Copper-64 radiolabeling and biological evaluation of bifunctional chelators for radiopharmaceutical development

IntroductionThe development of novel bifunctional chelates for attaching copper-64 to biomolecules has been an active area of research for several years. However, many of these 64Cu-chelates have poor in vivo stability or harsh radiolabeling conditions.MethodsIn this study, two triazacyclononane analogs; C-NE3TA (4-carboxymethyl-7-[2-(carboxymethyl-amino)-3-(4-nitro-phenyl)-propyl]-[1,4,7]triazo-nan-1-yl-acetic acid) and N-NE3TA (4-carboxymethyl-7-[2-[carboxymethyl-(4-nitro-benzyl)-amino]-ethyl]-[1,4,7]triazonan-1-yl-acetic acid) were evaluated for their labeling efficiency with 64Cu at room temperature and evaluated in vitro and in vivo. In vitro studies included complexation kinetics with Cu(II) using a spectrophotometric method and rat serum stability, while the in vivo biodistribution was evaluated using SCID mice.ResultsC-NE3TA and N-NE3TA were labeled at > 95% efficiency up to ~ 3.4 Ci/μmol. Both C-NE3TA and N-NE3TA formed complexes with Cu(II) almost immediately, with the Cu(II) complexation by C-NE3TA being faster than the formation of Cu(II)-N-NE3TA. Both 64Cu-N-NE3TA and 64Cu-C-NE3TA were 96.1% and 90.5% intact after 48 h incubation in rat serum, respectively. This is compared to 64Cu complexes of the control chelators, p-NH2-Bn-DOTA and p-NH2-Bn-NOTA, with 93.9% and 97.9% retention of 64Cu in the complex, respectively. In vivo evaluation of 64Cu-N-NE3TA and 64Cu-C-NE3TA demonstrates good clearance from normal tissues except for the liver, where 59% and 51% of the radioactivity is retained at 24 h compared to 1 h for 64Cu-N-NE3TA and 64Cu-C-NE3TA, respectively. This compares to 78% and 3% retention for 64Cu-p-NH2-Bn-DOTA and 64Cu-p-NH2-Bn-NOTA.ConclusionsThese studies demonstrate that while N-NE3TA and C-NE3TA appear to be superior chelators for 64Cu than p-NH2-Bn-DOTA, they are not better than p-NH2-Bn-NOTA. Nevertheless, it may still be interesting to evaluate these chelators after conjugation to biomolecules.