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 (64)Cu-DOTA and diamsar (two bifunctional Cu-64 chelators (BFCs)) conjugated DGEA peptides were evaluated. The in vitro stabilities of (64)Cu-DOTA-DGEA, (64)Cu-DOTA-Ahx-DGEA, and (64)Cu-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 (64)Cu-DOTA-DGEA and (64)Cu-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. (64)Cu(2+) labeling was performed with high radiochemical yields (>98%) for all tracers after 1 h incubation. Stability experiments revealed that (64)Cu-DOTA-DGEA had unexpectedly high (64)Cu(2+) dissociation when incubated in PBS (>55% free (64)Cu(2+) was observed at 48 h time point). The (64)Cu(2+) dissociation was significantly reduced in the carboxyl-protected (64)Cu-DOTA-DGEA complex but not in the (64)Cu-DOTA-Ahx-DGEA complex, which suggests the presence of competitive binding for (64)Cu(2+) between DOTA and the carboxyl groups of the DGEA peptide. In contrast, no significant (64)Cu(2+) dissociation was observed for (64)Cu-Z-E(diamsar)-Ahx-DGEA in PBS. For microPET imaging, the PC-3 tumors were clearly visualized with both (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA tracers. However, (64)Cu-DOTA-DGEA demonstrated 5× higher liver uptake than (64)Cu-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 (64)Cu-Z-E(diamsar)-Ahx-DGEA and (64)Cu-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 (64)Cu(2+) binding and therefore reduce the complex stability.