Proof-of-Concept Study of the NOTI Chelating Platform: Preclinical Evaluation of 64 Cu-Labeled Mono- and Trimeric c(RGDfK) Conjugates.

We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for the preparation of 68 Ga- and 64 Cu-based radiopharmaceuticals. Based on this platform, the chelator scaffold NOTI-TVA with three additional carboxylic acid groups for bioconjugation was synthesized and characterized. The primary aims of this proof-of-concept study were (1) to evaluate if trimeric radiotracers on the basis of the NOTI-TVA 6 scaffold can be developed, (2) to determine if the additional substituents for bioconjugation at the non-coordinating NH atoms of the imidazole residues of the building block NOTI influence the metal binding properties, and (3) what influence multiple targeting vectors have on the biological performance of the radiotracer. The cyclic RGDfK peptide that specifically binds to the α v ß 3 integrin receptor was selected as the biological model system. Two different synthetic routes for the preparation of NOTI-TVA 6 were explored. Three c(RGDfK) peptide residues were conjugated to the NOTI-TVA 6 building block by standard peptide chemistry providing the trimeric bioconjugate NOTI-TVA-c(RGDfK) 3 9. Labeling of 9 with [ 64 Cu]CuCl 2 was performed manually at pH 8.2 at ambient temperature. Binding affinities of Cu-8, the Cu 2+ complex of the previously described monomer NODIA-Me-c(RGDfK) 8, and the trimer Cu-9 to integrin α v ß 3 were determined in competitive cell binding experiments in the U-87MG cell line. The pharmacokinetics of both 64 Cu-labeled conjugates [ 64 Cu]Cu-8 and [ 64 Cu]Cu-9 were determined by small-animal PET imaging and ex vivo biodistribution studies in mice bearing U-87MG xenografts. Depending on the synthetic route, NOTI-TVA 6 was obtained with an overall yield up to 58 %. The bioconjugate 9 was prepared in 41 % yield. Both conjugates [ 64 Cu]Cu-8 and [ 64 Cu]Cu-9 were radiolabeled quantitatively at ambient temperature in high molar activities of A m ~ 20 MBq nmol -1 in less than 5 min. Competitive inhibitory ...