⁶⁴Cu labeled ambasar-RGD2 for micro-PET imaging of integrin α_vβ₃expression

Integrin α_vβ₃plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a ⁶⁴Cu-AmBaSar-RGD monomer with high in vivo stability compared with ⁶⁴Cu-DOTA-RGD was developed for integrin α_vβ₃3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated ⁶⁴Cu-labeled AmBaSar dimeric RGD conjugates (⁶⁴Cu-AmBaSar-RGD2) for PET imaging of integrin α_vβ₃ expression.The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with ⁶⁴Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of ⁶⁴Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4)and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]⁺ M: C₈₁H₁₂₅N₂₇O₁₉ 1779.9).⁶⁴Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥98.0%) under mild conditions(pH 5.0~5.5, 23~37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22GBq/μmol at the end of synthesis. Based on microPET imaging and biodistribution studies, ⁶⁴Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than ⁶⁴Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for ^64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for ⁶⁴Cu-AmBaSar-RGD2, respectively. The integrin α_vβ₃ targeting specificity was confirmed by blocking experiments. Therefore, the new tracer ⁶⁴Cu-AmBaSar-RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the ⁶⁴Cu labeled RGD monomer due to the polyvalency effect.