Evaluation of 64Cu-Labeled Bifunctional Chelate-Bombesin Conjugates

Several bifunctional chelates (BFCs) were investigated as carriers of Cu-64 for PET imaging. The most widely used chelator for Cu-64 labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N', N '',N '''-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with Cu-64, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-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 H2N-Aoc-[D-Tyr(6),beta Ala(11), Thi(13), Nle(14)]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 N-terminus of BBN. The partially protected aniline-BFC (p-H2N-Bn-PCTA(Ot-Bu)(3) or p-H2N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with Cu-64 using [Cu-64]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 degrees C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 degrees C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR All BFC-BBN Cu-64-conjugates were stable for over 20 h when incubated at 37 degrees C; in mouse plasma samples. However, in vivo, only 37% of the Cu-64/Oxo-DO3A complex remained intact after 20 h while the Cu-64/DOTA-BBN complex was completely demetalated. In contrast, both Cu-64/NOTA- and Cu-64/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 Cu-64-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.