Influence of a novel, versatile bifunctional chelator on theranostic properties of a minigastrin analogue

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 ⁶⁸Ga for positron emission tomography (PET) but also ¹⁷⁷Lu and ¹¹¹In 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.

An AAZTA derivative with an aliphatic C₉ chain as linker was coupled to a minigastrin, namely [AAZTA⁰, D-Glu¹, desGlu^2–6]-minigastrin (AAZTA-MG), and labelled with ⁶⁸Ga, ¹⁷⁷Lu and ¹¹¹In. 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.

AAZTA-MG showed high radiochemical yields for ⁶⁸Ga (>95 %), ¹⁷⁷Lu (>98 %) and ¹¹¹In (>98 %). The logD value of −3.7 for both [⁶⁸Ga]- and [¹⁷⁷Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [⁶⁸Ga]- and transchelation towards DTPA for and [¹⁷⁷Lu]-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 [⁶⁸Ga]-AAZTA-MG and >9.5 % for [¹⁷⁷Lu]-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 ⁶⁸Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The ¹¹¹In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [¹⁷⁷Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g).

Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both ⁶⁸Ga 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.