Purpose

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 ⁶⁸Ga- and ⁶⁴Cu-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_vß₃ integrin receptor was selected as the biological model system.

Procedures

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 [⁶⁴Cu]CuCl₂ was performed manually at pH 8.2 at ambient temperature. Binding affinities of Cu-8, the Cu²⁺ complex of the previously described monomer NODIA-Me-c(RGDfK) 8, and the trimer Cu-9 to integrin α_vß₃ were determined in competitive cell binding experiments in the U-87MG cell line. The pharmacokinetics of both ⁶⁴Cu-labeled conjugates [⁶⁴Cu]Cu-8 and [⁶⁴Cu]Cu-9 were determined by small-animal PET imaging and ex vivo biodistribution studies in mice bearing U-87MG xenografts.

Results

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 [⁶⁴Cu]Cu-8 and [⁶⁴Cu]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 constants IC₅₀ of c(RDGfK) 7, Cu-8, and Cu-9 were determined to be 159.5 ± 1.3 nM, 256.1 ± 2.1 nM, and 99.5 ± 1.1 nM, respectively. In small-animal experiments, both radiotracers specifically delineated α_vß₃ integrin-positive U-87MG tumors with low uptake in non-target organs and rapid blood clearance. The trimer [⁶⁴Cu]Cu-9 showed a ~ 2.5-fold higher tumor uptake compared with the monomer [⁶⁴Cu]Cu-8.

Conclusions

Functionalization of NOTI at the non-coordinating NH atoms of the imidazole residues for bioconjugation was straightforward and allowed the preparation of a homotrimeric RGD conjugate. After optimization of the synthesis, required building blocks to make NOTI-TVA 6 are now available on multi-gram scale. Modifications at the imidazole groups had no measurable impact on metal binding properties in vitro and in vivo suggesting that the NOTI scaffold is a promising candidate for the development of ⁶⁴Cu-labeled multimeric/multifunctional radiotracers.