In Vitro Evaluation of the Squaramide-Conjugated Fibroblast Activation Protein Inhibitor-Based Agents AAZTA(5).SA.FAPi and DOTA.SA.FAPi

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA(5m).SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [(68)Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA(5) chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA(5).SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA(5).SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC(50) values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA(5).SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA(5).SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.