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

Introduction: The 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 Cu-64-chelates have poor in vivo stability or harsh radiolabeling conditions. Methods: In this study, two triazacyclononane analogs: C-NE3TA (4-carboxymethyl-7-[2-(carboxymethylamino)-3-(4-nitro-phenyl)-propy]-[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 Cu-64 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 SOD mice. Results: C-NE3TA and N-NE3TA were labeled at >95% efficiency up to similar to 3.4 Ci/mu mol. 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 Cu-64-N-NE3TA and Cu-64-C-NE3TA were 96.1% and 90.5% intact after 48 h incubation in rat serum, respectively. This is compared to Cu-64 complexes of the control chelators, p-NH2-Bn-DOTA and p-NH2-Bn-NOTA, with 93.9% and 97.9% retention of Cu-64 in the complex, respectively. In vivo evaluation of Cu-64-N-NE3TA and Cu-64-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 Cu-64-N-NE3TA and Cu-64-C-NE3TA, respectively. This compares to 78% and 3% retention for Cu-64-p-NH2-Bn-DOTA and Cu-64-p-NH2-Bn-NOTA. Conclusions: These studies demonstrate that While N-NE3TA and C-NE3TA appear to be superior chelators for Cu-64 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. (C) 2012 Elsevier Inc. All rights reserved.