Article,
Evaluation of <sup>64</sup>Cu-Labelled Bifunctional Chelate-Bombesin Conjugates.
Bioconjug Chem, (July 2011)
DOI: 10.1021/bc2002665
Abstract
Several bifunctional chelates (BFCs) were investigated as carriers of <sup>64</sup>Cu for PET imaging. The most widely used chelator for <sup>64</sup>Cu labelling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-<i>N</i>,<i>N</i>',<i>N</i>'',<i>N</i>'''-tretraacetic acid), even though this complex exhibits only moderate <i>in vivo</i> stability. In this study we prepared a series of alternative chelator-peptide conjugates labelled with <sup>64</sup>Cu, measured <i>in vitro</i> receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing protein receptor (GRPR) and compared their <i>in vivo</i> stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo9.3.1pentadeca-1(15),11,13-triene-3,6,9-triacetic acid) and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H<sub>2</sub>N-Aoc-D-Tyr<sup>6</sup>,ßAla<sup>11</sup>,Thi<sup>13</sup>,Nle<sup>14</sup>bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide-bond, a succinyl spacer was introduced at the <i>N</i>-terminus of BBN. The partially protected aniline-BFC (p-H<sub>2</sub>N-Bn-PCTA(Ot-Bu)<sub>3</sub> or p-H<sub>2</sub>N-Bn-DO3A(Ot-Bu)<sub>3</sub>) was then coupled to the resulting <i>N</i>-terminal carboxylic acid pre-activated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labelled with <sup>64</sup>Cu using <sup>64</sup>CuCu(OAc)<sub>2</sub> in 0.1M ammonium acetate buffer at 100°C for 15 min. Labelling efficacy was > 90\% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100°C to achieve this high yield. Specific activities varied from 76-101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN <sup>64</sup>Cu-conjugates were stable for over 20h when incubated at 37°C in mouse blood samples. However <i>in vivo</i>, only 37\% of the <sup>64</sup>Cu/Oxo-DO3A complex remained intact after 20h while the <sup>64</sup>Cu/DOTA-BBN complex was completely demetallated. In contrast both <sup>64</sup>Cu/NOTA- and <sup>64</sup>Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that <sup>64</sup>Cu-labelled NOTA- and PCTA-BBN peptide-conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.
