Evaluation of Copper-64 Labeled AmBaSar Conjugated Cyclic RGD Peptide for Improved MicroPET Imaging of Integrin αvβ3 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-l-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron emission tomography (PET) tracer Cu-64-AmBaSar-RGD. In this study, we further evaluate the biological property of this new AmBaSar chelator by using Cu-64-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 Cu-64-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer Cu-64-DOTA-RGD. The Cu-64-AmBaSar-RGD was obtained with high radiochemical yield (>= 95%) and purity (>= 99%) under mild conditions (pH 5.0-5.5 and 23-37 degrees C) in less than 30 min. For in vitro studies, the radiochemical purity of Cu-64-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 Cu-64-AmBaSar-RGD was -2.44 +/- 0.12. For in vivo studies, Cu-64-AmBaSar-RGD and Cu-64-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on the basis of microPET imaging. The integrin alpha(v)beta(3) receptor specificity was confirmed by blocking experiments for both tracers. Compared with Cu-64-DOTA-RGD, Cu-64-AmBaSar-RGD demonstrated much lower liver accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported the observation that Cu-64-AmBaSar-RGD was more stable in vivo than Cu-64-DOTA-ROD. In summary, the in vitro and in vivo evaluations of the Cu-64-AmBaSar-RGD have demonstrated its improved Cu-chelation stability compared with that of the established tracer Cu-64-DOTA-RGD. The AmBaSar chelator will also have general applications for Cu-64 labeling of various bioactive molecules in high radiochemical yield and high in vivo stability.