Evaluation of copper-64 labeled AmBaSar conjugated cyclic RGD peptide for improved MicroPET imaging of integrin α_vβ₃ expression

Recently, we have developed a new cage-like bifunctional chelator 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron emission tomography (PET) tracer ⁶⁴Cu-AmBaSar-RGD. In this study, we further evaluate the biological property of this new AmBaSar chelator by using ⁶⁴Cu-AmBaSar-RGD as the model compound. In vitro and in vivo stability, lipophilicity, cell binding and uptake, microPET imaging, receptor blocking experiments, and biodistribution studies of ⁶⁴Cu-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer ⁶⁴Cu-DOTA-RGD. The ⁶⁴Cu-AmBaSar-RGD was obtained with high radiochemical yield (≥95%) and purity (≥99%) under mild conditions (pH 5.0-5.5 and 23-37 °C) in less than 30 min. For in vitro studies, the radiochemical purity of ⁶⁴Cu-AmBaSar-RGD was more than 97% in PBS or FBS and 95% in mouse serum after 24 h of incubation. The log P value of ⁶⁴Cu-AmBaSar-RGD was-2.44 ± 0.12. For in vivo studies, ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on the basis of microPET imaging. The integrin α_vβ₃ receptor specificity was confirmed by blocking experiments for both tracers. Compared with ⁶⁴Cu-DOTA-RGD, ⁶⁴Cu-AmBaSar-RGD demonstrated much lower liver accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported the observation that ⁶⁴Cu-AmBaSar-RGD was more stable in vivo than ⁶⁴Cu-DOTA- RGD. In summary, the in vitro and in vivo evaluations of the ⁶⁴Cu-AmBaSar-RGD have demonstrated its improved Cu-chelation stability compared with that of the established tracer ⁶⁴Cu-DOTA-RGD. The AmBaSar chelator will also have general applications for ⁶⁴Cu labeling of various bioactive molecules in high radiochemical yield and high in vivo stability.