably low serum protein binding were achieved, whereas lower logD7,4 values compared to reference compound were determined. Compared to the cell uptake of radiolabelled PP11 determined on the A431-CCK2R+ cell line, the selected radiolabelled CCK2R antagonists exhibited higher or comparable cell uptake and retention (at comparable values of the constant of dissociation (Kd)). For radiolabelled antagonists significantly higher Bmax values were achived compared to the radiolabelled agonists PP11. The differences observed between the radiolabelled antagonists and agonist in in vitro setting translated also to the differences in in vivo PET/CT imaging performed for gallium-68 radiolabelled compounds and biodistribution. The two selected antagonists radiolabelled with lutetium-177 had considerably higher tumour uptake compared to the [177Lu]Lu-PP11 uptake. Based on the results of the in vitro and in vivo testing, we can conclude that the radiolabelled CCK2R antagonists should be considered as suitable for further development of CCK2R radiolabelled compounds, as the agonistic behaviour does not seem to be prerequisite to achive high uptake in CCK2R expressing tumours.
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