Assessment of PSMA targeting ligands bearing novel chelates with application to theranostics: Stability and complexation kinetics of ⁶⁸Ga^{3 +}, ¹¹¹In^{3 +}, ¹⁷⁷Lu^{3 +} and ²²⁵Ac^{3 +}

Introduction Recent successes in the treatment of metastatic castration-resistant prostate cancer (mCRPCa) by systemic endoradiotherapy has sparked renewed interest in developing small molecule ligands targeting prostate-specific membrane antigen (PSMA) and chelators capable of stable complexation of metal radionuclides for imaging and therapy. As the size and coordination number of metals for imaging, such as ⁶⁸Ga^{3 +}, and for targeted therapy, such as ¹⁷⁷Lu^{3 +} and ²²⁵Ac^{3 +}, are substantially different, they may show a preference for macrocycles of different denticity. We have prepared three simple conjugates that target PSMA and form radiometal complexes through coordination by either octa-, deca-, or dodecadentate tetraazacyclododecane chelators. The complex formation and metal ion selectivity of these constructs were determined at two relevant temperatures, complex stability was examined in vitro, and tumor targeting was demonstrated in preclinical PCa models with a view towards identifying a candidate with potential value as a theranostic agent for the imaging and therapy of mCRPCa. Methods Three bifunctional chelates with high denticity, including the octadentate chelate DOTA, the decadentate 3p-C-DEPA and a novel dodecadentate analogue of DEPA, were synthesized and conjugated to a glutamate-urea-lysine (EuK) pharmacophore (EuK-DOTA, EuK-107 and EuK-106, respectively) to enable targeting of PSMA. The metal ion selectivity for each construct was determined by incubation at 25 °C and 95 °C with the trivalent radiometals ⁶⁸Ga^{3 +}, ¹¹¹In^{3 +}, ¹⁷⁷Lu^{3 +} and ²²⁵Ac^{3 +}. PSMA binding affinity was determined by competitive binding using LNCaP cells, while in vivo tumor targeting of the ⁶⁸Ga-labeled constructs was examined by positron emission tomography (PET) in LNCaP xenograft tumor-bearing mice. Results PMSA affinities (IC₅₀ values) were 13.3 ± 0.9 nM for EuK-DOTA, 18.0 ± 3.7 nM for EuK-107 and 42.6 ± 6.6 nM for EuK-106. EuK-107 and EuK-DOTA proved to rapidly and near quantitatively complex ⁶⁸Ga^{3 +}, ¹¹¹In^{3 +}, ¹⁷⁷Lu^{3 +} and ²²⁵Ac^{3 +} at 95 °C, with EuK-107 also rapidly complexing ¹¹¹In^{3 +} and ¹⁷⁷Lu^{3 +} at 25 °C. The inability of EuK-106 to chelate ¹⁷⁷Lu^{3 +} and ²²⁵Ac^{3 +} suggests that size of the cavity of the macrocylic ring may be more critical than the number of donor groups for the chelation of larger radiometals. In vivo, ⁶⁸Ga-EuK-107 proved to have similar uptake to ⁶⁸Ga-DKFZ-PSMA-617, a theranostic ligand currently in clinical evaluation, in a PSMA positive xenograft tumor model. Conclusions The broad metal ion selectivity, good in vitro affinity for PSMA and good in vivo tumor targeting suggest that EuK-107, with the 3p-C-DEPA chelator, merits further evaluation as a theranostics construct in prostate cancer.