Biological stability evaluation of the α2β1 receptor imaging agents: Diamsar and DOTA conjugated DGEA peptide

Robust chelating stability under biological condi-tions is critical for the design of copper-based radiopharmaceuticals. In this study, the stabilities of ⁶⁴Cu-DOTA and diamsar (two bifunctional Cu-64 chelators (BFCs)) conjugated DGEA peptides were evaluated. The in vitro stabilities of ⁶⁴Cu-DOTA-DGEA, ⁶⁴Cu-DOTA-Ahx-DGEA, and ⁶⁴Cu-Z-E(diamsar)-Ahx-DGEA were evaluated in PBS. A carboxyl-protected DOTA-DGEA was also synthesized to study the potential inter- and intramolecular interactions between DOTA and the carboxylate groups of DGEA peptide. microPET imaging of ⁶⁴Cu-DOTA-DGEA and ⁶⁴Cu-Z- E(diamsar)-Ahx-DGEA were performed in PC-3 prostate tumor model to further investigate the in vivo behavior of the tracers. DOTA-DGEA, DOTA-Ahx-DGEA, Z-E(diamsar)-Ahx-DGEA, and protected DOTA-DGEA peptides were readily obtained, and their identities were confirmed by MS. ⁶⁴Cu²⁺ labeling was performed with high radiochemical yields (>98%) for all tracers after 1 h incubation. Stability experiments revealed that ⁶⁴Cu-DOTA-DGEA had unexpectedly high ⁶⁴Cu²⁺ dissociation when incubated in PBS (>55% free ⁶⁴Cu²⁺ was observed at 48 h time point). The ⁶⁴Cu²⁺ dissociation was significantly reduced in the carboxyl-protected ⁶⁴Cu-DOTA-DGEA complex but not in the ⁶⁴Cu-DOTA-Ahx-DGEA complex, which suggests the presence of competitive binding for ⁶⁴Cu²⁺ between DOTA and the carboxyl groups of the DGEA peptide. In contrast, no significant ⁶⁴Cu²⁺ dissociation was observed for ⁶⁴Cu-Z- E(diamsar)-Ahx-DGEA in PBS. For microPET imaging, the PC-3 tumors were clearly visualized with both ⁶⁴Cu-DOTA-DGEA and ⁶⁴Cu-Z-E(diamsar)- Ahx-DGEA tracers. However, ⁶⁴Cu-DOTA-DGEA demonstrated 5× higher liver uptake than ⁶⁴Cu-Z-E(diamsar)-Ahx-DGEA. This biodistribution variance could be attributed to the chelating stability difference between these two tracers, which correlated well with the PBS stability experiments. In summary, the in vitro and in vivo evaluations of ⁶⁴Cu-Z-E(diamsar)-Ahx-DGEA and ⁶⁴Cu-DOTA-DGEA have demonstrated the significantly superior Cu-chelation stability for the diamsar derivative compared with the established DOTA chelator. The results also suggest that diamsar may be preferred for Cu chelation especially when multiple carboxylic acid groups are present. Free carboxyl groups may naturally compete with DOTA for ⁶⁴Cu²⁺ binding and therefore reduce the complex stability.