BACKGROUND: 6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane (AAZTA ) is a promising chelator with potential advantages over 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiopharmaceutical applications. Its mesocyclic structure enables fast radiolabelling under mild conditions with trivalent metals including not only (68)Ga for positron emission tomography (PET) but also (177)Lu and (111)In for single-photon emission computed tomography (SPECT) and radionuclide therapy. Here, we describe the evaluation of a bifunctional AAZTA derivative conjugated to a model minigastrin derivative as a potential theranostic agent. METHODS: An AAZTA derivative with an aliphatic C9 chain as linker was coupled to a minigastrin, namely [AAZTA(0), D-Glu(1), desGlu(2-6)]-minigastrin (AAZTA-MG), and labelled with (68)Ga, (177)Lu and (111)In. The characterisation in vitro included stability studies in different media and determination of logD (octanol/PBS). Affinity determination (IC50) and cell uptake studies were performed in A431-CCK2R cells expressing the human CCK2 receptor. μPET/CT and ex vivo biodistribution studies were performed in CCK2 tumour xenograft-bearing nude mice and normal mice. RESULTS: AAZTA-MG showed high radiochemical yields for (68)Ga (>95 %), (177)Lu (>98 %) and (111)In (>98 %). The logD value of -3.7 for both [(68)Ga]- and [(177)Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [(68)Ga]- and transchelation towards DTPA for and [(177)Lu]-AAZTA-MG. An IC50 value of 10.0 nM was determined, which indicates a high affinity for the CCK2 receptor. Specific cell uptake after 60 min was >7.5 % for [(68)Ga]-AAZTA-MG and >9.5 % for [(177)Lu]-AAZTA-MG, comparable to other DOTA-MG-analogues. μPET/CT studies in CCK2 receptor tumour xenografted mice not only revealed high selective accumulation in A431-CCK2R positive tumours of (68)Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The (111)In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [(177)Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g). CONCLUSION: Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both (68)Ga and trivalent metals having advantages over the currently used chelator DOTA. Studies are ongoing to further investigate in vivo targeting properties and stability issues and the influence of spacer length on biodistribution of AAZTA.

CAGE Chemicals srl Novara Italy

Department of Nuclear Medicine Innsbruck Medical University Anichstrasse 35 A 6020 Innsbruck Austria

Division of Nuclear Medicine Istituto Nazionale Tumori Fondazione G Pascale IRCCS Napoli Italy

DSF Università del Piemonte Orientale A Avogadro Novara Italy

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc Olomouc Czech Republic

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Developing Targeted Hybrid Imaging Probes by Chelator Scaffolding